Documents, The $100,000 gallery of art

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Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Water quality at Bakun HEP reservior, Belaga, Sarawak

Since the water supply at Bakun Hydroelectric Dam reached its full supply level, only one previous study has been done during the filling phase of the dam which was 3 years and 10 months earlier than this study. As water in the reservoir is very important for the aquatic organisms in the reservoir and downstream of the dam, a study was conducted at 3 stations to determine the selected water quality parameters 5 years 6 months after impoundment started. At each station, water in triplicate samples were collected at 6 levels, which is the subsurface, at 10m, 20m, 30m, 40m and 50m depth. Results showed that thermocline occurred at 3m to 10m depth at all stations. DO at the subsurface (4.99 -6.10 mg/L) dropped drastically to anoxic level starting from 2m to 10m depth at all stations. Water conductivity and turbidity increases while pH decreased as depth increased. The highest chlorophyll-a (11.68 J-lg/L) was recorded at 10m depth with positive correlation with turbidity. Increasing levels of ammonia-nitrogen (0.0533 -0.6400 mg/L), total suspended solids (3.30 -63.06 mg/L), and five-day biochemical oxygen demand (1.86 -4.30 mg/L) were observed while depth increases. Nitrate (0.01 -0.22 mg/L), nitrite (0.0020 -0.1170 mg/L), silica (0.39 -1.54 mg/L) and orthophosphate (0.0900 -1.7067 mg/L) showed different variations with depth. This present study showed that the water quality at Bakun Hydroelectric Dam was improving and still changing compared to the previous study during the filling phase and has not stabilize even after 5 years 6 months after impoundment started

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120▿ †

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B′/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC50) and IC90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design

Structure of the llama heavy chain antibody fragment V_HH D7.

<p>(<b>A</b>) Ribbon representation of D7; the complementarity determining regions (CDR) are highlighted in yellow (CDR1), orange (CDR2) and salmon (CDR3). The first and last residue of each CDR is shown together with the side chain of Trp⁹⁶ critical for gp120 interaction and neutralization. The dotted line indicates CDR3 residues lacking continuous main chain density for residues Arg¹⁰⁰ to Ser^100B. (<b>B</b>) A close-up of the CDR interaction network reveals multiple polar interactions between CDR1 and CDR3 as well as CDR2 and CDR3.</p

X-ray data collection and refinement statistics.

<p>X-ray data collection and refinement statistics.</p

Solvent accessible areas of CDR3 D7 residues.

<p>Solvent accessible areas of CDR3 D7 residues.</p

Binding affinities of D7 wild type and D7 mutants to gp120 (IIIB).

<p>Binding affinities of D7 wild type and D7 mutants to gp120 (IIIB).</p