Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. SUMMARY: Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

Clinicopathological features of narrow-band imaging endoscopy and immunohistochemistry in ultraminute esophageal squamous neoplasms.

To reveal clinicopathological features of narrow-band imaging (NBI) endoscopy and immunohistochemistry in ultraminute esophageal squamous neoplasms. If a lesion diameter was smaller or same compared with a width of closed biopsy forceps, a lesion was defined to be an ultraminute lesion. Twenty-five consecutive patients with 33 ultraminute esophageal lesions that were removed by endoscopic mucosal resection were included in the present study. We conducted two questionnaire surveys of six endoscopists by their retrospective review of endoscopic still images. The six endoscopists evaluated the endoscopic findings of the ultraminute lesions on still images taken by conventional white-light imaging endoscopy and non-magnified NBI endoscopy in the first questionnaire, and taken by magnified NBI endoscopy in the second questionnaire. An experienced pathologist who was unaware of any endoscopic findings made histological diagnosis and evaluated immunoexpression of p53 and Ki67. The 33 ultraminute lesions were all determined to be either 11 high-grade intraepithelial neoplasias (HGIENs) or 22 low-grade intraepithelial neoplasias (LGIENs). The tumor diameters were histologically confirmed to be <3 mm. All of the ultraminute tumors were visualized as unstained areas and brownish areas by real-time endoscopy with Lugol dye staining and non-magnified NBI endoscopy, respectively. All of the ultraminute IENs were visualized as brownish areas by real-time non-magnified NBI endoscopy. Three of the 25 patients with the ultraminute IENs (12%) had multiple brownish areas (more than several areas) in the esophagus on real-time non-magnified NBI endoscopy. All of the ultraminute IENs were visualized as unstained areas by real-time Lugol chromoendoscopy. Twenty of the 25 patients (80%) had multiple unstained areas (more than several areas) in the esophagus on real-time Lugol chromoendoscopy. The first questionnaire survey revealed that a significantly higher detection rate of the ultraminute IENs on non-magnified NBI endoscopy images compared with conventional white-light imaging endoscopy ones (100% vs. 72%, respectively: P < 0.0001). The second questionnaire survey revealed that presence rates of any magnified NBI endoscopy findings were not significantly different between HGIENs and LGIENs. Proliferation, dilation, and various shapes of intrapapillary capillary loops indicated remarkably high presence rates of more than 90% in both HGIENs and LGIENs. Six of 22 LGIENs (27%) and 3 of 11 HGIENs (27%) show a positive expression for p53. None of peri-IEN epithelia was positive for p53. A mean of Ki67 labeling index of LGIENs was 33% and that of HGIENs 36%. Ki67 labeling index was significantly greater in the LGIENs and HGIENs compared with that in the peri-IEN epithelia. There were no significant differences in p53 expression and Ki67 labeling index between the HGIENs and LGIENs. Non-magnified/magnified NBI endoscopy could facilitate visualization and characterization of ultraminute esophageal squamous IENs. The ultraminute HGIENs and LGIENs might have comparable features of magnified NBI endoscopy and immunohistochemistry.

Can mechanical balloon dissection be applied to cleave fibrotic submucosal tissues? A pilot study in a porcine model.

BACKGROUND AND STUDY AIMS: Removal of a lesion containing an ulcer scar is one of the most challenging applications of endoscopic submucosal dissection (ESD). The present study examined whether a novel balloon dissector could cleave fibrotic submucosal tissue beneath ulcer scars. METHODS: Six pigs were studied. Endoscopic mucosal resection (EMR) with ligation was performed at 7 or 8 sites in the stomach for each animal; 4 weeks later, 23 sites with a visible scar were selected for submucosal dissection. The procedure involved first creating a submucosal fluid cushion (SFC) by injecting either saline mixed with mesna or pure saline. A slender, compliant balloon with a diameter of 8, 13, or 18 mm was inserted into the SFC. The balloon was unfolded and thrust forward to cleave the fibrotic submucosa over approximately 5 cm. RESULTS: Fibrotic submucosa was dissected within 90 seconds in 17 of 23 attempts. Isolating the ulcer scar from the muscularis with the SFC prior to balloon dissection and using a thinner balloon catheter both ensured a better dissection. CONCLUSIONS: The fibrotic submucosa underlying post-EMR scars can be dissected with the novel balloon dissector, although the technique is less effective in cases with no sign of lifting.

Esophageal submucosal dissection under steady pressure automatically controlled endoscopy (SPACE): a randomized preclinical trial.

BACKGROUND AND STUDY AIMS: A new overtube system has been developed for steady pressure automatically controlled endoscopy (SPACE) in the gastrointestinal tract. The objectives of this study were to validate the feasibility and safety of SPACE in the esophagus, and to evaluate its potential advantages over conventional (manually insufflating) endoscopy in endoscopic submucosal dissection (ESD). METHODS: This was a multicenter preclinical trial using acute porcine models (n = 20). In Experiment 1 (feasibility/safety study), SPACE was attempted in the esophagus with continuous monitoring of cardiopulmonary parameters and intraluminal pressures in the downstream bowel. Different insufflation pressures were tested to optimize the insufflation condition. Each session was video-recorded and scored by blinded reviewers. In Experiment 2 (randomized trial), esophageal ESD was attempted using either SPACE or conventional endoscopy, and results were compared. RESULTS: In Experiment 1, SPACE was performed safely without intraluminal pressure elevation in the downstream bowel. According to video review, SPACE provided more stable, reproducible, and rapid visualization than conventional endoscopy. The insufflation pressure was optimized at 14 mmHg for esophageal SPACE. In Experiment 2, ESD was completed in all animals. The ESD time was significantly shorter with SPACE compared with conventional endoscopy (1326 vs. 1616 seconds; P = 0.009). Responses to questionnaires showed that 94 % - 100 % of participants considered SPACE to provide improved exposure and more uniform tissue tension than conventional endoscopy. Other data were comparable. CONCLUSIONS: SPACE is feasible, safe, and potentially effective for complicated endoscopic procedures, such as ESD. SPACE improves and standardizes endoscopic exposure and tissue tension. A clinical study is required to further confirm its safety and clinical effectiveness.

Initial evaluation of a novel multibending backward-oblique viewing duodenoscope in endoscopic retrograde cholangiopancreatography.

A novel multibending backward-oblique viewing duodenoscope was developed to overcome the difficult technical aspect of deep cannulation into the bile duct during endoscopic retrograde cholangiopancreatography (ERCP). The aim of the present study was to evaluate the initial experience of a novel multibending backward-oblique viewing duodenoscope (M-D scope) for ERCP. This was a retrospective review of 23 patients with native papilla who received biliary ERCP with the M-D scope between April and December 2010. The procedures were performed by two well-experienced endoscopists. In all patients, biliary cannulation and therapeutic procedure were successfully completed. In two patients with Billroth I gastrectomy, ERCP were initially attempted with a conventional single-bending duodenoscope, but biliary cannulations were unsuccessful. However, with the use of the M-D scope, biliary cannulation and therapeutic procedures were successfully completed. A novel multibending backward-oblique viewing duodenoscope is safe and feasible for therapeutic and diagnostic ERCP.

Chemically assisted submucosal injection facilitates endoscopic submucosal dissection of gastric neoplasms.

BACKGROUND AND STUDY AIMS: A randomized in vivo animal study previously demonstrated that topical injection of mesna solution (sodium-2-mercaptoethanesulfonate) chemically softened submucosal connective tissues and facilitated mechanical dissection of the submucosal tissue plane. The present study evaluated the technical feasibility and safety of chemically assisted endoscopic submucosal dissection (CA-ESD) using mesna in 20 consecutive patients who underwent endoscopic excision of gastric neoplasm. MATERIALS AND METHODS: Following the margination of the lesion with a mucosal circumcision, 4 - 12 mL of 10 % mesna solution was injected into the submucosal layer. Mechanical submucosal dissection was then performed by bluntly cleaving the chemically treated submucosal layer with the tip of a cap-fitted gastroscope. The use of cautery was restricted to prophylactic hemostasis, dissection of the coagulated vessels and persistent submucosal tissues, and the final snare resection. Post-therapeutic ulceration repair and adverse events were followed up during a 1-week hospitalization and by repeat endoscopies at 1 day, 1 week, and 1 month after the procedure. RESULTS: Sixteen gastric cancers and four adenomas were treated in this study. The sampled tissue measured 38.25 +/- 14.53 mm, with an en bloc resection rate of 100 %. Mean operation time was 21.17 +/- 11.6 minutes. The time spent using cautery was limited to 26.1 % of the total submucosal dissection time. Ulcerations healed normally without complications. CONCLUSIONS: This preliminary study demonstrates that submucosal injection of mesna facilitates and expedites mechanical submucosal dissection. The major limitations in this study include the single-arm study design and a small patient population.

A pilot study of EUS-guided hot saline injection for induction of pancreatic tissue necrosis.

BACKGROUND AND STUDY AIMS: Hot saline may be potentially useful for inducing necrosis of pancreatic tissue. However, the local and systemic effects are largely unknown. This pilot study aimed to evaluate the feasibility and safety of EUS-guided injection of hot saline into the pancreas in the porcine model. METHODS: Boiling hot saline was injected into the tail of normal porcine pancreas under EUS guidance in six pigs via a transgastric approach. Three pigs were killed 4 hours later to study the acute effect of the hot saline injection (acute study). The remaining three pigs were killed after 7 days of clinical observation (survival study). RESULT: Injection of 5 mL, 2 mL and 1 mL of hot saline produced localized necrosis (7 - 10 mm) of pancreatic tissue in the acute study. However, there was pooling of hot saline on the surface of the pancreas when 5mL was injected. On the basis of the results of the acute study, the volume of hot saline injected in the survival study was 1 mL. One milliliter of hot saline produced localized or sporadic necrosis of pancreatic tissue without any signs of pancreatitis in all three pigs in the survival study; hot saline was observed to pool on the pancreatic surface of one pig. There was no histological evidence of necrosis in the pancreatic tissue adjacent to the pooled hot saline in either the acute or the survival study. CONCLUSION: EUS-guided hot saline injection of pancreatic tissue in the porcine model was technically successful and led to localized necrosis of pancreatic tissue without any sign of pancreatitis.

Magnifying endoscopy with narrow band imaging for predicting the invasion depth of superficial esophageal squamous cell carcinoma.

The invasion depth of superficial esophageal squamous cell carcinoma is important in determining therapeutic strategy. The aim of this study was to prospectively investigate the clinical utility of magnifying endoscopy with narrow band imaging compared with that of non-magnifying high-resolution endoscopy or high-frequency endoscopic ultrasonography in predicting the depth of superficial esophageal squamous cell carcinoma. The techniques were carried out in 72 patients with 101 superficial esophageal squamous cell carcinomas, which were then resected by either endoscopic mucosal resection or esophagectomy. The histological invasion depth was divided into two: mucosal or submucosal carcinoma. We investigated the relationship between endoscopic staging and histology of tumor depth. Non-magnifying high-resolution endoscopy, magnifying endoscopy with narrow band imaging, and high-frequency endoscopic ultrasonography had overestimation/underestimation rates of 7/5, 4/4 and 8/3%, respectively. The sensitivity rates for the three techniques were 72, 78, and 83%, respectively, and the specificity rates were 92, 95, and 89%, respectively. There were no statistically significant differences among the three endoscopic techniques. Clinical utility of magnifying endoscopy with narrow band imaging does not seem to be significantly different from that of non-magnifying high-resolution endoscopy or high-frequency endoscopic ultrasonography in predicting the depth of superficial esophageal squamous cell carcinoma. Magnifying endoscopy with narrow band imaging may have potential to reduce overestimation risks of non-magnifying high-resolution endoscopy or high-frequency endoscopic ultrasonography.

A novel double-channel therapeutic endoscope ("R-scope") facilitates endoscopic submucosal dissection of superficial gastric neoplasms.

BACKGROUND AND STUDY AIM: Endoscopic submucosal dissection (ESD) is a new and radical treatment for superficial gastrointestinal neoplasms that provides high rates of en bloc resection compared with treatment by conventional mucosal resection. However, ESD is a complex procedure that is associated with long operating times and a higher complication rate. This feasibility study assessed the use of a novel double-channel therapeutic endoscope for performing en-bloc ESD in order to assess whether the procedure time could be shortened. PATIENTS AND METHODS: The therapeutic endoscope we used (the "R-scope") is equipped with a multibending system and has two movable instrument channels: one moves a grasping forceps vertically for lesion countertraction; the other swings a cutting knife horizontally for dissection. Twenty consecutive patients (18 men, 2 women; mean age 63 years, range 54 - 80 years) with superficial gastric neoplasms in the distal two-thirds of the stomach underwent resection of their tumor by ESD using the R-scope. Forty size- and location-matched gastric neoplasms resected by conventional ESD were reviewed retrospectively for the purposes of comparison. RESULTS: The rates of curative en-bloc resection, complications, and local recurrence using the two ESD methods were comparable. The mean +/- SD operating time was significantly shorter for ESD using the R-scope than for conventional ESD (57.9 +/- 29.7 minutes vs. 92.8 +/- 58.9 minutes, P = 0.016). CONCLUSION: The R-scope appears to shorten the operating time of ESD with comparable efficacy and complication rates.

Clinical and endoscopic characterization of depressed gastric adenoma.

BACKGROUND AND STUDY AIM: Depressed gastric adenoma remains poorly characterized because it is rare, and is infrequently detected by endoscopy. The aim of this study was to elucidate clinical and endoscopic characteristics of depressed adenoma of the stomach. PATIENTS AND METHODS: 95 consecutive patients who underwent endoscopic resection of gastric adenomas were studied. Gastric adenomas, diagnosed according to the Vienna classification, were endoscopically classified into two types: depressed and protruding adenomas. In order to clarify endoscopic features of gastric adenomas, we performed indigo carmine chromoendoscopy as well as magnifying endoscopy with narrow band imaging, which yields clear images of mucosal microvasculature. RESULTS: 12% of 100 gastric adenomas resected from 95 patients were depressed adenomas. Age and gender were comparable between patients with each type. Depressed adenomas (15.9 +/- 6.2 mm) were significantly larger in diameter than protruding adenomas (10.6 +/- 8.0 mm) (P = 0.01). Half of depressed adenomas were reddish in color, whereas only 18% of protruding adenomas were reddish. Magnifying endoscopy with narrow band imaging showed that 71% of depressed adenomas had a regular ultrafine network pattern of mucosal microvasculature, which was not seen in protruding adenomas. Intramucosal carcinomas were more frequently found in depressed adenomas (25%) than in protruding adenomas (4.5%). CONCLUSIONS: In comparison with protruding adenomas, depressed adenomas were rare and appeared endoscopically as large and reddish with a specific regular ultrafine network pattern of mucosal microvasculature. Depressed adenomas should be endoscopically resected because intramucosal carcinomas were found in a quarter of them.

Use of the curved linear-array echo endoscope to identify gastrorenal shunts in patients with gastric fundal varices.

BACKGROUND AND STUDY AIMS: : Balloon-occluded retrograde transvenous obliteration (B-RTO) has emerged as an effective, minimally invasive treatment for fundal varices. B-RTO requires a spontaneously developed gastrorenal shunt as a pathway for the balloon catheter to reach the fundal varices. We used a curved linear-array (CLA) echo endoscope in patients with fundal varices to identify gastrorenal shunts, and compared the detection rate with the gold standard, contrast-enhanced computed tomography (CECT). PATIENTS AND METHODS: A total of 40 patients with fundal varices were examined with both CLA echo endoscopy and CECT. The CECT images were retrospectively and independently evaluated by two gastroenterologists who were unaware of the clinical details, including the results of the CLA echo endoscopy. RESULTS: CLA echo endoscopy identified gastrorenal shunts in 26/40 patients with fundal varices. It visualized the shunt in a longitudinal direction and provided images of the connections of the shunt at both ends, the fundal varices and the left renal vein/branch of the inferior adrenal vein. The kappa index for CLA echo endoscopy and CECT for the identification of gastrorenal shunt was 0.9 (95 % CI, 0.6 to 1.0). When the cutoff point for the diameter of the gastrorenal shunt detected by the CLA echo endoscope was set at equal to or greater than 5 mm, the kappa index was 1.0 (95 % CI, 0.7 to 1.0). CONCLUSIONS: These results suggest that CLA echo endoscopy can successfully identify gastrorenal shunt and provide detailed morphological information. It also efficiently identifies patients suitable for B-RTO, particularly in cases of acute bleeding. It also has considerable potential for providing detailed information with regard to the treatment of gastric varices.

Hemodynamic analysis of esophageal varices using color Doppler endoscopic ultrasonography to predict recurrence after endoscopic treatment.

BACKGROUND AND STUDY AIMS: The time to recurrence of esophageal varices may vary greatly between patients even after the same endoscopic therapy. To clarify the factors which contribute to recurrence after endoscopic treatment, the hemodynamics and morphology of the left gastric vein (LGV) were investigated using color Doppler endoscopic ultrasonography (EUS). PATIENTS AND METHODS: A total of 31 patients with high-risk esophageal varices underwent color Doppler-EUS before receiving endoscopic variceal ligation and endoscopic injection sclerotherapy combined therapy. Endoscopic examination was performed every 3 months after the treatment to evaluate recurrence of varices. RESULTS: A total of 18 patients responded to the therapy, while 13 patients did not respond, and had recurrence within 12 months. The hepatofugal flow velocity in the LGV trunk was significantly lower in the responders (9.9 vs. 13.9 cm/sec; P = 0.02). The branch pattern of the LGV was categorized into three groups: anterior branch dominant, posterior branch dominant, and no-dominant type. The incidence of the anterior branch dominant type was significantly less in responders (17 vs. 70 %; P = 0.01). There was no significant difference in the LGV trunk diameter and the size of the paraesophageal vein between the two groups. CONCLUSION: Risk factors for recurrence can be analyzed in detail using color Doppler-EUS. Further investigation using color Doppler-EUS may enable us to select the optimal way to treat esophageal varices to prevent recurrence.

Low power diode laser treatment using indocyanine green for eradication of esophageal varices.

BACKGROUND AND STUDY AIMS: Endoscopic variceal ligation (EVL) is an alternative to sclerotherapy for the treatment of esophageal varices, but is associated with higher rates of recurrence and subsequent bleeding than sclerotherapy. To prevent recurrence of varices after EVL, we have developed a low-dose diode laser therapy combined with the injection of indocyanine green, which allows enhanced tissue absorption of the laser beam selectively around varices. In this study we investigated the efficacy and safety of this technique. PATIENTS AND METHODS: Eight patients with F2 or F3 esophageal varices were enrolled. At 1 week after EVL, indocyanine green solution (1 mg/ml) was injected submucosally around the remaining varices. A diode laser (power 10 watts) was applied to the surface from the esophagogastric junction to 5 cm above it. The spot size was kept to 5 mm in diameter. RESULTS: Laser irradiation was performed safely, without bleeding from the varices, or perforation. There were no major complications. Endoscopy 1 month later showed F0 forms in seven patients, F1 in one patient, and no red color sign in any patient. No recurrence of varices has been observed in any of the patients during the follow-up period of at least 12 months. CONCLUSION: This technique may provide a simple, safe and effective procedure, as an additional treatment to EVL, for the prevention of recurrence of esophageal varices.

Collaborated surgical works (surgical planning) in virtual space with tactile sensation between Japan and Germany.

Surgeons in Japan and Germany applied tele-virtual surgery and a force feedback device during a hepatectomy simulation. Using this system, surgeons in each country were able to perform various surgical maneuvers upon the same patient. They palpated abdominal skin, made electrical scalpel incisions and widened the incision line by using surgical tools in virtual space. While surgeons performed a virtual operation, the force feedback device conveyed tactile sensations. In each location, the force feedback devices and two graphic workstations of equal capability were employed. As each workstation communicated only event signals through an ISDN (64 Kb) line, it was possible to obtain real time tele-virtual surgery without a large capacity communication infrastructure.

An efficient cis-element discovery method using multiple sequence comparisons based on evolutionary relationships.

The discovery of cis-element control motifs in noncoding DNA poses a difficult problem in genome analysis. Functional analysis by means of reporter constructs expressed in transgenic organisms is the most reliable method, but is by itself time-consuming and expensive. Searching noncoding DNA for known control motifs by sequence analysis is problematic, since protein binding motifs are short, in the range of 8-10 bp, and occur frequently by chance. Heretofore, the most reliable sequence analysis method has been the comparison of homologous sequence domains in related but moderately evolutionarily divergent species such as, for example, mouse and human. In such pairwise combinations, control regions are conserved because they serve a vital function and can be identified by their similar sequences. Single pairwise comparisons, however, allow the discovery of conserved sequence strings only at low resolution and without specific identity. We have investigated the possibility of using multiple sequence comparisons to correct these shortcomings. We applied this method to the Hoxc8 early enhancer region that has been previously analyzed in depth by functional methods and through its application successfully identified known protein binding cis-element motifs. Candidate protein binding sites could also be identified. This method, based on evolutionarily related sequence comparisons, should be quite useful as a prescreening step prior to functional analysis with corresponding savings in time and resources.

Gene diversity of chimpanzee ABO blood group genes elucidated from intron 6 sequences.

The human and nonhuman primate ABO blood group gene shows relatively large numbers of nucleotide differences around the exon 7 region. In this study we determined intron 6 sequences for 9 alleles of common chimpanzee and for 3 alleles of bonobo to estimate nucleotide diversities among them. Sequence length polymorphisms are observed in this region as a repeat appears one to five times. From a phylogenetic network of intron 6 sequences of ABO blood group genes for humans, common chimpanzee, and bonobo, parallel substitutions and/or some kinds of convergent events are predicted in the chimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes; these values were 0.219% and 0.208%, respectively.

Gene diversity of chimpanzee ABO blood group genes elucidated from exon 7 sequences.

Human and non-human primate ABO blood group genes show relatively large numbers of nucleotide differences. In this study, we determined exon 7 sequences for 10 individuals of common chimpanzee and for four individuals of bonobo to estimate nucleotide diversities among them. Sequence data showed the existence of chimpanzee specific 9-base deletion in the beginning of the exon 7 coding region. From a phylogenetic network of exon 7 sequences of ABO blood group genes for human, common chimpanzee, bonobo and gorilla, effects of parallel substitutions and/or some kinds of convergent events are inferred in the chimpanzee lineage. We also estimated nucleotide diversities for common chimpanzee and bonobo ABO blood group genes, and these values were 0.4% and 0.2%, respectively. These values are higher than that of most human genes.

Conserved evolution of the Rh50 gene compared to its homologous Rh blood group gene.

We have sequenced the complete coding region of the Rh blood group gene for mouse and rat and that of Rh-related 50 kD glycoprotein (Rh50) for mouse, rat, and crab-eating macaque. Phylogenetic analyses of Rh and Rh50 amino acid sequences indicate that the Rh50 gene has been evolving about two times more slowly than the Rh blood group gene in both primates and rodents. This conservative nature of the Rh50 gene suggests its relative importance to the Rh blood group gene. The time of gene duplication that produced the Rh and Rh50 genes was estimated to be about 240-310 million years ago. We also conducted window analyses of synonymous and nonsynonymous nucleotide substitutions for those two genes. Some peaks where nonsynonymous substitutions are higher than synonymous ones were located on outer membrane regions. This suggests the existence of positive Darwinian selection on Rh and Rh50 genes through host-parasite interactions.