Cyclin D1 Induction by Benzo[a]pyrene-7,8-diol-9,10-epoxide via the Phosphatidylinositol 3-Kinase/Akt/MAPK- and p70s6k-dependent Pathway Promotes Cell Transformation and Tumorigenesis

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), the major metabolite of B[a]P, has been well recognized as one ubiquitous carcinogen, but the molecular mechanism involved in its carcinogenic effect remains obscure. In the present study, we found that bronchial epithelial cells (Beas-2B) and hepatocytes treated with B[a]PDE presented a significant increase of cyclin D1 expression. Moreover, Akt, p70^s6k, and MAPKs including JNK, Erks, and p38 were notably activated in B[a]PDE-treated Beas-2B cells, whereas NF-κB, NFAT, and Egr-1 were not. Our results demonstrated that JNK and Erks were required in B[a]PDE-induced cyclin D1 expression because the inhibition of JNK or Erks by a selective chemical inhibitor or dominant negative mutant robustly impaired the cyclin D1 induction by B[a]PDE. Furthermore, we found that overexpression of the dominant negative mutant of p85 (regulatory subunit of phosphatidylinositol 3-kinase) or Akt dramatically suppressed B[a]PDE-induced JNK and Erk activation as well as cyclin D1 expression, suggesting that cyclin D1 induction by B[a]PDE is via the phosphatidylinositol 3-kinase/Akt/MAPK-dependent pathway. In addition, we clarified that p70^s6k is also involved in B[a]PDE-induced cyclin D1 expression because rampamycin pretreatment dramatically reduced cyclin D1 induction by B[a]PDE. More importantly, we demonstrated that up-regulated cyclin D1 by B[a]PDE plays a critical role in oncogenic transformation and tumorigenesis of Beas-2B cells. These results not only broaden our knowledge of the molecular mechanism of B[a]PDE carcinogenicity but also lead to the further study of chemoprevention of B[a]PDE-associated human cancers.     

一种实用可靠的古代 DNA 获取方法

古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题．改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列．实验利用距今4 300～3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下．这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法．     

Modifications of nonwoven polyethylene terephthalate fibrous matrices via NaOH hydrolysis: Effects on pore size, fiber diameter, cell seeding and proliferation

A simple NaOH treatment method was developed for fabricating nonwoven fibrous matrices of polyethylene terephthalate (PET) with predictable porosity, pore size, and fiber diameter. Matrices with various porosities (90–97%), fiber diameters (13.5–25 μm), and pore sizes (54–65 μm) were prepared by treating with 1N NaOH at 70 °C for up to 120 h, resulting in up to 70% hydrolysis of the PET polymer. The hydrolysis of PET polymer by NaOH was found to follow a second-order kinetics with respect to the fiber surface area. Accordingly, mathematical models were developed to predict matrix porosity, fiber diameter, and apparent pore size of the PET matrices. The exponential decay coefficient of PET polymer was found to be 0.0147 h⁻¹. The matrices were used to study the effects of pore size and fiber diameter on cell seeding and proliferation. The seeding study demonstrated that cell adhesion on PET fibers can be enhanced, largely due to the increased surface roughness of the PET fibers. Decreasing the fiber diameter increases the surface curvature of the fibers and decreases available surface area for cell attachment, which, however, only resulted in a small decrease in the cell growth rate.     

The interstrand amino acid pairs play a significant role in determining the parallel or antiparallel orientation of β-strands

It is widely considered that it is not appropriate to treat β-pairs in isolation, since other secondary structural models (such as helices, coils), protein topology and protein tertiary structures would limit β-strand pairing. However, to understand the underlying mechanisms of β-sheet formation, studies ought to be performed separately on more concrete aspects. In this study, we focus on the parallel or antiparallel orientation of β-strands. First, statistical analysis was performed on the relative frequencies of the interstrand amino acid pairs within parallel and antiparallel β-strands. Consequently, features were extracted by singular value decomposition from the statistical results. By using the support vector machine to distinguish the features extracted from the two types of β-strands, high accuracy was achieved (up to 99.4%). This suggests that the interstrand amino acid pairs play a significant role in determining the parallel or antiparallel orientation of β-strands. These results may provide useful information for developing other useful algorithms to examine to the β-strand folding pathways, and could eventually lead to protein structure predictions.     

Epigenetic inactivation of SLIT2 in human hepatocellular carcinomas

Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene. In addition, hypermethylation of its promoter region has been detected in various cancers, including breast and lung cancer, colorectal carcinoma, and gliomas. Here, we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma (HCC). Downregulation of SLIT2 was detected in 6 of 8 (75%) HCC cell lines by quantitative real-time RT-PCR (qRT-PCR), and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore, expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC). Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples (83.3%), and the decrease in expression was significantly correlated with CpG island hypermethylation. This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC. Moreover, overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth, migration, and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC. Thus, SLIT2 may be useful as a therapeutic target in the treatment of HCC.     

Physiological integration in an introduced, invasive plant increases its spread into experimental communities and modifies their structure

What determines the invasiveness of introduced plants is still poorly known. Many of the most invasive plant species are clonal, and physiological integration between connected individuals (ramets) of clonal plants may contribute to their ability to spread into communities and reduce performance of existing species. This contribution of integration to the invasiveness of clonal plants may be greater in denser communities. A greenhouse study was conducted to test these two hypotheses. High- and low-density communities were created by sowing seeds of eight grassland species. Each community was planted with three ramets of the stoloniferous, introduced plant Alternanthera philoxeroides that were disconnected from or left connected to ramets growing on bare soil. Connection increased the spread of Alternanthera within a community, but did not reduce community biomass. Alternanthera grew less in high-density communities, but connection did not improve its growth more than in low-density communities. Low-density communities had higher evenness when Alternanthera was connected than when it was disconnected because shoot mass was lower in the more abundant species in the community and higher in the less abundant ones. These results partly supported the first hypothesis, but not the second. The effect of integration on community structure could be due to higher resource import by the ramets of Alternanthera closer to the dominant species. Integration therefore can increase the initial spread of new clonal plant species into communities and modify the effects of this spread on community structure.     

Copy-Number Mutations on Chromosome 17q24.2-q24.3 in Congenital Generalized Hypertrichosis Terminalis with or without Gingival Hyperplasia

Congenital generalized hypertrichosis terminalis (CGHT) is a rare condition characterized by universal excessive growth of pigmented terminal hairs and often accompanied with gingival hyperplasia. In the present study, we describe three Han Chinese families with autosomal-dominant CGHT and a sporadic case with extreme CGHT and gingival hyperplasia. We first did a genome-wide linkage scan in a large four-generation family. Our parametric multipoint linkage analysis revealed a genetic locus for CGHT on chromosome 17q24.2-q24.3. Further two-point linkage and haplotyping with microsatellite markers from the same chromosome region confirmed the genetic mapping and showed in all the families a microdeletion within the critical region that was present in all affected individuals but not in unaffected family members. We then carried out copy-number analysis with the Affymetrix Genome-Wide Human SNP Array 6.0 and detected genomic microdeletions of different sizes and with different breakpoints in the three families. We validated these microdeletions by real-time quantitative PCR and confirmed their perfect cosegregation with the disease phenotype in the three families. In the sporadic case, however, we found a de novo microduplication. Two-color interphase FISH analysis demonstrated that the duplication was inverted. These copy-number variations (CNVs) shared a common genomic region in which CNV is not reported in the public database and was not detected in our 434 unrelated Han Chinese normal controls. Thus, pathogenic copy-number mutations on 17q24.2-q24.3 are responsible for CGHT with or without gingival hyperplasia. Our work identifies CGHT as a genomic disorder.     

Hypoxia inhibits maturation and trafficking of hERG K channel protein: Role of Hsp90 and ROS

We previously reported that reactive oxygen species (ROS) generated during hypoxia decrease hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we investigated the molecular mechanisms involved in hypoxia-induced downregulation of hERG protein. Culturing cells at low temperatures and addition of chemical chaperones during hypoxia restored hERG expression and currents to normoxic levels while antiarrhythmic drugs, which selectively block hERG channels, had no effect on hERG protein levels. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Co-immunoprecipitation experiments revealed that hypoxia inhibited interaction of hERG with Hsp90 chaperone required for maturation, which was restored in the presence of ROS scavengers. These results demonstrate that ROS generated during hypoxia prevents maturation of the hERG protein by inhibiting Hsp90 interaction resulting in decreased protein expression and currents.     

Inertial sensor-based knee flexion/extension angle estimation

A new method for estimating knee joint flexion/extension angles from segment acceleration and angular velocity data is described. The approach uses a combination of Kalman filters and biomechanical constraints based on anatomical knowledge. In contrast to many recently published methods, the proposed approach does not make use of the earth's magnetic field and hence is insensitive to the complex field distortions commonly found in modern buildings. The method was validated experimentally by calculating knee angle from measurements taken from two IMUs placed on adjacent body segments. In contrast to many previous studies which have validated their approach during relatively slow activities or over short durations, the performance of the algorithm was evaluated during both walking and running over 5 minute periods. Seven healthy subjects were tested at various speeds from 1 to 5 mile/h. Errors were estimated by comparing the results against data obtained simultaneously from a 10 camera motion tracking system (Qualysis). The average measurement error ranged from 0.7 degrees for slow walking (1 mph) to 3.4 degrees for running (5 mph). The joint constraint used in the IMU analysis was derived from the Qualysis data. Limitations of the method, its clinical application and its possible extension are discussed.