Catalytic trajectory of a dimeric nonribosomal peptide synthetase subunit with an inserted epimerase domain.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique "H"-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and L-cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents

Gold Nanorods Conjugated Porous Silicon Nanoparticles Encapsulated in Calcium Alginate Nano Hydrogels Using Microemulsion Templates

Porous silicon nanoparticles (PSiNPs) and gold nanorods (AuNRs) can be used as biocompatible nanocarriers for delivery of therapeutics but undesired leakage makes them inefficient. By encapsulating the PSiNPs and AuNRs in a hydrogel shell, we create a biocompatible functional nano carrier that enables sustained release of therapeutics. Here, we report the fabrication of AuNRs-conjugated PSi nanoparticles (AuNRsPSiNPs) through two-step chemical reaction for high capacity loading of hydrophobic and hydrophilic therapeutics with photothermal property. Furthermore, using water-in-oil microemulsion templates, we encapsulate the AuNRsPSiNPs within a calcium alginate hydrogel nanoshell, creating a versatile biocompatible nanocarrier to codeliver therapeutics for biomedical applications. We find that the functionalized nanohydrogel effectively controls the release rate of the therapeutics while maintaining a high loading efficiency and tunable loading ratios. Notably, combinations of therapeutics coloaded in the functionalized nanohydrogels significantly enhance inhibition of multidrug resistance through synergism and promote faster cancer cell death when combined with photothermal therapy. Moreover, the AuNRs can mediate the conversion of near-infrared laser radiation into heat, increasing the release of therapeutics as well as thermally inducing cell damage to promote faster cancer cell death. Our AuNRsPSiNPs functionalized calcium alginate nanohydrogel holds great promise for photothermal combination therapy and other advanced biomedical applications.Peer reviewe

Coexistence of multiuser entanglement distribution and classical light in optical fiber network with a semiconductor chip

Building communication links among multiple users in a scalable and robust way is a key objective in achieving large-scale quantum networks. In realistic scenario, noise from the coexisting classical light is inevitable and can ultimately disrupt the entanglement. The previous significant fully connected multiuser entanglement distribution experiments are conducted using dark fiber links and there is no explicit relation between the entanglement degradations induced by classical noise and its error rate. Here we fabricate a semiconductor chip with a high figure-of-merit modal overlap to directly generate broadband polarization entanglement. Our monolithic source maintains polarization entanglement fidelity above 96% for 42 nm bandwidth with a brightness of 1.2*10^7 Hz/mW. We perform a continuously working quantum entanglement distribution among three users coexisting with classical light. Under finite-key analysis, we establish secure keys and enable images encryption as well as quantum secret sharing between users. Our work paves the way for practical multiparty quantum communication with integrated photonic architecture compatible with real-world fiber optical communication network

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. © 2016 Ye et al

Functional differences in the allometry of the water, carbon and nitrogen content of gelatinous organisms

We have supplemented available, concurrent measurements of fresh weight (W, g) and body carbon (C, g) (46 individuals, 14 species) and of body nitrogen (N, g) and C (11individuals, 9 species) of marine gelatinous animals with data obtained during the global ocean expedition MALASPINA 2010 (totalling 267 individuals and 33 species for the W vs. C data; totalling 232 individuals and 31 species for the N vs. C data). We then used those data to test the allometric properties of the W vs. C and N vs. C relationships. Overall, gelatinous organisms contain 1.13±1.57% of C (by weight, mean±SD) in their bodies and show a C:N of 4.56±2.46, respectively, although estimations can be improved by using separate conversion coefficients for the carnivores and the filter feeders. Reduced Major Axis Regression (RMA) indicates that W grows isometrically with C in the carnivores (cnidarians and ctenophores), implying that their water content can be described by a single conversion coefficient of 173.78 gW[gC]-1, or a C content of 1.17±1.90% by weight, although there is much variability due to the existence of carbon-dense species. In contrast, W grows faster than C in the filter feeders (salps and doliolids), according to the power relationship W=446.68)C1.54. This exponent is not significantly different from 1.2, which is consistent with the idea that the watery bodies of gelatinous animals represent an evolutionary response towards increasing food capture surfaces, i.e. a bottom up rather than a top down mechanism. Thus, the available evidence negates a bottom up mechanism in the carnivores, but supports it in the filter feeders. Last, N grows isometrically with C in both carnivores and herbivores, with C:N ratios of 3.89±1.34 and 4.38±1.21, respectively. These values are similar to those of compact, non-gelatinous organisms and reflect a predominantly herbivorous diet in the filter feeders, what is confirmed by a difference of one trophic level between filter feeders and carnivores, according to stable N isotope enrichment data.MALASPINA (CSD2008-00077)Versión del editor1,749

Copper Requirement and Acquisition by Marine Microalgae

Copper is a critical metal nutrient required by marine microalgae but may be toxic when supplied in excess. Maintaining an optimal intracellular Cu content is thus fundamentally necessary for microalgae and relies on cellular regulatory metabolisms and the process of Cu uptake that buffers the variation in environmental Cu availability. In this article the current progress in understanding the Cu requirements and acquisition mechanisms of marine microalgae is reviewed. Cu requirement by microalgae is primarily determined by the amount of Cu-dependent enzymes involved in cellular metabolisms and can be adjusted by Cu-sparing pathways. Decrease in metabolic Cu quotas caused a decline in the abundance of cuproenzymes and the dependent cellular metabolisms, and an induction of Cu acquisition pathways. Conventional models of Cu uptake describe the dependence of Cu uptake rate on free Cu2+ ions or kinetically labile species. A reductive, high-affinity Cu uptake system in marine microalgae is identified which enables cells to directly utilize organically complexed Cu, highlighting the importance of cell surface Cu reduction in the marine Cu cycle. This review provides new insights into Cu uptake models that may update the existing knowledge of Cu availability in the ocean

Molecular and physiological responses of an oceanic diatom to copper deficiency

Copper (Cu) is one of about ten known essential metals required for phytoplankton growth and a limiting resource in parts of the open sea. It functions primarily as a cofactor in key enzymes and redox proteins in photosynthesis and respiration and is involved in high affinity Fe uptake. Growth of marine diatoms is limited by Cu at environmentally-relevant concentrations, suggesting diatom production in the Cu-depleted ocean may be compromised. The molecular mechanisms of how diatoms respond to Cu deficiency and maintain cellular Cu homeostasis are not well known. This thesis examines molecular and physiological responses of an oceanic diatom, Thalassiosira oceanica 1005, to Cu deficiency and characterizes a Cu(I)-dependent uptake pathway. Proteomic analysis of T. oceanica identified Cu-regulated proteins involved in light harvesting, photosynthetic electron transport, carbon and nitrogen assimilation, fatty acid oxidation, and a putative Cu-binding transcription factor. Using RNAseq to explore the transcriptome of the same species, I validated the proteomic results and identified metabolic processes that were regulated by Cu. These included down-regulation of photosynthesis, nitrate assimilation and glycolysis and up-regulation of ammonium assimilation, pentose phosphate pathways, fatty acid metabolism and oxidative stress defense pathways. Acclimation to low Cu resulted in a slowing down of most cellular metabolic processes and an increase in stress defense systems to combat oxidative damage. A major finding of the thesis was that T. oceanica contains a Cu(I)-dependent uptake system that functions at limiting Cu concentrations and proceeds through a two-step reaction: extracellular Cu(II) reduction followed by Cu(I) internalization. Four CTR-type Cu(I) transporters were identified; two of which complemented a yeast Cu transport mutant and increased uptake rates of both Cu(II) and Cu(I). Two putative Cu reductase (FRE-type) genes were identified and shown to catalyze Cu(II) reduction. Inhibition of Cu(II) reduction and trapping Cu(I) produced by the reductase with a Cu(I)-complexing agent reduced Cu uptake rate and inhibited cell growth. Thus, the results provide the first experimental evidence for a Cu(I)-dependent Cu uptake pathway in Thalassiosira oceanica and show that Cu(II) reduction is a necessary first step in Cu uptake.Le cuivre (Cu) fait partie d'une dizaine de métaux essentiels qui sont requis pour la croissance de phytoplancton. Ce métal est aussi une ressource limitante dans certaines parties de l'océan. Il fonctionne principalement comme cofacteur à l'intérieur d'enzymes clés, ainsi que dans les protéines oxydo-réductrices, de la photosynthèse et de la respiration. De plus, il est impliqué dans l'assimilation du fer. La croissance de diatomées marines est limitée par le Cu retrouvé à des concentrations environnementales actuelles. Cela suggère que la production de diatomée dans un océan pauvre en Cu pourrait être compromise. Cependant, les mécanismes moléculaires de diatomées en réponse à des déficiences de Cu, ainsi qu'au maintien de leur homéostase cellulaire en Cu, sont mal compris. Ma thèse examine les réponses moléculaires et physiologiques de la diatomée océanique Thalassiosira oceanica 1005 à des déficiences de Cu, et caractérise une assimilation Cu(I)-dépendante. L'analyse protéomique de T. oceanica a identifié des protéines régulées par le Cu et impliquées dans le captage de lumière, le transport photosynthétique d'électrons, l'assimilation de carbone et d'azote, l'oxydation d'acides gras, ainsi que dans un facteur de transcription putatif qui lie le Cu. Utilisant RNAseq pour explorer le transcriptome de la même espèce, j'ai validé les résultats protéomiques et identifié les processus métaboliques régulés par le Cu. Ceux-ci incluent des inhibiteurs de photosynthèse, d'assimilation de nitrates et de glycolyse, ainsi que des activateurs d'assimilation d'ammonium, de voies de pentose phosphates, de métabolisme d'acide gras, et de voies de défense de stress oxydatif. Lorsque la diatomée s'acclimatait à des faibles concentrations de Cu, la majorité de ses processus métaboliques ralentissaient et les systèmes de défense du stress augmentaient pour combattre des dommages oxydatifs. Ma thèse a permis de découvrir la présence d'un système d'assimilation Cu(I)-dépendant chez T. oceanica qui fonctionne à des faibles concentrations de Cu. Ce système procède avec une réaction en deux temps : une réduction de Cu(II) extracellulaire suivie de l'assimilation du Cu(I). Quatre transporteurs de Cu(I) de type CTR furent identifiés; deux desquels complémentaient une levure mutante de transport du Cu, ainsi qu'augmentaient des taux d'assimilation du Cu(II) et Cu(I). J'ai identifié deux gènes réductases putatifs de Cu (type FRE) qui catalysent la réduction de Cu(II). La réductase qui utilise l'agent de complexation du Cu(I) trappe le Cu(I) et inhibe la réduction de Cu(II), menant à une réduction dans le taux d'assimilation du Cu ainsi qu'à une inhibition du développement cellulaire. Ainsi, ces résultats repoussent les limites de la frontière en recherche, car ils constituent la première évidence expérimentale de la voie assimilatrice du Cu Cu(I)-dépendante chez Thalassiosira oceanica et montrent que la réduction de Cu(II) est la première étape nécessaire dans l'assimilation du Cu

Diversity and distribution of microbes involved in marine nitrogen cycle

Marine nitrogen (N) fixation and anaerobic ammonium oxidation (anammox) represent by far the major N input and loss pathways in marine environments. Together, these two ecologically important N cycling processes can regulate the total N contents in the ocean. Previous studies have detected diverse microbes involved in these processes with functional genes based molecular analysis. However, the spatial or/and temporal variations in composition and distribution of these microbial communities in some special environments remain unknown. In this study, the phylogenetic diversity and abundance of diazotrophs were examined along a transect from Pearl River plume to oceanic waters of the South China Sea (SCS) in both summer and winter, where biological N fixation is thought to be an important N source to its oligotrophic surface waters. Highly diverse diazotrophs were recovered, including proteobacteria, cyanobacteria and cluster III diazotrophs, with γ-proteobacteria dominating the entire diazotrophic community. Cyanobacterial diazotrophs were only detected in oceanic stations and the disappearance of cyanobacterial diazotrophs in the plume influenced station was suggested to be driven by low salinity and high nutrient concentrations. Compared with spatial variations, seasonal variations of diazotrophic communities were less apparent. Trichodesmium spp. was the most abundant cyanobacterial diazotroph and potentially the most important nitrogen fixer in this environment. Marine oxygen minimum zone (OMZ) represents another habitat for active microbial N transformations, where substantial N loss through anammox occurs. Novel hzo sequences in cluster 2 were detected in the OMZ of the Costa Rica Dome (CRD), with the simultaneous presence of the well-known hzo cluster 1 sequences. The novel hzo sequences were much more abundant and more widespread than cluster 1. More cDNA transcripts of the novel sequences were also obtained, indicating their potentiality to expressing the hydrazine oxidoreductase. The novel cluster 2 hzo sequences are suggested to be more ecologically important due to their higher abundance and wider distribution. This study firstly provided insight on the community compositions and distributions of diazotrophs in SCS and anammox bacteria in the CRD-OMZ, which is essential for understanding and predicting local and global N budget

Modeling and Application of Vehicular Cyber Physical System Based Petri Nets

Mobile cyber physical system (MCPS) has been a hot research area, where mobile nodes can mobile, and communicate with each other. As a typical MCPS, vehicular cyber physical system (VCPS) plays an important role in intelligent transportation, especially in collision avoidance. There is no, however, a formal modeling and analysis method for VCPS. In the paper, the modeling method based Petri nets (PN) is presented. Furthermore, the behavior expression analysis method is also presented which can deal with arbitrary distribution timed transitions. Finally, a case is introduced to verify the effectiveness about proposed method, and the results show that VCPS can greatly reduce the reaction time of vehicles behind when emergent accident occurs and then enhance the traffic safety

Functional CTR-type Cu(I) transporters in an oceanic diatom

Copper concentration is so low in some remote parts of the sea it limits phytoplankton growth, but may be high enough in coastal and estuarine regions to be toxic. Acclimation to variations in Cu concentration thus requires a tightly regulated Cu transport system to help maintain Cu homeostasis. In marine species, the molecular mechanisms of Cu transport are not known. We studied Cu-responsive genes and uptake in Thalassiosira oceanica at environmentally relevant Cu concentrations varying between 0.012 and 12 900 pmol Cu' L-1 . Copper uptake rate assessed at high Cu concentration was 3-fold faster in Cu-limited than in Cureplete cells, confirming the existence of an inducible uptake pathway in this diatom. Four putative CTR-type Cu transporters (ToCTR1, ToCTR2, ToCTR3a and ToCTR3b) identified in the transcriptome shared conserved features with known high-affinity Cu(I) transporters. Expression of the CTR genes was upregulated as Cu concentration declined and cells maintained maximum rates of growth. Further decreases in Cu led to decreased growth rate and increased abundance of ToCT3a/b. Both ToCTR3a and 3b restored growth of a Cu transport mutant, Saccharomyces cerevisiae ctr1?ctr3?, in Cu-deficient medium and increased the uptake rates of Cu(I) and Cu(II). Thus, ToCTR3a/3b is a high-affinity Cu(I) transporter that, in conjunction with the other ToCTRs, may enable T. oceanica to survive in Cu-deplete ocean environments and respond to natural variation in Cu availabilit