The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored Hepatitis B virus (HBV) is the prototypic member of the Hepadnavirdae family, which contains a group of closely related hepatotropic small DNA viruses that infect their respective animal hosts (13,29). The HBV genome is a 3.2-kb, circular, partially double-stranded DNA molecule with four overlapping open reading frames (ORFs) named PC-C, PS-S, P, and X (35, 38). Upon HBV infection of the hepatocyte, the HBV genome is converted to covalently closed circular DNA in the nucleus. The covalently closed circular DNA serves as the template for transcription by the host RNA polymerase II, generating the 3.5-, 2.4-, 2.1-, and 0.7-kb viral transcripts that encode the HBV core and polymerase polypeptides, the large surface antigen polypeptide, the middle and major surface antigen polypeptides, and the HBx polypeptide. The expression of those transcripts are directed by four HBV promoters (Cp, PS1p, Sp, and Xp), respectively, and influenced by two HBV enhancers (Enh I and Enh II) (14,29,35,38,43). HBV replicates by reverse transcription of the viral pregenomic 3.5-kb RNA (pgRNA) using the HBV polymerase that catalyzes RNA-dependent DNA synthesis and DNA-dependent DNA synthesis (36,40). Besides encoding for the HBV core polypeptide and HBV DNA polymerase that compose the viral capsid, the greater-than-genome-length 3.5-kb pgRNA is also encapsidated and serves as the template for reverse transcription. The encapsidated pgRNA is converted into the 3.2-kb partially double-stranded genomic DNA inside the viral capsid in the cytoplasm of the hepatocytes (35,36,38,40). There are several putative regulatory steps for HBV replication, e.g., pgRNA synthesis, encapsidation of pgRNA, and reverse transcription of pgRNA. As the pgRNA encodes both the HBV polymerase and core polypeptides and serves an additional function as the replication template, regulation of the synthesis of this RNA is therefore a critical step in the viral life cycle (35,38,39).HBV infection is a worldwide health problem and is one of the major causes of hepatocellular carcinoma (HCC). The crucial role of HBV in hepatocarcinogenesis is established, while the mechanism by which HBV causes transformation of hepatocytes remains unclear (1, 2, 6). HBV X protein (HBx) has long been suspected of playing a positive role in hepatocarcinogenesis, as avian hepadnaviruses missing the X ORF seem not to be associated with HCC, and some HBx transgenic mice appear to develop HCC (15, 44) or be more sensitive to a car...

SummaryDrought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H2O2 production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild‐type) controls. Moreover, up‐regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA‐induced stomatal closure caused by hydrogen peroxide (H2O2) production in transgenic poplar plants.

Exosomes, as important signal transmitters, play a key role in intercellular communication, especially in cancer metastasis. There is considerable evidence that exosomes can be used as an indicator of cancer. However, convenient and sensitive methods for detecting exosomes are still technically challenging. Here, we present a convenient and highly sensitive surface-enhanced Raman scattering (SERS) based method by combining immunoaffinity, SERS nanoprobes, and portable Raman devices for specific isolation and accurate quantification of exosomes. To construct the SERS-based biosensor, the surfaces of gold nanostar@4-mercaptobenzoic acid@nanoshell structures (AuNS@4-MBA@Au) are modified with a bivalent cholesterol (B-Chol)-labeled DNA anchor to prepare SERS nanoprobes. Exosomes are specifically captured by immunomagnetic beads, and then SERS nanoprobes are fixed on the surface of exosomes by hydrophobic interactions between cholesterol and lipid membranes, thus forming a sandwich-type immunocomplex. The immunocomplex can be magnetically captured and produce enhanced SERS signals. In the absence of exosomes, the sandwich-type immunocomplex cannot be formed, and thus negligible SERS signals are detected. The degree of immunocomplex assembly and the corresponding SERS signals are positively correlated with the exosome concentration over a wide linear range of 40 to 4 × 107 particles per μL and the limit of detection is as low as 27 particles per μL. Consequently, a sensitive and simple strategy for detection of exosomes is successfully constructed. We believe that our biosensor has considerable potential as a convenient and highly sensitive quantification tool to detect exosomes in biological samples.

Exosomes, a class of small extracellular vesicles, play important roles in various physiological and pathological processes by serving as vehicles for transferring and delivering membrane and cytosolic molecules between cells. Since exosomes widely exist in various body fluids and carry molecular information on their originating cells, they are being regarded as potential noninvasive biomarkers. Nevertheless, the development of convenient and quantitative exosome analysis methods is still technically challenging. Here, we present a low-cost assay for direct capture and rapid detection of exosomes based on a copper-mediated signal amplification strategy. The assay involves three steps. First, bulk nanovesicles are magnetically captured by cholesterol-modified magnetic beads (MB) via hydrophobic interaction between cholesterol moieties and lipid membranes. Second, bead-binding nanovesicles of exosomes with a specific membrane protein are anchored with aptamer-modified copper oxide nanoparticles (CuO NPs) to form sandwich complexes (MB-exosome-CuO NP). Third, the resultant sandwich complexes are dissolved by acidolysis to turn CuO NP into copper(II) ions (Cu), which can be reduced to fluorescent copper nanoparticles (CuNPs) by sodium ascorbate in the presence of poly(thymine). The fluorescence emission of CuNPs increases with the increase of Cu concentration, which is directly proportional to the concentration of exosomes. Our method allows quantitative analysis of exosomes in the range of 7.5 × 10 to 1.5 × 10 particles/μL with a detection of limit of 4.8 × 10 particles/μL in biological sample. The total working time is about 2 h. The assay has the potential to be a simple and cost-effective method for routine exosome analysis in biological samples.

This study was aimed to explore the role of miR‐29b‐3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR‐29b‐3p and PGRN were up‐regulated in cartilage tissue from patients with OA. Transfection of miR‐29b‐3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR‐29b‐3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration‐related molecules were also altered by miR‐29b‐3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR‐29b‐3p. The fact that recombinant PGRN or shPGRN‐mediated PGRN interference abolished miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition suggested miR‐29b‐3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR‐29b‐3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR‐29b‐3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR‐29b‐3p antagomir, demonstrated by TUNEL and safranin O‐fast green staining. This work showed miR‐29b‐3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR‐29b‐3p or PGRN may be the potential target for OA treatments.

Exosomes, as an important subpopulation of extracellular vesicles (EVs), play an important role in intercellular communications in various important pathophysiological processes, especially cancer-related. However, reliable and convenient quantitative methods for their determination are still technically challenging. In this study, we developed an efficient and direct method by combining immunoaffinity and lipid membrane surface modification into a single platform for specific isolation and accurate quantification of exosomes. Exosomes are specifically captured by immunomagnetic beads, and then a bivalent-cholesterol (B-Chol)-labeled DNA anchor with high affinity is spontaneously inserted into the exosome membrane. The rationally designed sticky end of the anchor acts as the initiator for the subsequent horseradish peroxidase (HRP)-linked hybridization chain reaction (HCR) for signal amplification. Detection is based on the color change of HRP-catalyzed HO-mediated oxidation of 3,3',5,5'- tetramethyl benzidine (TMB), which can be conveniently observed by the naked eye and monitored by UV-vis spectrometry. This proposed method enables sensitive detection of 2.2 × 10 exosomes per microliter with a relative standard deviation of <5.6%, with 100-fold higher sensitivity compared to conventional ELISA. We believe that our assay has considerable potential as a routine bioassay (cost-efficient, reliable, and easy to operate) for the accurate quantification of exosomes in clinical samples.

The hostile hypoxic microenvironment takes primary responsibility for the rapid expansion of breast cancer tumors. However, the underlying mechanism is not fully understood. Here, using RNA sequencing (RNA-seq) analysis, we identified a hypoxia-induced long noncoding RNA (lncRNA) KB-1980E6.3, which is aberrantly upregulated in clinical breast cancer tissues and closely correlated with poor prognosis of breast cancer patients. The enhanced lncRNA KB-1980E6.3 facilitates breast cancer stem cells (BCSCs) self-renewal and tumorigenesis under hypoxic microenvironment both in vitro and in vivo. Mechanistically, lncRNA KB-1980E6.3 recruited insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to form a lncRNA KB-1980E6.3/IGF2BP1/c-Myc signaling axis that retained the stability of c-Myc mRNA through increasing binding of IGF2BP1 with m6A-modified c-Myc coding region instability determinant (CRD) mRNA. In conclusion, we confirm that lncRNA KB-1980E6.3 maintains the stemness of BCSCs through lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis and suggest that disrupting this axis might provide a new therapeutic target for refractory hypoxic tumors.

Previous studies documented significant behavioral changes in the offspring of cocaine-exposed mothers. We now explore the hypothesis that maternal cocaine exposure could alter the fetal epigenetic machinery sufficiently to cause lasting neurochemical and functional changes in the offspring. Pregnant CD1 mice were administered either saline or 20 mg/kg cocaine twice daily on gestational days 8–19. Male pups from each of ten litters of the cocaine and control groups were analyzed at 3 (P3) or 30 (P30) days postnatum. Global DNA methylation, methylated DNA immunoprecipitation followed by CGI2 microarray profiling and bisulfite sequencing, as well as quantitative real-time RT-PCR gene expression analysis, were evaluated in hippocampal pyramidal neurons excised by laser capture microdissection. Following maternal cocaine exposure, global DNA methylation was significantly decreased at P3 and increased at P30. Among the 492 CGIs whose methylation was significantly altered by cocaine at P3, 34% were hypermethylated while 66% were hypomethylated. Several of these CGIs contained promoter regions for genes implicated in crucial cellular functions. Endogenous expression of selected genes linked to the abnormally methylated CGIs was correspondingly decreased or increased by as much as 4–19-fold. By P30, some of the cocaine-associated effects at P3 endured, reversed to opposite directions, or disappeared. Further, additional sets of abnormally methylated targets emerged at P30 that were not observed at P3. Taken together, these observations indicate that maternal cocaine exposure during the second and third trimesters of gestation could produce potentially profound structural and functional modifications in the epigenomic programs of neonatal and prepubertal mice.