Space and genealogy determine inter-individual differences in siderophore gene expression in bacterial colonies.

Heterogeneity in gene expression is common among clonal cells in bacteria, although the sources and functions of variation often remain unknown. Here, we track cellular heterogeneity in the bacterium Pseudomonas aeruginosa during colony growth by focusing on siderophore gene expression (pyoverdine versus pyochelin) important for iron nutrition. We find that the spatial position of cells within colonies and non-genetic yet heritable differences between cell lineages are significant sources of cellular heterogeneity, while cell pole age and lifespan have no effect. Regarding functions, our results indicate that cells adjust their siderophore investment strategies along a gradient from the colony center to its edge. Moreover, cell lineages with below-average siderophore investment benefit from lineages with above-average siderophore investment, presumably due to siderophore sharing. Our study highlights that single-cell experiments with dual gene expression reporters can identify sources of gene expression variation of interlinked traits and offer explanations for adaptive benefits in bacteria.

[1-11C]-Butanol Positron Emission Tomography reveals an impaired brain to nasal turbinates pathway in aging amyloid positive subjects.

BACKGROUND: Reduced clearance of cerebrospinal fluid (CSF) has been suggested as a pathological feature of Alzheimer's disease (AD). With extensive documentation in non-human mammals and contradictory human neuroimaging data it remains unknown whether the nasal mucosa is a CSF drainage site in humans. Here, we used dynamic PET with [1-11C]-Butanol, a highly permeable radiotracer with no appreciable brain binding, to test the hypothesis that tracer drainage from the nasal pathway reflects CSF drainage from brain. As a test of the hypothesis, we examined whether brain and nasal fluid drainage times were correlated and affected by brain amyloid. METHODS: 24 cognitively normal subjects (≥ 65 years) were dynamically PET imaged for 60 min. using [1-11C]-Butanol. Imaging with either [11C]-PiB or [18F]-FBB identified 8 amyloid PET positive (Aß+) and 16 Aß- subjects. MRI-determined regions of interest (ROI) included: the carotid artery, the lateral orbitofrontal (LOF) brain, the cribriform plate, and an All-turbinate region comprised of the superior, middle, and inferior turbinates. The bilateral temporalis muscle and jugular veins served as control regions. Regional time-activity were used to model tracer influx, egress, and AUC. RESULTS: LOF and All-turbinate 60 min AUC were positively associated, thus suggesting a connection between the brain and the nose. Further, the Aß+ subgroup demonstrated impaired tracer kinetics, marked by reduced tracer influx and slower egress. CONCLUSION: The data show that tracer kinetics for brain and nasal turbinates are related to each other and both reflect the amyloid status of the brain. As such, these data add to evidence that the nasal pathway is a potential CSF drainage site in humans. These data warrant further investigation of brain and nasal contributions to protein clearance in neurodegenerative disease.

An LRR receptor kinase controls ABC transporter substrate preferences during plant growth-defense decisions.

The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions.

Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Unique Initiation and Termination Mechanisms Involved in the Biosynthesis of a Hybrid Polyketide-Nonribosomal Peptide Lyngbyapeptin B Produced by the Marine Cyanobacterium Moorena bouillonii.

Lyngbyapeptin B is a hybrid polyketide-nonribosomal peptide isolated from particular marine cyanobacteria. In this report, we carried out genome sequence analysis of a producer cyanobacterium Moorena bouillonii to understand the biosynthetic mechanisms that generate the unique structural features of lyngbyapeptin B, including the (E)-3-methoxy-2-butenoyl starter unit and the C-terminal thiazole moiety. We identified a putative lyngbyapeptin B biosynthetic (lynB) gene cluster comprising nine open reading frames that include two polyketide synthases (PKSs: LynB1 and LynB2), four nonribosomal peptide synthetases (NRPSs: LynB3, LynB4, LynB5, and LynB6), a putative nonheme diiron oxygenase (LynB7), a type II thioesterase (LynB8), and a hypothetical protein (LynB9). In vitro enzymatic analysis of LynB2 with methyltransferase (MT) and acyl carrier protein (ACP) domains revealed that the LynB2 MT domain (LynB2-MT) catalyzes O-methylation of the acetoacetyl-LynB2 ACP domain (LynB2-ACP) to yield (E)-3-methoxy-2-butenoyl-LynB2-ACP. In addition, in vitro enzymatic analysis of LynB7 revealed that LynB7 catalyzes the oxidative decarboxylation of (4R)-2-methyl-2-thiazoline-4-carboxylic acid to yield 2-methylthiazole in the presence of Fe2+ and molecular oxygen. This result indicates that LynB7 is responsible for the last post-NRPS modification to give the C-terminal thiazole moiety in lyngbyapeptin B biosynthesis. Overall, we identified and characterized a new marine cyanobacterial hybrid PKS-NRPS biosynthetic gene cluster for lyngbyapeptin B production, revealing two unique enzymatic logics.

Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases.

Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (kcat < 1 h-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.

Development of a quantification method for routine analysis of glucosinolates and camalexin in brassicaceous small-sized samples by simultaneous extraction prior to liquid chromatography determination.

Glucosinolates and camalexin are secondary metabolites that, as phytoanticipins and phytoalexins, play a crucial role in plant defence. The present work proposes an improved analytical method for routine analysis and quantification of glucosinolates and camalexin in brassicaceous small-sized samples by using the very specific desulfation process of glucosinolates analysis and the specificity of fluorescence detection for camalexin analysis. The approach is based on a simultaneous ultrasound-assisted extraction followed by a purification on an anion-exchange column. Final analyses are conducted by HPLC-UV-MS for desulfo-glucosinolates and HPLC coupled to a fluorescence detector (HPLC-FLD) for camalexin. The method is linear for glucosinolates (50-3500 µM) and camalexin (0.025-5 µg.mL-1) with an LOD/LOQ of 3.8/12.6 µM and 0.014/0.046 µg.mL-1 respectively. The method demonstrated adequate precision, accuracy and trueness on certified reference rapeseed. A practical application of our approach was conducted on different Brassicaceae genera (Barbarea vulgaris, Brassica nigra, Capsella bursa-pastoris, Cardamine hirsuta, Coincya monensis, Sinapis arvensis, and Sisymbrium officinale) and Arabidopsis thaliana genotypes (Columbia and Wassilewskija). Futhermore, different plant organs (seeds and leaves) were analysed, previously inoculated or not with the pathogenic fungus Alternaria brassicicola.

Exchange of Vitamin B1 and Its Biosynthesis Intermediates Shapes the Composition of Synthetic Microbial Cocultures and Reveals Complexities of Nutrient Sharing.

Microbial communities occupy diverse niches in nature, and community members routinely exchange a variety of nutrients among themselves. While large-scale metagenomic and metabolomic studies shed some light on these exchanges, the contribution of individual species and the molecular details of specific interactions are difficult to track. In this study, we follow the exchange of vitamin B1 (thiamin) and its intermediates between microbes within synthetic cocultures of Escherichia coli and Vibrio anguillarum. Thiamin contains two moieties, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole (THZ), which are synthesized by distinct pathways using enzymes ThiC and ThiG, respectively, and then coupled by ThiE to form thiamin. Even though E. coli ΔthiC, ΔthiE, and ΔthiG mutants are thiamin auxotrophs, we observed that cocultures of ΔthiC-ΔthiE and ΔthiC-ΔthiG mutants are able to grow in a thiamin-deficient medium, whereas the ΔthiE-ΔthiG coculture does not. Further, the exchange of thiamin and its intermediates in V. anguillarum cocultures and in mixed cocultures of V. anguillarum and E. coli revealed that there exist specific patterns for thiamin metabolism and exchange among these microbes. Our findings show that HMP is shared more frequently than THZ, concurrent with previous observations that free HMP and HMP auxotrophy is commonly found in various environments. Furthermore, we observe that the availability of exogenous thiamin in the media affects whether these strains interact with each other or grow independently. These findings collectively underscore the importance of the exchange of essential metabolites as a defining factor in building and modulating synthetic or natural microbial communities. IMPORTANCE Vitamin B1 (thiamin) is an essential nutrient for cellular metabolism. Microorganisms that are unable to synthesize thiamin either fully or in part exogenously obtain it from their environment or via exchanges with other microbial members in their community. In this study, we created synthetic microbial cocultures that rely on sharing thiamin and its biosynthesis intermediates and observed that some of them are preferentially exchanged. We also observed that the coculture composition is dictated by the production and/or availability of thiamin and its intermediates. Our studies with synthetic cocultures provide the molecular basis for understanding thiamin sharing among microorganisms and lay out broad guidelines for setting up synthetic microbial cocultures by using the exchange of an essential metabolite as their foundation.

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module.

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

Reprogramming the Biosynthesis of Precursor Peptide to Create a Selenazole-Containing Nosiheptide Analogue.

Nosiheptide (NOS), a potent bactericidal thiopeptide, belongs to a class of natural products produced by ribosomal synthesis and post-translational modifications, and its biosynthetic pathway has largely been elucidated. However, the central trithiazolylpyridine structure of NOS remains inaccessible to structural changes. Here we report the creation of a NOS analogue containing a unique selenazole ring by the construction of an artificial system in Streptomyces actuosus ATCC25421, where the genes responsible for the biosynthesis of selenoprotein from Escherichia coli and the biosynthetic gene cluster of NOS were rationally integrated to produce a selenazole-containing analogue of NOS. The thiazole at the fifth position in NOS was specifically replaced by a selenazole to afford the first selenazole-containing "unnatural" natural product. The present strategy is useful for structural manipulation of various RiPP natural products.

Nitazoxanide and related thiazolides induce cell death in cancer cells by targeting the 20S proteasome with novel binding modes.

Nitazoxanide and related thiazolides are a novel class of anti-infectious agents against protozoan parasites, bacteria and viruses. In recent years, it is demonstrated that thiazolides can also induce cell cycle arrest and apoptotic cell death in cancer cells. Due to their fast proliferating nature, cancer cells highly depend on the proteasome system to remove aberrant proteins. Many of these aberrant proteins are regulators of cell cycle progression and apoptosis, such as the cyclins, BCL2 family members and nuclear factor of κB (NF-κB). Here, we demonstrate at both molecular and cellular levels that the 20S proteasome is a direct target of NTZ and related thiazolides. By concurrently inhibiting the multiple catalytic subunits of 20S proteasome, NTZ promotes cell cycle arrest and triggers cell death in colon cancer cells, either directly or as a sensitizer to other anti-tumor agents, especially doxorubicin. We further show that the binding mode of NTZ in the ß5 subunit of the 20S proteasome is different from that of bortezomib and other existing proteasome inhibitors. These findings provide new insights in the design of novel small molecular proteasome inhibitors as anti-tumor agents suitable for solid tumor treatment in an oral dosing form.

Monitoring residue levels and dietary risk assessment of thiamethoxam and its metabolite clothianidin for Chinese consumption of Chinese kale.

BACKGROUND: Thiamethoxam is widely used to control pests in Chinese kale, popularly consumed leafy vegetables. The potential risk to the environment and human health has aroused much public concern. Therefore, it is important to investigate the degradation behavior, residue distribution and dietary risk assessment of thiamethoxam in Chinese kale. RESULTS: A sensitive analytical method for determination of thiamethoxam and its metabolite clothianidin residue in Chinese kale was established and validated through a quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The recoveries were 85.4-101.2% for thiamethoxam and 79.5-108.1% for clothianidin, with the relative standard deviations (RSDs) of 0.9-10.2% and 1.8-6.0%, respectively. For the dissipation kinetics, the data showed that thiamethoxam in Chinese kale was degraded with the half-lives of 4.1 to 4.5 days. In the terminal residue experiments, the residues of thiamethoxam were 0.017-0.357 mg kg-1 after application 2-3 times with a preharvest interval (PHI) of 7 days under the designed dosages. The chronic and acute dietary exposure assessment risk quotient (RQ) values of thiamethoxam in Chinese kale for different Chinese consumers were 0.08-0.19% and 0.05-0.12%, respectively, and those of clothianidin were 0.01-0.04% and 0.02-0.04%, respectively, all of the RQ values were lower than 100%. CONCLUSION: Thiamethoxam in Chinese kale was rapidly degraded following first-order kinetics models. The dietary risk of thiamethoxam and clothianidin through Chinese kale was negligible to consumers. The results from this study are important reference for Chinese governments to developing criteria for the safe and rational use of thiamethoxam, setting maximum residue levels (MRLs), monitoring the quality safety of agricultural products and protecting consumer health. © 2021 Society of Chemical Industry.

In Silico Docking of Novel Phytoalkaloid Camalexin in the Management of Benomyl Induced Parkinson's Disease and its In Vivo Evaluation by Zebrafish Model.

BACKGROUND: Parkinson's Disease (PD) exhibits the extrapyramidal symptoms caused due to the dopaminergic neuronal degeneration in the substantia nigra of the brain and depletion of Aldehyde Dehydrogenase (ALDH) enzyme. OBJECTIVE: This study was designed to enlighten the importance of the Aldehyde dehydrogenase enzyme in protecting the dopamine levels in a living system. Camalexin, a potentially active compound, has been evaluated for its dopamine enhancing and aldehyde dehydrogenase protecting role in pesticide-induced Parkinson's disease. METHODS: AutoDock 4.2 software was employed to perform the docking simulations between the ligand camalexin and standard drugs Alda-1, Ropirinole with three proteins 4WJR, 3INL, 5AER. Consequently, the compound was evaluated for its in vivo neuroprotective role in the zebrafish model by attaining Institutional Animal Ethical Committee permission. The behavioral assessments and catecholamine analysis in zebrafish were performed. RESULTS: The Autodock result shows that the ligand camalexin has a lower binding energy (-3.84) that indicates a higher affinity with the proteins when compared to the standard drug of proteins (-3.42). In the zebrafish model, behavioral studies provided evidence that camalexin helps in the improvement of motor functions and cognition. The catecholamine assay has proved that there is an enhancement in dopamine levels, as well as an improvement in aldehyde dehydrogenase enzyme. CONCLUSION: The novel compound, camalexin, offers a protective role in Parkinson's disease model by its interaction with neurochemical proteins and also in alternative in vivo model.

A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole.

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase whose inhibitors are useful for the regulation of the actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole scaffold. The reversible trans-cis isomerization by visible light stimuli enabled us to manipulate ROCK activities in vitro and in cells.

Effect of drug metabolism in the treatment of SARS-CoV-2 from an entirely computational perspective.

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.

Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase.

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

The hidden effects of agrochemicals on plant metabolism and root-associated microorganisms.

Agrochemicals are commonly used in agriculture to protect crops and ensure yields. Several of them are mobile within the plant and, being perceived as xenobiotics regardless of their protective/curative roles, they induce a reprogramming of secondary metabolism linked to the detoxification processes even in the absence of phenotype symptoms. Moreover, it is well documented that plants are able to shape the microbial population at the rhizosphere and to significantly affect the processes occurring therein thanks to the root exudation of different metabolites. Here we show that plant metabolic response to foliarly-applied pesticides is much broader than what previously thought and includes diverse and compound-specific hidden processes. Among others, stress-related metabolism and phytohormones profile underwent a considerable reorganization. Moreover, a distinctive microbial rearrangement of the rhizosphere was recorded following foliar application of pesticides. Such effects have unavoidably energetic and metabolic costs for the plant paving the way to both positive and negative aspects. The understanding of these effects is crucial for an increasingly sustainable use of pesticides in agriculture.

A novel bivalent chromatin associates with rapid induction of camalexin biosynthesis genes in response to a pathogen signal in Arabidopsis.

Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, trimethylation of lysine 27 of histone 3 (H3K27me3) (repression) and acetylation of lysine 18 of histone 3 (H3K18ac) (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27me3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name as kairostat, plays an important role in controlling the timely induction of gene expression upon stress stimuli in plants.

In the fight against harmful fungi and bacteria, plants have an arsenal of chemicals at their disposal. For instance, species in the crucifer family ­ which includes mustard, cabbages and the model plant Arabidopsis thaliana ­ can defend themselves with camalexin, a compound produced soon after the plant receives signals from its attacker. What controls this precise timing, however, is still unclear. For the genes that rule the production of camalexin to be 'read', interpreted, and ultimately converted into proteins, their DNA sequences first need to be physically accessible to the cell. This availability is controlled, in part, by adding or removing chemical groups onto histones, the spool-like structures which DNA wraps around. These precisely controlled modifications ultimately help to activate or repress a gene. Sometimes, activating and inhibiting chemical groups can be present in the same location, creating what is known as a bivalent chromatin domain. Zhao et al. investigated whether histone modifications regulate when A. thaliana produces camalexin in response to an attack. A combination of bioinformatics and experimental approaches highlighted two chemical modifications (one repressive, the other activating) which were present on the same histone, forming a previously unknown bivalent chromatin domain. Mutant plants which did not carry these modifications could not produce camalexin at the right time. Further experiments showed that under normal conditions, both histone modifications were present. However, when the plant was under attack, the level of repressive and activating modifications respectively decreased and increased, leading to gene activation. Together, the results by Zhao et al. suggest that both histone modifications are required for camalexin genes to respond appropriately to signals from a harmful agent. A deeper understanding of this new mechanism could, in turn, allow scientists to engineer crops that are better at resisting disease.

Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.