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In vivo pathogenesis studies of wild bird H5N6 influenza viruses were designed as previously described (Zhang et al., 2008, 2009; Pu et al., 2015). In brief, groups of 12 6-week-old SPF chickens were intranasally inoculated with 0.2 mL of 105 EID50 of SW8 or ZH283, while a control group of 12 chickens was inoculated with 0.2 mL of phosphate buffered saline (PBS) using the same route. Three days later, six inoculated chickens from each group were humanely euthanized to test for viral replication in lung, kidney, spleen, cecal tonsils, bursa of Fabricius, trachea, pancreas, liver, heart, brain, duodenum, ileum, descending colon, and jejunum tissue. The remaining chickens were observed twice daily, at 8:00 and 20:00, for clinical symptoms, morbidity, and mortality for 14 days according to the protocol provided by the World Organisation for Animal Health2.
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Direct contact virus transmission experiments in chickens were conducted per the procedure of (Yuan et al., 2014). Briefly, the chickens of inoculated groups (n = 6) were intranasally inoculated with 0.2 mL of 105 EID50 of either the SW8 or ZH283 virus in a ABSL-3 laboratory, and after 24 h, additional naïve contact groups (n = 3) were also intranasally inoculated with 0.2 mL of PBS and placed in physical contact in the same cage to share feed and water with chickens inoculated with the virus. At 3 days post-infection (DPI), three inoculated chickens were humanely euthanized, and target tissues (i.e., brain, lung, spleen, and bursa of Fabricius) were harvested to determine viral titers and for RNA extraction. At 3, 5, 7, 9, and 11 DPI, oropharynx and cloacal swabs samples were collected for the detection of viral shedding and suspended in 1 mL of PBS. All tested tissues and swabs samples were harvested for viral detection and titration in SPF chick embryos. All surviving chickens were euthanized at 14 DPI, and the serum was harvested and tested for seroconversion by hemagglutination inhibition testing using 1% turkey erythrocytes (Stephenson et al., 2004).
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Total RNA was extracted from the brain, lung, spleen, and bursa of Fabricius of H5N6-inoculated chickens and mock-infected chickens at 3 DPI using the Takara MiniBEST Universal RNA Extraction Kit (Takara Bio Inc., Tokyo, Japan) following the manufacturer’s instructions. Total RNA (1 μg) was reverse-transcribed with the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (Takara Bio Inc.) and stored at -20°C for further study.
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Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a FastStart Universal SYBR Green Master kit (Roche Diagnostics, Shanghai, China). qRT-PCR primers (Table 1) were designed from published target sequences and previously reported (Adams et al., 2009) with Primer Premier 7.0 software (Premier Biosoft, Palo Alto, CA, United States). qRT-PCR was performed on a LightCycler480 (Roche Applied Science, Mannheim, Germany), the products of which were purified by using a DNA gel extraction kit (Takara Bio Inc., Tokyo, Japan). For the purposes of assay validation, purified products were cloned into pMD19-T and sequenced to verify correct target amplification.
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The relative expression ratios of target genes in tested tissues vs. those in control tissues were calculated by the 2-ΔΔCT method using the chicken housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (NM_204305) as the endogenous reference gene in order to normalize the level of target gene expression (Livak and Schmittgen, 2001). Standard deviations were determined by using the relative expression ratios of three replicates for each gene measured. Differences of virus titers and mRNA expression levels were statistically analyzed with an unpaired non-parametric test and paired Student’s t-test, respectively, using GraphPad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, United States) software. Compared to the mock-infected control, p < 0.05 and p < 0.01 were considered to indicate a statistically significant difference unless stated otherwise.
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In previous research, we characterized the two H5N6 influenza viruses isolated from apparently healthy wild birds in 2014 and 2015 in Guangdong Province, China. On the one hand, SW8 was isolated from an oriental magpie-robin, and its PB2 gene with poultry H5N6 viruses shared the highest nucleotide similarity with that of A/chicken/Dongguan/2690/2013 (H5N6). On the other hand, ZH283 was isolated from a Pallas’s sandgrouse, and its PB2 gene shared the highest nucleotide similarity with that of A/Guangdong/ZQ874/2015 (H5N6) isolated from a 40-year-old woman who reported exposure to domestic poultry3.
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To determine the pathogenicity of the viruses in chickens, we intranasally inoculated 6-week-old SPF white leghorn chickens with 105 EID50 of either H5N6 virus (i.e., SW8 or ZH283). All inoculated chickens exhibited clinical signs of illness, including severe depression, cloudy eye, and intermittent head-shaking, and died within 5 DPI, with a mean death time (MDT) of 3.3 to 4.0 d (Figure 1A). SW8 and ZH283 replicated systemically in chickens and at 3 DPI was detectable in all tested organs, including the respiratory tract (i.e., lung and trachea), kidney, lymphoid tissues (i.e., spleen, cecal tonsils, and bursa of Fabricius), pancreas, liver, brain, intestinal tract (i.e., duodenum, ileum, descending colon, and jejunum), and heart. SW8 and ZH283 replicated efficiently in the lower respiratory tract; high viral titers were detected in the lung, with mean titers of 6.33 log10EID50 and 8.58 log10EID50, respectively (Figure 1B). The two novel viruses also replicated in the brain, spleen, and bursa of Fabricius, with mean titers of 4.83–7.17 log10EID50, 5.83–7.33 log10EID50, and 6.08–7.58 log10EID50, respectively (Figure 1B). Overall, the two novel H5N6 influenza viruses of wild bird origin showed high pathogenicity in chickens and could replicate systematically in them.
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Pathogenesis of H5N6 viruses in specific pathogen-free chickens. (A) Percentage of survival of SW8 and ZH283 in chickens. (B) Comparison of two A(H5N6) influenza virus titers of wild bird origin in chickens. Groups of 12 6-week-old chickens were intranasally inoculated with 0.2 mL of 105 EID50 of SW8, ZH283, or PBS; six chickens in each group were euthanized at 3 days post-infection, and lung, kidney, spleen, cecal tonsils, bursa of Fabricius, trachea, pancreas, liver, heart, brain, duodenum, ileum, descending colon, and jejunum tissues were collected. The remaining chickens were observed for clinical signs of illness and lethality for 2 weeks. Virus titers were determined in eggs and expressed as log10 EID50/g of tissue. Data are expressed as M ± SD. Dashed black lines indicate the lower limit of detection. Differences were analyzed with a paired Student’s t-test and were considered statistically significant at ∗p < 0.05 compared to control.
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To evaluate the horizontal intraspecies transmissibility of the two novel H5N6 viruses, three SPF chickens were intranasally inoculated with 0.2 mL PBS and introduced into the same cage as a naïve contact group, which were then housed with chickens inoculated with SW8 or ZH283. Shedding of SW8 could be detected from both oropharyngeal and cloacal swabs within 3 DPI, with viral titers in the ranges of 2.42–3.83 log10EID50 in oropharyngeal swab samples and of 1.52–3.79 log10EID50 in cloacal swab samples. ZH283 could also be detected from oropharyngeal and cloacal swabs within 5 DPI, with viral titers in the range of 4.58–4.75 log10EID50 in oropharyngeal swabs and of 3.50–3.90 log10EID50 in cloacal swabs (Figures 2A,B). Naïve contact chickens co-housed with chickens inoculated with SW8 did not die during the observation time, but all contact group chickens seroconverted and exhibited high titers (9.33 ± 0.58 log2), as shown in Table 2. Viral shedding was observed in both oropharyngeal and cloacal swabs, and viral titers of 1.50–1.83 log10EID50 within 5 DPI were detected in oropharyngeal swabs (Figure 2A); however, viral titers of the cloacal swabs could be detected (1.08 log10EID50) at 3 DPI (Figure 2B). Naïve contact chickens co-housed with chickens inoculated with ZH283 exhibited 100% lethality and mortality, with a MDT of 5.0 days (Table 2), and exhibited clinical signs of illness, including coughing, cloudy eye, and dyspnea. All surviving chickens in the naïve contact group co-housed with ZH283-infected chickens shed virus from the oropharynx and cloaca within 7 DPI, with mean viral titers of 2.75–3.75 log10EID50 in oropharyngeal swabs and of 1.75–4.50 log10EID50 in cloacal swabs (Figures 2A,B). In short, results demonstrate that the two novel H5N6 influenza viruses replicated efficiently in chickens and exhibited efficient transmission via direct contact in the chicken model.
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Direct contact transmissibility of H5N6 influenza viruses of wild bird origin among chickens. Viral titers of ZH283 and SW8 in oropharyngeal swabs (A) and cloacal swabs (B) in H5N6 influenza virus-inoculated and physical contact chickens. Three chickens were inoculated intranasally with 105 EID50 of SW8 or ZH283, whereas three naïve chickens were placed in the cage of H5N6-infected chickens at 24 h post-infection to initiate contact. Oropharyngeal and cloacal swabs were collected from infected and naïve contact chickens at indicated time points; virus titers were titrated and are expressed as log10EID50/0.1 mL. Data are expressed as M ± SD. The proportion of chicken swabs presenting infectious virus from all detected swabs at indicated time points appears in the figure above each group. Dashed black lines indicate the lower limit of detection.
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Toll-like receptors are PRRs with a unique and essential physiological function in host immune systems activated by pathogen-associated molecular patterns (Medzhitov, 2001). Expression profiles of two PRRs—TLR3 and TLR7—were examined in the target tissues of H5N6-infected chickens. As shown in Figure 3A, in contrast to mock-infected chickens, their expression level of TLR3 in the brain and lung was significantly elevated when induced by both viruses, with a fold increase of 2.80–78.56 in the brain or lung. In the spleen, the expression level of TLR3 was downregulated in response to SW8 infection, yet upregulated following infection with ZH283. In the bursa of Fabricius, the expression level of TLR3 was markedly downregulated when induced by both viruses. The expression level of TLR7 was upregulated in the lung when induced by SW8 or ZH283, by 1.79- and 19.41-fold, respectively. However, the expression level of TLR7 in the brain, spleen, and bursa of Fabricius showed different expression patterns when induced by the viruses; TLR7 expression level was downregulated when induced by both viruses compared to the control, with a fold change of 0.003–0.78 in all tested tissues except lung tissue. In particular, TLR7 expression remained low and was no longer visible in the bursa of Fabricius when triggered by both viruses. Notably, the expression levels of TLR3 and TLR7 in target tissues induced by ZH283 were generally greater than those induced by SW8 (Figures 3A,B).
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Toll-like receptors (TLRs) and sphingosine-1-phosphate-1 receptor (S1RP1) expression profiles in the target tissues of chickens infected with H5N6. At 3 days post-infection, the target tissues (i.e., brain, lung, spleen, and bursa of Fabricius) of H5N6-infected chickens were harvested for TLR and S1RP1 mRNA level detection via qRT-PCR method. (A) TLR3, (B) TLR7, (C) S1PR1. Data are expressed as M ± SD. Differences were analyzed with a paired Student’s t-test and were considered statistically significant at ∗p < 0.05, ∗∗p < 0.01 compared to control. B, Brain; L, Lung; S, Spleen; BF, Bursa of Fabricius.
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As an indispensable regulator of inflammation activation, S1PR1 plays a crucial role in immune cell trafficking and immune response (Rivera et al., 2008). When induced by SW8, S1PR1 expression was upregulated in the brain, lung, and spleen—by 5.51-, 2.29-, and 1.16-fold, respectively—but not in the bursa of Fabricius (0.17-fold). However, the expression level S1PR1 showed different tendencies when infected by ZH283. Unlike the expression level of TLR3 and TLR7, S1PR1 expression in the tested tissues after infection with ZH283 was lower than that in response to infection with SW8 (Figure 3C). Our data indicate that the engagement of PRRs and S1PR1 by the H5N6 influenza virus occurs in a tissue-dependent manner.
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The engagement of TLRs by influenza virus in specific target tissues initiated animal immunity via the production of proinflammatory cytokines and chemokines, including IL-1β, IL-6, IL-8, IL-10, TNF-α, and CCL5. As shown in Figures 4A,B,D, the expression level of IL-1β, IL-6, and IL-10 were remarkably unregulated in the lungs of tested chickens when infected by SW8 and ZH283 compared to those of mock-infected chickens. On the contrary, in the brain, spleen, and bursa of Fabricius, the expression levels of IL-1β, IL-6, and IL-10 were downregulated when induced by both viruses. However, the expression levels of IL-8, TNF-α, and CCL5 in the tested tissues of infected chickens showed a different expression patterns. As illustrated in Figure 4F, ZH283 induced an upregulated expression level of CCL5 in all tested tissues, whereas SW8 induced an upregulated expression level of CCL5 in the brain and spleen, but a downregulated one in the lung and bursa of Fabricius. Notably, ZH283-induced expression levels of IL-1β, IL-8, TNF-α, IL-6, and IL-10 were greater than those induced by SW8 in all tested tissues of chickens (Figures 4A–E).
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Proinflammatory cytokines and chemokines expression profiles in the target tissues of chickens when infected by H5N6. At 3 days post-infection, the target tissues (i.e., brain, lung, spleen, and bursa of Fabricius) of H5N6-infected chickens were harvested for proinflammatory cytokines and chemokines mRNA level detection via quantitative real-time polymerase chain reaction. (A) IL-1β, (B) IL-6, (C) IL-8, (D) IL-10, (E) TNF-α, (F) CCL5, (G) IFN-α, (H) IFN-β, (I) IFN-γ. Data are expressed as M ± SD. Differences were analyzed with a paired Student’s t-test and considered statistically significant at ∗p < 0.05, ∗∗p < 0.01 compared to the control. B, Brain; L, Lung; S, Spleen; BF, Bursa of Fabricius.
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The activation of TLRs also mediated the activation of IFN regulatory factor 3/7, primarily by recruiting MyD88 or TNF receptor-associated factor 6, which ultimately activated I and II IFNs (i.e., IFN-α, IFN-β, and IFN-γ). In the lungs of tested chickens, both ZH283 and SW8 induced significantly upregulated expression levels of IFN-α, IFN-β, and IFN-γ by 7.55- and 75.97-fold, 68.23- and 362.80-fold, 30.11- and 85.31-fold, respectively (p < 0.05) compared to uninoculated chickens (Figures 4G–I). In contrast to the lung, the brain, spleen, and bursa of Fabricius showed different expression patterns in the levels of IFN-α, IFN-β, and IFN-γ in response to ZH283 and SW8 infection. However, ZH283 induced the expression levels of IFN-α, IFN-β, and IFN-γ to a greater extent than SW8 in the tested tissues of infected chickens.
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To investigate whether MHC classes I and II molecules were involved in the host innate immune response to H5N6 influenza virus infection, we examined their expression levels in the lung, brain, spleen, and bursa of Fabricius in chickens at 3 DPI. As illustrated in Figures 5A,B, MHC classes I and II molecule expression levels were upregulated in the brain, spleen, and bursa of Fabricius when infected by both viruses. In the lung, in contrast to the mock-infected control, the expression level of the MHC class I molecule was remarkably downregulated (0.063- and 0.20-fold, respectively, p < 0.05); however, that of the MHC class II molecule was significantly upregulated when induced by SW8 and ZH283 (12.83- and 99.08-fold, respectively, p < 0.05). Those results demonstrated that MHC classes I and II molecules could play a significant role in the course of host innate immune response to H5N6 influenza virus infection in chickens.
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Major histocompatibility complex (MHC) classes I and II molecule expression profiles in the target tissues of chickens infected by H5N6. At 3 days post-infection, target tissues (i.e., brain, lung, spleen, and bursa of Fabricius) of H5N6-infected chickens were harvested for MHC classes I and II molecule mRNA level detection via quantitative real-time polymerase chain reaction. (A) MHC-I, (B) MHC-II. Data are expressed as M ± SD. Differences were analyzed with a paired Student’s t-test and considered statistically significant at ∗p < 0.05, ∗∗p < 0.01 compared to the control. B, Brain; L, Lung; S, Spleen; BF, Bursa of Fabricius.
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The first case of human infection with H5N6 AIVs was reported in southwest China’s Sichuan Province in 2013 (Pan et al., 2016). Results of epidemiological surveillance show that the viruses have recently been isolated from humans (Shen et al., 2016), domestic poultry (Bi et al., 2015; Butler et al., 2016; Du et al., 2017; Li et al., 2017), pigs (Li et al., 2015), environmental samples (Yuan et al., 2016), cats (Yu et al., 2015), and wild birds (Bi et al., 2016b) and resulted in heavy losses in the poultry industry. However, the pathogenicity and transmissibility of H5N6 AIVs have remained unclear. In the current research, we systematically investigated the pathogenicity, transmissibility and the host immune-related gene in the target tissues of infected chickens when challenged by those of wild bird-origin H5N6 AIVs. Our findings provide insights into understanding the host innate immune response of chickens to infection with different pathogenicities of wild bird-origin H5N6 AIVs.
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Importantly, we found that both H5N6 viruses isolated from wild birds were highly pathogenic and could efficiently be transmitted in chickens. Both viruses were shed from the oropharynx and cloaca in inoculated chickens and could be efficiently transmitted from infected chickens to naïve contact groups, the latter of which also shed viruses from both the cloaca and oropharynx throughout the experimental period. That the H5N6 HPAIVs isolated from wild birds could infect and be transmitted in chickens suggests that they may co-circulate in poultry and thus pose a great threat to the poultry industry.
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Notably, chickens inoculated with SW8 showed high pathogenicity, whereas naïve contact chickens infected showed no deaths. By contrast, chickens inoculated with ZH283 showed high pathogenicity, with a mortality rate of 100% within 2–3 days and efficient horizontal transmission in chickens. The mechanisms of lethality and transmissibility might be associated with mutations at positions K52T, I155T, and A544V of the HA protein, at positions K207R and Y436H of the PB1 protein, and at position T515A of the PA protein (Hulse-Post et al., 2007; Li et al., 2014). However, with the exception of position I155T of the HA protein, no mutations were observed in ZH283, which suggests that differences in the pathogenicity and transmissibility of H5N6 influenza viruses in chickens correlate with the probability of their being at position I155T of the HA protein. In addition, the transmissibility of H5N6 AIV in different birds may also depend on the stability of viral particles and the difference of viral protein structure, relative humidity, and temperature (Webster et al., 1992; Lowen et al., 2007). However, our experiment posed several limitations, meaning that more viral strains isolated from different animals and species need to be tested in order to investigate the correlation between pathogenicity and host immunity. Further investigation is also clearly needed to elucidate the differences of pathogenicity, transmissibility, and host innate immune response to infection with H5N6 AIVs in chickens.
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Remarkably, the expression levels of TLR3 and S1PR1 were upregulated in the brain following infection with SW8 and ZH283, yet showed different expression patterns in lymphoid tissues. Similarly, the production of TLR7, IL-1β, IL-6, IL-10, and IFN-γ were upregulated in the lung but downregulated in brain, spleen, and bursa of Fabricius in response to both viruses. Such results suggest that the engagement of the TLRs and cytokines are involved in a tissue-dependent manner. Previous studies have revealed tissue-specific immune responses following infection with H5N1 (Wei et al., 2013), H5N2 (Vanderven et al., 2012), and H7N1 (Cornelissen et al., 2012). The difference of cell types could be associated with immune responses and virus titers in the tissues tested for infection.
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The robust production of proinflammatory cytokines and chemokines such as IL-1β, IL-6, IL-8, IL-10, TNF-α, MCP-1, IFN-α, IFN-β, and IFN-γ in mammals during influenza virus infection, referred to as cytokine storms, have been confirmed to contribute to the severity of pathological damage via immune-mediated mechanisms (Walsh et al., 2011; Teijaro et al., 2014). In our study, the expression levels of IL-1β, IL-10, and IFN-β in the lungs and MHC-II in the brain were upregulated to a remarkably high level after infection with ZH283 and SW8, although were greater for ZH283 than SW8. Moreover, the expression level of S1PR1 in tested tissues following infection with ZH283 was less than that following infection with SW8. Consistent with the results of other studies (Walsh et al., 2011; Teijaro et al., 2014), our results demonstrated that the activation of S1PR1 can suppress the induction of cytokines, chemokines, and PRRs, meaning reducing morbidity and mortality, in chickens infected with H5N6. However, the specific mechanism of action remains to be determined.
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In sum, both H5N6 AIVs were highly pathogenic to chickens, caused multiple systemic infections in tissues, and were efficiently and rapidly transmitted in chickens. Those results indicate that H5N6 viruses could be transmitted to domestic poultry, which represents a serious threat to the poultry industry and both human and animal health. Furthermore, the expression profiles of PRRs, proinflammatory cytokines, chemokines, and MHC molecules in the tested tissues of H5N6-infected chickens were involved in a tissue-dependent manner. Lastly, our experiments demonstrated that ZH283 was associated with greater pathogenicity in chickens, for high virus titers appeared in tested tissues early in the infection process and were accompanied by the excessive expression of cytokines. Such data provide new insights into the relationship between the pathogenicity of H5N6 AIVs and host immune responses to them in chickens.
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Cerebral ischemia initiates a cascade of cytotoxic molecules responsible for the death of neural cells as well as the damage of the blood brain barrier (BBB) at the injury site (Doyle et al., 2008). In more than one decade, ischemia-associated neuronal injury has been a topic of intensive investigation, which leads to the identification of several mechanisms accounting for cerebral ischemia injury, such as apoptosis, oxidative damage, inflammatory injury, mitochondrial dysfunction and dysregulated protein degradation (Caldeira et al., 2014; Kalogeris et al., 2014; Palencia et al., 2015). The ubiquitin-proteasome system (UPS) is the major intracellular machinery for protein degradation, which is responsible for maintaining cellular homeostasis by regulating several important processes such as cell death, cell division, cell signal transduction, cell cycle progression and transmembrane transport (Wagner et al., 2011). Emerging evidence has suggested a role of suppressed proteasome activity in contributing to neuronal death in ischemic brain injury. However, little is known about the UPS components whose activities are suppressed under brain ischemia conditions (Caldeira et al., 2014).
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Neuregulin receptor degradation protein-1 (Nrdp1, also known as FLRF or RNF41), is a ring finger E3 ubiquitin ligase and primarily expressed in the brain, heart, prostate and skeletal muscle (Diamonti et al., 2002). Several studies have demonstrated an important role of Nrdp1 in regulating cell growth, apoptosis, oxidative stress and inflammation, in which Nrdp1 promotes the ubiquitination of ubiquitin-specific protease 8 (USP8), ErbB3, ErbB4, BRUCE/Apollon, MyD88 and Parkin (Qiu et al., 2004; Yen et al., 2006; Yu and Zhou, 2008; Wang et al., 2009; De Ceuninck et al., 2013; Sun et al., 2017). In a previous study, we have shown that Nrdp1 is implicated in ischemic cardiomyocyte death, in which overexpression of Nrdp1 augmented ischemia–reperfusion (I/R)-induced cardiomyocyte apoptosis while inhibition of endogenous Nrdp1 could protect cardiomyocytes against I/R injury (Zhang et al., 2011b). In the brain, Nrdp1 is found to be involved in suppressing brain tumor formation and promoting lipopolysaccharide (LPS)-induced neuroinflammation via its pro-apoptotic action (Shen et al., 2015; Shi et al., 2015; Wu et al., 2016). It is well known that neuronal apoptosis is a major event in ischemic stroke, however, the role of Nrdp1 in ischemic neuronal death has not yet been investigated. Our preliminary data shows that cerebral ischemia induces Nrdp1 mRNA expression in ischemic cerebral cortex in a rat model of middle cerebral artery occlusion (MCAO). However, the exact role of Nrdp1 in ischemia-induced neuronal damage remains to be determined.
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USP8 is a substrate of Nrdp1, and interestingly, it is also a de-ubiquitination enzyme (Wu et al., 2004; De Ceuninck et al., 2013). This de-ubiquitination activity has made USP8 a stabilizing molecule for HIF-1α protein, in which USP8 prevents HIF-1α from pVHL-mediated degradation (Troilo et al., 2014). It is well known that HIF-1α plays a vital role in attenuating brain tissue damage through promoting adaptive response during ischemic stroke (Helton et al., 2005; Baranova et al., 2007; Fan et al., 2009; Singh et al., 2012; Zhang et al., 2014; Yang Y. et al., 2017). These data raise an important hypothesis that ischemia-induced Nrdp1 upregulation may contribute to ischemic neuronal injury via downregulating USP8 (via degradation) and thus destabilizing HIF-1α in ischemic neurons.
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In the present study, we tested this hypothesis in cultured primary cerebral cortical neurons and PC12 cells (pheochromocytoma of the rat adrenal medulla) using an in vitro ischemic model of oxygen-glucose deprivation (OGD). We chose relative short OGD durations as the ischemic stimulus in this study because we have been focusing on early ischemic BBB damage that occurs within the first 4.5 h after ischemia onset (i.e., the therapeutic time window of tPA thrombolysis for ischemic stroke; Hacke et al., 2008; Jin et al., 2012; Liu et al., 2012, 2016), and in these studies, we observed substantial neuronal death in the ischemic brain within several hours after stroke onset (Jin et al., 2012; Liu et al., 2012). Our data showed that OGD treatment significantly increased Nrdp1 expression in neuronal cells, and knockdown or overexpression of Nrdp1 augmented or attenuated OGD-induced neuronal death, respectively. Moreover, Nrdp1 upregulation was accompanied by increased ubiquitinization of USP8 and its degradation, and this change was associated with decreased HIF-1α levels in ischemic neurons.
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The Laboratory Animal Care and Use Committee of Shenzhen University approved all animal related experimental protocols. Male Sprague–Dawley rats (purchased from the Experimental Animal Center Southern Medical University, Guangzhou, Guangdong, China) weighing 300 g to 400 g were anesthetized with isoflurane (4% for induction, 1.75% for maintenance) in N2O:O2 (70%:30%) during surgical procedures and the body temperature was maintained through a heated pad. A 4–0 silicone-coated monofilament nylon suture was introduced into the right intra-carotid artery to occlude the opening of the MCA as we previously described (Liu et al., 2009). MCAO was lasted for 3 h, and the animals were then deeply anesthetized with isoflurane and euthanized by decapitation. Successful MCAO was confirmed by 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MI, USA) staining of the 2-mm-thick brain coronal section 6 mm away from the tip of the front lobe as we previously described (Liu et al., 2008).
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Rat primary cortical neurons were cultured using a method as we described previously (Liu et al., 2007). Briefly, cerebral cortices were removed from the embryos of Sprague–Dawley pregnant rats at 15–18 days gestation (Shouthern Medical University Experimental Animal Center). After removing the meninges, the cortical tissue was minced and incubated with 0.05% trypsin for 30 min at 37°C with gentle trituration. After digestion, the neurons were achieved and suspended in neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine. Before seeding, culture vessels including 96-well plates, 1.2 cm glass slides or 6 cm dishes were coated with poly-L-lysine (PLL; 50 μg/mL, Sigma-Aldrich) at room temperature overnight. Neurons were maintained at 37°C in a humidified 5% CO2 incubator and half of the culture medium was changed every 3 days. The neurons were subjected to experiments 8 days after seeding.
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PC12 cells, a rat PC12 cell line, were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Peking Union Medical College/Chinese Academy of Medical Sciences (Beijing, China). PC12 cells were grown as a monolayer in dulbecco’s modified eagle medium (DMEM) supplemented with 10% horse serum and 5% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator gassed with 5% CO2 and 95% room air. For neuronal differentiation, PC12 cells were seeded in PLL pre-coated plates and allowed to adhere for 24 h. Following adherence, the culture medium was replaced with nerve growth factor (NGF, 50 ng/ml; New England Biolabs, MA, USA) containing medium. The NGF-containing medium was replaced every other day. Seven days after the supplement of NGF, the NGF-induced differentiation of PC12 cells were determined using immunofluorescence staining with antibodies against the neuron-specific marker microtubule associated protein 2 (MAP2; Supplementary Figure S1).
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Recombinant adenoviral constructs, including Ad-control (control construct), Ad-Nrdp1 (overexpressing Nrdp1), Ad-si-control (expressing non-targeting control siRNA) and Ad-si-Nrdp1 (expressing Nrdp1 siRNA) were generated as described previously (Zhang et al., 2011a). Eight days after plating, cells were infected with Ad-control, Ad-Nrdp1, Ad-si-control or Ad-si-Nrdp1 for 24 h before OGD treatment.
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To mimic ischemic condition in vitro, primary neurons or PC12 cells were exposed to OGD as described previously (Liu et al., 2012). In brief, neurons or PC12 cells were subjected to OGD by replacing the normal growth medium with glucose free medium (DMEM without glucose) pre-equilibrated with 95% N2 and 5% CO2. The cells were then incubated in a humidified airtight chamber (Biospherix Ltd., Lacona, NY, USA) for 1 h, 3 h, or 6 h. Control cultures were incubated with normal DMEM medium without FBS at 37°C in 5% CO2/95% air. OGD was terminated by removing cells from the hypoxic chamber and the cells were collected separately for further measurement.
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After OGD treatment, cell cytotoxicity was determined by the release of lactate dehydrogenase (LDH), a cytoplasmic enzyme released from cells. LDH release into the culture medium was detected using a CytoTox 96®Non-Radioactive Cytotoxicity Assay Kit (Promega Corporation. Madison, WI, USA). Briefly, 50 μl of each sample medium (i.e., pure culture medium for measuring background LDH release, culture media collected from control or OGD-treated cells for measuring experimental LDH release and lysis buffer-treated cells for measuring maximum LDH release) was collected to assay LDH release. The samples were incubated with reduced form of nicotinamide adenine dinucleotide and pyruvate for 30 min at room temperature and the reaction was terminated by adding Stop Solution. LDH release was assessed by measuring the absorbance of supernatants at 490 nm. Cell death rate was calculated as follows: cell death rate = (experimental LDH release-background LDH release) /(maximum LDH release-background LDH release) × 100%. The results were presented as fold increase of the control cells.
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| 100.0 |
Total RNA was isolated from neurons using Trizol reagents (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA samples (2 μg) were reverse-transcribed to generate first-strand cDNA. After reverse transcription using TaqMan® Reverse Transcription Kits (Applied Biosystems), reverse-transcribed products were amplified with the 7900HT real-time PCR System (Applied Biosystems) using SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) under the following conditions: 30 s at 95°C, followed by a total of 40 cycles of two temperature cycles (15 s at 95°C and 1 min at 60°C). Primer sequences were as follows: rat Nrdp1 forward: 5′-ATGGGGTATGATGTAACCCGG-3′ and reverse: 5′-GATGCAGGCGTTGCAGAAG-3′; Rat GAPDH served as endogenous control, and the primers were forward: 5′-CAATGTGTCCGTCGTGGATCT-3′; reverse: 5′-GTCCTCAGTGTAGCCCAAGATG-3′. The Ct value was calculated by the comparative ∆∆Ct method using the SDS Enterprise Database software (Applied Biosystems).
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| 99.94 |
Apoptosis was analyzed by TUNEL assay using Click-iT® Plus TUNEL Assay (Life Technologies, Inc., Carlsbad, CA, USA) according to manufacturer’s instruction. Briefly, at the end of the indicated treatments, primary rat cerebral cortical neurons grown on coverslips were incubated with TdT reaction mixture for 2 h at 37°C, followed by 30-min incubation with the Alexa Fluor® 594 dye. Then, the cells were counterstained with DAPI (Sigma-Aldrich) for 20 min and observed under a fluorescence microscope (Leica, Germany). The TUNEL-positive nuclei of six non-overlapping fields per coverslip were counted by a researcher blinded to treatment, and these counts were converted to percentages by comparing the TUNEL-positive counts to the total number of cell nuclei as determined by DAPI counterstaining, that is TUNEL-positive ratio = (number of red nuclei/number of blue nuclei) × 100%.
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| 99.94 |
Protein samples were prepared from cultured neurons or PC12 cells using extraction buffer as described previously (Liu et al., 2012). The protein samples were electrophoresed on SDS-PAGE gels and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with the indicated primary antibodies overnight and then with horseradish peroxidase-conjugated secondary antibody for 1 h. The blots were developed using a chemiluminescent system, and the bands were scanned, and densitometry analysis was performed with Gel-pro 4.5 Analyzer (Media Cybernetics, Silver Spring, MD, USA). The primary antibodies were anti-cleaved-PARP, anti-PARP, anti-Bcl-2, anti-Bax, anti-USP8 and anti-β-actin. Anti-Nrdp1 and anti-HIF-1α antibodies were purchased from BETHYL Laboratories and Novus Biologicals, respectively, and the rest primary antibodies were purchased from Santa Cruz Biotech. Relative protein levels were quantified after normalization to the loading control β-actin.
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| 100.0 |
After the indicated treatments, PC12 cells were lysed in RIPA buffer and centrifuged at 12,000× g for 10 min at 4°C. The supernatant were incubated overnight at 4°C with 4 μg of anti-USP8 (Santa Cruz Biotech, Chicago, IL, USA), followed by precipitation with 50 μl of Dynabeads protein A (Pierce Biotechnology, Rockford, IL, USA) for 10 min at room temperature. The protein A were then washed extensively with binding buffer, resuspended in SDS-PAGE buffer, and boiled for 5 min. Samples of 30 μg total cell lysate were used as an input control. The precipitated complexes were separated on SDS-PAGE gels, and transferred to nitrocellulose membranes, and immunoblotted with anti-Nrdp1 (BETHYL Laboratories, Montgomery, TX, USA), K48-linkage-specific anti-ubiquitin antibody (Abcam, Cambridge, MA, USA) or anti-HIF-1α antibody (Novus Biological, Littleton, CO, USA) to detect the presence of these proteins in the complex. Normal rabbit IgG (Santa Cruz Biotech) was used as a loading control.
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| 100.0 |
All data were expressed as means ± SEM. Differences between groups were evaluated by either an unpaired Student’s t test or one-way ANOVA followed by Tukey’s post hoc test as indicated in the Figure Legends. P < 0.05 was regarded as statistically significant.
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| 99.94 |
Nrdp1 is found to be widely expressed in the brain and is implicated in ischemic damage to the heart (Zhang et al., 2011b). To determine whether Nrdp1 plays a role in ischemic brain injury, we examined the change of Nrdp1 expression in the cerebral cortex isolated from the rats that were subjected to 3-h MCAO without reperfusion. Nrdp1 mRNA expression was analyzed in isolated hemispheric cortex by real time RT-PCR and found that 3-h MCAO induced a significant increase (~1-fold) of Nrdp1 mRNA expression in ischemic hemispheric cortex compared to non-ischemic cortical tissue (Figure 1A, P < 0.05). Consistent with its mRNA change, Nrdp1 protein levels were also significantly increased in ischemic cerebral cortex (Figure 1B). These results demonstrate that Nrdp1 is upregulated in the ischemic brain cortex. To further demonstrate a role of Nrdp1 in ischemic neuron injury and the underlying mechanisms involved, we chose the widely used in vitro model of ischemia (i.e., OGD) for the rest of this study.
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| 100.0 |
Middle cerebral artery occlusion (MCAO) induces neuregulin receptor degradation protein-1 (Nrdp1) upregulation in cerebral cortex. Rats were subjected to 3-h MCAO before isolating hemispheric cerebral cortex. The mRNA and protein levels of Nrdp1 in cerebral cortex from nonischemic (Non-I) and ischemic (I) hemispheric tissue were analyzed by real-time RT-PCR and western blot. (A) Real-time RT-PCR analysis showed that Nrdp1 mRNA expression was significantly increased in ischemic hemispheric cortex. *P < 0.05 vs. Non-I, ANOVA; n = 6. (B) Western blot analysis revealed increased levels of Nrdp1 protein in ischemic hemispheric cortex. Upper panel: representative immunoblots of Nrdp1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin. *P < 0.05 vs. Non-I, ANOVA; n = 6.
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| 100.0 |
To determine the functional role of cerebral Nrdp1 in response to OGD treatment, we examined the expression of Nrdp1 in OGD-treated primary rat cerebral cortical neurons. The cells were exposed to OGD for 1, 3, or 6 h before analyzing Nrdp1 mRNA and protein levels. Real time RT-PCR analysis showed that Nrdp1 mRNA expression was increased in cerebral cortical neurons after exposing to OGD for 1 h and was further increased at 6-h OGD, while no significant difference was seen between 1-h OGD and 3-h OGD (Figure 2A). Western blot analysis showed that Nrdp1 protein levels were significantly increased in cerebral cortical neurons after exposing to OGD for 3 h and 6 h, but not for 1 h (Figure 2B). To further verify the above findings, we assayed the expression of Nrdp1 in PC12 cells exposed to the same OGD treatment as above. Real time RT-PCR analysis showed that Nrdp1 mRNA expression was increased in PC12 cells after exposing to OGD for 3-h and 6-h OGD, but not for 1 h (Figure 2C). Western blot analysis showed that Nrdp1 protein levels were significantly increased in PC12 cells after exposing to OGD for 6 h, but not for 1 h and 3 h (Figure 2D). These data demonstrate that OGD induces Nrdp1 upregulation in cerebral cortical neurons as well as PC12 cells in a time-dependent manner.
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| 100.0 |
Oxygen-glucose deprivation (OGD) induces Nrdp1 upregulation in primary rat cerebral cortical neurons and PC12 cells. Cells were subjected to OGD treatment or normoxia (Control, Con) for 1, 3, or 6 h before analyzing Nrdp1 mRNA and protein expression using real-time RT-PCR and western blot. (A) Real time RT-PCR analysis showed that Nrdp1 mRNA expression was significantly increased in primary rat cerebral cortical neurons at 1 h after OGD treatment and was further increased when OGD was prolonged to 6 h. *P < 0.05 vs. Con, ANOVA; n = 4. (B) Western blot analysis showed that Nrdp1 protein levels were increased in 3-h OGD- and 6-h OGD-treated primary rat cerebral cortical neurons, but not in 1-h OGD-treated cells. Upper panel: representative immunoblots of Nrdp1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin. *P < 0.05 vs. Con, ANOVA; n = 4. (C) Real time RT-PCR analysis showed that Nrdp1 mRNA expression was significantly increased in PC12 cells at 3 h and 6 h after OGD treatment, but not 1 h. *P < 0.05 vs. Con, ANOVA; n = 4. (D) Western blot analysis showed that Nrdp1 protein levels were increased in 6-h OGD-treated PC12 cells, but not in 1-h OGD- and 3-h OGD-treated cells. Upper panel: representative immunoblots of Nrdp1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin. *P < 0.05 vs. Con, ANOVA; n = 4.
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study
| 100.0 |
To investigate whether Nrdp1 is implicated in OGD-induced apoptosis in neurons, we transfected cerebral cortical neurons with Ad-control, Ad-Nrdp1 or Ad-si-Nrdp1. As shown in Figure 3, the transfection efficiency reached more than 90% at 24 h after transfection (Figure 3A), and western blot analysis showed that incubating the neurons with Ad-si-Nrdp1 and Ad-Nrdp1 for 48 h significantly reduced (~90% reduction) and increased (~2-fold increase) Nrdp1 protein levels, respectively (Figures 3B,C). We assessed the effect of Ad-si-Nrdp1 and Ad-Nrdp1 on OGD-induced neuronal death by measuring LDH release (indicating late apoptosis and necrosis) and TUNEL staining (indicating apoptosis). The data showed that cell death and apoptosis did not differ across the groups under basal conditions (Figures 3D,E and Supplementary Figure S2). However, after OGD treatment, inhibition of Nrdp1 significantly attenuated neuronal death and apoptosis as compared with the Ad-control, while transfection with the Ad-Nrdp1 greatly enhanced OGD-induced neurons death and apoptosis (Figures 3D,E and Supplementary Figure S2).
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study
| 100.0 |
Effects of Nrdp1 on OGD-induced apoptosis in primary rat cerebral cortical neurons. (A) The infection efficiency of neurons with Ad-control, Ad-Nrdp1, Ad-si-control and Ad-si-Nrdp1 was visualized for green fluorescent protein (GFP) 24 h later using fluorescence microscopy (magnification, ×400). (B) Western blot analysis showed that incubation neurons with Ad-si-control and Ad-si-Nrdp1 for 48 h significantly (~90%) reduced Nrdp1 protein levels. Upper panel: representative immunoblots of Nrdp1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin. *P < 0.05 vs. Ad-si-control, ANOVA; n = 4. (C) Western blot analysis showed that incubation neurons with Ad-control and Ad-Nrdp1 for 48 h significantly increased Nrdp1 protein levels. Upper panel: representative immunoblots of Nrdp1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin. *P < 0.05 vs. Ad-control, ANOVA; n = 4. (D) Neurons were infected by with Ad-control, Ad-Nrdp1 or Ad-si-Nrdp1 and then treated with OGD for 6 h. Cell death rate was assessed by lactate dehydrogenase (LDH) release. *P < 0.05 vs. Ad-control. #P < 0.05 vs. Ad-control + OGD, ANOVA; n = 4. (E) Apoptosis was detected using TUNEL assay. Quantitative analysis of TUNEL-positive cells from three independent experiments. *P < 0.05 vs. Ad-control. #P < 0.05 vs. Ad-control + OGD, ANOVA; n = 4. (F) Neurons were infected and treated with OGD as in (D). Western blots analysis of expression of cleaved PARP protein (upper panel). Quantitative analysis of cleaved PARP was shown in the bottom panel. *P < 0.05 vs. Ad-control. #P < 0.05 vs. Ad-control + OGD, ANOVA; n = 4. (G) Western blots analysis of expression of Bax and Bcl-2 proteins (upper panel). Quantitative analysis of the ratio of Bax/Bcl-2 was shown in the bottom panel. *P < 0.05 vs. Ad-control. #P < 0.05 vs. Ad-control + OGD, ANOVA; n = 4.
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study
| 100.0 |
To further verify a role of Nrdp1 in OGD-induced apoptosis in neurons, we assessed the effect of Nrdp1 on several key apoptosis-associated signal proteins including cleaved-PARP and Bax/Bcl-2. As shown in Figures 3F,G, 6-h OGD induced a significant increase in cleaved PARP levels (PARP activation) and a greater ratio of Bax/Bcl-2 in cerebral cortical neurons, and transfection with Ad-si-Nrdp1 abolished these changes. Accordingly, overexpression of Nrdp1 augmented OGD-induced increases in cleaved PARP and Bax/Bcl-2 ratio. As expected, Ad-si-Nrdp1 or Ad-Nrdp1 alone did not affect these apoptosis-associated signal proteins (Figures 3F,G). Taken together, these results clearly indicate that Nrdp1 plays an important role in ischemia-induced apoptosis in cerebral cortical neurons.
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study
| 100.0 |
HIF-1α acts as an intracellular sensor for hypoxia and promotes the cells to adapt to hypoxic/ischemic conditions (Zis et al., 2015), thus we hypothesized that Nrdp1 might interact with or suppress HIF-1α to promote neuronal cell death under OGD conditions. To test this possibility, we transfected primary cortical neurons and PC12 cells with Ad-control, Ad-Nrdp1, Ad-si-control or Ad-si-Nrdp1 before exposing to OGD for 6 h. HIF-1α protein levels were analyzed by western blot. As shown in Figures 4A,B, 6-h OGD induced a significant increase in the accumulation of HIF-1α protein in both primary neurons and PC12 cells transfected with Ad-si-control, and of note, this increase was further augmented or attenuated when Nrdp1 was knocked down by Ad-si-Nrdp1 or overexpressed by Ad-Nrdp1, respectively (Figures 4C,D). These data suggest that Nrdp1 may act as a negative regulator for HIF-1α expression in neurons under OGD conditions.
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study
| 100.0 |
Effects of Nrdp1 expression on hypoxia inducible factor-1α (HIF-1α) and USP8 in vitro after OGD treatment. (A,B) Primary rat cerebral cortical neurons and PC12 cells were infected with Ad-si-control or Ad-si-Nrdp1 and exposed to 6-h OGD respectively. Western blot analysis of protein levels of HIF-1α and USP8 of ischemic neurons (top panels). Histograms show relative intensity of HIF-1α and USP8 (bottom panels). *P < 0.05 vs. Ad-si-control. #P < 0.05 vs. Ad-si-control + OGD; n = 4. (C,D) Primary rat cerebral cortical neurons and PC12 cells were infected with Ad-control or Ad-Nrdp1 and exposed to 6-h OGD respectively. Western blot analysis of protein levels of HIF-1α and USP8 of ischemic neurons (top panels). Histograms show relative intensity of HIF-1α and USP8 (bottom panels). *P < 0.05 vs. Ad-control. #P < 0.05 vs. Ad-control + OGD; n = 4.
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study
| 100.0 |
The fact that Nrdp1’s substrate USP8 can protect HIF-1α from pVHL-mediated degradation (Troilo et al., 2014) led us to hypothesize that Nrdp1 may act on USP8 to regulate HIF-1α expression under OGD condition. To test this, we investigated the change of USP8 in OGD-treated neurons and the impact of Ad-Nrdp1 and Ad-si-Nrdp1 on USP8 expression. As shown in Figures 4A,B, 6-h OGD induced a significant decrease in the level of USP8 proteins in the cells transfected with Ad-si-control. Importantly, this reduction was partially reversed when Nrdp1 was knocked down by Ad-si-Nrdp1, and was exacerbated when Nrdp1 was overexpressed by Ad-Nrdp1 (Figures 4C,D). Collectively, these results suggest that Nrdp1 may contribute to OGD-induced neuronal cell death via suppressing HIF-1α and USP8 expression.
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study
| 100.0 |
Lastly, to ambiguously demonstrate the interactions between Nrdp1 and USP8 and between USP8 and HIF-1α in OGD-treated neurons, we performed co-immunoprecipitation experiments. Since Nrdp1 targets USP8 for ubiquitylation, we speculated that overexpression of Nrdp1 could enhance protein ubiquitylation and USP8 degradation in PC12 cells. To test this, we pulled down ubiquitylated species from PC12 cell extracts, and then detected the levels of protein ubiquitilytion in the presence of proteasome inhibitor MG132 as well as the protein levels of Nrdp1 and USP8. As shown in Figure 5A, overexpression of Nrdp1 by Ad-Nrdp1 significantly increased the whole levels of protein ubiquitylation in comparison to Ad-green fluorescent protein (GFP) control, and this change was accompanied by decreased USP8 protein levels.
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study
| 100.0 |
Nrdp1 accelerates ubiquitin-mediated degradation of USP8 and decreases its interaction with HIF-1α. (A) The lysates from Ad-GFP/Ad- Nrdp1 adenovirus PC12 cells were immune-precipitated with anti-USP8 antibody and analyzed by immunoblotted with anti-ubiquitin antibody to detect ubiquitylated forms of USP8 in vitro. (B) The interactions of USP8 with HIF-1α were detected with co-immunoprecipitation in PC12 cells under OGD treatment or (C) after transfected Ad-si-control/Ad-si-Nrdp1/Ad-Nrdp1 adenovirus. PC12 cells lysates were immune-precipitated with anti-USP8 antibody or control IgG, and the immune-precipitates were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-USP8 and anti-HIF-1α antibody.
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study
| 100.0 |
To demonstrate a direct interaction between USP8 and HIF-1α in OGD-treated PC12 cells, we performed co-immunoprecipitation assays and found that HIF-1α was precipitated by antibody against USP8, but not by control rabbit IgG (Figure 5B). Moreover, under OGD conditions, overexpression of Nrdp1 by Ad-Nrdp1 reduced co-immunoprecipitation between USP8 and HIF-1α, while knockdown of Nrdp1 by Ad-si-Nrdp1 enhanced the interaction between these two proteins (Figure 5C). These data indicate that under ischemic conditions, Nrdp1 upregulation may hinder the stabilization of HIF-1α in neurons via promoting ubiquitin-mediated degradation of USP8, thus attenuating cellular adaptive response to hypoxia/ischemia.
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study
| 100.0 |
The E3 ligase Nrdp1 has been extensively investigated on cell growth, apoptosis and inflammation in cancer cells and other cell types (Qiu et al., 2004; Wang et al., 2009; Ingalla et al., 2010; Byun et al., 2015). In the present study, we investigated Nrdp1’s role in ischemic neuronal injury. The major findings include: (1) Nrdp1 is significantly upregulated in the ischemic brain tissue and in OGD-treated neuronal cells; (2) overexpression or knockdown of Nrdp1 enhances or attenuates OGD-induced apoptosis in neurons, respectively, and these changes are accompanied by the downregulation or upregulation of Nrdp1’s substrate USP8; and (3) USP8 may directly interact with HIF-1α to prevent its degradation, and under OGD conditions, Nrdp1 may interfere with HIF-1α stabilization via promoting USP8 degradation. These data suggest that Nrdp1 may attenuate neuron’s adaptive response to hypoxia/ischemia via interfering USP8-mediated HIF-1α stabilization, thus contributing to neuronal death under ischemic conditions.
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study
| 100.0 |
Apoptotic neuronal death is a common event accounting for neuron loss in ischemic stroke (Cao et al., 2004; Widiapradja et al., 2012; Wang et al., 2014). Therefore, the mechanism of neuronal apoptosis under ischemic conditions has been an important research focus in the past decades. Deregulation of the UPS is believed to be an important contributor to ischemic neuronal injury (Wojcik and Di Napoli, 2004; Doeppner et al., 2016). The E3 ligase Nrdp1 has been recently shown to mediate neuronal apoptosis through reducing BRUCE expression in LPS-induced neuroinflammation (Shen et al., 2015). Our previous study has also demonstrated a role of Nrdp1 in promoting cardiac myocyte apoptosis in experimental I/R (Zhang et al., 2011b). Here our in vivo and in vitro data show that ischemia induces Nrdp1 upregulation in cerebral cortical neurons. Of note, this change is quite rapid and persistent, as Nrdp1 mRNA expression is increased in neurons at 1 h after OGD treatment and remains high at the end of 6-h OGD exposure. Our data that knockdown of Nrdp1 with siRNA reduces OGD-induced cell death/apoptosis and overexpression of Nrdp1 by Ad-Nrdp1 increases neuronal death clearly supports a role of Nrdp1 in ischemic neuronal injury. Moreover, along with Nrdp1 knockdown or overexpression is the inhibition or activation of apoptosis-associated proteins, including caspase-3, PARP-1, Bax/Bcl-2 ratio, further supporting a proapoptotic action of Nrdp1 in OGD-induced neuron injury.
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study
| 99.94 |
Nrdp1 is inducible in cells in response to different stimuli, and its stability largely relies on its substrate USP8, a de-ubiquitinating enzyme (Wu et al., 2004). Thus, Nrdp1 and USP8 may restrict each other (De Ceuninck et al., 2013). When Nrdp1 is increased, more USP8 will be degraded by Nrdp1, and as a return, less USP8 will make Nrdp1 unstable, resulting in less Nrdp1 and more USP8 in the cells. Consistently, here our data also show that under OGD condition, Nrdp1 upregulation concurrently occurs with USB8 downregulation in neuronal cells. Moreover, Nrdp1 overexpression augments OGD-induced USP8 downregulation, while knockdown of Nrdp1 ameliorates this effect. Although we did not design experiments to verify USP8’s effect in stabilizing Nrdp1 protein in neurons under ischemic conditions, our data clearly demonstrate that the interaction between these two proteins is associated with OGD-induced neuronal death.
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study
| 100.0 |
Mounting evidence suggests that HIF-1α is an essential transcriptional regulator of various vital processes in neurons including the adaptation of cells to hypoxic environments (Barteczek et al., 2017), cell proliferation (Zhang et al., 2017), cell apoptosis (Yin J. et al., 2017) and metabolism (Carmeliet et al., 1998; Cho et al., 2015). In the brain, HIF-1α has been reported to act as a pivotal protective regulator in ischemic brain injury (Fan et al., 2009; Singh et al., 2012; Zhang et al., 2014). Baranova et al. (2007) found that knockdown of neuronal HIF-1α enhances ischemic brain injury. Activation of HIF-1α-associated signaling cascades, such as EPO pathway (Liu et al., 2006; Ryou et al., 2012) and VEGF pathway (Yin W. et al., 2017) in neurons could protect the brain from I/R damage through increasing microvascular density and/or restoring local blood flow and oxygen supply. Yang X. S. et al. (2017) found that HIF-1α involved in necroptosis contributed to ischemic brain injury induced by OGD and MCAO. Inhibition of the 20S proteasomal activity can protects HIF-1α from degradation and provides neuroprotection in cerebral ischemia (Badawi and Shi, 2017). Here we show that 6-h OGD without re-oxygenation induces HIF-1α protein accumulation in neuronal cells, and this change is enhanced or suppressed by overexpression or knockdown of Nrdp1, respectively. This important observation has evoked us to further explore the interaction between Nrdp1 and HIF-1α in OGD-treated neurons.
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study
| 100.0 |
USP8 can protect HIF-1α from degradation mediated by E3 ubiquitin ligase pVHL via de-ubiquitination (Troilo et al., 2014), which promoted us to hypothesize that USP8 may be an important bridge molecule that mediates the interaction between Nrdp1 and HIF-1α. Indeed, we observed two simultaneous changes in OGD-treated neurons that support our hypothesis. First, Nrdp1 overexpression leads to increased protein ubiquitylation and suppressed interaction between Nrdp1 and USP8 (due to increased USP8 degradation). Second, USP8 directly interacts with HIF-1α, and this interaction is increased when Nrdp1 is knocked down. The interaction between USP8 and HIF-1α has been previously reported by Troilo et al. (2014). Our data suggest that under ischemic conditions, Nrdp1 upregulation may lead to an accelerated degradation of USP8, which in turn attenuates USP8’s capability to protect HIF-1α against pVHL-mediated degradation, thus interfering neuronal cells to timely adapt to hypoxic/ischemic conditions. In addition, our data also suggest that HIF-1α is an important downstream effector molecule in the pathway of Nrdp1-mediated apoptosis during ischemic neuronal injury. Future studies are warranted to explore the mechanisms underlying enhanced Nrdp1 expression under ischemic conditions.
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study
| 100.0 |
It is worth pointing out one important fact, that is, whether HIF-1α is protective or detrimental in ischemic stroke depends on the stroke stage, ischemia severity and ischemia duration (Yang et al., 2013). For example, there are studies showing that HIF-1α knockdown protects the brain against ischemic damage (Helton et al., 2005). However, other studies have reported that inhibition of HIF-1α and HIF-2α is beneficial to the neurons in the very acute phase after ischemic stroke (Barteczek et al., 2017). Under our experimental conditions, HIF-1α may be more likely a good molecule in promoting neuronal cells to rapidly adapt hypoxic conditions. However, future experiments are needed to demonstrate this speculation.
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study
| 99.94 |
Taken together, the present study demonstrates that in response to ischemic stimuli, Nrdp1 is upregulated in neurons and contributes to ischemic neuronal death, and this effect may be associated with suppressed adaptive response to hypoxia/ischemia due to accelerated USP8 degradation and HIF-1α destabilization. Therapeutic strategies that target Nrdp1 activation may provide neuroprotection against ischemic brain injury.
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study
| 99.94 |
According to historical sources, the Hungarian tribal alliance conquered the eastern parts of the Carpathian Basin in 895 AD, and in successive campaigns occupied its central territories until 907 AD1. The mixed autochthonous population, which mostly spoke different Slavic, Turkic Avar, and German languages, integrated with variable speed with the newcomers, as we know from contemporaneous sources2. Whereas the Slavs lived mainly on the fringes, the successors of the Avars persisted in some inner territories of the Carpathian Basin. The Avars arrived in the Carpathian Basin in 568 AD, fleeing the westward-expanding influence of the Turkic Khaganate in Inner Asia3. The Avar population already included several folk elements at this time; and the population was uniform from neither a cultural nor a physical anthropological perspective. Over one hundred thousand excavated graves from the Avar period in the Carpathian Basin picture a heterogenic physical anthropological composition of this population, which contained mainly Europid characters and, only in certain regions and periods, was dominated by Asian craniometric indices4. The occupation policy of Avar and ancient Hungarian tribes were similar due to similar steppe-type husbandry and management of space and power. In the politically unified alliance of the Hungarian tribes, both the leader and the tributary folks influenced each other culturally. These interactions are easily seen from the changing material culture of the Hungarian conquerors, who began to use local types of jewels but also maintained steppe-like traditions during the 10th century5. It is difficult to estimate the size of the 10th–11th century population of the Carpathian Basin from ca. twenty-five thousand excavated graves56. Scholars estimate the Hungarian conqueror population in the Carpathian Basin between a few thousand and half a million, while the indigenous population size, which is also uncertain, is estimated at a few hundred thousand people7.
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other
| 99.56 |
Historical sources give evidence of the mixed ethnic composition of the Hungarians before the conquest of the Carpathian Basin28. The diverse origin of the Hungarian tribes has also been documented in physical anthropological research. Craniometrical analyses revealed that the Europid crania type was predominant in the conquerors, with smaller amounts of Europo-Mongoloid characters9. Regional groups of the ancient Hungarian anthropological series show morphometric parallels ranging from the Crimean Peninsula to the Kazakh steppe10.
|
other
| 99.9 |
The Finno-Ugric origin of the Hungarian language is well recorded by linguistic research, which lead to an assumption that there was a Uralic substrate of the ancient Hungarian population2. However, Turkic-speaking groups could also have had a significant role in the formation of the Hungarian people and political institutions, as suggested by ancient Turkic loanwords in the early layer of the Hungarian language and the Turkic origin of toponyms and person names of tribe leaders of the conquest-period11. After leaving the Central Uralic homeland, an obvious source of the Turkic influence was the Turkic-speaking political environment of the Bulgars (Onogurs) and Khazars in the 9th-century Eastern European steppe, where the Hungarians lived for a period of time. The exact route and chronology of the Hungarian migration between the Ural region and the Carpathian Basin is continually debated among archaeologists, linguists and historians.
|
other
| 99.9 |
The genetic origin of ancient Hungarians is still in question, although some modern and ancient DNA studies have focused on this issue. For example, Tömöry et al. have described the mitochondrial DNA (mtDNA) of a small group of ancient Hungarians from the 10th–12th century Carpathian Basin, where the ancient Hungarians’ affinity to modern day Central Asia has been demonstrated. Tömöry et al. concluded, without simulation tests, that there was no genetic continuity between the classical conquerors and modern day Hungarians12. A small 10th–12th century population from the northwestern Carpathian Basin has been reported with heterogeneous maternal genetic characteristics similar to modern Europeans13. On the other hand, ancient mitochondrial DNA data from the putative source region of the ancient Hungarians is still scarce, and concentrates only on the prehistory of Siberia and Central Asia14151617. Of four analyzed Y chromosomes from the conqueror population, two showed connections to Uralic peoples through N1c1 haplogroup marker Tat18.
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study
| 99.94 |
Genetic research of modern Hungarians has been a subject of four further mtDNA and Y chromosomal studies. Brandstätter et al. and Egyed et al. built the mtDNA control region and Y chromosomal microsatellite databases from different groups of modern Hungarians, including an “average” Hungarian group from Budapest and two groups of Hungarian minorities–Ghimes Csango and Szekler–living in modern Romania. Both Szeklers and Csangos were found to harbor some Asian genetic components, and the Csango population showed genetic signs of long term isolation, which differentiated them from the Szeklers and the population of Budapest192021. Asian genetic mtDNA and Y chromosome components are apparently rare in the modern Hungarian gene pool, which led Semino et al. to the conclusion that the Hungarian conquerors were in small number and that the Hungarian language could be an example of cultural dominance22. The pitfalls of the very hypothetical historical interpretation of modern day population genetic results have been critically reviewed by Bálint23.
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review
| 99.9 |
The archaeogenetic contribution to the historical era of the Avar and conquest-periods (6th–10th centuries) in the Carpathian Basin is still sparse. Our research approaches the questions of maternal genetic composition and the origin of the ancient Hungarians, analyzing a dataset four times larger than previous work has attempted. The connections of the conquerors to the previous Avar and contemporaneous Slavic-Hungarian contact zone population will be determined, as well as connections to other ancient populations of Eurasia that have previously been published. We also compare our dataset with modern day data from the Carpathian Basin and Eurasia, in order to better understand the maternal genetic origin and legacy of the 7th–11th century population of the Carpathian Basin.
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study
| 100.0 |
Reproduced hyper variable segment I (HVS-I) sequences were obtained from mtDNA of 111 individuals from the medieval Carpathian Basin: 31 mtDNA profiles from Avars, 76 from Hungarian conquerors and four from the southern Hungarian-Slavic contact zone (see Supplementary Table S3). The mtDNA of 111 individuals was extracted at least twice per individual from different skeletal elements (tooth and femur or other long bones, Supplementary Table S1), the HVS-I fragments were reproduced in subsequent PCR and sequencing reactions, at least twice per DNA extract. The sequence results of these replicates, spanning HVS-I nucleotide positions (np) 16040–16400, typing individual selections of 14 coding region positions and two fragments of the HVS-II (np 29–254) confirm the haplotypes to be authentic. Of the 144 processed samples, 33 had no amplifiable DNA yield, or the sequences gave ambiguous haplotype results.
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study
| 100.0 |
The Avar group from the southeastern Great Hungarian Plain (Alföld) had a mixed European-Asian haplogroup composition with four Asian haplogroups (C, M6, D4c1, F1b) at 15.3%, but a predominantly European (H, K, T, U), haplogroup composition (Fig. 2). In the conqueror population the most common Eurasian haplogroups were detected. West-Eurasian haplogroups (H, HV, I, J, K, N1a, R, T, U, V, X, W) were present at a frequency of 77%, and Central and East-Eurasian haplogroups (A, B, C, D, F, G, M) at 23%. The most widespread haplogroups of the conqueror population were H and U with frequencies 22% and 20% respectively (Supplementary Table S5). Five individuals from the 9th–10th centuries from the west Hungarian Vörs-Papkert site were excluded from any statistical analysis because of their offside geographical location and cultural differences from the Avar and Hungarian sites. Their mtDNA belonged to the common European J and H haplogroups, but with rare haplotype variants in ancient and modern mtDNA databases (see Supplementary Table S15 for database references). The number of typed mtDNA from the 10th–12th century contact zone metapopulation13 was enlarged by four 10th century samples from present-day north Croatia. One belonged to a characteristic European H10e haplotype; another belonged to U7 haplotype, mainly distributed in modern Southwest Asia and Southern Europe; a third belonged to the Southwest Asian N1b1 type; the fourth U5a2a haplotype was common in modern Eurasia (private database, see Material and Methods, Supplementary Table S15).
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| 100.0 |
The principal component analyses (PCAs) of ancient and modern-day populations were computed based on haplogroup frequencies (Supplementary Tables S5 and S6). PCA of 21 ancient populations showed a predominant difference between European and Asian populations, which indicates a clustering of the medieval populations of Europe, as well as the assembly of Avars, conquerors and further Mediterranean populations (Fig. 3a, Supplementary Fig. S1). Although the East Asian medieval populations were clearly separated from the European contemporaneous period on both PCA and Ward clustering, prehistoric Central Asian (Kazakhstan) and North Asian (Siberian Late Bronze Age Baraba) populations showed similarities to the conquest-period dataset in both analyses (Fig. 3b). The three Carpathian Basin populations were compared with populations from most of the ancient North European and medieval Asian populations, showing significant differences in haplogroup composition (p < 0.05). On the other hand, prehistoric Central Asian, south central Siberian (Minusinsk Hollow) and Baraba populations were not significantly different from the populations of the Carpathian Basin, and these affinities are also reflected in the clustering tree (Supplementary Table S5, Fig. 3b).
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| 100.0 |
The PCA of the investigated ancient and modern Eurasian populations demonstrated the clustering of most modern European populations by PC1, PC2, and PC3. Furthermore, their affinities to modern Near Eastern populations are represented by PC1 and PC3, whereas the modern Asian populations are dispersed along PC1. The conqueror population has a similar haplogroup composition to modern Central Asians and Finno-Ugric populations, which is also supported by Ward type clustering. While Avars rather showed modern European connections, the contact zone population had a Near Eastern type haplogroup composition (Supplementary Figs S2 and S3).
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| 100.0 |
The distance calculations based on high subhaplogroup resolution also showed that modern Central Asian populations were highly similar to the conqueror population. The maternal genetic connections of the Avar group concentrated on modern Eastern European populations, and the contact zone group showed Southwest Asian affinities on genetic distance maps (GDM) (Fig. 4a, Supplementary Figs S6A and S7A, see Supplementary Table S13 for references).
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| 100.0 |
The haplogroup frequency-based test of population continuity (TPC)24 rejected neither the null hypothesis of population continuity between the Avars and the southeastern Alföld group of conquest-period Hungarians, nor between Avars and all conquerors analyzed from the Carpathian Basin. Furthermore, the haplogroup frequency differences between the 10th–12th century populations and modern Hungarians, and also Hungarian minorities of Szeklers and Csangos living in Romania can be explained by genetic drift that occurred during the last millennium (Supplementary Table S7).
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study
| 100.0 |
Pairwise genetic distances were calculated between 21 ancient and 52 modern populations. Interestingly, pairwise FST values of Avars indicated non-significant differences among nearly all medieval European populations, and from Central Asia, as well as from many modern-day Europeans. The Hungarian conqueror population showed the lowest distances from modern-day Uzbeks and Turkmens (FST = 0.00335 and 0.00489 respectively) and from six ancient populations: medieval Poles (FST = −0.00018), Bronze and Iron Age in present-day Kazakhstan (FST = −0.00164), Bronze Age along the south central Siberian flow of Yenisey River (Minusinsk Hollow) (FST = −0.00208), Siberian Baraba population (FST = −0.01003), Avars (FST = 0.00233), and 6th century Lombards from Hungary (FST value 0.00762), these values were non-significant (p > 0.05). The distances from the ancient populations were visualized on an FST level plot (Fig. 5). The mixed contact zone population has the shortest distances from present-day Iraq (FST = 0.00817), Italy (FST = 0.00923), Czechs (FST = 0.01023) and Avars (FST = 0.01094). For the genetic FST values and their corresponding p-values, see Supplementary Tables S8 and S9.
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study
| 100.0 |
In order to visualize these genetic distances, linearized Slatkin FST values were displayed on multi-dimensional scaling (MDS) plots (Fig. 6, Supplementary Fig. S5 and Tables S8 and S9). The plot of ancient populations reflects the PCA and shows the connection between the south western Siberian Baraba population17, south central Siberian Minusinsk Depression and Kazakhstani prehistoric populations1416 and the conquerors. The Avar and contact zone populations show stronger affinities to the European medieval populations, similarly to the PCA results. On the modern population MDS plot, which also contains the three investigated medieval datasets, a very similar picture is observable to the modern PCA, except that the Southwest Asian populations do not separate from Europe along coordinate 2 (Supplementary Fig. S5).
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study
| 100.0 |
The sequence-based genetic distance maps, encompassing 141 modern populations, show congruently the Central Asian affinity to the conquerors, the European/Near Eastern characteristic populations to the Avar sequences, and predominant Near Eastern affinities to the contact zone group (Fig. 4b, Supplementary Figs S6B and S7B, see Supplementary Table S14 for references).
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study
| 99.94 |
The 101 ancient Hungarian samples belong to 75 HVS-I haplotypes (haplotype diversity Hd = 0.987). The haplotype diversity is highest in the Avar group, and lowest in the contact zone dataset (Table 1). The shared haplotype analysis (SHA) shows that medieval populations from Southern Europe (Spain and Italy) shared over 50% of haplotypes with the conqueror population (Fig. 5 and Supplementary Table S10). High proportions of shared lineages with the conquerors were detected in the contact zone population (43.5%), Vikings from Norway (39.3%), Iceland (39.7%), and 6th-century Lombards in Hungary (39.3%). The SHA analysis was strongly influenced by altering haplotype diversity and the high number of Cambridge Reference Sequence (rCRS) H lineages in medieval Spanish, Italian and Norwegian Viking groups, which caused high proportion of lineage sharing with only a small number (n = 4–5) of shared lineage types. Medieval populations from Italy and Spain shared many of their haplotypes (40–48%) with the Avar and contact zone populations as well. On the other hand, many lineages of the Bronze Age Andronovo, Baraba, and Bronze Age population of the region of today’s Kazakhstan were shared with the conquerors (37.5–29.4%), with some identical Asian lineages among them.
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| 100.0 |
We analyzed more deeply the sharing of the Eastern Eurasian haplotypes–found in the Carpathian Basin medieval datasets–with modern and ancient populations (Supplementary Table S11). Based on our updated Eurasian mtDNA database of 64,650 HVS-I sequences, the Asian lineages in the conqueror dataset showed diverse hits. Three Asian A haplotypes had no matches in our modern-day mtDNA database (see references in Supplementary Table S15). Other A11 and A12a haplotypes had parallels in present-day Uzbekistan, Kazakhstan, other Asian populations, in people of the Xiongnu confederation of the 3rd BC to 2nd AD century, in the late medieval Yakuts, and in medieval Scandinavia. Two B haplotypes were present in today’s China, Kazakhstan and spread as far as Thailand. The detected conqueror C-C4, F1b, and G2a haplotypes were widespread in modern Eurasia, and had parallels even in China and Korea. Six of these C, F, and G2a haplotypes had parallels in ancient populations of Asia. Among the five Asian D lineages, two were unique in the database and two were common in Central and East Asia. One D haplotype however (Der4.522) showed rare occurrence in Kazakhs, Uzbeks and Altaians, and Siberian populations. Among the Avars, three Asian haplotypes (C, M, D4c1) were found. One C haplotype had only one match in modern Kazakh population, the other M lineage was common in Central and East Asia, but also occurred in Southwest Asia and Europe. The third Asian haplotype was D4c1, which also occurred at low frequency in Central, North, and East Asia (Supplementary Table S11). It is noted that other lineages belonging to Western Eurasian type haplogroups could also be brought into the Carpathian Basin from Central/North Asia, for example, U4 or T types that were also frequent in ancient and modern Siberia17.
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study
| 100.0 |
We selected 23 modern populations from the GDM, MDS, and PCA datasets, which possibly had increased lineage-sharing with the conquerors and we compared them using a modern SHA (Supplementary Table S12). Populations speaking Uralic languages are not well studied for mtDNA, therefore we could only use Khantys, Mansis, Nenets, and Komis as references for Uralic peoples. The ancient conquest-period population had the highest lineage-sharing with the Tatars in Russian Tatarstan, and the Nenets and Komi groups (42–36%). They were followed by Hungarians, Russians in Bashkortostan, and three populations of almost identical percentages; Ukrainians, the Khanty and Mansi population, and Szeklers. When counting lineages, rather than the number of sequences, Csangos, Khantys and Mansis, and the population of the Russian Bashkortostan Republic were the third, fourth and fifth populations with the highest lineage-sharing (22.6–17%). Interestingly, the relatively low lineage sharing with Uzbeks and Turkmens did not reflect the high similarities visible on MDS and GDMs (Fig. 4, Supplementary Fig. S5).
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study
| 100.0 |
We typed the mtDNA of 111 medieval individuals and performed population genetic and statistical analyses, focusing on three populations that existed in the 7th–12th centuries in the Carpathian Basin. The earliest population under study is the 7th–8th century Avars from the southern part of modern Hungary (Fig. 1). The genetic results from the Avars demonstrate their predominant southern and eastern European maternal genetic composition, with some Asian elements. The local continuity of the Avar population on the southern Great Hungarian Plain to the Hungarian conquest-period cannot be rejected by haplogroup based simulation analyses (TPC, Supplementary Table S7) and was also demonstrated on PCA plots (Fig. 3a, Supplementary Fig. S1). However, sequence-based tests and shared haplotype analyses showed a low level of identical maternal lineage among the Avars and ancient Hungarians, even when including the geographically connecting southeastern group of the conquerors in the calculations (Fig. 5, Supplementary Table S10). The Avar dataset originates from a single micro regional group of the complex Avar society, who buried their dead in catacomb graves. Furthermore, anthropological results showed that this part of the Avar population represents mostly Europid, local morphological characters, and therefore it cannot be used as a proxy of the whole Avar population of the Carpathian Basin. Further regional groups should be analyzed from the late Avar period for a better estimation of the Avar-Hungarian continuity.
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study
| 100.0 |
The Hungarian conqueror genetic dataset from the 10th century showed more explicit connections toward Central Asian ancient and modern populations, in contrast to the preceding Avars. Asian haplogroups occurred among both male and female conquerors (Supplementary Tables S1 and S3), which can be an argument for a Hungarian settlement in which both men and women took part. It reflects the physical anthropological and archaeological data, which showed that, not only an armed population stratum, but a whole population arrived in the Carpathian Basin25. However, Asian lineages in the conqueror dataset can also be an argument for the continuity of the Avars, who could have mixed and acculturated during the Hungarian conquest-period26. We would need more Avar period genetic data, especially from the late Avar period to assess this hypothesis.
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study
| 99.9 |
In a previous study, Tömöry et al. presented mitochondrial genetic data of 26 Hungarian conquerors, who were divided into “commoners” (n = 15) and “high status” (n = 12) groups according to the excavated grave goods12. The latter group shows more heterogeneous haplogroup composition, and also some haplotypes that are rare in modern populations. We do not follow this concept in our current study, because grave goods cannot represent evidence of social status with a high level of certainty2627, and therefore levels of richness or status cannot be categorized precisely. Furthermore, people of low social status could also have been part of the conqueror community, who most probably arrived from the east of the Carpathian Basin as well. Chronological subdivision of the studied graves is also challenging, even 14C dating is not accurate enough for dating 9th–10th centuries AD.
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| 100.0 |
Most of the Asian mtDNA lineages occurred in 10th century cemeteries with small numbers of graves (7–18 graves), and identical lineages were found among cemeteries, rather than within them. This is especially interesting in light of the fact that seven analyzed cemeteries have been completely excavated (Kiskundorozsma, Balatonújlak, Harta, Makó-Igási járandó, Levice-Géňa, Szeged-Öthalom, Szentes-Derekegyháza graveyards). This phenomenon suggests that the conquerors had a mobile way of life or can be explained by the strong marriage connections of the Hungarian communities. The lack of, or small number of intra-cemetery maternal relations is striking at the sites Kiskundorozsma and Levice-Géňa (nine typed and maternally unrelated individuals in both cases), Szeged-Öthalom (eight unrelated people) and Harta. At the Harta site, fifteen women, three men and two children were excavated. We found only one pair of females with identical HVS-I sequences (a common rCRS H type), but in other cases the maternal kinship relation among the 16 typed individuals could be excluded by HVS-I analyses. Many academic archaeologists explain that the small conqueror graveyards were small family graveyards, and use the grave goods of the assumed generations in these graveyards as chronological horizons28. The example of Harta raises the possibility that family relations were not the sole rule of burial order. Mobile groups of people could use these cemeteries for a short period of time. These observations are relevant for the relative chronological and socio-archaeological assumptions about the 10th century Carpathian Basin. Nevertheless, other classic 10th-century graveyards, such as Balatonújlak, contained more signs of possible maternal relations within the cemetery (Supplementary Table S3). The unequal geographic distribution of the samples did not allow us to make further conclusions on the internal (geography or chronology based) genetic structure of the presented 10th-century population of the Carpathian Basin.
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study
| 100.0 |
We found genetic similarities of the conquerors with the Late Bronze Age population of the Baraba region, situated between the rivers Ob and Irtis17, and with Bronze Age and Iron Age populations that lived in Central Asia15 and south Siberia1416. Comparing the conqueror mtDNA dataset to a large modern-day population dataset, we also found comprehensive genetic affinities towards modern populations of Central Asia and Central Russia. The parallels of these Asian haplogroups are found in modern ethnic groups speaking both Ugric and Turkic languages. The historically and linguistically assumed homeland of the ancient Hungarians was in the Central Ural region, which is an easily accessible part of the mountain range. Finno-Ugric-speaking groups might have settled on both sides of the Urals during the early Medieval period29. Archeological records, for example, from central-eastern Uralic site Uelgi, indicate archaeological cultural mixture of northern Ugric and eastern steppic Turkic elements. These eastern components show cultural connections toward the region of the Emba River in today’s western Kazakhstan and toward the Srostki culture30, which indicates that the ancient Hungarian population could already have been reached in the Central Ural region by several cultural and genetic influences. Newly revised archaeological connections of the Central Urals and the Carpathian Basin suggest a quick migration from the forest steppe to the Carpathian Basin31, and during these events, the genetic make-up of the conquerors retained some Central Asian signatures.
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study
| 100.0 |
Modern-day Hungarians were very similar to their surrounding Central European populations from the maternal genetic point of view, as demonstrated by previous mtDNA studies1219. In our analyses, the Hungarian speaking Szekler, Ghimes, and Csango minorities in today’s Romania showed differing genetic connections from each other. Whereas the Szekler population was consistent with the Central and Eastern European maternal genetic diversity, the haplogroup and haplotype composition of the Csangos was more related to Near Eastern populations (Supplementary Fig. S4, Table S9). These results correspond to the fact that the Csangos, in the Romanian Ghimes region, are a genetically isolated population20, living separately from both Romanians and Szeklers.
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study
| 100.0 |
The maternal gene pool of Csangos, Szeklers and “average” Hungarians can be descended from 10th–12th century ancient Hungarians, and the differences in their haplogroup composition from the conquerors can be explained by genetic drift (Supplementary Table S7). It is an interesting phenomenon that some Asian haplogroups (A, B, C, G2a) that occured in the conquerors also occurred among Szeklers. This could suggest a sizeable legacy of the conquerors or it may mean that these Asian influences reached Romania in other time periods. Of the 76 detected conqueror haplotypes, 21 had matches in the modern Szekler and Hungarian populations (11.2–15.4% of all lineage types), but none were Asian (Supplementary Table S11). Fourteen conqueror lineages had matches in the Csango dataset, which represents a greater proportion (22.6%) of the total number of Csango lineage types, one of which belonged to the Asian C haplogroup. We would need more medieval samples from Romania and a reconsidered sampling of the current population in the Carpathian Basin in order to better estimate the genetic relations among past and present populations.
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study
| 100.0 |
The 10th century population of the Carpathian Basin had regionally different, but mostly heterogeneous physical anthropological and linguistic natures, which could be a consequence of the varied ethnic and linguistic composition of the conquerors. On the one hand, this parallels with the genetic diversity of the conquerors, and that the tribe alliance of the Hungarians was a culturally and linguistically mixed community in the steppe2. On the other hand, it could also be a consequence of the mixture of several populations, which had experienced the conquest-period in the Carpathian Basin and the geopolitical environment of the new homeland. The mixed nature of the newly founded Hungarian State was documented in the early 11th century, and described as a basic characteristic of a successful medieval state11. The samples from the 10th–12th century contact zone dataset from the fringes of the Hungarian territory originate from different geographic regions. They represent a mixed dataset within medieval Europe, which showed haplogroup-level connections to the conquerors and ancient Asia (Fig. 3B), but on the sequence level they had affinities with medieval Poles, Lombards, and Avars. Their subsisted maternal genetic signature was found today in Southern Europe and the Near East (Figs S2 and S7). Written sources document the diverse acculturation speed of local populations in the Carpathian Basin. For example, the population of the Čakajovce settlement slowly adopted items of Hungarian traditions to their culture32. This process could last 100–150 years, until burials with poor costume elements and jewels appeared, and Christian cemeteries became used. A new mixed culture began to form in the mid-10th century, which disseminated in the whole territory of the Hungarian Principality regardless of ethnicity.
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study
| 99.94 |
The results presented here provide a picture of the maternal gene pool of three medieval populations in the Carpathian Basin. Research should continue with the analysis of whole mitochondrial genomes for more exact haplogroup definitions, and Y chromosomal genetic diversity of these populations, in order to define the paternal genetic components of these populations, along with possible sex differences in migration and dispersal patterns. Furthermore, genome-wide sequencing of these samples and analyses of the comparatively ancient (early medieval) Eastern European, Central and North Asian data, which are currently still lacking, might reveal further signs of origin and admixture of the populations discussed here. Moreover, this may shed light on a complex population genetic structure of the first millennium BC of West, North, and Central Eurasia.
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study
| 99.94 |
This study contributes ancient mtDNA data to the research on Hungarian ethnogenesis and the conquest-period. We present the first described Avar-period ancient DNA dataset (n = 31), an almost four-fold enlargement of the existing Hungarian conquest-period dataset (with n = 76), and a magnified dataset from the Hungarian-Slavic contact zone of the 10–12th centuries (with n = 4). These together with the previously published 10th–12th century results were compared with published ancient and modern Eurasian mtDNA data. The results comprehensively demonstrate the conqueror maternal gene pool as a mixture of West Eurasian and Central/North Eurasian elements. Both the linguistically recorded Finno-Ugric roots and the Turkic, Central Asian influxes had possible genetic imprints in the conquerors’ mixed genetic composition. The small number of potential intra-site maternal relations compared to the number of detected inter-sites relations suggests that conqueror communities were mobile within the Carpathian Basin. Our data support the complex series of population genetic events before and during the formation of the 10th-century population of the Carpathian Basin. These processes might be defined by future ancient DNA studies focusing in the Ural region and in the Eastern European steppe using genome-wide sequencing techniques.
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| 100.0 |
The human skeletal remains (bones and teeth) used in this study were collected from 6th–10th century cemeteries excavated in the Carpathian Basin. The sampling was performed by co-workers of the Institute of Archaeology, considering various aspects: (1) geographical location; (2) chronology; (3) archaeological characteristics; (4) grave goods33.
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other
| 62.25 |
We investigated 144 medieval samples: from the Hungarian conquest-period 88 samples were analyzed from the cemeteries of Harta-Freifelt, Balatonújlak-Erdődűlő, Kiskundorozsma-Hosszúhát, Baks-Iskola, Szeged-Öthalom, Makó-Igási járandó, Szentes-Derekegyháza, Nyíregyháza, Kiszombor, Szentes-Borbásföld (all from Hungary), and Levice-Géňa (Slovakia). Furthermore, four Avar-period cemeteries were studied with 50 samples collected from Szegvár-Oromdűlő, Pitvaros-Víztározó, Székkutas-Kápolnadűlő, the 9th–10th century Vörs-Papkert (all from Hungary), and six samples from one site in the medieval Hungarian-Slavic contact zone, Zvonimirovo (located in present-day northern Croatia). Nine samples from these sites were already part of G. Tömöry’s PhD dissertation34 (Fig. 1, Supplementary Table S1). It is important to note that the graves of the conquest-period population are mainly dated to the 10th century. They were probably not the first generation of conquerors, which is very problematic to distinguish at the current state of research. One further point to note is that the Avar samples belong to a single micro region of the Avar Khaganate, and therefore do not represent the whole Avar population of the Carpathian Basin.
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| 100.0 |
Sampling was carried out using gloves, facemasks, and body suits, in order to minimize the risk of contamination by contributors. Two bone fragments, usually two compact bone tissues from different parts of long bones, or one tooth and one compact bone fragment of a femur were collected from each individual. All stages of work were performed under clean conditions in a dedicated ancient DNA laboratory at the Institute of Archaeology, Research Centre for the Humanities, Hungarian Academy of Sciences, Budapest, following published ancient DNA workflow protocols and authentication criteria121335. Laboratory rooms for pre-PCR and post-PCR works were strictly separated. All pre-PCR steps (bone cutting, surface removing, powdering, extraction, PCR set-up) were carried out in separate clean rooms. The laboratory work was carried out wearing clean overalls, facemasks and face-shields, gloves and over-shoes. All materials and work areas were bleached and irradiated with UV-C light. We used PCR-clean plastic wares and Milli-Q ultrapure water for reaction preparation. In order to detect possible contamination by exogenous DNA, one milling blank per sample, one extraction and amplification blanks per every five samples were used as negative controls. MtDNA haplotypes of all contributors (anthropologists, geneticists) in the sampling and laboratory work were determined in the post-PCR lab, and compared with the results obtained from the ancient bone samples. Only one haplotype match was found between an ancient sample (PitV124.436B) and an anthropologist, who had no contact with this specific sample (Supplementary Table S16).
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study
| 100.0 |
The specimens were prepared following the protocols described by Kalmár et al.36 and Szécsényi-Nagy et al.37. The bone and teeth samples were bleached, washed, and irradiated with UV-C light (1.0 J/cm2, 25 min). The surfaces of teeth samples were cleaned by sandblasting (Bego, EasyBlast), while the surfaces of bone samples were removed with a fresh drilling bit at slow speed, followed by UV exposure for 30 min on each side. Bone and tooth pieces were mechanically ground into fine powder in a sterile mixer mill (Retsch MM301).
|
study
| 99.94 |
Different DNA extraction methods were used, repeatedly validating the results per sample123638. MtDNA hypervariable segment I (HVS- I) and coding region positions were amplified in several PCRs in a total volume of 40 μl reaction mix, containing 5 μl DNA extract, 1× AmpliTaq Gold-Buffer; 2 U AmpliTaq Gold DNA polymerase (Applied Biosystems); 200 μM of each of the dNTP ; 25 pmolμl−1 primer; 1.5 mM MgCl2; 4mgml−1 BSA. The HVS-I region of mtDNA was amplified in two overlapping fragments with two sets of primers, and an additional 16 primer pairs were used to amplify haplogroup-diagnostic nucleotide positions in coding regions (see Supplementary Table S2). Cycling parameters were 98 °C for 10 min; followed by 39 cycles of denaturation at 98 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 40 s; and a final step of 72 °C for 5 min. PCR products were checked on 8% native polyacrylamide gel. The PCR products were purified using QIAquick® PCR Purification Kit (Qiagen) following the manufacturer’s protocol, or purified from 2% agarose gel with Bioline Isolate PCR & Gel Kit in a final volume of 15 μl. Sequencing reactions were performed using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and sequencing products were purified by ethanol precipitation. The sequences were determined on ABI PRISM 3100 (PE Applied Biosystems) in cooperation with BIOMI Ltd (Gödöllő, Hungary). The sequences were evaluated with Chromas Lite 2.4.1 and GeneDoc software39.
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study
| 100.0 |
The sequence polymorphisms in the nucleotide position range 16040–16400 were compared with the rCRS40 as well as the Reconstructed Sapiens Reference Sequence (RSRS, www.mtdnacommunity.org)41. Sequences were submitted to GenBank under the accession numbers KU739156–KU739266. Haplogroup determination was carried out according to the mtDNA phylogeny of PhyloTree build 17, accessed 18 February 201642, and these haplogroup definitions were checked in our mtDNA database of 78,000 samples (enlarged database of that reported in ref. 24), and in EMPOP (http://empop.online/).
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study
| 100.0 |
We could not determine the haplogroup classification of one sample (HAR1.56B), due to detection failure of U haplogroup-diagnostic at coding region position 12308. Therefore we included it only into shared haplotype analyses of HVS-I sequences, and excluded it from other statistical analyses.
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study
| 100.0 |
Of the typed 111 mtDNA profiles, we excluded the site Vörs-Papkert from the population genetic analyses, because it represents a 9th–10th-century late Avar-Slavic mixed population in Transdanubia (western-Hungary). On the other hand, we included 26 samples from medieval Hungary described by Tömöry et al.12, and 19 samples from medieval Slovakia13 into the population genetic analyses because of their similar historical, chronological and geographical traits to the new sample sets. We created three groups from a total of 150 analyzed Carpathian Basin samples for population genetics analyses: (1) conquest-period dataset (75 new samples and 26 samples described by Tömöry et al., 2007); (2) Avars in the southeastern part of today’s Hungary (26 samples) (3) “contact zone” (23 samples of the conquest-period derived from the outskirts of medieval Hungary: 4 new samples from today’s Croatia and 19 samples from the cemeteries of Nitra-Šindolka and Čakajovce (today’s Slovakia) described by Csákyová et al.13 (Supplementary Table S4).
|
study
| 100.0 |
The ancient datasets were compared with 57,098 published modern HVS-I sequences as well as 614 medieval sequences of European, Near Eastern and Asian populations: Lombards from Hungary and Italy, medieval population from northern Italy, medieval Basques from Spain, medieval populations from Poland, Iceland and Denmark, Vikings from Norway and Denmark, three ancient populations (3rd century BC–14th century AD) from Mongolia and Inner Mongolia (China), and late medieval Yakuts from Russia. In addition, in order to have a proxy for the genetically uncharacterized first millennium AD populations of Central and North Asia, we used prehistoric (Bronze Age and Iron Age) datasets from modern-day Russia, Kazakhstan and Mongolia. Their characteristics, abbreviations and references are described in Supplementary Tables S5, S6 and S15.
|
study
| 100.0 |
Standard statistical methods were used for comparisons and calculations of genetic distances between our investigated populations (conquerors, Avars, and contact zone) and a further 18 ancient and 53–157 modern populations. Diversity indices were calculated in DNASP v543 using sequence range np 16040–16400.
|
study
| 99.94 |
PCAs were carried out based on mtDNA haplogroup frequencies. We considered 31 mtDNA haplogroups in PCA of 21 ancient populations, while in PCA with the 3 medieval populations and 53 modern-day populations, 36 mtDNA haplogroups were considered (Supplementary Tables S5 and S6). All PCAs were performed using the prcomp function for categorical PCA, implemented in R 3.1.3 (R Foundation of Statistical Computing, 2015) and plotted in a two-dimensional space, displaying the first two or the first and third principal components, respectively (Fig. 3A, Supplementary Figs S1–S3).
|
study
| 100.0 |
Hierarchical clustering was performed using Ward type algorithm44 and Euclidean measurement method, where frequencies of the PCA haplogroups were used. The result was visualized as a dendrogram with the pvclust library in R.2.13.145 (Fig. 3b). Cluster significance was evaluated by 10,000 bootstrap replicates. Significance of each cluster was given as an AU (Approximately Unbiased) p-value, in percentage. Fisher tests based on absolute haplogroup frequencies used in ancient PCA (except that U1 and Y, Z remained separated) were performed using sqldf library and fisher.test function in R.3.1.3.
|
study
| 100.0 |
Population comparisons were estimated using Arlequin 3.5.146. Pairwise FST values were calculated based on 35,203 modern and 764 ancient HVS-I sequences (np 16050–16383) of 73 populations: 21 ancient and 52 modern-day populations from Eurasia. Tamura & Nei substitution model47 was assumed with a gamma value of 0.325 and 10,000 permutations were used for p-value calculation (Supplementary Tables S8 and 9). The FST values were analyzed using MDS and applied on the matrix of linearized Slatkin FST values48 (Supplementary Tables S8 and 9) and visualized in a two-dimensional space (Fig. 6) using the metaMDS function based on Euclidean distances implemented in the vegan library of R 3.1.345.
|
study
| 100.0 |
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