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We sequenced the genomes of five new Methylophilaceae strains isolated from Lake Washington sediment. We used the new sequences to sort these new strains into specific Methylophilaceae ecotypes, including one novel ecotype. The new genomes expand the known diversity of Methylophilaceae and provide new models for studying the ecology of methylotrophy.
All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.
The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55–62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 μg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 μg/ml. Furthermore, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.
Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ~35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. We report six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the “out” position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.
Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli , Pseudomonas aeruginosa , and Staphylococcus aureus . We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. Here, these findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation. Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.
Exploration of environmental microbiomes has shed light on the ecological and evolutionary principles at play in natural ecosystems and has been further accelerated through the reconstruction of population genomes to provide genome-centric context. Yet technical challenges with traditional shotgun metagenomics remain for computationally intense short-read assembly, strain heterogeneity within communities, and depth of coverage required for low-abundance microbes. In this Perspective, we highlight three main avenues for promising future developments, including coupling stable isotope probing and genome-resolved metagenomics, applying fluorescence-activated cell sorting approaches to target mini-metagenomes within a larger community, and utilizing single-molecule long-read and synthetic long-read technology to link mobile elements to host microbial cells. These developments on the horizon will undoubtedly advance genome-resolved metagenomic approaches and enable a better understanding of uncultivated microbes in their natural environments.
Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of Burkholderia thailandensis populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence.
The Western Interior of the USA was dominated by desert environments for much of the Jurassic. Of these desert environments, the most extensive by far was the Navajo Sandstone erg, which, in conjunction with its correlative Nugget and Aztec Sandstones, is thought to have covered well over 500,000 km2, and is one of the largest ergs preserved in the rock record. The Navajo Sandstone is also one of a few geologic units which show promise as a potential CO2 sequestration reservoir in Utah. It outcrops along the eastern and southern margin of the state, and subcrops throughout most of the central area. The Navajo Sandstone consists of stacked eolian deposits, composed of well sorted, clean sandstone, deposited in commonly tabular, high angle cross bedded paleodunes, which are on average 15 m thick. Interspersed between these paleodunes are interdune deposits. Interdune deposits consist of sandy to calcareous, predominantly planar bedded, lensoid units, ranging from <1 m up to 7 m thick. In some cases, these interdune deposits occur along more extensive stratigraphic bounding surfaces. Dating within the Navajo Sandstone has historically been very difficult, as a result of its predominantly clean, aeolian sandstone composition. In addition, complexity of internal stratigraphic surfaces coupled with a low structural dip and generally flat topography has resulted in a poor understanding of the internal stratigraphy of the Navajo Sandstone. Exceptional three-dimensional exposures of the Navajo Sandstone in the upper portions of Lake Powell permit us to relate interdune carbonates to a hierarchical bounding surfaces model. Here we use this combined approach to interpret the internal stratigraphy of the Navajo Sandstone in this region. This work forms part of a larger study of the Navajo Sandstone across Utah and its potential as a CO2 reservoir, by developing a more comprehensive stratigraphic framework, and increasing understanding of stratigraphic complexity within one of the most significant eolian systems in the world.
Pyroclastic density currents have been observed to both enter the sea, and to travel over water for tens of kilometers. Here, we identified a 1.2-m-thick, stratified pumice lapilli-ash cored at Site U1396 offshore Montserrat (Integrated Ocean Drilling Program [IODP] Expedition 340) as being the first deposit to provide evidence that it was formed by submarine deposition from pumice-rich pyroclastic density currents that traveled above the water surface. The age of the submarine deposit is ca. 4 Ma, and its magma source is similar to those for much younger Soufrière Hills deposits, indicating that the island experienced large-magnitude, sub-aerial caldera-forming explosive eruptions much earlier than recorded in land deposits. The deposit's combined sedimentological characteristics are incompatible with deposition from a submarine eruption, pyroclastic fall over water, or a submarine seafloor-hugging turbidity current derived from a subaerial pyroclastic density current that entered water at the shoreline. The stratified pumice lapilli-ash unit can be subdivided into at least three depositional units, with the lowermost one being clast supported. The unit contains grains in five separate size modes and has a >12 phi range. Particles are chiefly sub-rounded pumice clasts, lithic clasts, crystal fragments, and glass shards. Pumice clasts are very poorly segregated from other particle types, and lithic clasts occur throughout the deposit; fine particles are weakly density graded. We interpret the unit to record multiple closely spaced (< 2 d) hot pyroclastic density currents that flowed over the ocean, releasing pyroclasts onto the water surface, and settling of the various pyroclasts into the water column. Our settling and hot and cold flotation experiments show that waterlogging of pumice clasts at the water surface would have been immediate. The overall poor hydraulic sorting of the deposit resulted from mixing of particles from multiple pulses of vertical settling in the water column, attesting to complex sedimentation. Slow-settling particles were deposited on the seafloor together with faster-descending particles that were delivered at the water surface by subsequent pyroclastic flows. The final sediment pulses were eventually deflected upon their arrival on the seafloor and were deposited in laterally continuous facies. This study emphasizes the interaction between products of explosive volcanism and the ocean and discusses sedimentological complexities and hydrodynamics associated with particle delivery to water.
Three new succinate-containing complexes of uranyl with carbamide (Urea) and N,N'-dimethylurea (s-Dmur) are synthesized and studied by IR spectroscopy and X-ray diffraction. Structures of the same type, [UO{sub 2}(Urea){sub 4}(H{sub 2}O)][(UO{sub 2}){sub 2}(C{sub 4}H{sub 4}O{sub 4})3] · 3H{sub 2}О and [UO{sub 2}(Urea){sub 4}(H{sub 2}O)][(UO{sub 2}){sub 2}(C{sub 4}H{sub 4}O{sub 4})3] · 2Urea contain two sorts of uranium-containing complex groups, namely, mononuclear [UO{sub 2}(Urea){sub 4}(H{sub 2}O)]{sup 2+} cations and two-dimensional [(UO{sub 2}){sub 2}(C{sub 4}H{sub 4}O{sub 4}){sub 3}]{sup 2–} anions described by crystal-chemical formulas AМ{sub 5}{sup 1} and A{sub 2}Q{sub 3}{sup 02}, respectively (A = UO{sub 2}{sup 2+}, M{sup 1} = Urea or H{sub 2}O, Q{sup 02} = C{sub 4}H{sub 4}O{sub 4}{sup 2-}), and differ only in the nature of noncoordinated molecules—water and carbamide. The main structural groups of the [(UO{sub 2}){sub 2}(C{sub 4}H{sub 4}O{sub 4}){sub 2}(s-Dmur){sub 3}] crystals are [(UO{sub 2}){sub 2}(C{sub 4}H{sub 4}O{sub 4}){sub 2}(s-Dmur){sub 3}] chains belonging to the А{sub 2}Q{sub 2}{sup 02}M{sub 3}{sup 1} (A = UO{sub 2}{sup 2+}, Q{sup 02} = C{sub 4}H{sub 4}O{sub 4}{sup 2-}, M{sup 1} = s-Dmur) crystal-chemical group. Specific features of intermolecular interactions in the crystal structures are revealed using the Voronoi–Dirichlet method of molecular polyhedra.
The great advantage of the C{sub 60} molecule is its potential for polymerization, due to which the molecule can be the building block of new all carbon materials. In addition, it contains, both sp{sup 2} and sp{sup 3} hybridized carbon atoms, which allows synthesizing new carbon materials with desired physicochemical properties using both types of carbon bonding. The one- and two-dimensional polymeric phases of C{sub 60} are prototype materials of this sort. Their properties, especially polymerization under pressure and room temperature via covalent bonding between molecules belonging to adjacent polymeric chains or polymeric layers, can be used for further development of new materials. The present review focuses on the study of the pressure-induced polymerization and thermodynamic stability of these materials and their recovered new phases by in-situ high-pressure Raman and X-ray diffraction studies. The phonon spectra show that the fullerene molecular cage in the high-pressure phases is preserved, while these polymers decompose under heat treatment into the initial fullerene C{sub 60} monomer.
The problem of states of an electron system interacting with impurities that have a spin of 1/2 is considered. It is shown that in the calculation of the energy of the system, the electron spin-flip processes and the formation of electron–hole–impurity flip spin (hole against the background of electrons with another spin projection) play the major role. Such complexes are accumulated in the system (a sort of Bose condensate of complexes is formed); this reduces the energy of the system, which is a linear function of the initial interaction of an electron with the impurity spin (in contrast, for example, to the result obtained in perturbation theory). The hole-type excitation and the spin excitation have a gap in the spectrum. Small parameters of the problem are the interaction of electrons with impurity spins and the number of impurities. The electron–electron interaction is not taken into account. Impurities are assumed to be distributed at random, and calculations are performed using the known averaging over the positions of impurities.
The presence of substantial systematic discrepancies between the results of different experiments devoted to determining cross sections for partial photoneutron reactions-first of all, ({gamma}, n), ({gamma}, 2n), and ({gamma}, 3n) reactions-is a strong motivation for studying the reliability and authenticity of these data and for developing methods for taking into account and removing the discrepancies in question. In order to solve the first problem, we introduce objective absolute criteria involving transitional photoneutron-multiplicity functions F{sub 1}, F{sub 2}, F{sub 3}, Horizontal-Ellipsis ; by definition, their values cannot exceed 1.0, 0.5, 0.33, Horizontal-Ellipsis , respectively. With the aim of solving the second problem, we propose a new experimental-theoretical approach. In this approach, reaction cross sections are evaluated by simultaneously employing experimental data on the cross section for the total photoneutron yield, {sigma}{sup expt}({gamma}, xn) = {sigma}{sup expt}({gamma}, n) + 2{sigma}{sup expt}({gamma}, 2n) + 3{sigma}{sup expt}({gamma}, 3n) + Horizontal-Ellipsis , which are free from drawbacks plaguing experimental methods for sorting neutrons in multiplicity, and the results obtained by calculating the functions F{sub theor}{sup 1}, F{sub theor}{sup 2}, F{sub theor}{sup 3}, Horizontal-Ellipsis on the basis of the modern model of photonuclear reactions. The reliability and authenticity of data on the cross sections for ({gamma}, n), ({gamma}, 2n), and ({gamma}, 3n) partial reactions-{sigma}{sup eval}({gamma}, in) = F{sub i}{sup theor}{sigma}{sup expt}({gamma}, xn)-were evaluated for the {sup 90}Zr, {sup 115}In, {sup 112,114,116,117,118,119,120,122,124}Sn, {sup 159}Tb, and {sup 197}Au nuclei.
Systematic discrepancies between the results of various experiments devoted to determining cross sections for total and partial photoneutron reactions are analyzed by using objective criteria of reliability of data in terms of the transitional photoneutron-multiplicity function F{sub i} = {sigma}({gamma}, in)/{sigma}({gamma}, xn), whose values for i = 1, 2, 3, ... cannot exceed by definition 1.00, 0.50, 0.33, ..., respectively. It was found that the majority of experimental data on the cross sections obtained for ({gamma}, n), ({gamma}, 2n), and ({gamma}, 3n) reactions with the aid of methods of photoneutron multiplicity sorting do not meet objective criteria (in particular, F{sub 2} > 0.50 for a vast body of data). New data on the cross sections for partial reactions on {sup 181}Ta and {sup 208}Pb nuclei were obtained within a new experimental-theoretical method that was proposed for the evaluation of cross sections for partial reactions and in which the experimental neutron yield cross section {sigma}{sup expt}({gamma}, xn) = {sigma}({gamma}, n) + 2{sigma}({gamma}, 2n) + 3{sigma}({gamma}, 3n) + ..., which is free from problems associated with determining neutron multiplicities, is used simultaneously with the functions F{sub i}{sup theor} calculated within a combined model of photonuclear reactions.
Experimental data on {sup 91,94}Zr photodisintegration that were obtained in a beam of quasimonoenergetic annihilation photons by the method of neutron multiplicity sorting are analyzed. It is found that the cross sections for the (γ, 1n), (γ, 2n), and (γ, 3n) reactions on both isotopes do not meet the objective data-reliability criteria formulated earlier. Within the experimental–theoretical method for evaluating partial-reaction cross sections that satisfy these criteria, new data on the cross sections for the aforementioned partial reactions, as well as for the (γ, sn) = (γ, 1n) + (γ, 2n) + (γ, 3n) +... total photoneutron reaction, are obtained for the isotopes {sup 91,94}Zr.
Results of microscopic and semiphenomenological calculations of features of spherical nuclei lying near the stability line and far from it are presented. The reason why the nuclei being considered are spherical is that they are magic at least in one nucleon sort. The present analysis is performed for Z = 50 and Z = 28 isotopes and for N = 50 isotones, the region extending from neutron-rich to neutron-deficient nuclei being covered. The isotopic dependence of the mean-field spin–orbit nuclear potential is revealed; systematics of energies of levels and probabilities for electromagnetic transitions is examined; and root-mean-square radii of nuclei are calculated, along with the proton- and neutron-density distributions in them. Nuclei in the vicinity of closed shells are considered in detail, and the axial-vector weak coupling constant in nuclei is evaluated. A systematic comparison of the results of calculations with experimental data is performed.
The novel scheme of proton and deuteron polarization control in the NICA collider at Dubna is proposed. By means of two Siberian Shakes with solenoid magnetic field the beam spin tune is shifted to the “zero” spin resonance vicinity, whereas manipulation of the polarization is realized by “weak” field solenoids. The scheme makes it possible to obtain any desired direction of the polarization in the both MPD and SPD detectors for any sort of the particles. The possibility of the beam polarization control in the orbit plane at any azimuth of the collider magnetic arcs exists also. The last gives necessary flexibility of optimal matching the beam polarization at injection into collider and at the polarimetery monitor points.
The well-known significant systematic disagreements between data on partial photoneutron reaction cross sections obtained in experiments using quasimonoenergetic annihilation photons and bremsstrahlung were investigated using objective physical criteria of data reliability. It was shown that many data are not reliable because of significant systematic uncertainties of the photoneutron multiplicity sorting methods used. The experimental-theoretical method for evaluation of reliable partial reaction cross sections was proposed and many new data were obtained.