A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing

Background: Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. Methods: A multicenter study was conducted to validate an updated assay design for 454 Life Sciences’ GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940–447,400 copies/mL, two dilution series (52,129–1,340 and 25,130–734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10–99, RT codons 1–251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. Results: The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592–3,488) and 2,410 for V3 (IQR 786–3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P20% were missed, variants 2–10% were detected at most sites (even at low VT), and variants 1–2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. Conclusions: This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing

Analysis Of The Genes Involved In Organizing The Tail Segments Of The Drosophila Melanogaster Embryo

The metameric organization of the Drosophila melanogaster tail is obscured by developmental events that partially suppress or fuse some of its regions. To better define the developmental origins and segmental identities in the tail of the Drosophila embryo, we documented expression patterns and mutant phenotypes of several genes that play important roles in its morphogenesis. We documented the domains of engrailed (en), Abdominal-B (Abd-B) and caudal (cad) expression in the tail region. The staining pattern of cut (ct) was used to correlate the embryonic sense organs with their respective positions on the larval cuticle. The en patterns in different Bithorax-Complex (BX-C) Abd-B morphogenetic (m) and regulatory (r) mutants demonstrated that Abd-B functions to, among other things, suppress embryonic ventral epidermal structures on the posterior side of A8 to A9. Ventral epidermal structures were not added back into the en pattern in r- or BX-C- mutants, indicating that although the BX-C functions extend through A10, other non-BX-C genes must be required for development of this segment. © 1995

Development of the Drosophila-Melanogaster Caudal Segments Involves Suppression of the Ventral Regions of A8, A9 and A10

Whereas the segmental organization of the thorax and anterior abdomen is morphologically delineated in both the Drosophila larva and adult, segments in the head and caudal regions lack such well-defined boundaries. Consequently, the organization of these regions has been difficult to decipher. In this study, transformations caused by the bithorax-complex homeotic mutants 48, M3, Ultraabdominal-1 (Uab1) and tumorous-head-3 (tuh-3), as well as the patterns of engrailed gene expression have been analyzed to investigate the segmental organization of the caudal segments. A special emphasis was placed on sense organs appearing in abdominal segments 8, 9 and 10 (A8-A10): We find that: (1) transformations in the caudal segments obey parasegmental borders; (2) the sense organs on A8, A9, and A10 are probably homologous to the pits and hairs in anterior A1-A7; (3) except for the larval anal tuft and the anterior side of A8, all structures in larval segments A8, A9 and A10 are dorsal/lateral in origin; and (4) dorsalization of embryonic A8 and A9 cells leaves space ventrally for A10, as it follows the contracting ventral nervous system during the embryological process of germ band contraction

A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing

Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. 5A multicenter study was conducted to validate an updated assay design for 454 Life Sciences' GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940-447,400 copies/mL, two dilution series (52,129-1,340 and 25,130-734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10-99, RT codons 1-251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592-3,488) and 2,410 for V3 (IQR 786-3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P20% were missed, variants 2-10% were detected at most sites (even at low VT), and variants 1-2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing

Drug resistance mutation detection in limiting dilution series.

<p>Two HIV-1 subtype B samples were used for the dilution series (A, samples 40–44; B, samples 45–49). The four groups shown in the histogram are based on the median frequency of each drug resistance mutation in its respective <i>undiluted</i> sample: >20% (white bars), 10–20% (bright grey bars), 2–10% (dark grey bars), and 1–2% (black bars). The means and standard deviations are given of the percentage of sites reporting drug-resistance mutations in these categories. The number of mutations in each category is represented by n.</p

Triplicate sample analysis.

<p>A set of three samples was measured in triplicate at each of the 11 sites. Variant percentages measured in the triplicate samples are shown for each mutation.</p

The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis

Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes