ABI1 protein, human

RNA was extracted from patient samples that demonstrated the highest concentration of ZIKV RNA determined by the real-time assay, and for which sufficient sample volume was available (patients 824, 037, 830a, and 958). Briefly, RNA was extracted from 150 μL of serum by using the QIAamp Viral RNA Mini Kit (QIAGEN), and RNA was eluted with 75 μL of RNase-free water. A series of RT-PCRs was performed with each RNA preparation by using primer pairs designed to generate overlapping DNA fragments that spanned the entire polyprotein coding region of the virus. Primers were designed by using the ZIKV MR 766 prototype virus coding region sequence (GenBank accession no. AY632535) and the PrimerSelect software module of the LaserGene package (DNASTAR Inc., Madison, WI, USA). Several primers initially failed to amplify because of sequence mismatches between ZIKV MR 766 and ZIKV Yap 2007. Therefore, primers were redesigned by using newly generated DNA sequence data, and a “genome walking” approach was used to derive complete coding region sequence data. The complete list of amplification and sequencing primers is available upon request.
All RT-PCRs were performed with 10 μL of RNA by using the OneStep RT-PCR Kit (QIAGEN) following the manufacturer’s protocol. DNAs were analyzed by 2% agarose gel electrophoresis, and bands of the predicted size were excised from the gel and purified by using the QIAquick Gel Extraction Kit (QIAGEN). Purified DNAs were subjected to nucleic acid sequence analysis with sequencing primers spaced ≈500 bases apart on both strands of the DNA fragments by using the ABI BigDye Terminator V3.1 Ready Reaction Cycle Sequencing Mixture (Applied Biosystems). Nucleotide sequence was determined by capillary electrophoresis by using the ABI 3130 genetic analyzer (Applied Biosystems) following the manufacturer’s protcol. Raw sequence data were aligned and edited by using the SeqMan module of LaserGene (DNASTAR Inc.). Because of insufficient sample volume, no patient RNA was sufficient to generate DNA that included the entire coding region. Therefore, DNA data obtained from 4 patients was combined to generate a consensus sequence heretofore designated the ZIKV 2007 epidemic consensus (EC) sequence (GenBank accession no. EU545988).
The complete coding region of ZIKV 2007 EC or the nonstructural protein 5 (NS5) gene subregion was aligned with all available flavivirus sequences in GenBank by using the Clustal W algorithm within the MEGA version 4 software package (www.megasoftware.net). Phylogenetic trees were constructed by using either the complete coding region or the NS5 region because a large number of NS5 sequences were available in GenBank and trees for the NS5 region have been constructed (16 (link)). Additional ZIKV strains from the CDC/World Health Organization reference collection (strains 41662, 41524, and 41525) isolated from Aedes spp. mosquitoes collected in Senegal in 1984 were also amplified by RT-PCR in the NS5 region and subjected to nucleic acid sequencing as described above and included in the NS5 region analysis. Trees were constructed from coding region data or from NS5 data by MEGA 4 from aligned nucleotide sequences. We used maximum parsimony, neighbor-joining, or minimum evolution algorithms with 2,000 replicates for bootstrap support of tree groupings. All trees generated nearly identical topology; only the neighbor-joining NS5 tree is shown (Figure 1).

Primer pairs were designed for PCR amplification and sequencing of internal portions of seven housekeeping genes (Table 4). Three of these pairs (cpn60, gltA and recA) were designed by Bartual et al.[24] (link). Primer pairs for three other genes, which are present in most bacterial phyla (fusA, pyrG and rplB), were designed by adapting, using the A. baylyi and A. baumannii genome sequences, the primers initially proposed by Santos and Ochman [69] (link). Finally, primers for gene rpoB were designed previously [70] (link). The portion of rpoB that was amplified with these primers corresponds to positions 1,681 to 2,136. These genes represent seven distinct loci on the A. baumannii chromosome (Table 4). The internal gene portions chosen for MLST allele and profile definition ranged in length from 297 bp (pyrG) to 633 bp (fusA). Further details on this MLST scheme can be found at www.pasteur.fr/mlst. Nucleotide sequences were obtained using Big Dye version 1.1 chemistry on an ABI 3730XL apparatus.

The required part of genomic DNA was amplified using primers listed in S1 Table to determine the presence or the absence of the mutant allele for each analysed gene.
PCR mixture for each amplification reaction contained 2–2.5 mM MgCl₂, 1x Dream Taq buffer (Thermo Fisher Scientific), 1 μM¹ of both forward and reverse primer, 0.25 mM dNTPs (Thermo Fisher Scientific), 1 U Dream Taq polymerase (Thermo Fisher Scientific), approximately 50 ng of template DNA and H₂O.
We used PCR followed by the restriction fragment length polymorphism (RFLP) analysis to detect alleles of CMR1, DM, PRCD and SHT alleles. The restriction enzyme digestion was performed in a 20 μl reaction mixture which consisted of 2 U of the restriction endonuclease (HphI—CMR1, Eco57I - DM, SfaNI—HUU, AlwI and RsaI—PRCD, FD-Eco91I - SHT) (Thermo Fisher Scientific), 1x supplied buffer, 10 μl PCR product and distilled water. Fragments were separated by size using electrophoresis on 1.5% agarose gel or 10% polyacrylamide gel, depending on the product length.
Sequencing was used for HSF4 mutation screening and for verifying the results of RFLP methods. The five samples were chosen randomly from each genotype available, and the sequencing data were compared with the results of the RFLP analysis.
The PCR products were directly sequenced after ExoSAP-IT (Applied Biosystems) treatment using PCR primers with the ABI BigDye Terminator Sequencing Kit 3.1 (Applied Biosystems) on an ABI 3500 capillary sequencer. Sequence data were analysed with Vector NTI Advance 7.0 (Invitrogen).

To evaluate functional blood flow in the lower extremities, we conducted physiological measurements, including ankle–brachial pressure index (ABI) and toe–brachial pressure index (TBI). ABI and TBI were measured, as previously reported, using the ABI form (Colin, Co., Ltd., Komaki, Japan) [8 (link)]. Normal ABI and TBI were reported to be 0.9–1.3 and 0.6<, respectively, in the general population [24 (link)]. However, in our previous study, we confirmed that the best ABI cut-off value for detecting LEAD in HD patients was 1.06 [8 (link)]. Sensitivity and specificity of ABI values < 1.06 for detecting LEAD in dialysis patients were 80.0% and 98%, respectively [8 (link)]. Therefore, an ABI cut-off value of 1.06 was used in this study. The cut-off value for TBI was <0.6, as used in our previous study.

Genome DNA extractions were carried out from dried samples using the modified CTAB method protocol of Li [17 (link)]. The internal transcribed spacer (ITS) and 28S large subunit regions of ribosomal DNA were amplified with base primer pairs of ITS5/ITS4 and LROR/LR5 [31 (link)]. Partial 16S small subunit region of mitochondrial DNA was amplified with primer pair MS1/MS2 [31 (link)]. Partial largest and second largest RNA polymerase II regions (rpb1 and rpb2) were amplified with primer pairs of rpb1-Ac/rpb1-Cr and brpb2-6f/frpb2-7cR [32 (link),33 (link),34 (link)]. Partial translation elongation factor 1-α (tef1-α) gene was amplified with primer pair EF-983F/EF-1567R [35 (link)]. PCR conditions in aforementioned references were applied. Amplification products were detected by 1.5% agarose gel electrophoresis. Then PCR product depurations were performed using a Biomed Nucleic Purification Kit. Sanger sequencing was performed with the same primers as for PCRs in a ThermoFisher ABI 3730XL DNA Analyzer by Biomed Gene Technology Company (Beijing, China). An ABI BigDye 3.1 Terminator Cycle Sequencing Kit was also applied in sequencing procedure. The newly acquired sequences in this analysis were deposited in GenBank (bold text in Supplementary Tables S1 and S2).