Efficient palladium-catalyzed electrocarboxylation enables late-stage carbon isotope labelling.

Carbon isotope labelling of bioactive molecules is essential for accessing the pharmacokinetic and pharmacodynamic properties of new drug entities. Aryl carboxylic acids represent an important class of structural motifs ubiquitous in pharmaceutically active molecules and are ideal targets for the installation of a radioactive tag employing isotopically labelled CO2. However, direct isotope incorporation via the reported catalytic reductive carboxylation (CRC) of aryl electrophiles relies on excess CO2, which is incompatible with carbon-14 isotope incorporation. Furthermore, the application of some CRC reactions for late-stage carboxylation is limited because of the low tolerance of molecular complexity by the catalysts. Herein, we report the development of a practical and affordable Pd-catalysed electrocarboxylation setup. This approach enables the use of near-stoichiometric 14CO2 generated from the primary carbon-14 source Ba14CO3, facilitating late-stage and single-step carbon-14 labelling of pharmaceuticals and representative precursors. The proposed isotope-labelling protocol holds significant promise for immediate impact on drug development programmes.

Lignocellulose Conversion via Catalytic Transformations Yields Methoxyterephthalic Acid Directly from Sawdust.

Poly(ethylene terephthalate) polyester represents the most common class of thermoplastic polymers widely used in the textile, bottling, and packaging industries. Terephthalic acid and ethylene glycol, both of petrochemical origin, are polymerized to yield the polyester. However, an earlier report suggests that polymerization of methoxyterephthalic acid with ethylene glycol provides a methoxy-polyester with similar properties. Currently, there are no established biobased synthetic routes toward the methoxyterephthalic acid monomer. Here, we show a viable route to the dicarboxylic acid from various tree species involving three catalytic steps. We demonstrate that sawdust can be converted to valuable aryl nitrile intermediates through hydrogenolysis, followed by an efficient fluorosulfation-catalytic cyanation sequence (>90%) and then converted to methoxyterephthalic acid by hydrolysis and oxidation. A preliminary polymerization result indicates a methoxy-polyester with acceptable thermal properties.

Recent Advances in the Multistep Continuous Preparation of APIs and Fine Chemicals.

Over the last two decades, with the advent of continuous flow technologies, continuous processes have emerged as a major area in organic synthesis. In this context, continuous flow processes have been increasing in the preparation of Active Pharmaceutical Ingredients (APIs) and fine chemicals, such as complex synthetic intermediates, agrochemicals, and fragrances. Thus, the development of multi-step protocols has attracted special interest from the academic and industrial chemistry communities. In addition to the beneficial aspects intrinsically associated with continuous processes (e.g., waste reduction, optimal heat transfer, improved safety, and the possibility to work under harsh reaction conditions and with more dangerous reagents), these protocols also allow a rapid increase in molecular complexity. Moreover, in telescoped multi-step processes, isolation and purification steps are generally avoided or, if necessary, carried out in-line, presenting an important economy of time, solvents, reagents, and labor. Last, important synthetic strategies such as photochemical and electrochemical reactions are compatible with flow processes and are delivering relevant advances to the synthetic approaches. In this review, a general overview of the fundamentals of continuous flow processes is presented. Recent examples of multi-step continuous processes for the preparation of fine chemicals, including telescoped and end-to-end processes, are discussed, pointing out the possible advantages and/or limitations of each of these methodologies.

Hierarchical compound topology uncovers complex structure of species interaction networks.

Nestedness and modularity have been found in many species interaction networks. Despite being conceptually distinct, negatively correlated and having different causes, these patterns often co-occur. A realistic but seldom investigated alternative to these simple topologies is hierarchical compound networks, in which the entire network is modular, and modules are internally nested. In compound networks, nestedness is suppressed by modularity at higher network hierarchical levels, but prevails at lower levels, within modules. The aims of this study are (i) to evaluate the prevalence of simple and hierarchical compound topologies in binary and weighted networks describing different kinds of species interactions and (ii) to probe the relationships between modularity and nestedness at different network hierarchical levels. With a procedure that discriminates between simple and compound structures, we re-analysed the topology of 142 well-studied binary networks including seed dispersal, host-parasite, pollination and plant-herbivore interactions; 68 of these also had quantitative information. Additionally, we tested the relationship between robustness and topology of binary networks and compared the robustness of networks with compound topologies to different sequences of species removals. Compound topologies were detected in 34% of binary and 71% of weighted networks of all interaction kinds. These results establish the hierarchical compound topology as a widespread network architecture, often undetected without quantitative data. Furthermore, they disentangle an apparent paradox: despite conflicting with overall nestedness, modularity usually co-occurs with high values of low-level nestedness. Nestedness progressively decreased, while modularity increased, from seed dispersal to host-parasite, pollination and plant-herbivore networks. There were no consistent differences in the robustness of networks with nested and compound topologies. However, compound topologies were especially vulnerable to removal sequences that accelerate the exclusion of entire modules. Compound topologies improve the depiction of ecological networks and differentiate ecological and evolutionary processes that operate at different hierarchical levels, with the potential to advance our understanding of network dynamics, stability and response to species loss or change. Quantitative data often reveal specialization patterns that are indistinguishable in binary networks, strongly improving the detection of modular and compound topologies.

Prevalence of genetically similar Flavobacterium columnare phages across aquaculture environments reveals a strong potential for pathogen control.

Intensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria. Flavobacterium columnare causes columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126 F. columnare strains and 63 phages against F. columnare from Finland and Sweden in 2017. Bacterial isolates were virulent on rainbow trout (Oncorhynchus mykiss) and fell into four previously described genetic groups A, C, E and G, with genetic groups C and E being the most virulent. Phage host range studied against a collection of 227 bacterial isolates (from 2013 to 2017) demonstrated modular infection patterns based on host genetic group. Phages infected contemporary and previously isolated bacterial hosts, but bacteria isolated most recently were generally resistant to previously isolated phages. Despite large differences in geographical origin, isolation year or host range of the phages, whole-genome sequencing of 56 phages showed high level of genetic similarity to previously isolated F. columnare phages (Ficleduovirus, Myoviridae). Altogether, this phage collection demonstrates a potential for use in phage therapy.

Origin of Enantioselectivity in Chiral Phosphoric-Acid-Catalyzed Azlactone Dynamic Kinetic Resolution.

Theoretical calculations, associated with control experiments, were carried out to gain insights into the mechanism and origin of enantioselectivity in the phosphoric-acid-catalyzed dynamic kinetic resolution of azlactones. The results revealed a Münchnone intermediate as the key species involved in the isomerization of azlactone rings. The developed model was successfully employed in the comprehension and prediction of enantioselectivity under diverse of reaction conditions, including alcoholysis and aminolysis protocols.

Host migration and environmental temperature influence avian haemosporidians prevalence: a molecular survey in a Brazilian Atlantic rainforest.

Avian haemosporidians are parasites with great capacity to spread to new environments and new hosts, being considered a good model to host-parasite interactions studies. Here, we examine avian haemosporidian parasites in a protected area covered by Restinga vegetation in northeastern Brazil, to test the hypothesis that haemosporidian prevalence is related to individual-level traits (age and breeding season), species-specific traits (diet, foraging strata, period of activity, species body weight, migratory status, and nest shape), and climate factors (temperature and rainfall). We screened DNA from 1,466 birds of 70 species captured monthly from April 2013 to March 2015. We detected an overall prevalence (Plasmodium/Haemoproteus infection) of 22% (44 host species) and parasite's lineages were identified by mitochondrial cyt b gene. Our results showed that migration can be an important factor predicting the prevalence of Haemoproteus (Parahaemoproteus), but not Plasmodium, in hosts. Besides, the temperature, but not rainfall, seems to predict the prevalence of Plasmodium in this bird community. Neither individual-level traits analyzed nor the other species-specific traits tested were related to the probability of a bird becoming infected by haemosporidians. Our results point the importance of conducting local studies in particular environments to understand the degree of generality of factors impacting parasite prevalence in bird communities. Despite our attempts to find patterns of infection in this bird community, we should be aware that an avian haemosporidian community organization is highly complex and this complexity can be attributed to an intricate net of factors, some of which were not observed in this study and should be evaluated in future studies. We evidence the importance of looking to host-parasite relationships in a more close scale, to assure that some effects may not be obfuscated by differences in host life-history.

Mutual Information as a General Measure of Structure in Interaction Networks.

Entropy-based indices are long-established measures of biological diversity, nowadays used to gauge partitioning of diversity at different spatial scales. Here, we tackle the measurement of diversity of interactions among two sets of organisms, such as plants and their pollinators. Actual interactions in ecological communities are depicted as bipartite networks or interaction matrices. Recent studies concentrate on distinctive structural patterns, such as nestedness or modularity, found in different modes of interaction. By contrast, we investigate mutual information as a general measure of structure in interactive networks. Mutual information (MI) measures the degree of reciprocal matching or specialization between interacting organisms. To ascertain its usefulness as a general measure, we explore (a) analytical solutions for different models; (b) the response of MI to network parameters, especially size and occupancy; (c) MI in nested, modular, and compound topologies. MI varies with fundamental matrix parameters: dimension and occupancy, for which it can be adjusted or normalized. Apparent differences among topologies are contingent on dimensions and occupancy, rather than on topological patterns themselves. As a general measure of interaction structure, MI is applicable to conceptually and empirically fruitful analyses, such as comparing similar ecological networks along geographical gradients or among interaction modalities in mutualistic or antagonistic networks.

Electron-Donor-Acceptor Complex-Enabled Flow Methodology for the Hydrotrifluoromethylation of Unsaturated ß-Keto Esters.

A novel electron-donor-acceptor (EDA) complex-enabled flow photochemical hydrotrifluoromethylation of unsaturated ß-keto esters is described. The developed protocol has an easy experimental procedure and does not require the use of transition-metal-based photocatalysts, allowing the isolation of 14 new compounds in up to 86% yield. Control experiments and computational studies revealed that the reaction proceeds through a Michael-type 1,4-addition of a trifluoromethyl radical, followed by a proton transfer step. Furthermore, the reaction could be scaled up to 1 mmol, and the final product could be employed in the preparation of an isoxazolone and a pyrazolone as trifluoro-substituted heterocycles.

Bacteriophage Adherence to Mucus Mediates Preventive Protection against Pathogenic Bacteria.

Metazoans were proposed to host bacteriophages on their mucosal surfaces in a symbiotic relationship, where phages provide an external immunity against bacterial infections and the metazoans provide phages a medium for interacting with bacteria. However, scarce empirical evidence and model systems have left the phage-mucus interaction poorly understood. Here, we show that phages bind both to porcine mucus and to rainbow trout (Oncorhynchus mykiss) primary mucus, persist up to 7 days in the mucosa, and provide protection against Flavobacterium columnare Also, exposure to mucus changes the bacterial phenotype by increasing bacterial virulence and susceptibility to phage infections. This trade-off in bacterial virulence reveals ecological benefit of maintaining phages in the metazoan mucosal surfaces. Tests using other phage-bacterium pairs suggest that phage binding to mucus may be widespread in the biosphere, indicating its importance for disease, ecology, and evolution. This phenomenon may have significant potential to be exploited in preventive phage therapy.IMPORTANCE The mucosal surfaces of animals are habitat for microbes, including viruses. Bacteriophages-viruses that infect bacteria-were shown to be able to bind to mucus. This may result in a symbiotic relationship in which phages find bacterial hosts to infect, protecting the mucus-producing animal from bacterial infections in the process. Here, we studied phage binding on mucus and the effect of mucin on phage-bacterium interactions. The significance of our research is in showing that phage adhesion to mucus results in preventive protection against bacterial infections, which will serve as basis for the development of prophylactic phage therapy approaches. Besides, we also reveal that exposure to mucus upregulates bacterial virulence and that this is exploited by phages for infection, adding one additional layer to the metazoan-bacterium-phage biological interactions and ecology. This phenomenon might be widespread in the biosphere and thus crucial for understanding mucosal diseases, their outcome and treatment.

The Fate of Bacteriophages in Recirculating Aquaculture Systems (RAS)-Towards Developing Phage Therapy for RAS.

Aquaculture production has increased tremendously during the last decades, and new techniques have been developed, e.g., recirculating aquaculture systems (RAS). In RAS, the majority of water volume is circulated via mechanical and biological filters and reused in the tanks. However, the prevention and treatment of diseases in these systems are challenging, as the pathogens spread throughout the system, and the addition of chemicals and antibiotics disrupts the microbiome of the biofilters. The increasing antibiotic resistance has made phage therapy a relevant alternative for antibiotics in food production. Indeed, as host-specific and self-replicating agent they might be optimal for targeted pathogen eradication in RAS. We tested the survival and spread of Flavobacterium columnare -infecting phage FCL-2 in recirculating aquaculture fish farm with rainbow trout (Oncorhynchus mykiss) in a fully controlled study. After a single addition, phage persisted in water samples collected from tank, fixed bed, moving bed, and aeration unit up to 14 days, and in the water of rearing tanks, rainbow trout mucus, and bioreactor carrier media from the fixed and moving bed biofilters for 21 days. Furthermore, phage adsorbed preferentially to moving bed carrier media, which contained biofilm attached and from which higher phage numbers were recovered. This study shows phages as a potent strategy for maintaining biosecurity in RAS systems.

gem-Dichlorocyclopropanation of Dicarbonyl Derivatives.

A novel methodology for the 1,1-dichlorocyclopropanation of dicarbonyl conjugated olefins was described. The developed protocol is simple and uses readily accessible starting materials, allowing the isolation of the desired adducts in moderate to excellent yields (up to 99 %). Furthermore, the reaction tolerated scale up to the gram scale; thus highlighting the synthetic potential of this transformation. Control experiments and DFT studies revealed that the reaction proceeded through a Michael-initiated ring-closure process, in which reaction temperature played a crucial role. Finally, these gem-dichlorocyclopropanes were also employed in the preparation of a trisubstituted naphthyl derivative and a diastereoselective reduction was also demonstrated.

Quantum Chemical-Guided Steglich Rearrangement of Azlactones and Isoxazolones.

The theoretical-guided evaluation of the Steglich rearrangement of azlactones and isoxazolones allowed the determination of the reactivity patterns in these heterocycles, including the factors that drive the regioselectivity toward both possible sites. These results allowed the first experimental report on the regioselective Steglich rearrangement of isoxazolones, affording the nitrogen- or carbon-acyloxy adducts.

A new model explaining the origin of different topologies in interaction networks.

Nestedness and modularity have been recurrently observed in species interaction networks. Some studies argue that those topologies result from selection against unstable networks, and others propose that they likely emerge from processes driving the interactions between pairs of species. Here we present a model that simulates the evolution of consumer species using resource species following simple rules derived from the integrative hypothesis of specialization (IHS). Without any selection on stability, our model reproduced all commonly observed network topologies. Our simulations demonstrate that resource heterogeneity drives network topology. On the one hand, systems containing only homogeneous resources form generalized nested networks, in which generalist consumers have higher performance on each resource than specialists. On the other hand, heterogeneous systems tend to have a compound topology: modular with internally nested modules, in which generalists that divide their interactions between modules have low performance. Our results demonstrate that all real-world topologies likely emerge through processes driving interactions between pairs of species. Additionally, our simulations suggest that networks containing similar species differ from heterogeneous networks and that modules may not present the topology of entire networks.

Aquaculture as a source of empirical evidence for coevolution between CRISPR-Cas and phage.

So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution. Existing data from disease surveillance conducted in aquaculture, coupled with growing interest towards phage therapy, may have already resulted in large collections of bacterium and phage isolates. These data, combined with premeditated efforts, can provide empirical evidence on phage-bacterium dynamics such as the bacteriophage adherence to mucus hypothesis, phage life cycles and their relationship with CRISPR-Cas and other immune defences. Typing of CRISPR spacer content in pathogenic bacteria can also provide practical information on diversity and origin of isolates during outbreaks. In addition to providing information of CRISPR functionality and phage-bacterium dynamics, aquaculture systems can significantly impact perspectives on design of phage-based disease treatment at the current era of increasing antibiotic resistance. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma.

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label-free shotgun UDMSE approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728.

Theoretical Study on the Epimerization of Azlactone Rings: Keto-Enol Tautomerism or Base-Mediated Racemization?

Azlactones are versatile heterocycles employed in a diversity of transformations; the main drawback of these cycles consists in the epimerization of the α-carbonyl stereocenter during its preparation. We hereby present a theoretical study to explain how the racemization occurs. Two hypotheses were investigated: the keto-enol tautomerism and the base-mediated racemization, through an enolate intermediate. The results showed that the latter is consistent with the experimental data and can spontaneously occur at room temperature. The same pathway was evaluated for 2-alcoxy azlactone, showing a slower epimerization ratio, consistent with the literature data.

The major Alternaria alternata allergen, Alt a 1: A reliable and specific marker of fungal contamination in citrus fruits.

The ubiquitously present spores of Alternaria alternata can spoil a wide variety of foodstuffs, including a variety of fruits belonging to the Citrus genus. The major allergenic protein of A. alternata, Alt a 1, is a species-specific molecular marker that has been strongly associated with allergenicity and phytopathogenicity of this fungal species. This study aimed to evaluate the potential of the detection of Alt a 1 as a reliable indicator of A. alternata contamination in citrus fruits. To accomplish this aim, sixty oranges were artificially infected with a spore suspension of A. alternata. Internal fruit material was collected at different incubation times (one, two and three weeks after the fungal inoculation) and used for both total RNA extraction and protein extraction. Alt a 1 detection was then performed by polymerase chain reaction (PCR) amplification using Alt a 1 specific primers and by enzyme-linked immunosorbent assay (ELISA). The experimental model presented in this work was effective to simulate the typical Alternaria black rot phenotype and its progression. Although both PCR and ELISA techniques have been successfully carried out for detecting Alt a 1 allergen in A. alternata infected oranges, the PCR method was found to be more sensitive than ELISA. Nevertheless, ELISA results were highly valuable to demonstrate that considerable amounts of Alt a 1 are produced during A. alternata fruit infection process, corroborating the recently proposed hypothesis that this protein plays a role in the pathogenicity and virulence of Alternaria species. Such evidence suggests that the detection of Alt a 1 by PCR-based assay may be used as a specific indicator of the presence of pathogenic and allergenic fungal species, A. alternata, in fruits. This knowledge can be employed to control the fungal infection and mitigate agricultural losses as well as human exposure to A. alternata allergens and toxins.

Chemoselective Reduction of Azlactones Using Schwartz's Reagent.

Highly chemoselective addition of Schwartz's reagent to widely available azlactones is described. This method allows the preparation of challenged functionalized α-amino aldehydes, in good to high isolated yields at room temperature, after only 2 min reaction. The presence of sensitive functionalities or electronic factors does not compromise the potential of the method. The use of an excess of the reducing reagent gave a very functionalized allylic alcohol derivative in 86% yield.