Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120₃/gp41₃). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.Identification of conserved regions accessible on the HIV-1 envelope and design of immunogens that elicit broadly neutralizing antibodies against these sites continue to be major challenges in the development of an effective HIV-1 vaccine. The HIV-1 viral spike—composed of three exterior gp120 subunits and three transmembrane gp41 subunits—is highly protected, but a limited number of these conserved regions exist on the spike, identified primarily by the broadly neutralizing antibodies that target them. One region is quaternary in nature and appropriately formed only on the assembled viral spike (gp120₃/gp41₃). This region is targeted by a recently discovered (14) and fast expanding class of monoclonal antibodies (36, 40) that recognize epitopes with quaternary structural constraints, which are composed of portions of two gp120-variable loops, V2 and V3 (reviewed in reference 49). These quaternary structure-specific (or quaternary-specific) antibodies (also called quaternary-neutralizing epitope or “QNE” antibodies) are found in the sera of selected HIV-1-infected individuals who have broadly neutralizing serum antibodies (41); individual members of the class, however, vary greatly in their breadth of neutralization.Initial evidence for the existence of quaternary-specific antibodies arose in simian/human immunodeficiency virus-infected rhesus macaques and HIV-1-infected chimpanzees (6, 9, 13). Characterization of polyclonal sera from these infected animals suggested the presence of antibodies targeting a conformational epitope involving the variable loop regions of the gp120 viral envelope.Antibody 2909 was the first human monoclonal antibody against HIV-1 to be characterized as being specific for an epitope dependent on the quaternary interaction of envelope glycoproteins (14). It was identified by direct screening for neutralization activity against a pseudovirus derived from strain SF162 of HIV-1. It recognizes a quaternary epitope on the surface of native virions and infected cells but does not bind soluble gp120/gp140 envelope proteins or cell surface-expressed gp120 monomers (14, 20). Competition analysis and virological assays indicate that the 2909 epitope includes portions of the V2 and V3 loops of gp120 (14, 16), with the V2-V3 elements originating either from within a gp120 monomer or between gp120 protomers in the trimer context. Mapping of 2909 recognition identifies a particular anomaly in its recognition (16); neutralization by 2909 depends on the presence of a rare lysine at position 160 in the V2 loop rather than the conserved N-linked site of glycosylation found at this position in most HIV-1 isolates (providing a residue-specific explanation for the neutralization specificity of 2909 for the SF162 virus, which contains this rare lysine).Other strain-specific monoclonal antibodies like 2909 have been isolated from rhesus macaques infected with a chimeric simian/human immunodeficiency virus that contained an SF162 isolate-derived viral spike (SHIV_SF162P4) (36). These rhesus monoclonal antibodies exhibit properties similar to those of 2909 in their potent neutralization of SF162 and their recognition of V2-V3 only in the context of the functional viral spike (e.g., on virus particles) (36). Details from epitope mapping indicate that these rhesus antibodies and human antibody 2909 recognize overlapping epitopes, with some differences in requirements for V2 N-linked glycosylation (36).The somatically related human monoclonal antibodies, PG9 and PG16, were also identified by a direct screen for neutralization (40). They target a quaternary-specific V2-V3 epitope, but unlike 2909, they neutralize an extraordinary 70 to 80% of circulating primary HIV-1 isolates and appear to have some reactivity for monomeric gp120 (40). Much of their increased breadth of neutralization arises from their ability to recognize an N-linked glycan at position 160 in the V2 loop, a motif which is found in greater than 90% of HIV-1 group M isolates (25).Despite substantial differences in their neutralization breadth, antibodies 2909 and PG9/PG16 may be closely related. Notably, an N160K mutation in the V2 loop of typical primary HIV-1 isolates like YU2 and JR-FL can recover 2909 activity (16). Conversely, isolate SF162 can be converted to a PG9- and PG16-sensitive pseudovirus by the K160N mutation (40). Thus, a single N or K at position 160 appears to control much of the neutralization difference between 2909 and PG16. Together the results suggest that 2909 and PG9/PG16 antibodies recognize distinct immunotypes of a similar quaternary epitope.To gain insight into how antibodies achieve recognition of this epitope, we determined the crystal structure of the antigen-binding fragment (Fab) of 2909 at a 3.3-Å resolution and compared this structure to the previously determined structure of PG16 (31, 33). Mutational analysis was used to confirm structural hot spots, and chimeric analysis of domain swaps between 2909 and other quaternary-specific antibodies was used to refine assessments of functional similarity. By identifying structural features—shared between 2909 and PG16 but otherwise highly uncommon in antibodies—the results provide insight into conserved solutions by human antibodies for recognition of an important vaccine target on HIV-1.     

Pathogenomics of Xanthomonas: understanding bacterium-plant interactions

Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant hosts, including many economically important crops. Pathogenic species and pathovars within species show a high degree of host plant specificity and many exhibit tissue specificity, invading either the vascular system or the mesophyll tissue of the host. In this Review, we discuss the insights that functional and comparative genomic studies are providing into the adaptation of this group of bacteria to exploit the extraordinary diversity of plant hosts and different host tissues.     

Nano-stenciled RGD-gold patterns that inhibit focal contact maturation induce lamellipodia formation in fibroblasts

Cultured fibroblasts adhere to extracellular substrates by means of cell-matrix adhesions that are assembled in a hierarchical way, thereby gaining in protein complexity and size. Here we asked how restricting the size of cell-matrix adhesions affects cell morphology and behavior. Using a nanostencil technique, culture substrates were patterned with gold squares of a width and spacing between 250 nm and 2 μm. The gold was functionalized with RGD peptide as ligand for cellular integrins, and mouse embryo fibroblasts were plated. Limiting the length of cell-matrix adhesions to 500 nm or less disturbed the maturation of vinculin-positive focal complexes into focal contacts and fibrillar adhesions, as indicated by poor recruitment of α5-integrin. We found that on sub-micrometer patterns, fibroblasts spread extensively, but did not polarize. Instead, they formed excessive numbers of lamellipodia and a fine actin meshwork without stress fibers. Moreover, these cells showed aberrant fibronectin fibrillogenesis, and their speed of directed migration was reduced significantly compared to fibroblasts on 2 μm square patterns. Interference with RhoA/ROCK signaling eliminated the pattern-dependent differences in cell morphology. Our results indicate that manipulating the maturation of cell-matrix adhesions by nanopatterned surfaces allows to influence morphology, actin dynamics, migration and ECM assembly of adhering fibroblasts.     

Synthesis of propiophenone derivatives as new class of antidiabetic agents reducing body weight in db/db mice

A series of propiophenone derivatives (6-23) have been synthesized and evaluated for their in vivo antihyperglycemic activities in sucrose loaded model (SLM), sucrose challenged streptozotocin (STZ-S) induced diabetic rat model and C57BL/KsJ db/db diabetic mice model. Compound 15 and 16 were emerged as potent antihyperglycemics and lipid lowering agents. These compounds (15, 16) further validate the potency by reducing body weight and food intake in db/db mice model. Possible mechanism of action for the propiophenone derivatives was established by the evaluation in various in vitro models. Interestingly some of the compounds were efficiently inhibiting PTP-1B.     

Characterization of a Reuterin-Producing Lactobacillus reuteri BPL-36 Strain Isolated from Human Infant Fecal Sample

A reuterin (3-hydroxypropinaldehyde, 3-HPA)-producing isolate from a human infant fecal sample was identified as Lactobacillus reuteri BPL-36 strain. The organism displayed a broad-spectrum antimicrobial activity. The gene (gdh) encoding a glycerol dehydratase subunit was detected by PCR, thus confirming its reuterin-producing ability. Reuterin concentration of 89.63?mM/mL was obtained in the MRS?Cglycerol medium after 16?h of incubation at 37?°C. The reuterin concentration required to inhibit the growth of Pseudomonas aeruginosa, Escherichia coli O157: H7, Salmonella typhi, Staphylococcus aureus, and Listeria monocytogenes was found to be 1.0, 2.0, 2.0, 4.0, and 10.0?AU/mL, respectively. Antimicrobial efficiency test using BPL-36 cell-free supernatant co-incubated along with different test pathogens was done. Viability of all the tested pathogens decreased with increasing contact time with the cell-free supernatant. S. typhi was observed to be the most susceptible among the tested organisms, and the number of viable cells hugely declined as the contact with cell-free supernatant continued, resulting in a reduction of 6 log cycles (100?% inhibition) of the cells after 4?h of treatment. Production of biogenic amines and degradation of mucin by the reuterin-producing BPL-36 strain were not detected.     

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome.     

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in “real time” during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10³ spores and10² spores in talcum powder, respectively, whereas PCR could detect 10⁴ spores in soil and 10³ spores in talcum powder, respectively.     

Side Population in Human Non-Muscle Invasive Bladder Cancer Enriches for Cancer Stem Cells That Are Maintained by MAPK Signalling

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2^hi) relative to the non-SP (NSP) fraction (ABCG2^low). ABCG2^hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2^low NSP cells. In vivo, ABCG2^hi SP cells enriched for tumour growth compared with ABCG2^low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2^hi SP cells and MEK inhibition also inhibited the ABCG2^hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2^hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.