Mechanism of Hexane Displaced by Supercritical Carbon Dioxide: Insights from Molecular Simulations.

Supercritical carbon dioxide (sCO2) has great potential for displacing shale oil as a result of its high solubility and low surface tension and viscosity, but the underlying mechanisms have remained unclear up to now. By conducting equilibrium molecular dynamics (EMD) simulations, we found that the displacing process could be divided into three steps: the CO2 molecules were firstly injected in the central region of shale slit, then tended to adsorb on the SiO2-OH wall surface and mix with hexane, resulting in loose hexane layer on the shale surface, and finally displaced hexane from the wall due to strong interactions between CO2 and wall. In that process, the displacing velocity and efficiency of hexane exhibit parabolic and increased trends with pressure, respectively. To gain deep insights into this phenomenon, we further performed non-equilibrium molecular dynamics (NEMD) simulations and found that both the Onsager coefficients of CO2 and hexane were correlated to increase with pressure, until the diffusion rate of hexane being suppressed by the highly dense distribution of CO2 molecules at 12 MPa. The rapid transportation of CO2 molecules in the binary components (CO2 and hexane) actually promoted the hexane diffusion, which facilitated hexane flowing out of the nanochannel and subsequently enhanced oil recovery efficiency. The displacing process could occur effectively at pressures higher than 7.5 MPa, after which the interaction energies of the CO2-wall were stronger than that of the hexane-wall. Taking displacing velocity and efficiency and hexane diffusion rate into consideration, the optimal injection pressure was found at 10.5 MPa in this work. This study provides detailed insights into CO2 displacing shale oil and is in favor of deepening the understanding of shale oil exploitation and utilization.

The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (agomiRs) significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Juvenile hormone upregulates sugarbabe for vitellogenesis and egg development in the migratory locust Locusta migratoria.

Sugarbabe is a C2 H2 zinc-finger transcription factor that is sensitive to sugar and essential for lipid biosynthesis in larvae of Drosophila melanogaster. However, the role of Sugarbabe in adult insect development remains unexplored. Vitellogenesis is a nutrient-dependent process that is promoted by juvenile hormone (JH) in many insect species. Here, we cloned an ortholog gene of D. melanogaster Sugarbabe (DmSug) in the migratory locust Locusta migratoria. The locust Sugarbabe (LmSug) has five C2 H2 zinc-finger motifs similar to DmSug. LmSug was expressed at a low level in adult female locusts raised under poor nutrient conditions. JH treatment increased the expression level of LmSug. Knockdown of the JH receptor gene Met caused a reduction of LmSug expression. Depletion of the LmSug transcript level caused a significant reduction in vitellogenin expression in the fat body, resulting in impaired oocyte development and ovary growth. The results suggest that LmSug is expressed in response to JH, and plays an essential role in female insect reproduction.

Post-transcriptional regulation of insect metamorphosis and oogenesis.

Metamorphic transformation from larvae to adults along with the high fecundity is key to insect success. Insect metamorphosis and reproduction are governed by two critical endocrines, juvenile hormone (JH), and 20-hydroxyecdysone (20E). Recent studies have established a crucial role of microRNA (miRNA) in insect metamorphosis and oogenesis. While miRNAs target genes involved in JH and 20E-signaling pathways, these two hormones reciprocally regulate miRNA expression, forming regulatory loops of miRNA with JH and 20E-signaling cascades. Insect metamorphosis and oogenesis rely on the coordination of hormones, cognate genes, and miRNAs for precise regulation. In addition, the alternative splicing of genes in JH and 20E-signaling pathways has distinct functions in insect metamorphosis and oogenesis. We, therefore, focus in this review on recent advances in post-transcriptional regulation, with the emphasis on the regulatory role of miRNA and alternative splicing, in insect metamorphosis and oogenesis. We will highlight important new findings of miRNA interactions with hormonal signaling and alternative splicing of JH receptor heterodimer gene Taiman.

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.

Visualization and quantification of simian immunodeficiency virus-infected cells using non-invasive molecular imaging.

In vivo imaging can provide real-time information and three-dimensional (3D) non-invasive images of deep tissues and organs, including the brain, whilst allowing longitudinal observation of the same animals, thus eliminating potential variation between subjects. Current in vivo imaging technologies, such as magnetic resonance imaging (MRI), positron emission tomography-computed tomography (PET-CT) and bioluminescence imaging (BLI), can be used to pinpoint the spatial location of target cells, which is urgently needed for revealing human immunodeficiency virus (HIV) dissemination in real-time and HIV-1 reservoirs during suppressive antiretroviral therapy (ART). To demonstrate that in vivo imaging can be used to visualize and quantify simian immunodeficiency virus (SIV)-transduced cells, we genetically engineered SIV to carry different imaging reporters. Based on the expression of the reporter genes, we could visualize and quantify the SIV-transduced cells via vesicular stomatitis virus glycoprotein pseudotyping in a mouse model using BLI, PET-CT or MRI. We also engineered a chimeric EcoSIV for in vivo infection study. Our results demonstrated that BLI is sensitive enough to detect as few as five single cells transduced with virus, whilst PET-CT can provide 3D images of the spatial location of as few as 10 000 SIV-infected cells. We also demonstrated that MRI can provide images with high spatial resolution in a 3D anatomical context to distinguish a small population of SIV-transduced cells. The in vivo imaging platform described here can potentially serve as a powerful tool to visualize lentiviral infection, including when and where viraemia rebounds, and how reservoirs are formed and maintained during latency or suppressive ART.

The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks.

During their circulation in nature, H5N1 avian influenza viruses (AIVs) have acquired the ability to kill their natural hosts, wild birds and ducks. The genetic determinants for this increased virulence are largely unknown. In this study, we compared two genetically similar H5N1 AIVs, A/duck/Hubei/49/05 (DK/49) and A/goose/Hubei/65/05 (GS/65), that are lethal for chickens but differ in their virulence levels in ducks. To explore the genetic basis for this difference in virulence, we generated a series of reassortants and mutants of these two viruses. The virulence of the reassortant bearing the PA gene from DK/49 in the GS/65 background increased 10(5)-fold relative to that of the GS/65 virus. Substitution of two amino acids, S224P and N383D, in PA contributed to the highly virulent phenotype. The amino acid 224P in PA increased the replication of the virus in duck embryo fibroblasts, and the amino acid 383D in PA increased the polymerase activity in duck embryo fibroblasts and delayed the accumulation of the PA and PB1 polymerase subunits in the nucleus of virus-infected cells. Our results provide strong evidence that the polymerase PA subunit is a virulence factor for H5N1 AIVs in ducks.

Continued evolution of H5N1 influenza viruses in wild birds, domestic poultry, and humans in China from 2004 to 2009.

Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51 representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to 2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition, antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in nature have complex biological characteristics and pose a continued challenge for disease control and pandemic preparedness.

Immunogenicity and protective efficacy of a live attenuated H5N1 vaccine in nonhuman primates.

The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. A cold-adapted, live attenuated vaccine with the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N1 virus A/VN/1203/2004 (clade 1) was protective against the pulmonary replication of homologous and heterologous wild-type H5N1 viruses in mice and ferrets. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H5N1 vaccine (AH/AAca) that contains HA and NA genes from a recent H5N1 isolate, A/Anhui/2/05 virus (AH/05) (clade 2.3), and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus (AAca). AH/AAca was attenuated in chickens, mice, and monkeys, and it induced robust neutralizing antibody responses as well as HA-specific CD4+ T cell immune responses in rhesus macaques immunized twice intranasally. Importantly, the vaccinated macaques were fully protected from challenge with either the homologous AH/05 virus or a heterologous H5N1 virus, A/bar-headed goose/Qinghai/3/05 (BHG/05; clade 2.2). These results demonstrate for the first time that a cold-adapted H5N1 vaccine can elicit protective immunity against highly pathogenic H5N1 virus infection in a nonhuman primate model and provide a compelling argument for further testing of double immunization with live attenuated H5N1 vaccines in human trials.

Clustered miR-2, miR-13a, miR-13b and miR-71 coordinately target Notch gene to regulate oogenesis of the migratory locust Locusta migratoria.

MicroRNAs (miRNAs), ∼22-nt small noncoding RNAs with a crucial role in various biological processes of organisms, are usually clustered in the genome. However, little is known about the miRNA clusters involved in insect reproduction. By small RNA sequencing and quantification followed by qRT-PCR, we found that the expression of invertebrate-specific miR-2/13/71 cluster including miR-2, miR-13a, miR-13b and miR-71 significantly decreased after adult ecdysis of the migratory locust, Locusta migratoria. Luciferase reporter assay and RNA immunoprecipitation demonstrated that miR-2/13/71 bound to the protein coding sequence of Notch and downregulated its expression. Injection of miR-2/13/71 agomiRs led to significant decrease of Notch expression as well as markedly reduced levels of Vitellogenin mRNA, suppressed oocyte maturation and impaired ovarian growth. Moreover, the expression of miR-2/13/71 was repressed by juvenile hormone (JH). Our results thus point to a previously unidentified mechanism by which JH-repressed miR-2/13/71 coordinately downregulates Notch to modulate insect reproduction. The increase of JH and decrease of miR-2/13/71 expression in both previtellogenic and vitellogenic stages of adult females ensure a high level of Notch expression, critically contributing to JH-dependent vitellogenesis and oogenesis.

An isoform of Taiman that contains a PRD-repeat motif is indispensable for transducing the vitellogenic juvenile hormone signal in Locusta migratoria.

Taiman (Tai) has been recently identified as the dimerizing partner of juvenile hormone (JH) receptor, Methoprene-tolerant (Met). However, the role of Tai isoforms in transducing vitellogenic signal of JH has not been determined. In this study, we show that the migratory locust Locusta migratoria has two Tai isoforms, which differ in an INDEL-1 domain with the PRD-repeat motif rich in histidine and proline at the C-terminus. Tai-A with the INDEL-1 is expressed at levels about 50-fold higher than Tai-B without the INDEL-1 in the fat body of vitellogenic adult females. Knockdown of Tai-A but not Tai-B results in a substantial reduction of vitellogenin expression in the fat body accompanied by the arrest of ovarian development and oocyte maturation, similar to that caused by depletion of both Tai isoforms. Either Tai-A or Tai-B combined with Met can induce target gene transcription in response to JH, but Tai-A appears to mediate a significantly higher transactivation. Our data suggest that the INDEL-1 domain plays a critical role in Tai function during reproduction as Tai-A appears be more active than Tai-B in transducing the vitellogenic JH signal in L. migratoria.

Selection of antigenically advanced variants of seasonal influenza viruses.

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

Synergistic Effect of S224P and N383D Substitutions in the PA of H5N1 Avian Influenza Virus Contributes to Mammalian Adaptation.

The adaptation of H5N1 avian influenza viruses to human poses a great threat to public health. Previous studies indicate the adaptive mutations in viral polymerase of avian influenza viruses are major contributors in overcoming the host species barrier, with the majority of mammalian adaptive mutations occurring in the PB2 protein. However, the adaptive mutations in the PA protein of the H5N1 avian influenza virus are less defined and poorly understood. In this study, we identified the synergistic effect of the PA/224P + 383D of H5N1 avian influenza viruses and its ability to enhance the pathogenicity and viral replication in a mammalian mouse model. Interestingly, the signature of PA/224P + 383D mainly exists in mammalian isolates of the H5N1 influenza virus and pdmH1N1 influenza virus, providing a potential pathway for the natural adaptation to mammals which imply the effects of natural adaptation to mammals. Notably, the mutation of PA/383D, which is highly conserved in avian influenza viruses, increases the polymerase activity in both avian and human cells, and may have roles in maintaining the avian influenza virus in their avian reservoirs, and jumping species to infect humans.

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells.

Krüppel-homolog 1 mediates juvenile hormone action to promote vitellogenesis and oocyte maturation in the migratory locust.

Juvenile hormone (JH) prevents insect larval metamorphosis and stimulates processes for adult reproduction. Krüppel-homolog 1 (Kr-h1), a zinc finger transcription factor, is shown to mediate the anti-metamorphic effect of JH in both holometabolous and hemimetabolous insects. However, the role of Kr-h1 in JH-mediated reproduction has not been determined. Using the migratory locust, Locusta migratoria, we showed here that Kr-h1 was expressed in response to JH in female adults, and Kr-h1 transcription was directly regulated by the JH-receptor complex comprised of Methoprene-tolerant (Met) and steroid receptor co-activator. We demonstrated that Kr-h1 RNAi phenocopied Met RNAi and JH-deprived condition during post-eclosion development and vitellogenesis of female locusts. Knockdown of Kr-h1 resulted in substantial reduction of Vg expression in the fat body and lipid accumulation in the primary oocytes, accompanied by blocked follicular epithelium development, oocyte maturation and ovarian growth. Our data therefore reveal a crucial role of Kr-h1 in insect vitellogenesis and egg production. This study suggests that JH-Met-Kr-h1 signaling pathway is also functional in insect reproduction.

Mutations in NA that induced low pH-stability and enhanced the replication of pandemic (H1N1) 2009 influenza A virus at an early stage of the pandemic.

An influenza A virus that originated in pigs caused a pandemic in 2009. The sialidase activity of the neuraminidase (NA) of previous pandemic influenza A viruses are stable at low pH (≤5). Here, we identified the amino acids responsible for this property. We found differences in low-pH stability at pH 5.0 among pandemic (H1N1) 2009 viruses, which enhanced the replication of these viruses. Low-pH-stable NA enhancement of virus replication may have contributed to the rapid worldwide spread and adaptation to humans of pandemic (H1N1) 2009 viruses during the early stages of the 2009 pandemic.

Argonaute 1 is indispensable for juvenile hormone mediated oogenesis in the migratory locust, Locusta migratoria.

Juvenile hormone (JH) is the primary hormone controlling vitellogenesis and oocyte maturation in the migratory locust Locusta migratoria, an evolutionarily primitive insect species with panoistic ovaries. However, molecular mechanisms of locust oogenesis remain unclear and the role of microRNA (miRNA) in JH mediated locust vitellogenesis and oocyte maturation has not been explored. Using miRNA sequencing and quantification with small RNA libraries derived from fat bodies of JH-deprived versus JH analog-exposed female adult locusts, we have identified 83 JH up-regulated and 60 JH down-regulated miRNAs. QRT-PCR validation has confirmed that transcription of selected miRNAs responded to JH administration and correlated with changes in endogenous hemolymph JH titers. Depletion of Argonaute 1 (Ago1), a key regulator of miRNA biogenesis and function by RNAi in female adult locusts dramatically decreased the expression of vitellogenin (Vg) and severely impaired follicular epithelium development, terminal oocyte maturation and ovarian growth. Our data indicate that Ago1 and Ago1-dependent miRNAs play a crucial role in locust vitellogenesis and egg production.

A single amino acid V4I substitution in VP1 attenuates virulence of very virulent infectious bursal disease virus (vvIBDV) in SPF chickens and increases replication in CEF cells.

Infectious bursal disease virus (IBDV) is a birnavirus that causes immunosuppressive disease in chickens. The emergence of very virulent IBDV (vvIBDV) has brought new challenges for this disease. The molecular determinants for the high pathogenicity of vvIBDV are not fully understood. Previous studies focused mostly on the VP2 protein on segment A, but recent evidence suggests that segment B also plays an important role. Previously we identified eight amino acid changes in the VP1 protein of vvIBDV. In this study, we investigated effect of amino acids substitutions in VP1 on viral replication and pathogenicity. We identified a Valine to Isoleucine substitution at amino acid position 4 (V4I) of VP1 that attenuates viral pathogenicity and reduces viral replication in SPF chickens but increases viral replication in CEF cells. This study confirms that VP1 of segment B play an important role in viral replication and pathogenicity of vvIBDV.

An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer.

Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice.

A/duck/Guangxi/53/2002 (DKGX/53) and A/duck/Fujian/01/2002 (DKFJ/01) are H5N1 avian influenza viruses that are lethal in chickens. In mice, however, DKFJ/01 is highly pathogenic, whereas DKGX/53 displays low pathogenicity. In this study, we used reverse genetics to demonstrate that two amino acid residues at positions 30 and 215 of the M1 protein of these two viruses are important determinants for pathogenicity in mice. We thus firstly prove the M1 protein contributes to the virulence of H5N1 viruses in mice, and the amino acid residues shown to attenuate the virulence could be targeted in influenza virus candidates for live vaccine development.