Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs

FIGURE 1 in Pamphilius ishikawai feeds on Astilbe: the first record of Pamphiliidae (Hymenoptera) associated with Saxifragaceae

FIGURE 1. Larval abodes (leaf-rolls) on Astilbe formosa (A–F), A. odontophylla (G), A. thunbergii var. thunbergii (H–K). Photographed by A. Shinohara (A, B), H. Kojima (C–F), S. Ibuki (G–K). A, Probably fourth instar (1 in Table 1), underside, Takamine-onsen, August 7, 2013; B, late instar (one of 17–20 in Table 2), Takamine-onsen–Ikenotaira, August 29, 2014; C, probably fourth instar (21 in Table 2), upper side, Mt. Kasadake, August 21, 2014; D, the same, underside, enlarged; E, probably second instar (4 in Table 2), upper side, Takamine-onsen–Ikenotaira, August 15, 2014; F, the same, underside; G, middle instar (12 in Table 5), underside, Wami, June 5, 2015; H, early instar (1 in Table 5), upper side, Sukusukunomori, June 1, 2015; I, the same, underside; J, early or middle instar (7 in Table 5), underside, Bicchuzawa, June 5, 2015; K, late instar (2 in Table 5), underside, Bicchuzawa, June 2, 2015. White or black arrows: larval abodes (leaf-rolls). Yellow arrows: remains of egg shells

A case of laparoscopic surgery for endometrial cancer in a patient previously treated with a transvaginal mesh procedure for pelvic organ prolapse

Transvaginal mesh (TVM) surgery is an effective treatment option for women with pelvic organ prolapse (POP). Because the TVM procedure preserves the uterus, it is possible for endometrial cancer to occur at a later date. We herein present the first report of such an endometrial cancer, diagnosed well after TVM surgery for POP, and the use of laparoscopic surgery to conduct a simple total hysterectomy to treat it. Keywords: endometrial cancer, laparoscopic surgery, pelvic organ prolapse, transvaginal mes

カリキュラムCS2013とCC2001の比較・考察

2013 年 12 月，米国にて ACM/IEEE-CS によるコンピュータ科学カリキュラム標準 CS2013 が公表された．それまでの標準だった CC2001 の刊行後 10 年以上が経ち，その後継として作成されたものである．日本では，情報専門学科カリキュラム標準 「J07」 が 2008 年に公表されているが，現在，改訂への活動が始まりつつある．J07 は，CC2001 を元として作られたものであるので，CC2001 が CS2013 にどのように改訂されたかを知ることは重要である．本報告では，CS2013 と CC2001 を比べ，知識体系 (BOK) を中心に，どのように改訂されたのかを比較，考察する

Electrophoretogram of PCR products from multiplexes N-I to N-VI.

<p>The primer sets of multiplexes N-I to N-VI are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158463#pone.0158463.t002" target="_blank">Table 2</a>.Yellow, light blue, light green, red, green, and purple frames indicate multiplexes N-I, II, III, IV, V, and VI, respectively. The color coding corresponds to that given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158463#pone.0158463.g002" target="_blank">Fig 2</a>. LM indicates the 10-bp ladder marker.</p

Sensitivity test using progressively diluted crude DNA with known mtDNA haplogroups.

<p>Samples 1 and 2 correspond to mtDNA haplogroups D4j and F2, respectively. Results obtained using multiplexes M-I and N-I are framed in yellow, while results obtained using multiplexes M-III and N-VI are framed in light green and purple, respectively. Dotted lines indicate detection limits. NC indicates negative PCR control. LM indicates the 10-bp ladder marker.</p

Electrophoretograms of the multiplex PCR products for mtDNA of ancient skeletons.

<p>Each lane gives results for a single sample: lane LM, 10-bp ladder marker; lane 1, B516C; lane 2, B516D; lane 3, B202C; lane 4, B228D; lane 5, B192; lane 6, B202A; lane 7, B202B; lane 8, B509A; lane 9, B516A; lane 10, B228C; lane 11, B511; lane 12, B585. Using the conventional APLP system, 3 out of 12 samples could be assigned to relevant haplogroups (B192 to N9b, B516C to M7a, and B516D to M7a). Arrows indicate subsequent haplogrouping flows based on the results obtained using multiplexes M-I and N-I. Yellow frames identify results obtained using multiplexes M-I and N-I, while results obtained using mutiplexes M-V and N-III are framed in green and light green, respectively (color coding corresponds to that given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158463#pone.0158463.g002" target="_blank">Fig 2</a>).</p

Primers of multiplexes M-I to M-VIII used for haplogrouping mtDNAs that stem from macro-haplogroup M.

<p>Primers of multiplexes M-I to M-VIII used for haplogrouping mtDNAs that stem from macro-haplogroup M.</p

Electrophoretogram of PCR products from multiplexes M-I to M-VIII.

<p>The primer sets of multiplexes M-I to M-VIII are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158463#pone.0158463.t002" target="_blank">Table 2</a>. Yellow, light blue, light green, red, green, purple, orange, and blue frames indicate multiplexes M-I, II, III, IV, V, VI, VII, and VIII, respectively. This color coding corresponds to that given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158463#pone.0158463.g002" target="_blank">Fig 2</a>. LM indicates the 10-bp ladder marker.</p