Genetic analysis of a Yayoi individual from the Doigahama site provides insights into the origins of immigrants to the Japanese Archipelago.

Mainland Japanese have been recognized as having dual ancestry, originating from indigenous Jomon people and immigrants from continental East Eurasia. Although migration from the continent to the Japanese Archipelago continued from the Yayoi to the Kofun period, our understanding of these immigrants, particularly their origins, remains insufficient due to the lack of high-quality genome samples from the Yayoi period, complicating predictions about the admixture process. To address this, we sequenced the whole nuclear genome of a Yayoi individual from the Doigahama site in Yamaguchi prefecture, Japan. A comprehensive population genetic analysis of the Doigahama Yayoi individual, along with ancient and modern populations in East Asia and Northeastern Eurasia, revealed that the Doigahama Yayoi individual, similar to Kofun individuals and modern Mainland Japanese, had three distinct genetic ancestries: Jomon-related, East Asian-related, and Northeastern Siberian-related. Among non-Japanese populations, the Korean population, possessing both East Asian-related and Northeastern Siberian-related ancestries, exhibited the highest degree of genetic similarity to the Doigahama Yayoi individual. The analysis of admixture modeling for Yayoi individuals, Kofun individuals, and modern Japanese respectively supported a two-way admixture model assuming Jomon-related and Korean-related ancestries. These results suggest that between the Yayoi and Kofun periods, the majority of immigrants to the Japanese Archipelago originated primarily from the Korean Peninsula.

Diversity in matrilineages among the Jomon individuals of Japan.

BACKGROUND: The Jomon period of Japan is characterised by a unique combination of sedentary and hunting/gathering lifestyles, spanning for more than 10,000 years from the final Pleistocene to the Holocene. The transition from the preceding Palaeolithic period to the Jomon period is known to have begun with the appearance of pottery usage. However, knowledge of the genetic background of the Jomon people is still limited. AIM: We aimed to determine the population-scale complete mitogenome sequences of the Initial Jomon human remains and compare the occurrence of mitochondrial haplogroups in the Jomon period from temporal and regional perspectives. SUBJECTS AND METHODS: For human remains dated to 8200-8600 cal BP, we determined their complete mitogenome sequences using target enrichment-coupled next-generation sequencing. RESULTS: We successfully obtained the complete mitogenome sequences with high depth of coverage and high concordance on consensus sequences. These sequences differed by more than three bases each, except for two individuals having completely identical sequences. Co-existence of individuals with haplogroups N9b and M7a was first observed at the same archaeological site from the Initial Jomon period. CONCLUSION: The genetic diversity within the population was not found to be low even in the Initial Jomon period.

Bioarchaeological study of ancient Teotihuacans based on complete mitochondrial genome sequences and diet isotopes.

BACKGROUND: The Teotihuacan civilisation was the largest one in ancient Mesoamerica. The Teotihuacan city was born in the north-eastern Basin of Mexico around the second century BC, reached its peak in the fourth century AD, and had cultural influence throughout Mesoamerica. At its peak, the size of the city reached more than 20 km2, and the total population is estimated to have increased from 100,000 to 200,000. However, knowledge of the genetic background of the Teotihuacan people is still limited. AIM: We aimed to determine the mitogenome sequences of the Teotihuacan human remains and compare the ancient and present Mesoamericans. In addition, we aimed to identify the food habits of ancient Teotihuacans. SUBJECTS AND METHODS: We determined the mitogenome sequences of human remains dated to 250-636 cal AD using target enrichment-coupled next generation sequencing. We also performed stable isotope analysis. RESULTS: We successfully obtained nearly full-length sequences newly unearthed from a civilian dwelling in the Teotihuacan site. Teotihuacan mitochondrial DNA was classified into the haplogroups in present and ancient Mesoamericans. In addition, Teotihuacan individuals had a diet dependent on C4 plants such as maize. CONCLUSION: Genetic diversity varied among the Teotihuacans.

A study of 8,300-year-old Jomon human remains in Japan using complete mitogenome sequences obtained by next-generation sequencing.

Ancient human remains have been assigned to their mitochondrial DNA (mtDNA) haplogroups. To obtain efficiently deep and reliable nucleotide sequences of ancient DNA of interest, we achieved target enrichment followed by next-generation sequencing (NGS). Complete mitochondrial genome (mitogenome) sequences were obtained for three human remains from the Iyai rock-shelter site of the Initial Jomon Period in Japan. All the Jomon mitogenomes belong to haplogroup N9b, but no sequences among them were identical. High genetic diversity was clarified even among the Jomon human remains belonging to haplogroup N9b, which has been described as a haplogroup representing the Jomon people.

Imputation approach for deducing a complete mitogenome sequence from low-depth-coverage next-generation sequencing data: application to ancient remains from the Moon Pyramid, Mexico.

It is considered that more than 15 depths of coverage are necessary for next-generation sequencing (NGS) data to obtain reliable complete nucleotide sequences of the mitogenome. However, it is difficult to satisfy this requirement for all nucleotide positions because of problems obtaining a uniform depth of coverage for poorly preserved materials. Thus, we propose an imputation approach that allows a complete mitogenome sequence to be deduced from low-depth-coverage NGS data. We used different types of mitogenome data files as panels for imputation: a worldwide panel comprising all the major haplogroups, a worldwide panel comprising sequences belonging to the estimated haplogroup alone, a panel comprising sequences from the population most closely related to an individual under investigation, and a panel comprising sequences belonging to the estimated haplogroup from the population most closely related to an individual under investigation. The number of missing nucleotides was drastically reduced in all the panels, but the contents obtained by imputation were quite different among the panels. The efficiency of the imputation method differed according to the panels used. The missing nucleotides were most credibly imputed using sequences of the estimated haplogroup from the population most closely related to the individual under investigation as a panel.

Characterization of complete mitochondrial genomes of indigenous Mayans in Mexico.

BACKGROUND: The authors have previously published the complete mitochondrial genome (mitogenome) sequences of two indigenous Mesoamerican populations, Mazahua (n = 25) and Zapotec (n = 88). METHODS: This study determined the complete mitogenome sequences of nine unrelated individuals from the indigenous Maya population living in Mexico. RESULTS: Their mitogenome sequences could be classified into either of the haplogroups A2 and C1. Surprisingly, there were no mitogenome sequences (haplotypes) that the Maya, Mazahua, and Zapotec people share in common. CONCLUSIONS: This indicates that no genetic exchange, at least matrilineally, has occurred among them.

Analysis of cardiohemodynamic and electrophysiological effects of morphine along with its toxicokinetic profile using the halothane-anesthetized dogs.

Although morphine has been used for treatment-resistant dyspnea in end-stage heart failure patients, information on its cardiovascular safety profile remains limited. Morphine was intravenously administered to halothane-anesthetized dogs (n=4) in doses of 0.1, 1 and 10 mg/kg/10 min with 20 min of observation period. The low and middle doses attained therapeutic (0.13 µg/mL) and supratherapeutic (0.97 µg/mL) plasma concentrations, respectively. The low dose hardly altered any of the cardiovascular variables except that the QT interval was prolonged for 10-15 min after its start of infusion. The middle dose reduced the preload and afterload to the left ventricle for 5-15 min, then decreased the left ventricular contractility and mean blood pressure for 10-30 min, and finally suppressed the heart rate for 15-30 min. Moreover, the middle dose gradually but progressively prolonged the atrioventricular conduction time, QT interval/QTcV, ventricular late repolarization period and ventricular effective refractory period without altering the intraventricular conduction time, ventricular early repolarization period or terminal repolarization period. A reverse-frequency-dependent delay of ventricular repolarization was confirmed. The high dose induced cardiohemodynamic collapse mainly due to vasodilation in the initial 2 animals by 1.9 and 3.3 min after its start of infusion, respectively, which needed circulatory support to treat. The high dose was not tested further in the remaining 2 animals. Thus, intravenously administered morphine exerts a rapidly appearing vasodilator action followed by slowly developing cardiosuppressive effects. Morphine can delay the ventricular repolarization possibly through IKr inhibition in vivo, but its potential to develop torsade de pointes will be small.

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry.

Dextromethorphan was extracted from human plasma samples (100 µL) using MonoTip C(18) tips, which are packed with C(18)-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200 µL of distilled water and 50 µL of 1 mol/L glycine-sodium hydroxide buffer (pH 10). The mixture was extracted to the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.5 µm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320 ng/mL of plasma, and the limit of detection was 1.25 ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.

Existence of a threshold for hydroxyl radical generation independent of hypoxia in rat striatum during carbon monoxide poisoning.

We examined the role of hypoxia in the carbon monoxide (CO)-induced generation of the hydroxyl radical (•OH) in the striatum, which could contribute to brain damage due to CO poisoning. Exposure of free-moving rats to 1,000 and 3,000 ppm CO or 8 and 5% O2 for 40 min caused concentration-dependent hypoxic conditions in terms of carboxyhemoglobin (COHb), deoxyhemoglobin, oxyhemoglobin, and O2 contents in arterial blood. The hypoxic conditions seemed comparable between 3,000 ppm CO and 5% O2, although alterations of pH and partial O2 pressure (PO2) were complex and concentration independent. In the striatum, CO and low O2 decreased tissue PO2 levels in a concentration-dependent and concentration-independent manner, respectively, but levels at the end of exposure were comparable among all groups. This was also the case for the increase in striatal blood flow. Although the increases in extracellular glutamate (excitatory), taurine (inhibitory), and alanine (non-neurotransmitter), in the striatum in response to CO and low O2 were complex, 3,000 ppm CO and 5% O2 had comparable effects. Thus, 3,000 ppm CO and 5% O2 seemed to induce comparable hypoxic conditions. Nevertheless, the former more strongly stimulated (•OH generation in the striatum than the latter. In addition, in contrast to low O2 which caused a concentration-dependent increase in (•OH, 1,000 ppm CO had no effect. The findings suggest that striatal •OH generation due to CO poisoning may be independent of hypoxia per se and that a threshold might exist.

The number of SNPs required for distinguishing Japanese from other East Asians.

In some cases, it is necessary to estimate the national origin of an unknown subject in forensic medicine. The use of single nucleotide polymorphism (SNP) markers appears to be very effective for this purpose, since genome-wide SNP genotype data of many human populations are publicly available. In this study, we examined the number of SNPs that could objectively and accurately distinguish Japanese subjects (1KG-JPT) from the other East Asians (1KG-CDX, -CHB, -CHS, and -KHV) using the combination of principal component analysis and hierarchical cluster analysis. A computer simulation showed that approximately 3000 randomly selected SNPs were enough for the discrimination. Our results suggest that at least a 0.024% coverage is needed in the next generation sequencing experiment to objectively determine whether an unknown person is Japanese or not if the amount of DNA sample from him/her is insufficient or the quality is low.

Population dynamics in the Japanese Archipelago since the Pleistocene revealed by the complete mitochondrial genome sequences.

The Japanese Archipelago is widely covered with acidic soil made of volcanic ash, an environment which is detrimental to the preservation of ancient biomolecules. More than 10,000 Palaeolithic and Neolithic sites have been discovered nationwide, but few skeletal remains exist and preservation of DNA is poor. Despite these challenging circumstances, we succeeded in obtaining a complete mitogenome (mitochondrial genome) sequence from Palaeolithic human remains. We also obtained those of Neolithic (the hunting-gathering Jomon and the farming Yayoi cultures) remains, and over 2,000 present-day Japanese. The Palaeolithic mitogenome sequence was not found to be a direct ancestor of any of Jomon, Yayoi, and present-day Japanese people. However, it was an ancestral type of haplogroup M, a basal group of the haplogroup M. Therefore, our results indicate continuity in the maternal gene pool from the Palaeolithic to present-day Japanese. We also found that a vast increase of population size happened and has continued since the Yayoi period, characterized with paddy rice farming. It means that the cultural transition, i.e. rice agriculture, had significant impact on the demographic history of Japanese population.

2-aminophenoxazine-3-one prevents pulmonary metastasis of mouse B16 melanoma cells in mice.

2-Aminophenoxazine-3-one (Phx-3) induced cellular apoptosis in mouse melanoma B16 cells as detected by DNA laddering and upregulated Fas expression in the cells in vitro. Next, the anti-metastatic effects of Phx-3 were investigated in C56BL/6 mice. When B16 melanoma cells were injected into the tail veins of mice, significant metastasis of the cells was indicated in the lungs, 14 days after treatment. In contrast, when 0.5 mg/kg Phx-3 was administered to mice through the tail veins, once simultaneously with or every three days after the administration of B16 melanoma cells, the number of metastasized pulmonary cells was extremely reduced. Moderate reduction of the number of metastasized pulmonary cells was indicated in the mice with a single dose of Phx-3 on day 3 after injection of the cells. However, when Phx-3 was administered in a single dose, 6 or 9 days after the injection of the cells, the number of metastasized pulmonary cells remained the same. The present results indicate that the metastasis of mouse B16 melanoma cells to the lung was significantly inhibited in mice administered Phx-3, which activated the intrinsic and extrinsic apoptotic pathways. The present study suggests that Phx-3 might be a potential anti-metastatic agent as well as an anticancer agent.

A biomolecular anthropological investigation of William Adams, the first SAMURAI from England.

William Adams (Miura Anjin) was an English navigator who sailed with a Dutch trading fleet to the far East and landed in Japan in 1600. He became a vassal under the Shogun, Tokugawa Ieyasu, was bestowed with a title, lands and swords, and became the first SAMURAI from England. "Miura" comes from the name of the territory given to him and "Anjin" means "pilot". He lived out the rest of his life in Japan and died in Hirado, Nagasaki Prefecture, in 1620, where he was reportedly laid to rest. Shortly after his death, graveyards designated for foreigners were destroyed during a period of Christian repression, but Miura Anjin's bones were supposedly taken, protected, and reburied. Archaeological investigations in 1931 uncovered human skeletal remains and it was proposed that they were those of Miura Anjin. However, this could not be confirmed from the evidence at the time and the remains were reburied. In 2017, excavations found skeletal remains matching the description of those reinterred in 1931. We analyzed these remains from various aspects, including genetic background, dietary habits, and burial style, utilizing modern scientific techniques to investigate whether they do indeed belong to the first English SAMURAI.

Role of nitric oxide system in hydroxyl radical generation in rat striatum due to carbon monoxide poisoning, as determined by microdialysis.

We explored the possible role of the nitric oxide (NO) system in hydroxyl radical (*OH) generation induced by carbon monoxide (CO) poisoning in rat striatum by means of microdialysis with the use of NO synthase (NOS) inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA), as well as L-arginine (L-Arg; the NOS substrate) and D-arginine (D-Arg). The CO-induced *OH generation was suppressed by both L-Arg and D-Arg. It was also suppressed by L-NAME, which inhibits generation of reactive oxygen species (ROS) via neuronal NOS (nNOS) and inducible NOS, but not via endothelial NOS. In contrast, L-NMMA, which inhibits only ROS generation via inducible NOS, potentiated the *OH generation. L-Arg completely reversed the L-NAME effect and partly reversed the L-NMMA effect. D-Arg reversed the L-NAME effect more potently than did L-Arg, resulting in much more *OH generation than was observed with CO alone, and also potentiated the L-NMMA effect. On the other hand, W-7, an antagonist of calmodulin, which is critical for nNOS activity, had no effect on the CO-induced *OH generation. These findings suggest that complex mechanisms operate in *OH generation in rat striatum upon CO poisoning and that the NO system might not be included among those mechanisms.

Gene expression in rat striatum following carbon monoxide poisoning.

Carbon monoxide (CO) poisoning causes brain damage, which is attenuated by treatment with hydrogen [1], [2], a scavenger selective to hydroxyl radical (•OH) [3]. This suggests a role of •OH in brain damage due to CO poisoning. Studies have shown strong enhancement of •OH production in rat striatum by severe CO poisoning with a blood carboxyhemoglobin (COHb) level > 70% due to 3000 ppm CO, but not less severe CO poisoning with a blood COHb level at approximately 50% due to 1000 ppm CO [4]. Interestingly, 5% O2 causes hypoxia comparable with that by 3000 ppm CO and produces much less •OH than 3000 ppm CO does [4]. In addition, cAMP production in parallel with •OH production [5] might contribute to •OH production [6]. It is likely that mechanisms other than hypoxia contribute to brain damage due to CO poisoning [7]. To search for the mechanisms, we examined the effects of 1000 ppm CO, 3000 ppm CO and 5% O2 on gene expression in rat striatum. All array data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE94780.

Right ventricular free wall dissection as a rupture tract in left ventricular rupture during acute myocardial infarction.

Three rare cases of cardiac rupture with right ventricular wall dissection during acute myocardial infarction (AMI) were reported. The cases comprised 2% among our 148 previously reported postinfarction cardiac ruptures with sudden death. The dissections occurred in hearts with biventricular inferior wall AMI and developed between the superficial layers and the deeper layers of inferior wall of the right ventricle. All had an endocardial tear at the basal septum where it meets the inferior free wall of the left ventricle, and had an epicardial tear on the middle inferior wall of the right ventricle. Based on the evidence of the ages of the thrombi of the rupture tracts, delayed epicardial rupture was found besides that soon after the right ventricular dissection.

Overexpression and gene amplification of EGFR, HER2, and HER3 in biliary tract carcinomas, and the possibility for therapy with the HER2-targeting antibody pertuzumab.

BACKGROUND: Pertuzumab is a humanized monoclonal antibody that binds to HER2 at an epitope that prevents HER2 from dimerizing with ligand-activated HER-family receptors. To assess the potential of pertuzumab as a new therapy, the expression status of HER family members was determined in biliary tract carcinoma (BTC), and the antitumor activity of pertuzumab was investigated by assessing the inhibition of BTC cell growth. METHODS: The expression status of HER family members in 113 archival specimens of BTC was analyzed by using immunohistochemistry and fluorescence in situ hybridization. Using ten BTC cell lines, heregulin-alpha (HRG-α) stimulated cell proliferation and its inhibition by pertuzumab was tested in vitro. The phosphorylated HER family proteins and their respective downstream molecules were analyzed. In vivo antitumor activity of pertuzumab was evaluated in a xenograft model. RESULTS: Protein overexpression of HER2 and/or HER3 was observed in 23-34 % of the specimens and gene amplification in 17-27 %. Seven of the ten cell lines showed HER2 and/or HER3 protein overexpression and gene amplification, and HRG-α stimulated cell proliferation was observed in four of the ten cell lines. In a BTC cell line co-overexpressing HER2 and HER3, pertuzumab potently inhibited the HRG-α stimulated cell proliferation in a dose-dependent manner, and completely blocked the phosphorylation of HER3. Suppression of downstream pathway molecules including p-AKT was also observed. Pertuzumab inhibited the in vivo growth of subcutaneous tumors, and increased the number of apoptotic cancer cells. CONCLUSIONS: Pertuzumab exerts potent antitumor activity in BTC cells co-overexpressing HER2 and HER3. Pertuzumab provides a new therapeutic option against BTC.

Characterization of hydroxyl radical generation in the striatum of free-moving rats due to carbon monoxide poisoning, as determined by in vivo microdialysis.

Carbon monoxide (CO) poisoning caused by CO exposure at 3000 ppm for 40 min resulted in stimulation of hydroxyl radical (*OH) generation (estimated by measuring 2,3-dihydroxybenzoic acid (2,3-DHBA) production from salicylic acid) in the striatum of free-moving rats, as determined by means of brain microdialysis. Pretreatment with a voltage-dependent Na+ channel blocker, tetrodotoxin (TTX), lowered the basal level of 2,3-DHBA and strongly suppressed the increase in 2,3-DHBA induced by CO poisoning. CO poisoning significantly, though only slightly, increased extracellular glutamate in the striatum, and glutamate (Glu) receptor antagonists, such as MK-801 (dizocilpine) and NBQX, failed to suppress the CO-induced increase in 2,3-DHBA. These findings suggest that CO poisoning may induce Na+ influx via the voltage-dependent Na+ channels, resulting in stimulation of *OH generation in rat striatum. This effect may be independent of Glu receptor activation by increased extracellular Glu.

Characterization of suppression of nitric oxide production by carbon monoxide poisoning in the striatum of free-moving rats, as determined by in vivo brain microdialysis.

We examined the effect of carbon monoxide (CO) poisoning on the nitric oxide (NO) system in the striatum of free-moving rats by means of in vivo brain microdialysis. The extracellular levels of the oxidative NO products, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), decreased during exposure to CO at 3000 ppm for 40 min, a condition which causes CO poisoning. The extracellular levels of citrulline (Cit; a by-product of NO production) and arginine (Arg; an NO precursor) also decreased during CO exposure. Following reoxygenation by withdrawal of CO, the NO(2)(-) and NO(3)(-) levels gradually recovered to the control values, though Arg and Cit remained at lower levels, except for a rapid, but transient, recovery shortly before and after reoxygenation, respectively. Simultaneous application of exogenous L-Arg (50 and 100 mM) with CO exposure attenuated the decreases in NO(2)(-) and NO(3)(-) during the CO exposure and accelerated their recovery following reoxygenation. However, D-Arg (100 mM) had no effect on the decrease in NO(2)(-) and NO(3)(-), except for slight and transient attenuation shortly after reoxygenation. Exogenous L-Cit (10 and 100 mM) failed to attenuate the CO-induced decrease in NO(2)(-) and NO(3)(-) levels. The decrease in the NO(2)(-) and NO(3)(-) levels during 8% O(2) exposure for 40 min, which was comparable with that in response to 3000 ppm CO, was resistant to exogenous 100 mM L-Arg, but the recovery of the NO(2)(-) and NO(3)(-) levels following reoxygenation was strongly accelerated. These findings suggest that CO poisoning suppresses NO production in rat striatum in vivo though a mechanism which may not be common with that in hypoxic hypoxia.

Different response to exogenous L-arginine in nitric oxide production between hippocampus and striatum of conscious rats: a microdialysis study.

We previously showed that systemic administration of a nitric oxide (NO) precursor, L-arginine (L-Arg), failed to reverse suppression by NO synthase (NOS) inhibitors of chemically induced shaking behavior in rats, leading to the hypothesis that exogenous L-Arg might be non-uniformly supplied to brain regions susceptible to NOS inhibitors. In the present study, therefore, we examined the effect of exogenous L-Arg on the extracellular levels of the oxidative nitric oxide (NO) products, nitrite (NO2-) and nitrate (NO3-), in two different brain regions, the hippocampus and the striatum, of conscious rats by means of in vivo brain microdialysis. The basal NO2- levels in the two brain regions were comparable, while the NO3- level was significantly lower in the hippocampus than the striatum. The addition of 10 mM L-Arg, but not D-Arg, to the perfusing solution significantly increased NO2- and NO3- in the hippocampus and NO2- alone in the striatum. These increases were abolished by 1 mM N(omega)-nitro-L-arginine, an NOS inhibitor. L-Arg at 1mM was able to significantly increase NO2-, but not NO3-, in the hippocampus to a level comparable with that at 10 mM L-Arg, while it had no effect in the striatum. L-Arg (500 mg/kg, i.p.) induced a significant increase in NO2- and NO3- in the hippocampus, but not in the striatum. These results suggest that the striatum may have a lower ability to enhance NO production by utilising exogenous L-Arg than the hippocampus, despite higher basal NO production.