Molecular authentication of commercial "Qian-hu" through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature.

BACKGROUND: Peucedani Radix, also known as "Qian-hu" is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging. MATERIALS AND METHODS: We initially examined and compared 104 commercial "Qian-hu" samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial "Qian-hu". The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences. RESULTS: The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic "Qian-hu", while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5'-ATTGTCGTACGAATCCTCGTCGTC-3') revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation. CONCLUSIONS: We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

Plastome evolution in the genus Sium (Apiaceae, Oenantheae) inferred from phylogenomic and comparative analyses.

BACKGROUND: Sium L. (Apiaceae) is a small genus distributed primarily in Eurasia, with one species also occurring in North America. Recently, its circumscription has been revised to include 10 species, however, the phylogenetic relationships within its two inclusive clades were poorly supported or collapsed in previous studies based on nuclear ribosomal DNA ITS or cpDNA sequences. To identify molecular markers suitable for future intraspecific phylogeographic and population genetic studies, and to evaluate the efficacy of plastome in resolving the phylogenetic relationships of the genus, the complete chloroplast (cp) genomes of six Sium species were sequenced. RESULTS: The Sium plastomes exhibited typical quadripartite structures of Apiaceae and most other higher plant plastid DNAs, and were relatively conserved in their size (153,029-155,006 bp), gene arrangement and content (with 114 unique genes). A total of 61-67 SSRs, along with 12 highly divergent regions (trnQ, trnG-atpA, trnE-trnT, rps4-trnT, accD-psbI, rpl16, ycf1-ndhF, ndhF-rpl32, rpl32-trnL, ndhE-ndhG, ycf1a and ycf1b) were discovered in the plastomes. No significant IR length variation was detected showing that plastome evolution was conserved within this genus. Phylogenomic analysis based on whole chloroplast genome sequences produced a highly resolved phylogenetic tree, in which the monophyly of Sium, as well as the sister relationship of its two inclusive clades were strongly supported. CONCLUSIONS: The plastome sequences could greatly improve phylogenetic resolution, and will provide genomic resources and potential markers useful for future studies of the genus.

KIF18B promotes hepatocellular carcinoma progression through activating Wnt/ß-catenin-signaling pathway.

This study aimed to investigate the functional roles of kinesin family member 18B (KIF18B) in hepatocellular carcinoma (HCC) development, as well as the related molecular mechanisms. Tissue specimens were collected from 105 patients with HCC, and the messenger RNA (mRNA) and protein levels of KIF18B were detected using quantitative real-time polymerase chain reaction and immunohistochemistry assays, respectively. The χ2 test was performed to estimate the association of KIF18B with clinical characteristics of patients with HCC. Effects of KIF18B expression on biological behaviors of HCC cells were detected by clone formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and transwell assays. The expression patterns of proteins were investigated using Western blot analysis. HCC tissues and cell lines showed significant upregulation of KIF18B at both mRNA and protein levels (p > .05, for all). Furthermore, the elevated KIF18B expression was positively correlated with the tumor-node-metastasis stage (p = .015) and lymph node metastasis (p = .007). Knockdown of KIF18B might suppress HCC cell clone formation, proliferation, migration, and invasion in vitro. Besides, the activity of Wnt/ß-catenin pathway was also significantly inhibited after the KIF18B knockdown. However, the antitumor actions caused by KIF18B knockdown might be reversed by lithium chloride treatment, which was the inducer of Wnt/ß-catenin-signaling pathway. KIF18B may serve as an oncogene in HCC through enhancing the activity of Wnt/ß-catenin pathway.

Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway.

Innate lymphoid cells (ILCs) are severely depleted during chronic HIV-1 infection by unclear mechanisms. We report here that human ILC1s comprising of CD4+ and CD4- subpopulations were present in various human lymphoid organs but with different transcription programs and functions. Importantly, CD4+ ILC1s expressed HIV-1 co-receptors and were productively infected by HIV-1 in vitro and in vivo. Furthermore, chronic HIV-1 infection activated and depleted both CD4+ and CD4- ILC1s, and impaired their cytokine production activity. Highly active antiretroviral (HAART) therapy in HIV-1 patients efficiently rescued the ILC1 numbers and reduced their activation, but failed to restore their functionality. We also found that blocking type-I interferon (IFN-I) signaling during HIV-1 infection in vivo in humanized mice prevented HIV-1 induced depletion or apoptosis of ILC1 cells. Therefore, we have identified the CD4+ ILC1 cells as a new target population for HIV-1 infection, and revealed that IFN-I contributes to the depletion of ILC1s during HIV-1 infection.

Relationships between Tertiary relict and circumboreal woodland floras: a case study in Chimaphila (Ericaceae).

BACKGROUND AND AIMS: Tertiary relict and Arctic/circumboreal distributions are two major patterns of Northern Hemisphere intercontinental disjunctions with very different histories. Each has been well researched, but members of one biome have generally not been incorporated in the biogeographical analyses of the other, and links or transitions between these two biomes have rarely been addressed. METHODS: Phylogenies of Chimaphila were generated based on cpDNA and nuclear ITS, using Bayesian and maximum likelihood methods. A time-calibrated phylogeny was generated using BEAST. Ancestral area reconstruction was inferred using both statistical dispersal-vicariance analysis and a dispersal-extinction-cladogenesis model. KEY RESULTS: The Chimaphila crown group was estimated to have originated in the early Miocene. The lineages of C. umbellata diverged early, but its present circumboreal distribution was not achieved until around the middle Pliocene or later. Sister to this is a clade of four species with Tertiary relict distribution. Among these, two expansions occurred from North America to Asia, probably via the Bering Land Bridge, generating its current disjunctions. CONCLUSIONS: Our data concur with a few other studies, indicating that the circumboreal woodland biome has an older origin than most true Arctic-alpine taxa, having gradually recruited taxa since the early Oligocene. For the origin of Asia-North America disjunctions in Chimaphila, an 'out-of-America' migration was supported. It is not clear in which direction Pyroloideae lineages moved between Tertiary relict disjunctions and Arctic/circumboreal distributions; each biome might have recruited species from the other.

[Application of DNA metabarcoding technology in identification of Chinese patent medicines].

Metabarcoding technology is a research method derived from the combination of traditional DNA barcodes and highthroughput sequencing technologies. It can quickly,easily and efficiently identify and restore biological samples from multiple species.Biological species are currently widely used in environmental biology research. In the market of traditional Chinese medicines,adulteration and quality instability have severely restricted the sustainable development of the related industries. This article introduced the background of the metabarcoding technology and its preliminary application in the identification of Chinese patent medicines. It also outlined the possible problems in the research process and prospected to the development of the DNA metabarcoding technology.

[Collection and sorting of medicinal plants in Chinese Apiaceae( Umbelliferae)].

The family Apiaceae( Umbelliferae) includes some of the world's most important medicinal plants,with more than 100 species recorded in the traditional Chinese medicine,of which more than ten species are commonly used medicinal materials. However,due to morphological similarities,high market demands and regional factors,substitutes and adulterants are often mixed with genuine in the medicinal market. Therefore,a comprehensive sorting for these poorly known plants has been done in this study by combining market survey with literature review,including its species,distribution,price and substitutes. According to the statistics,there are 65 genera and 262 species of medicinal plants of Apiaceae in China,with medicinal part mostly from radix and rhizoma. Sichuan province is the most abundant in distribution and planting resources,with about 137 species,followed by Yunnan,Hubei and Gansu provinces.Furthermore,we summarized the genuine and substitutes of 11 medicinal plants,e. g. Bupleurum,Angelica and Peucedanum etc.,which found that the medicinal plants of Apiaceae were substituted or mixed in different taxonomic ranks. This study would contribute to reduce the risk of medicine misuse,as well as explore other plants of Apiaceae with potential medicinal value,to achieve sustainable development of related industries.

[The clinical analysis of orthotopic liver transplantation for primary biliary cirrhosis].

OBJECTIVE: To analyze the clinical outcome and complications of orthotopic liver transplantation (OLT) for patients with primary biliary cirrhosis(PBC). METHODS: Clinical data of 25 patients with PBC who underwent OLT were analyzed retrospectively. The postoperative cumulative survival rate of the patients was calculated. The postoperative recurrence of PBC, de novo diseases and other complications were analyzed. RESULTS: A total of 25 patients were recruited including 22 females and 3 males. The average age was 49.1 years (range from 40 to 64 years). The score of model for end-stage liver disease (MELD) was 21.80±5.49, and the Mayo score 8.01±1.38. Four patients died after liver transplantation. The 1-year, 2-year and 7-year patient cumulative survival rates were 92.0%, 87.8% and 75.3%, respectively. Eight cases (32.0%) developed recurrent PBC based on histological evidence. The median time to recurrence was 17.5 months. One of the eight patients with recurrent PBC was diagnosed with de novo auto-immune hepatitis (AIH) simultaneously, who eventually died. Acute rejection and de novo hepatitis B virus (HBV) infection developed in thirteen (52.0%) patients and five (20.0%) patients, respectively. Two cases (8.0%) of de novo AIH were diagnosed, one of which was fatal. CONCLUSIONS: OLT is an effective procedure for end-stage PBC. Much attention needs to be paid to the post-transplantation complications, including recurrence of primary disease, combined de novo HBV infection, de novo AIH, etc.

[Clinical analysis of liver transplantation for autoimmune hepatitis].

OBJECTIVE: To describe the outcomes and recurrence of autoimmune hepatitis (AIH) after liver transplantation. METHODS: Clinical data of 16 patients with AIH who underwent liver transplantation were analyzed retrospectively. The postoperative cumulative survival rate of the patients was calculated. The postoperative rejections and AIH recurrence were analyzed. The Kaplan-Meier method was used for statistical analysis of survival. RESULTS: All patients were female, with an average age of 52.6 years (range: 41-66 years), and an average MELD score of 21.4. According serological analysis, 15 patients were AIH type 1 and 1 patient was AIH type 2. Three patients died, including 2 of pulmonary infection and 1 of graft dysfunction.The 1-, 2-and 5-year cumulative survival rates were 93.8%, 87.1% and 79.1%, respectively. Five cases (31.3%) of recurrent AIH were diagnosed based on histological evidence. Acute rejection occurred in 6 (37.5%) patients, and de novo HBV infection occurred in 1 (6.3%) patient. CONCLUSION: Liver transplantation is an effective treatment for end-stage AIH. Recurrence and rejection were commonly associated with AIH, but did not negatively impact patient survival.

Impairment of CD4+ cytotoxic T cells predicts poor survival and high recurrence rates in patients with hepatocellular carcinoma.

UNLABELLED: The role of CD4(+) cytotoxic T cells (CTLs) in hepatocellular carcinoma (HCC) remains obscure. This study characterized CD4(+) CTLs in HCC patients and further elucidated the associations between CD4(+) CTLs and HCC disease progression. In all, 547 HCC patients, 44 chronic hepatitis B (CHB) patients, 86 liver cirrhosis (LC) patients, and 88 healthy individuals were enrolled in the study. CD4(+) CTLs were defined by flow cytometry, immunohistochemistry, and lytic granule exocytosis assays. A multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Circulating and liver-infiltrating CD4(+) CTLs were found to be significantly increased in HCC patients during early stage disease, but decreased in progressive stages of HCC. This loss of CD4(+) CTLs was significantly correlated with high mortality rates and reduced survival time of HCC patients. In addition, the proliferation, degranulation, and production of granzyme A, granzyme B, and perforin of CD4(+) CTLs were inhibited by the increased forkhead/winged helix transcription factor (FoxP3(+) ) regulatory T cells in these HCC patients. Further analysis showed that both circulating and tumor-infiltrating CD4(+) CTLs were independent predictors of disease-free survival and overall survival after the resection of the HCC. CONCLUSION: The progressive deficit in CD4(+) CTLs induced by increased FoxP3(+) regulatory T cells was correlated with poor survival and high recurrence rates in HCC patients. These data suggest that CD4(+) CTLs may represent both a potential prognostic marker and a therapeutic target for the treatment of HCC.

Multiple origins of circumboreal taxa in Pyrola (Ericaceae), a group with a Tertiary relict distribution.

BACKGROUND AND AIMS: In the Northern Hemisphere, Tertiary relict disjunctions involve older groups of warm affinity and wide disjunctions, whereas circumboreal distributions in Arctic-Alpine taxa tend to be younger. Arctic-Alpine species are occasionally derived from Tertiary relict groups, but Pyrola species, in particular, are exceptional and they might have occurred multiple times. The aim of this study was to reconstruct the biogeographic history of Pyrola based on a clear phylogenetic analysis and to explore how the genus attained its circumboreal distribution. METHODS: Estimates of divergence times and ancestral geographical distributions based on neutrally evolving DNA sequence variation were used to develop a spatio-temporal model of colonization patterns for Pyrola. KEY RESULTS: Pyrola originated and most diversification occurred in Asia; North America was reached first by series Scotophyllae in the late Miocene, then by sub-clades of series Pyrola and Ellipticae around the Pliocene. The three circumboreal taxa, P. minor, P. chlorantha and the P. rotundifolia complex, originated independently of one another, with the last two originating in Asia. CONCLUSIONS: Three circumboreal Pyrola lineages have arisen independently and at least two of these appear to have originated in Asia. The cool, high-altitude habitats of many Pyrola species and the fact that diversification in the genus coincided with global cooling from the late Miocene onwards fits a hypothesis of pre-adaptation to become circumboreal within this group.

Clinical-pathological analysis of hepatitis B virus recurrence after liver transplantation in Chinese patients.

BACKGROUND AND AIM: Liver transplantation (LT) for hepatitis B virus (HBV)-related disease can be complicated by HBV recurrence. The aim of this study was to evaluate the risk factors, prophylaxis treatment, and histological characteristics of HBV recurrence after LT when using long-term, low-dose hepatitis B immunoglobulin (HBIG) plus nucleoside analog (lamivudine [LAM] or entecavir [ETV]). METHODS: Retrospective data from 253 adult LT patients using long-term, low-dose HBIG plus nucleoside analog after LT, for a mean treatment duration of 1-72 months, were collected from a single center in Beijing, China. Univariate analyses were conducted to determine the association among gender, age, hepatocellular carcinoma, hepatitis B e antigen-positive status, HBV-DNA level and tyrosine-methionine-aspartate-aspartate (YMDD) mutations on HBV recurrence in these patients. RESULTS: Overall, the HBV recurrence rate was 6.32% (16/253). There was no significant difference in the survival rate between the HBV recurrence and non-recurrence groups. Risk factors for HBV recurrence were: hepatitis B e antigen positivity, HBV-DNA > 10(5) copies/mL, hepatocellular carcinoma, and YMDD mutation. Sixteen patients receiving LAM had HBV recurrence (16/169; mean treatment duration: 61.8 ± 18.3 months). No HBV recurrence occurred in patients receiving ETV after LT (0/84; mean treatment duration: 57.1 ± 15.9 months). Differences in rate of mortality and HBV recurrence were not significant between the two groups. CONCLUSIONS: LT is an effective treatment for HBV-related end-stage liver disease. The combination of ETV and intramuscular HBIG for HBV recurrence prophylaxis after LT was more effective than LAM, especially in Chinese patients with HBV recurrence risk factors.

DNA barcoding of Notopterygii Rhizoma et Radix (Qiang-huo) and identification of adulteration in its medicinal services.

Safety concerns, stemming from the presence of complex and unpredictable adulterants, permeate the entire industrial chain of traditional Chinese medicines (TCMs). The Notopterygii Rhizoma et Radix (NReR) from the Apiaceae family, commonly known as "Qiang-huo", is a widely used herbal medicine. The recent surge in its demand has given rise to a proliferation of counterfeit and substituted products in the market. Traditional identification presents inherent limitations, while DNA mini-barcoding, reliant on sequencing a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In this study, we constructed a comprehensive Internal Transcribed Spacer 2 (ITS2) matrix encompassing genuine NReR and their commonly found adulterants for the first time. Leveraging this matrix, we conducted a thorough assessment of the genetic profiles and sources of NReR available in the Chinese herbal medicine market. Following established DNA barcoding protocols, the intra-specific genetic divergences within NReR species were found to be lower than the inter-specific genetic divergences from other species. Among the 120 samples that were successfully amplified, ITS2 exhibits an outstanding species-level identification efficiency of 100% when evaluated using both the BLASTN and neighbor-joining (NJ) tree methods. We concluded that ITS2 is a mini-barcode that has shown its potential and may become a universal mini-barcode for the quality control of "Qiang-huo", thereby ensuring the safety of clinical medication.

Circumscription of the East Asia clade (Apiaceae subfamily Apioideae) and the taxonomic placements of several problematic genera.

The East Asia (or Physospermopsis) clade was recognized in previous molecular phylogenetic investigations into the higher-level relationships of Apiaceae subfamily Apioideae. The composition of this clade, the phylogenetic relationships among its constituent taxa, and the placement of species previously determined to be problematic have yet to be resolved. Herein, nrDNA ITS sequences were obtained for 150 accessions of Apioideae, representing species whose distributions are in East Asia or genera having one or more species included within the East Asia clade. These data, along with published ITS sequences from other Apioideae (for 3678 accessions altogether), were subjected to maximum likelihood and Bayesian inference analyses. The results show that the East Asia clade contains representatives of 11 currently recognized genera: Hansenia, Hymenolaena, Keraymonia, Sinolimprichtia, Acronema, Hymenidium, Physospermopsis, Pimpinella, Sinocarum, Tongoloa, and Trachydium. However, the latter seven genera have members falling outside of the East Asia clade, including the generic types of all except Tongoloa. Within the clade, the species comprising these seven genera are widely intermingled, greatly increasing confusion among relationships than previously realized. The problematic species Physospermopsis cuneata is confirmed as falling within the East Asia clade, whereas P. rubrinervis allies with the generic type in tribe Pleurospermeae. Physospermopsis kingdon-wardii is confirmed as a member of the genus Physospermopsis, whereas the generic attributions of P. cuneata and Tongoloa stewardii remain unclear. Two species of Sinocarum (S. filicinum and S. wolffianum) are transferred into the genus Meeboldia. This is the most comprehensive molecular phylogenetic investigation of the East Asia clade to date, and while the results increase systematic understanding of the clade, they also highlight the need for further studies of one of the most taxonomically intractable groups in Apioideae.

Comparative genomic analysis of Sanghuangporus sanghuang with other Hymenochaetaceae species.

Sanghuangporus sanghuang is a medicinal macrofungus with antioxidant and antitumor activities, and it is enriched with secondary metabolites such as polysaccharides, terpenes, polyphenols, and styrylpyrone compounds. To explore the putative core genes and gene clusters involved in sanghuang biosynthesis, we sequenced and assembled a 40.5-Mb genome of S. sanghuang (SH1 strain). Using antiSMASH, local BLAST, and NCBI comparison, 12 terpene synthases (TPSs), 1 non-ribosomal peptide synthase, and five polyketide synthases (PKSs) were identified in SH1. Combining the transcriptome analysis with liquid chromatography mass spectrometry-ion trap-time of flight analysis, we determined that ShPKS1, one phenylalanine aminolyase (ShPAL), and one P450 monooxygenase (ShC4H1) were associated with hispidin biosynthesis. Structural domain comparison indicated that ShPKS2 and ShPKS3 are involved in the biosynthesis of orsellinic acid and 2-hydroxy-6-methylbenzoic acid, respectively. Furthermore, comparative genomic analysis of SH1 with 14 other fungi from the Hymenochaetaceae family showed variation in the number of TPSs among different genomes, with Coniferiporia weirii exhibiting only 9 TPSs and Inonotus obliquus having 20. The number of TPSs also differed among the genomes of three strains of S. sanghuang, namely Kangneng (16), MS2 (9), and SH1 (12). The type and number of PKSs also varied among species and even strains, ranging from two PKSs in Pyrrhoderma noxium to five PKSs in S. sanghuang SH1. Among the three strains of S. sanghuang, both the structural domains and the number of PKSs in strains MS2 and SH1 were consistent, whereas strain Kangneng exhibited only four PKSs and lacked the PKS with the structural domain KS-AT-DH-KR-ACP. Additionally, Sanghuangporus species exhibited more similar PKSs to Inonotus, with higher gene similarity around five PKSs, while showing differences from those of other fungi in the same family, including Phellinus lamaoensis. This result supports the independent taxonomic significance of the genus Sanghuangporus to some extent.

[Clinical manifestation and autoantibody profile in 123 patients with primary biliary cirrhosis].

OBJECTIVE: To investigate the autoantibody profile and its clinical implication in the patients with primary biliary cirrhosis. METHODS: During the period of 2008 to 2010,123 patients with primary biliary cirrhosis (PBC) in our hospital were enrolled in this study, of whom, 70 patients were with cirrhosis and 53 without cirrhosis, The autoantibody profile was tested for each patient by using immunoblotting and indirect immunofluorescence. RESULTS: Of the 123 PBC patients with liver cirrhosis, 49% were positive with serum ANA positive; 47%, 51%, 54%, 31% and 49% were positive with serum anti-nuclear antibodies (ANA), anti-mitochondrial antibodies-M2 (AMA-M2), anti-promyelocytic leukemia (anti-PML), anti-sp100 antibodies (anti-sp100), anti-Ro-52 antibody (anti-52KD), respectively. By contrast, of the PBC patients without liver cirrhosis, only 38%, 37%, 51%, 60%, 30% and 51% were positive with serum ANA, AMA, AMA-M2, anti-PML, anti-sp100 and anti-52KD, respectively.There was the statistical difference between the two groups. In addition, it was also found that the anti-gp210 antibody positive group had a higher Mayo risk score,lower serum albumin and severe cholestasis and impaired liver function when compared with anti-gp210 antibody negative patients. CONCLUSION: Our data indicate that serum AMA is helpful for early diagnosis of PBC, and in particular, serum ANA positivity can help make a diagnosis for the AMA-negative patients. These indicate that anti-gp210 antibodies appear in the late course of PBC.Anti-gp210 positive PBC patients have more severe cholestasis and liver dysfunction.

The complete chloroplast genome of Ligusticopsis acaulis (Shan et Sheh) Pimenov (Apiaceae), an endemic species from China.

Ligusticopsis acaulis, belonging to the family Apiaceae (Umbelliferae), is endemic to China. The complete chloroplast genome sequence of L. acaulis was assembled and annotated for the first time in this study. The results showed that the plastome was 148,509 bp in length and consisted of a pair of inverted repeat regions (IRs: 19,468 bp), a large single-copy region (LSC: 91,902 bp), and a small single-copy region (SSC: 17,671 bp). A total of 114 unique genes were annotated, including 80 protein-coding, 30 tRNA, and four rRNA genes. According to the phylogenetic analysis, L. acaulis belongs to the tribe Selineae, with a close relationship to Ligusticum hispidum (Franch.) Wolff.

TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs.

The pregnane X receptor (PXR) is an important regulator of hepatocellular carcinoma cellular resistance to antitumor drugs. Activation of PXR was modulated by the co-regulators. The target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2) known as TPX2 that was previously considered as a tubulin regulator, also functions as the regulator of some transcription factors and pro-oncogenes in human malignances. However, the actions of TPX2 on PXR and HCC cells are still unclear. In the present study, our results demonstrate that the high expression of endogenous mRNA level of TPX2 not only correlated with the poor prognosis of advanced HCC patients who received sorafenib treatment but also with expression of PXR's downstream genes, cyp3a4 and/or mdr-1. Results from luciferase and real-time polymerase chain reaction (qPCR) showed that TPX2 leads to enhancement of the transcription factor activation of PXR. Protein-protein interactions between PXR and TPX2 were identified using co-immunoprecipitation. Mechanically, overexpression of TPX2 led to enhancement of PXR recruitment to its downstream gene cyp3a4's promoter region (the PXRE region) or enhancer region (the XREM region). Treatment of HCC cells with paclitaxel, a microtubule promoter, led to enhancement of the effects of TPX2, whereas vincristine, a microtubule depolymerizing agent caused a decrease in TPX2-associated effects. TPX2 was found to cause acceleration of the metabolism or clearance of sorafenib, a typical tyrosine kinase inhibitor (TKI) in HCC cells and in turn led to the resistance to sorafenib by HCC cells. By establishing novel actions of TXP2 on PXR in HCC cells, the results indicate that TPX2 could be considered a promising therapeutic target to enhance HCC cells sensitivity to antitumor drugs.

Behavioral responses of two captive bottlenose dolphins (Tursiops truncatus) to a continuous 50 kHz tone.

At present, the fundamental frequencies of signals of most commercially available acoustic alarms to deter small cetaceans are below 20 kHz, but it is not well ascertained whether higher frequencies have a deterrent effect on bottlenose dolphins (Tursiops truncatus). Two captive bottlenose dolphins housed in a floating pen were subjected to a continuous pure tone at 50 kHz with a source level of 160 ± 2 dB (re 1 µPa, rms). The behavioral responses of dolphins were judged by comparing surfacing distance relative to the sound source, number of surfacings, and number of echolocation clicks produced, during forty 15 min baseline periods with forty 15 min test periods (four sessions per day, 40 sessions in total). On all 10 study days, surfacing distance and the number of surfacings increased while click production decreased during broadcasts of test sound. The avoidance threshold sound pressure level for a continuous 50 kHz tone for the bottlenose dolphins, in the context of this study, was estimated to be 144 ± 2 dB (re 1 µPa, rms). The results indicated that a continuous 50 kHz tonal signal can deter bottlenose dolphins from an area.

Development of 16 microsatellite markers for Prince's Pine, Chimaphila japonica (Pyroleae, Monotropoideae, Ericaceae).

The perennial evergreen herb, Chimaphila japonica is found exclusively in East Asian temperate coniferous or sometimes in deciduous forests. By using the Fast Isolation by Amplified Fragment Length Polymorphism (AFLP) of Sequences Containing repeats (FIASCO) protocol, 20 microsatellite primer sets were identified in two wild populations. Of these primers, 16 displayed polymorphisms and 4 were monomorphic. The number of alleles per locus ranged from one to six among populations, values for expected and observed heterozygosities ranged from 0.000 to 0.848 and from 0.000 to 1.000, respectively. The new SSR markers will be useful in obtaining estimates of population-level genetic diversity and in phylogeographic studies of C. japonica.