A multi-ethnic meta-analysis identifies novel genes, including ACSL5, associated with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating progressive motor neuron disease that affects people of all ethnicities. Approximately 90% of ALS cases are sporadic and thought to have multifactorial pathogenesis. To understand the genetics of sporadic ALS, we conducted a genome-wide association study using 1,173 sporadic ALS cases and 8,925 controls in a Japanese population. A combined meta-analysis of our Japanese cohort with individuals of European ancestry revealed a significant association at the ACSL5 locus (top SNP p = 2.97 × 10−8). We validated the association with ACSL5 in a replication study with a Chinese population and an independent Japanese population (1941 ALS cases, 3821 controls; top SNP p = 1.82 × 10−4). In the combined meta-analysis, the intronic ACSL5 SNP rs3736947 showed the strongest association (p = 7.81 × 10−11). Using a gene-based analysis of the full multi-ethnic dataset, we uncovered additional genes significantly associated with ALS: ERGIC1, RAPGEF5, FNBP1, and ATXN3. These results advance our understanding of the genetic basis of sporadic ALS

Cell-to-Cell Transformation in Escherichia coli: A Novel Type of Natural Transformation Involving Cell-Derived DNA and a Putative Promoting Pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as ‘cell-to-cell transformation’. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10–5–10–6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria

Comparison of cell-to-cell transformation with artificial transformation.

<p>Strain names: CAG, CAG18439; HB, HB101; and MC, MC4100. Cell-to-cell transformation (A) was performed with three combinations including donor cells harbouring pHSG299. Artificial transformations by the CaCl₂ method (B) and PEG method (C) was performed as described in Methods. In artificial transformation, plasmid (pHSG299) was used in a semi-saturating amount (500 ng/sample) or the roughly estimated amount of leaked plasmid DNA in co-culture [CAG: 2×10^–10; HB: 1×10^–8; MC: 1×10^–9 ng per recipient ( = competent) cells]. The latter values were calculated on the assumptions that free plasmid DNA was supplied from dead donor cells (maximum 5% of total population) to co-existing recipient cells in each corresponding co-culture of cell-to-cell transformation, and that the pHSG299 copy number is ∼200 per cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016355#pone.0016355-Hong1" target="_blank">[38]</a>. Transformation frequency and transformation efficiency were calculated as described in Methods. Data are presented as mean and S.D. (<i>n</i> = 3).</p

Lateral plasmid transfer with various combinations of strains and plasmids in colony biofilm culture.

<p>Frequency of plasmid transfer (mean, <i>n</i> = 3) in each combination is presented in decimal ranges as follows: ++++++, 1E–4 to 1E–5; +++++, 1E–5 to 1E–6; +++, 1E–7 to 1E–8; ++, 1E–8 to 1E–9; +, 1E–9 to 1E–10; – : below detection limit; n.d., not determined or unable to examine because of coincidence of the same antibiotic resistance between strains and plasmids. Samples with plasmid transfer frequency >1E–6 are indicated by .</p

Characterization of putative active factor in CM of CAG18439.

<p>Effects of heat exposure and protease treatment of CM of CAG18439 on cell-to-cell transformation and cell growth in co-culture of MG1655 harbouring pHSG299 and MC4100. Heat treatment of CM was performed at 121°C for 20 min. Treatment of CM with trypsin (100 µg/mL) and proteinase K (200 µg/mL) was performed at 37°C for 120 min. CM was used for culture at 50% (v/v) in heat experiments and at 1% (v/v) in protease experiments. Data are presented as mean and S.D. (*: <i>t</i>-test: <i>P</i><0.05, <i>n</i> = 4; †: <i>t</i>-test: <i>P</i><0.005, <i>n</i> = 4).</p

Measurement of the dead cell ratio in liquid and colony biofilm cultures.

<p>*Measured at 24 h from culture start.</p

Lateral plasmid transfer with various combinations of strains and plasmids in liquid culture.

<p>Frequency of plasmid transfer (mean, <i>n</i> = 3) in each combination is presented in decimal ranges as follows: ++++++, 1E–4 to 1E–5; +++++, 1E–5 to 1E–6; ++++, 1E–6 to 1E–7; +++, 1E–7 to 1E–8.</p

Effects of dilution of CM of CAG18439 on cell-to-cell transformation and cell growth.

<p>Effects of dilution of CM of CAG18439 on cell-to-cell transformation (A) and cell growth (B). Co-culture of MG1655 harbouring pHSG299 and MC4100 was performed in the presence of CM. Data are presented as mean and S.D. (<i>n</i> = 3).</p