Genome-wide identification of interferon-sensitive mutations enables influenza vaccine design

<p>In conventional attenuated viral vaccines, immunogenicity is often suboptimal. Here we present a systematic approach for vaccine development that eliminates interferon (IFN)&ndash;modulating functions genome-wide while maintaining virus replication fitness. We applied a quantitative high-throughput genomics system to influenza A virus that simultaneously measured the replication fitness and IFN sensitivity of mutations across the entire genome. By incorporating eight IFN-sensitive mutations, we generated a hyper&ndash;interferon-sensitive (HIS) virus as a vaccine candidate. HIS virus is highly attenuated in IFN-competent hosts but able to induce transient IFN responses, elicits robust humoral and cellular immune responses, and provides protection against homologous and heterologous viral challenges. Our approach, which attenuates the virus and promotes immune responses concurrently, is broadly applicable for vaccine development against other pathogens.</p>

A self-assembled nanoscale robotic arm controlled by electric fields

<p>The use of dynamic, self-assembled DNA nanostructures in the context of nanorobotics requires fast and reliable actuation mechanisms. We therefore created a 55-nanometer&ndash;by&ndash;55-nanometer DNA-based molecular platform with an integrated robotic arm of length 25 nanometers, which can be extended to more than 400 nanometers and actuated with externally applied electrical fields. Precise, computer-controlled switching of the arm between arbitrary positions on the platform can be achieved within milliseconds, as demonstrated with single-pair Fo&#x0308;rster resonance energy transfer experiments and fluorescence microscopy. The arm can be used for electrically driven transport of molecules or nanoparticles over tens of nanometers, which is useful for the control of photonic and plasmonic processes. Application of piconewton forces by the robot arm is demonstrated in force-induced DNA duplex melting experiments.</p>

Quantum liquid droplets in a mixture of Bose-Einstein condensates

<p>Quantum droplets are small clusters of atoms self-bound by the balance of attractive and repulsive forces. Here, we report on the observation of droplets solely stabilized by contact interactions in a mixture of two Bose-Einstein condensates. We demonstrate that they are several orders of magnitude more dilute than liquid helium by directly measuring their size and density via in situ imaging. We show that the droplets are stablized against collapse by quantum fluctuations and that they require a minimum atom number to be stable. Below that number, quantum pressure drives a liquid-to-gas transition that we map out as a function of interaction strength. These ultradilute isotropic liquids remain weakly interacting and constitute an ideal platform to benchmark quantum many-body theories.</p>

Hydraulic fracturing volume is associated with induced earthquake productivity in the Duvernay play

<p>A sharp increase in the frequency of earthquakes near Fox Creek, Alberta, began in December 2013 in response to hydraulic fracturing. Using a hydraulic fracturing database, we explore relationships between injection parameters and seismicity response. We show that induced earthquakes are associated with completions that used larger injection volumes (10<sup>4</sup> to 10<sup>5</sup> cubic meters) and that seismic productivity scales linearly with injection volume. Injection pressure and rate have an insignificant association with seismic response. Further findings suggest that geological factors play a prominent role in seismic productivity, as evidenced by spatial correlations. Together, volume and geological factors account for ~96% of the variability in the induced earthquake rate near Fox Creek. This result is quantified by a seismogenic index&ndash;modified frequency-magnitude distribution, providing a framework to forecast induced seismicity.</p>

Chiromagnetic nanoparticles and gels

<p>Chiral inorganic nanostructures have high circular dichroism, but real-time control of their optical activity has so far been achieved only by irreversible chemical changes. Field modulation is a far more desirable path to chiroptical devices. We hypothesized that magnetic field modulation can be attained for chiral nanostructures with large contributions of the magnetic transition dipole moments to polarization rotation. We found that dispersions and gels of paramagnetic Co<SUB>3</SUB>O<SUB>4</SUB> nanoparticles with chiral distortions of the crystal lattices exhibited chiroptical activity in the visible range that was 10 times as strong as that of nonparamagnetic nanoparticles of comparable size. Transparency of the nanoparticle gels to circularly polarized light beams in the ultraviolet range was reversibly modulated by magnetic fields. These phenomena were also observed for other nanoscale metal oxides with lattice distortions from imprinted amino acids and other chiral ligands. The large family of chiral ceramic nanostructures and gels can be pivotal for new technologies and knowledge at the nexus of chirality and magnetism.</p>

Digitization of multistep organic synthesis in reactionware for on-demand pharmaceuticals

<p>Chemical manufacturing is often done at large facilities that require a sizable capital investment and then produce key compounds for a finite period. We present an approach to the manufacturing of fine chemicals and pharmaceuticals in a self-contained plastic reactionware device. The device was designed and constructed by using a chemical to computer-automated design (ChemCAD) approach that enables the translation of traditional bench-scale synthesis into a platform-independent digital code. This in turn guides production of a three-dimensional printed device that encloses the entire synthetic route internally via simple operations. We demonstrate the approach for the -aminobutyric acid receptor agonist, (&plusmn;)-baclofen, establishing a concept that paves the way for the local manufacture of drugs outside of specialist facilities.</p>

A global atlas of the dominant bacteria found in soil

<p>The immense diversity of soil bacterial communities has stymied efforts to characterize individual taxa and document their global distributions. We analyzed soils from 237 locations across six continents and found that only 2% of bacterial phylotypes (~500 phylotypes) consistently accounted for almost half of the soil bacterial communities worldwide. Despite the overwhelming diversity of bacterial communities, relatively few bacterial taxa are abundant in soils globally. We clustered these dominant taxa into ecological groups to build the first global atlas of soil bacterial taxa. Our study narrows down the immense number of bacterial taxa to a "most wanted" list that will be fruitful targets for genomic and cultivation-based efforts aimed at improving our understanding of soil microbes and their contributions to ecosystem functioning.</p>

Improving refugee integration through data-driven algorithmic assignment

<p>Developed democracies are settling an increased number of refugees, many of whom face challenges integrating into host societies. We developed a flexible data-driven algorithm that assigns refugees across resettlement locations to improve integration outcomes. The algorithm uses a combination of supervised machine learning and optimal matching to discover and leverage synergies between refugee characteristics and resettlement sites. The algorithm was tested on historical registry data from two countries with different assignment regimes and refugee populations, the United States and Switzerland. Our approach led to gains of roughly 40 to 70%, on average, in refugees&rsquo; employment outcomes relative to current assignment practices. This approach can provide governments with a practical and cost-efficient policy tool that can be immediately implemented within existing institutional structures.</p>

Dicer uses distinct modules for recognizing dsRNA termini

<p>Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and <I>Drosophila</I> Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, we used cryo&ndash;electron microscopy to solve structures of <I>Drosophila</I> Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate&ndash;dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers.</p>

Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose

<p>Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that <I>Escherichia coli</I> produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The <I>bcsEFG</I> operon is present in many bacteria, including <I>Salmonella</I> species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.</p>

Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

<p>Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by the kinetochore protein CENP-N. We report cryo&ndash;electron microscopy (cryo-EM), biophysical, biochemical, and cell biological studies of the interaction between the CENP-A nucleosome and CENP-N. We show that human CENP-N confers binding specificity through interactions with the L1 loop of CENP-A, stabilized by electrostatic interactions with the nucleosomal DNA. Mutational analyses demonstrate analogous interactions in <I>Xenopus</I>, which are further supported by residue-swapping experiments involving the L1 loop of CENP-A. Our results are consistent with the coevolution of CENP-N and CENP-A and establish the structural basis for recognition of the CENP-A nucleosome to enable kinetochore assembly and centromeric chromatin organization.</p>

Multiplexed gene synthesis in emulsions for exploring protein functional landscapes

<p>Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene&rsquo;s assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in <I>Escherichia coli</I>. We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout <I>E. coli</I> strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.</p>

Toward dynamic structural biology: Two decades of single-molecule Fo&#x0308;rster resonance energy transfer

<p>Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Fo&#x0308;rster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.</p>

Membrane protein insertion through a mitochondrial {beta}-barrel gate

<p>The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of &beta;-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane insertion of &beta;-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by &beta;-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of &beta;-barrel proteins.</p>

The epigenetic control of stemness in CD8+ T cell fate commitment

<p>After priming, nai&#x0308;ve CD8<sup>+</sup> T lymphocytes establish specific heritable transcription programs that define progression to long-lasting memory cells or to short-lived effector cells. Although lineage specification is critical for protection, it remains unclear how chromatin dynamics contributes to the control of gene expression programs. We explored the role of gene silencing by the histone methyltransferase Suv39h1. In murine CD8<sup>+</sup> T cells activated after <I>Listeria monocytogenes</I> infection, Suv39h1-dependent trimethylation of histone H3 lysine 9 controls the expression of a set of stem cell&ndash;related memory genes. Single-cell RNA sequencing revealed a defect in silencing of stem/memory genes selectively in <I>Suv39h1</I>-defective T cell effectors. As a result, <I>Suv39h1</I>-defective CD8<sup>+</sup> T cells show sustained survival and increased long-term memory reprogramming capacity. Thus, Suv39h1 plays a critical role in marking chromatin to silence stem/memory genes during CD8<sup>+</sup> T effector terminal differentiation.</p>

Coherent band excitations in CePd3: A comparison of neutron scattering and ab initio theory

<p>In common with many strongly correlated electron systems, intermediate valence compounds are believed to display a crossover from a high-temperature regime of incoherently fluctuating local moments to a low-temperature regime of coherent hybridized bands. We show that inelastic neutron scattering measurements of the dynamic magnetic susceptibility of CePd<SUB>3</SUB> provides a benchmark for ab initio calculations based on dynamical mean field theory. The magnetic response is strongly momentum dependent thanks to the formation of coherent f-electron bands at low temperature, with an amplitude that is strongly enhanced by local particle-hole interactions. The agreement between experiment and theory shows that we have a robust first-principles understanding of the temperature dependence of f-electron coherence.</p>

Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics

<p>Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 <I>Plasmodium falciparum</I> parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target&ndash;inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the <I>P. falciparum</I> resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.</p>

Exposed subsurface ice sheets in the Martian mid-latitudes

<p>Thick deposits cover broad regions of the Martian mid-latitudes with a smooth mantle; erosion in these regions creates scarps that expose the internal structure of the mantle. We investigated eight of these locations and found that they expose deposits of water ice that can be &gt;100 meters thick, extending downward from depths as shallow as 1 to 2 meters below the surface. The scarps are actively retreating because of sublimation of the exposed water ice. The ice deposits likely originated as snowfall during Mars&rsquo; high-obliquity periods and have now compacted into massive, fractured, and layered ice. We expect the vertical structure of Martian ice-rich deposits to preserve a record of ice deposition and past climate.</p>

Detection of the aromatic molecule benzonitrile (c-C6H5CN) in the interstellar medium

<p>Polycyclic aromatic hydrocarbons and polycyclic aromatic nitrogen heterocycles are thought to be widespread throughout the universe, because these classes of molecules are probably responsible for the unidentified infrared bands, a set of emission features seen in numerous Galactic and extragalactic sources. Despite their expected ubiquity, astronomical identification of specific aromatic molecules has proven elusive. We present the discovery of benzonitrile (<I>c</I>-C<SUB>6</SUB>H<SUB>5</SUB>CN), one of the simplest nitrogen-bearing aromatic molecules, in the interstellar medium. We observed hyperfine-resolved transitions of benzonitrile in emission from the molecular cloud TMC-1. Simple aromatic molecules such as benzonitrile may be precursors for polycyclic aromatic hydrocarbon formation, providing a chemical link to the carriers of the unidentified infrared bands.</p>

Ordered macro-microporous metal-organic framework single crystals

<p>We constructed highly oriented and ordered macropores within metal-organic framework (MOF) single crystals, opening up the area of three-dimensional&ndash;ordered macro-microporous materials (that is, materials containing both macro- and micropores) in single-crystalline form. Our methodology relies on the strong shaping effects of a polystyrene nanosphere monolith template and a double-solvent&ndash;induced heterogeneous nucleation approach. This process synergistically enabled the in situ growth of MOFs within ordered voids, rendering a single crystal with oriented and ordered macro-microporous structure. The improved mass diffusion properties of such hierarchical frameworks, together with their robust single-crystalline nature, endow them with superior catalytic activity and recyclability for bulky-molecule reactions, as compared with conventional, polycrystalline hollow, and disordered macroporous ZIF-8.</p>

Antagonism toward the intestinal microbiota and its effect on Vibrio cholerae virulence

<p>The bacterial type VI secretion system (T6SS) is a nanomachine that delivers toxic effector proteins into target cells, killing them. In mice, we found that the <I>Vibrio cholerae</I> T6SS attacks members of the host commensal microbiota in vivo, facilitating the pathogen&rsquo;s colonization of the gut. This microbial antagonistic interaction drives measurable changes in the pathogenicity of <I>V. cholerae</I> through enhanced intestinal colonization, expression of bacterial virulence genes, and activation of host innate immune genes. Because ablation of mouse commensals by this enteric pathogen correlated with more severe diarrheal symptoms, we conclude that antagonism toward the gut microbiota could improve the fitness of <I>V. cholerae</I> as a pathogen by elevating its transmission to new susceptible hosts.</p>

Spatial representations of self and other in the hippocampus

<p>An animal&rsquo;s awareness of its location in space depends on the activity of place cells in the hippocampus. How the brain encodes the spatial position of others has not yet been identified. We investigated neuronal representations of other animals&rsquo; locations in the dorsal CA1 region of the hippocampus with an observational T-maze task in which one rat was required to observe another rat&rsquo;s trajectory to successfully retrieve a reward. Information reflecting the spatial location of both the self and the other was jointly and discretely encoded by CA1 pyramidal cells in the observer rat. A subset of CA1 pyramidal cells exhibited spatial receptive fields that were identical for the self and the other. These findings demonstrate that hippocampal spatial representations include dimensions for both self and nonself.</p>

Social place-cells in the bat hippocampus

<p>Social animals have to know the spatial positions of conspecifics. However, it is unknown how the position of others is represented in the brain. We designed a spatial observational-learning task, in which an observer bat mimicked a demonstrator bat while we recorded hippocampal dorsal-CA1 neurons from the observer bat. A neuronal subpopulation represented the position of the other bat, in allocentric coordinates. About half of these "social place-cells" represented also the observer&rsquo;s own position&mdash;that is, were place cells. The representation of the demonstrator bat did not reflect self-movement or trajectory planning by the observer. Some neurons represented also the position of inanimate moving objects; however, their representation differed from the representation of the demonstrator bat. This suggests a role for hippocampal CA1 neurons in social-spatial cognition.</p>

Rapid hybrid speciation in Darwins finches

<p>Homoploid hybrid speciation in animals has been inferred frequently from patterns of variation, but few examples have withstood critical scrutiny. Here we report a directly documented example, from its origin to reproductive isolation. An immigrant Darwin&rsquo;s finch to Daphne Major in the Gal&aacute;pagos archipelago initiated a new genetic lineage by breeding with a resident finch (<I>Geospiza fortis</I>). Genome sequencing of the immigrant identified it as a <I>G. conirostris</I> male that originated on Espa&ntilde;ola &gt;100 kilometers from Daphne Major. From the second generation onward, the lineage bred endogamously and, despite intense inbreeding, was ecologically successful and showed transgressive segregation of bill morphology. This example shows that reproductive isolation, which typically develops over hundreds of generations, can be established in only three.</p>

Structure of the human TRPM4 ion channel in a lipid nanodisc

<p>Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage-dependent manner but, unlike many other TRP channels, is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3-angstrom resolution, as determined by single-particle cryo&ndash;electron microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium-binding site within the intracellular side of the S1-S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.</p>

CX3CR1+ mononuclear phagocytes control immunity to intestinal fungi

<p>Intestinal fungi are an important component of the microbiota, and recent studies have unveiled their potential in modulating host immune homeostasis and inflammatory disease. Nonetheless, the mechanisms governing immunity to gut fungal communities (mycobiota) remain unknown. We identified CX3CR1<sup>+</sup> mononuclear phagocytes (MNPs) as being essential for the initiation of innate and adaptive immune responses to intestinal fungi. CX3CR1<sup>+</sup> MNPs express antifungal receptors and activate antifungal responses in a Syk-dependent manner. Genetic ablation of CX3CR1<sup>+</sup> MNPs in mice led to changes in gut fungal communities and to severe colitis that was rescued by antifungal treatment. In Crohn&rsquo;s disease patients, a missense mutation in the gene encoding CX3CR1 was identified and found to be associated with impaired antifungal responses. These results unravel a role of CX3CR1<sup>+</sup> MNPs in mediating interactions between intestinal mycobiota and host immunity at steady state and during inflammatory disease.</p>

Structure of the cold- and menthol-sensing ion channel TRPM8

<p>Transient receptor potential melastatin (TRPM) cation channels are polymodal sensors that are involved in a variety of physiological processes. Within the TRPM family, member 8 (TRPM8) is the primary cold and menthol sensor in humans. We determined the cryo&ndash;electron microscopy structure of the full-length TRPM8 from the collared flycatcher at an overall resolution of ~4.1 &aring;ngstroms. Our TRPM8 structure reveals a three-layered architecture. The amino-terminal domain with a fold distinct among known TRP structures, together with the carboxyl-terminal region, forms a large two-layered cytosolic ring that extensively interacts with the transmembrane channel layer. The structure suggests that the menthol-binding site is located within the voltage-sensor&ndash;like domain and thus provides a structural glimpse of the design principle of the molecular transducer for cold and menthol sensation.</p>

Gene therapy comes of age

<p>After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches.</p>

Fatty acyl recognition and transfer by an integral membrane S-acyltransferase

<p>DHHC (Asp-His-His-Cys) palmitoyltransferases are eukaryotic integral membrane enzymes that catalyze protein palmitoylation, which is important in a range of physiological processes, including small guanosine triphosphatase (GTPase) signaling, cell adhesion, and neuronal receptor scaffolding. We present crystal structures of two DHHC palmitoyltransferases and a covalent intermediate mimic. The active site resides at the membrane-cytosol interface, which allows the enzyme to catalyze thioester-exchange chemistry by using fatty acyl&ndash;coenzyme A and explains why membrane-proximal cysteines are candidates for palmitoylation. The acyl chain binds in a cavity formed by the transmembrane domain. We propose a mechanism for acyl chain&ndash;length selectivity in DHHC enzymes on the basis of cavity mutants with preferences for shorter and longer acyl chains.</p>

Phase separation of a yeast prion protein promotes cellular fitness

<p>Despite the important role of prion domains in neurodegenerative disease, their physiological function has remained enigmatic. Previous work with yeast prions has defined prion domains as sequences that form self-propagating aggregates. Here, we uncovered an unexpected function of the canonical yeast prion protein Sup35. In stressed conditions, Sup35 formed protective gels via pH-regulated liquid-like phase separation followed by gelation. Phase separation was mediated by the N-terminal prion domain and regulated by the adjacent pH sensor domain. Phase separation promoted yeast cell survival by rescuing the essential Sup35 translation factor from stress-induced damage. Thus, prion-like domains represent conserved environmental stress sensors that facilitate rapid adaptation in unstable environments by modifying protein phase behavior.</p>