Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens.     

Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing

The prototypical 5′-nuclease, flap endonuclease-1 (FEN1), catalyzes the essential removal of single-stranded flaps during DNA replication and repair. FEN1 hydrolyzes a specific phosphodiester bond one nucleotide into double-stranded DNA. This specificity arises from double nucleotide unpairing that places the scissile phosphate diester on active site divalent metal ions. Also related to FEN1 specificity is the helical arch, through which 5′-flaps, but not continuous DNAs, can thread. The arch contains basic residues (Lys-93 and Arg-100 in human FEN1 (hFEN1)) that are conserved by all 5′-nucleases and a cap region only present in enzymes that process DNAs with 5′ termini. Proline mutations (L97P, L111P, L130P) were introduced into the hFEN1 helical arch. Each mutation was severely detrimental to reaction. However, all proteins were at least as stable as wild-type (WT) hFEN1 and bound substrate with comparable affinity. Moreover, all mutants produced complexes with 5′-biotinylated substrate that, when captured with streptavidin, were resistant to challenge with competitor DNA. Removal of both conserved basic residues (K93A/R100A) was no more detrimental to reaction than the single mutation R100A, but much less severe than L97P. The ability of protein-Ca²⁺ to rearrange 2-aminopurine-containing substrates was monitored by low energy CD. Although L97P and K93A/R100A retained the ability to unpair substrates, the cap mutants L111P and L130P did not. Taken together, these data challenge current assumptions related to 5′-nuclease family mechanism. Conserved basic amino acids are not required for double nucleotide unpairing and appear to act cooperatively, whereas the helical cap plays an unexpected role in hFEN1-substrate rearrangement.     

Analysis of the anticancer drugs VP 16-213 and VM 26 and their metabolites by high-performance liquid chromatography

A rapid and convenient high-performance liquid chromatographic procedure for the analysis of the clinically useful anticancer agents VP 16-213 and VM 26 is described. The drugs, which are semi-synthetic derivatives of the natural product podophyllotoxin, are extracted from plasma with chloroform. The extracts are evaporated to dryness, reconstituted in methanol, and chromatographed on a reversed-phase microparticle C₁₈ column using isocratic elution with a mixture of methanol—water (60:40). Each drug is used as the internal standard for the other. Quantitation to 500 ng/ml (0.85 nmole/ml) plasma is based on peak height ratios using UV detection at 254 nm. Patient plasma concentration vresus time data agree well with previously published data obtained using radiolabelled drug.Investigations into the nature of the hydroxy acid metabolite of VP 16-213, carried out using paired-ion chromatography with tetrabutylammonium bromide and fluorescence detection, are described. Also, a unique separation of VP 16-213 and a possible metabolite, the isomer, picro VP 16-213, is described.     

Significant Association between Sulfate-Reducing Bacteria and Uranium-Reducing Microbial Communities as Revealed by a Combined Massively Parallel Sequencing-Indicator Species Approach

Massively parallel sequencing has provided a more affordable and high-throughput method to study microbial communities, although it has mostly been used in an exploratory fashion. We combined pyrosequencing with a strict indicator species statistical analysis to test if bacteria specifically responded to ethanol injection that successfully promoted dissimilatory uranium(VI) reduction in the subsurface of a uranium contamination plume at the Oak Ridge Field Research Center in Tennessee. Remediation was achieved with a hydraulic flow control consisting of an inner loop, where ethanol was injected, and an outer loop for flow-field protection. This strategy reduced uranium concentrations in groundwater to levels below 0.126 μM and created geochemical gradients in electron donors from the inner-loop injection well toward the outer loop and downgradient flow path. Our analysis with 15 sediment samples from the entire test area found significant indicator species that showed a high degree of adaptation to the three different hydrochemical-created conditions. Castellaniella and Rhodanobacter characterized areas with low pH, heavy metals, and low bioactivity, while sulfate-, Fe(III)-, and U(VI)-reducing bacteria (Desulfovibrio, Anaeromyxobacter, and Desulfosporosinus) were indicators of areas where U(VI) reduction occurred. The abundance of these bacteria, as well as the Fe(III) and U(VI) reducer Geobacter, correlated with the hydraulic connectivity to the substrate injection site, suggesting that the selected populations were a direct response to electron donor addition by the groundwater flow path. A false-discovery-rate approach was implemented to discard false-positive results by chance, given the large amount of data compared.Massively parallel sequencing has increased our ability to study microbial communities to a greater depth and at decreased sequencing costs to an extent that replication and gradient interrogation are now reasonably attainable. However, this massive throughput has mostly been used in exploratory studies, given the challenges to analysis of the big data sets generated and the relative novelty of the technique. To date, no report of a study that has used this method to describe the microbial community over a large area influenced by complicated hydrogeochemical factors during bioremediation has been published. Here, we used pyrosequencing technology complemented with a hypothesis-based approach to identify bacteria associated with biostimulation of U(VI) reduction at Area 3 of the U.S. Department of Energy''s (DOE''s) Oak Ridge Field Research Center (FRC) at Oak Ridge, TN.The Oak Ridge FRC is one of the most-studied sites for uranium bioremediation (2, 8, 19-22, 27, 37, 45-48). Previously used as a uranium enrichment plant, the site remains contaminated with depleted uranium, nitrate, and acidity. To deal with uranium contamination, dissimilatory metal reduction has been studied as an alternative that reduces risk by converting toxic soluble metals and radionuclides to insoluble, less toxic forms (2, 3, 16, 21, 26, 45). For example, some microbes can use metals such as Cr(VI), Se(VI), and the radionuclides U(VI) and Tc(VII) as final electron acceptors, producing a reduced insoluble species, thus blocking dispersal and reducing bioavailability.The ability to reduce U(VI) to U(IV) has been found in several unrelated phylogenetic groups, i.e., Delta-, Beta-, and Gammaproteobacteria, Firmicutes, Deinococci, and Actinobacteria, among others (42). Most previous studies have focused on the Fe(III)-reducing bacteria (FRB), especially Geobacter, and the sulfate-reducing bacteria (SRB), especially Desulfovibrio. Uranium(VI) reduction for bioremediation purposes has been tested and confirmed in laboratory-scale experiments using serum bottles (13, 18, 48), microcosms (23, 32), sediment columns (14, 43), and in situ field studies (3, 21, 41, 45), with the last one demonstrating the feasibility of U(VI) remediation and the correlation of U(VI) reduction with FRB (3, 6, 18, 31, 41) or SRB (40), or both (8, 19, 49).During field studies at Area 3 of the Oak Ridge site, a hydraulic control system together with ethanol injection successfully promoted U(VI) reduction from 5 μM to levels below U.S. Environmental Protection Agency (EPA) maximum contaminant levels (MCLs) for drinking water (0.126 μM) over a 2-year period (46). Reduction of U(VI) to U(IV) was confirmed by X-ray absorption near edge structure (XANES) (22, 46). Previous microbial surveys of sediments and groundwater from Area 3 wells by the use of 16S rRNA gene clone libraries detected genera known to harbor U(VI)-reducing members, such as Geobacter, Desulfovibrio, Anaeromyxobacter, Desulfosporosinus, and Acidovorax, after U(VI) reduction was established (8, 19). In one study, microbial counts from sediments were correlated with the hydraulic path, suggesting differences in organic carbon availability throughout Area 3 (8). The study that tracked the groundwater microbial communities of four locations of Area 3 over a 1.5-year period during ethanol stimulation found that nitrate, uranium, sulfide, and ethanol were correlated with particular bacterial populations and that the engineering control of dissolved oxygen and delivered nutrients was also significant in explaining the microbial community variability (19). However, the analysis of communities has been focused on limited wells and the community of the entire test area has not been characterized.On the basis of the previous results, we further hypothesized that the hydrological control strategy employed for the remediation of the site constrained the geochemistry of the site by controlling the distribution of organic carbon substrates and other nutrients and that this in turn selected a characteristic microbial community that was distinguishable from its surrounding community. We used massively parallel sequencing of 16S rRNA genes from sediments of 15 wells to characterize the microbial communities along hydrological gradients from the microbiologically active and hydraulically protected inner-loop zone to less active and still contaminated areas outside the treatment area and downgradient. Our sediment-sampling strategy allows a more precise spatial characterization than the use of groundwater samples, where filtering large volumes of water is often required, and also because samples of the attached communities can differ from the planktonic ones, as expected in oligotrophic aquifers (15), such as this site. The deeper sequencing allowed a more extensive survey of the communities, higher confidence in the detection of less dominant but significant members, and a more statistically robust indicator species assessment. We were able to detect groups significantly associated with U(VI) reduction and to explain differences in community structure with hydrogeochemical conditions.     

Demographic features of Bartonella infections in Richardson's ground squirrels (Spermophilus richardsonii)

The epidemiology of Bartonella infections in Richardson's ground squirrels (Spermophilus richardsonii) was studied at multiple sites in Saskatchewan, Canada, from 2002 to 2004. The overall prevalence of Bartonella infection was 48%. Juvenile squirrels were significantly more likely to be infected with Bartonella than were adults (58% and 37%, respectively), and juvenile animals also were significantly more likely to have high levels of bacteremia compared to adult animals. Prevalence of Bartonella infection appeared to decrease with age; only 24% of animals known to be > or = 2 yr old were infected with Bartonella. Prevalence of infection was lowest in May (27%) and highest in late summer and early autumn (71%). The prevalence of fleas also varied seasonally, and animals were more likely to have fleas in the late summer and early autumn than in early summer. We found no relationship between Bartonella prevalence and host density or flea prevalence.     

Structural changes of bacteriophage phi29 upon DNA packaging and release

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.     

Discovery of highly selective EP4 receptor agonists that stimulate new bone formation and restore bone mass in ovariectomized rats

Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.