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Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.
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Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Epigenómica , Genómica , Glioblastoma/genética , Glioblastoma/patología , Análisis de la Célula Individual , Microambiente Tumoral , Heterogeneidad GenéticaRESUMEN
We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.
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Neoplasias/genética , Adulto , Niño , Análisis por Conglomerados , ADN Polimerasa II/genética , ADN Polimerasa III/genética , Replicación del ADN , Humanos , Mutación , Neoplasias/clasificación , Neoplasias/patología , Neoplasias/terapia , Proteínas de Unión a Poli-ADP-Ribosa/genéticaRESUMEN
Heterosynaptic plasticity, along with Hebbian homosynaptic plasticity, is an important mechanism ensuring the stable operation of learning neuronal networks. However, whether heterosynaptic plasticity occurs in the whole brain in vivo, and what role(s) in brain function in vivo it could play, remains unclear. Here, we used an optogenetics approach to apply a model of intracellular tetanization, which was established and employed to study heterosynaptic plasticity in brain slices, to study the plasticity of response properties of neurons in the mouse visual cortex in vivo. We show that optogenetically evoked high-frequency bursts of action potentials (optogenetic tetanization) in the principal neurons of the visual cortex induce long-term changes in the responses to visual stimuli. Optogenetic tetanization had distinct effects on responses to different stimuli, as follows: responses to optimal and orthogonal orientations decreased, responses to null direction did not change, and responses to oblique orientations increased. As a result, direction selectivity of the neurons decreased and orientation tuning became broader. Since optogenetic tetanization was a postsynaptic protocol, applied in the absence of sensory stimulation, and, thus, without association of presynaptic activity with bursts of action potentials, the observed changes were mediated by mechanisms of heterosynaptic plasticity. We conclude that heterosynaptic plasticity can be induced in vivo and propose that it may play important homeostatic roles in operation of neural networks by helping to prevent runaway dynamics of responses to visual stimuli and to keep the tuning of neuronal responses within the range optimized for the encoding of multiple features in population activity.
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Manganese complexes [(arene)Mn(CO)3]+ were prepared in one step from arenes and Mn(CO)5Br. They were found to be efficient catalysts in the carbonyl cyanation with TMSCN, CO2 fixation by epoxides, and direct reductive amination in the presence of syngas. The amination reaction tolerated various reducible functional groups. The synergy of carbon monoxide and hydrogen in syngas provides high efficiency of the catalytic system. The developed protocols do not require an inert atmosphere, and the catalysts can be handled in air.
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We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.
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Antibacterianos , Calcio , Bacterias Grampositivas , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Calcio/metabolismo , Estructura Molecular , Bacterias Grampositivas/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Daptomicina/farmacología , Daptomicina/química , Membrana Dobles de Lípidos/química , MicelasRESUMEN
The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the ß-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the ß3-ß4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.
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Ceftriaxona , Simulación de Dinámica Molecular , Neisseria gonorrhoeae , Proteínas de Unión a las Penicilinas , Ceftriaxona/farmacología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/metabolismo , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/metabolismo , Antibacterianos/farmacología , Mutación , Farmacorresistencia Bacteriana/genética , Acilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , D-Ala-D-Ala Carboxipeptidasa de Tipo SerinaRESUMEN
Synaptic plasticity is currently considered the main mechanism underlying the plastic modification of neural networks. The vast majority of studies of synaptic plasticity are carried out on reduced preparations, but the situation in vivo is fundamentally different from that in vitro. In this work, we used the Hebbian paradigm, which is known to induce long-term changes in synaptic strength in vitro, to manipulate the properties of a single pyramidal neuron in the mouse visual cortex. We have shown that optogenetic stimulation of a ChR2-expressing pyramidal neuron in the primary visual cortex of Thy-ChR2 mice paired with the presentation of a visual stimulus of non-optimal orientation induces long-term changes in the properties of the receptive field, manifested in alteration of the orientation selectivity of the cell. Non-paired stimulation did not lead to changes in the properties of the receptive field of the neuron during the experiment. Thus, we have demonstrated the role of associative plasticity in the dynamic organization of the receptive fields of neurons in the visual cortex.
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Optogenética , Corteza Visual , Ratones , Animales , Estimulación Luminosa , Plasticidad Neuronal/fisiología , Neuronas/fisiologíaRESUMEN
Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.
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Emulsiones/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN/genética , Cartilla de ADN/genética , Biblioteca de Genes , Genoma/genética , Humanos , Modelos Teóricos , Técnicas de Amplificación de Ácido Nucleico/métodos , Moldes GenéticosRESUMEN
Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.
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Natural compounds continue to serve as the most fruitful source of new antimicrobials. Analysis of bacterial genomes have revealed that the biosynthetic potential of antibiotic producers by far exceeds the number of already discovered structures. However, due to the repeated discovery of known substances, it has become necessary to change both approaches to the search for antibiotics and the sources of producer strains. The pressure of natural selection and the diversity of interactions in symbiotic communities make animal microbiomes promising sources of novel substances. Here, microorganisms associated with various animals were examined in terms of their antimicrobial agents. The application of alternative cultivation techniques, ultrahigh-throughput screening, and genomic analysis facilitated the investigation of compounds produced by unique representatives of the animal microbiota. We believe that new strategies of antipathogen defense will be discovered by precisely studying cell-cell and host-microbe interactions in microbiomes in the wild.
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Antibacterianos , Microbiota , Animales , Antibacterianos/farmacología , Frutas , Genoma Bacteriano , GenómicaRESUMEN
ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.
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Proteasa La , Proteasas ATP-Dependientes/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Péptido Hidrolasas/metabolismo , Proteasa La/genética , Proteasa La/metabolismo , Proteoma/metabolismoRESUMEN
The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.
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Animales Salvajes/microbiología , Antibacterianos/administración & dosificación , Bacillus pumilus/química , Escherichia coli/crecimiento & desarrollo , Microbiota , Probióticos/administración & dosificación , Staphylococcus aureus/crecimiento & desarrollo , Animales , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/efectos de los fármacos , Genoma Bacteriano , Metaboloma , Familia de Multigenes , Probióticos/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacosRESUMEN
Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.
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Antibacterianos/farmacología , Cumarinas/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Metabolómica/métodos , Animales , Antibacterianos/metabolismo , Bacillus pumilus/efectos de los fármacos , Bacillus pumilus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cumarinas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Farmacorresistencia Bacteriana/fisiología , Microbioma Gastrointestinal/fisiología , Perfilación de la Expresión Génica , Voluntarios Sanos , Humanos , Dispositivos Laboratorio en un Chip , Proteómica/métodos , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Análisis de la Célula Individual/métodos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Ursidae/microbiologíaRESUMEN
Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.
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Hongos/genética , Proteínas Luminiscentes/genética , Secuencia de Aminoácidos , Animales , Vías Biosintéticas/genética , Ácidos Cafeicos , Línea Celular , Línea Celular Tumoral , Femenino , Duplicación de Gen/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Alineación de Secuencia , Xenopus laevisRESUMEN
Placozoa are small disc-shaped animals, representing the simplest known, possibly ancestral, organization of free-living animals. With only six morphological distinct cell types, without any recognized neurons or muscle, placozoans exhibit fast effector reactions and complex behaviors. However, little is known about electrogenic mechanisms in these animals. Here, we showed the presence of rapid action potentials in four species of placozoans (Trichoplax adhaerens [H1 haplotype], Trichoplax sp.[H2], Hoilungia hongkongensis [H13], and Hoilungia sp. [H4]). These action potentials are sodium-dependent and can be inducible. The molecular analysis suggests the presence of 5-7 different types of voltage-gated sodium channels, which showed substantial evolutionary radiation compared to many other metazoans. Such unexpected diversity of sodium channels in early-branched metazoan lineages reflect both duplication events and parallel evolution of unique behavioral integration in these nerveless animals.
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Placozoa/metabolismo , Canales de Sodio/metabolismo , Sodio/metabolismo , Potenciales de Acción , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Evolución Molecular , Variación Genética , Modelos Moleculares , Filogenia , Placozoa/clasificación , Placozoa/genética , Conformación Proteica , Canales de Sodio/química , Canales de Sodio/genéticaRESUMEN
A majority of thousands of intracellular mammalian proteins are recognized by proteasome only being conjugated with ubiquitin (Ub), representing a universal degradation signal operated by the ubiquitination system. Ub-independent proteasome targeting is rationalized by the existence of 2 types of direct proteasome signals (DPSs), specific amino acid sequences or post-translational modifications, which are recognized by proteasome regulatory subunits. Historically, the first type was shown to exist in ornithine decarboxylase, whereas acetylation of core histones recently was reported as a second type of DPS. Here we declare a third type, representing charge-mediated DPS. This discovered DPS may be classified as a monopartite composition- but not sequence-dependent element of â¼70 Å in length enriched in basic and flexible amino acids. This type of degradation signal, which may be provided by cationic chemicals, is most efficiently engaged by proteasomes capped with regulator (REG)α or REGγ in an ATP-independent manner. Taken together, our findings suggest a novel modality of proteasome-substrate interrelation bypassing ubiquitination.-Kudriaeva, A., Kuzina, E. S., Zubenko, O., Smirnov, I. V., Belogurov, A. Charge-mediated proteasome targeting.
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Autoantígenos/metabolismo , Cationes/metabolismo , Proteína Básica de Mielina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Cationes/química , Células HEK293 , Humanos , Hígado/enzimología , Ratones Endogámicos BALB C , Proteína Básica de Mielina/química , Procesamiento Proteico-Postraduccional , Proteolisis , Especificidad por Sustrato , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite d-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients mutant IDH1 and 2HG are not required for later clonal expansions.
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Epigenómica , Amplificación de Genes , Glioma/genética , Isocitrato Deshidrogenasa/genética , Mutación , Recurrencia Local de Neoplasia/genética , Eliminación de Secuencia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Variaciones en el Número de Copia de ADN , Metilación de ADN , Perfilación de la Expresión Génica , Glioma/patología , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Células Tumorales CultivadasRESUMEN
Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
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Butirilcolinesterasa/química , Ensayos Analíticos de Alto Rendimiento/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Paraoxon/química , Análisis de la Célula Individual/instrumentación , Antibiosis , Biodiversidad , Comunicación Celular , Emulsiones , Citometría de Flujo , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Aceites Volátiles/química , Fenotipo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Agua/químicaRESUMEN
In this paper, the structural characteristics of a W-Ta-Mo-Nb-V-Cr-Zr-Ti non-equiatomic refractory metal alloy obtained by spark plasma sintering (SPS) of a high-energy ball-milled powder mixture are reported. High-energy ball milling resulted in the formation of particle agglomerates ranging from several tens to several hundreds of micrometers. These agglomerates were composed of micrometer and submicrometer particles. It was found that, during ball milling, a solid solution of A2 structure formed. The grains of the sintered material ranged from fractions of a micrometer to several micrometers. During SPS, the phase transformations in the alloy led to the formation of a Laves phase of C15 structure and ZrO and ZrO2 nanoparticles. The microhardness of the ball-milled alloy and sintered material was found to be 9.28 GPa ± 1.31 GPa and 8.95 GPa ± 0.42 GPa, respectively. The influence of the processing conditions on the structure, phase composition, and microhardness of the alloy is discussed.
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PURPOSE: Telomere length-associated SNPs have been associated with incidence and survival rates for malignant brain tumors such as glioma. Here, we study the influence of genetically determined lymphocyte telomere length (LTL) by comparing telomerase associated SNPs between the most common non-malignant brain tumor, i.e. meningioma, and healthy controls. METHODS/PATIENTS: One thousand fifty-three (1053) surgically treated meningioma patients and 4437 controls of Western European ancestry were included. Germline DNA was genotyped for 8 SNPs previously significantly associated with LTL. Genotypically-estimated LTL was then calculated by summing each SNP's genotypically-specified telomere length increase in base pairs (bp) for each person. Odds ratios for genotypically-estimated LTL in meningioma cases and controls were evaluated using logistic regression with the first two ancestral principal components and sex as covariates. RESULTS: Three out of the eight evaluated LTL SNPs were significantly associated with increased meningioma risk (rs10936599: OR 1.14, 95% CI 1.01-1.28, rs2736100: OR 1.13, 95% CI 1.03-1.25, rs9420907: OR 1.22, 95% CI 1.07-1.39). Only rs9420907 remained significant after correction for multiple testing. Average genotypically-estimated LTL was significantly longer for those with meningioma compared to controls [mean cases: 560.2 bp (standard error (SE): 4.05 bp), mean controls: 541.5 bp (SE: 2.02 bp), logistic regression p value = 2.13 × 10-5]. CONCLUSION: Increased genotypically-estimated LTL was significantly associated with increased meningioma risk. A role for telomere length in the pathophysiology of meningioma is novel, and could lead to new insights on the etiology of meningioma.