Characterization of a full-length infectious clone of bovine foamy virus 3026

The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid(AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Diffusion coefficients of natural gas in foamy oil systems under high pressures

The diffusion coefficient of natural gas in foamy oil is one of the key parameters to evaluate the feasibility of gas injection for enhanced oil recovery in foamy oil reservoirs. In this paper, a PVT cell was used to measure diffusion coefficients of natural gas in Venezuela foamy oil at high pressures, and a new method for deter- mining the diffusion coefficient in the foamy oil was de- veloped on the basis of experimental data. The effects of pressure and the types of the liquid phase on the diffusion coefficient of the natural gas were discussed. The results indicate that the diffusion coefficients of natural gas in foamy oil, saturated oil, and dead oil increase linearly with increasing pressure. The diffusion coefficient of natural gas in the foamy oil at 20 MPa was 2.93 times larger than that at 8.65 MPa. The diffusion coefficient of the natural gas in dead oil was 3.02 and 4.02 times than that of the natural gas in saturated oil and foamy oil when the pressure was 20 MPa. However, the gas content of foamy oil was 16.9 times higher than that of dead oil when the dissolution time and pressure were 20 MPa and 35.22 h, respectively.

A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection.A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL.Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection.These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

Prostatic Foamy Adenocarcinoma:A Case Report.

A 58-year-old male patient, complaining of dysuresia, which increased over a period of 2 months, had a history of urine retention that did not respond to treatment administered in an outpatient clinic. Upon admission to the hospital on August 2, 2005, examination showed that his prostate was midrange size by rectal palpation, and without pain or prostate nodus. An ultrasound examination indicated the prostate size was 6.1 cm×4.7 cm×3.6 cm, without an occupying lesion in the prostate.

Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.

Making and Study of Moderate Pressure Glow Discharge on the Basis of Electrolytic Foamy Cathode, Prepared as the Aqueous Solution of NaHCO3

A method to generate the dynamic moderate pressure dc glow discharge on the basis of electrolytic cathode in the form of aqueous solution of sodium bicarbonate NaHCO3 is described. Photo and video images of the discharge are presented as well as the synchronized therewith “oscillograms” of current and voltage. Different phases of the discharge were discovered, one of which is a quasi-stationary glow discharge with the foamy cathode, was recorded for the first time. It was shown, that in this phase the discharge is supported by a so-called three-dimensional cathode spot, having the finite volume. The time-spatial diagram was plotted for the discharge. The Rayleigh-Taylor instability in the two-layered electrolytic cathode was recorded.

Borf-1 protein identified as a transcriptional trans-activator of bovine foamy virus

Bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses,contains two open reading frames (ORF-1 and ORF-2) in addition to the genes coding for gag,po/and env. Borf-1 protein, encoded by BFV ORF-1, is identified as a transcriptional transactivator, which augments gene expression directed by the viral long terminal repeat (LTR).Further investigations in transient expression assays reveal that the Borf-1 responsive elements are located in the U3 domain of the LTR, upstream from position -140 ( + 1 represents the transcription initiation site), and the BFV RU5 region has an inhibitory effect in LTR-directed gene expression.

cis-acting element located in the bovine foamy virus internal promoter possesses the properties of a transcrip-tional enhancer

Bovine foamy virus encodes a transcriptional transactivitor, Tas or Borf-1, which governs the level of viral transcripts initiated by both the promoter in the long terminal repeat (LTR) and the internal promoter (IP) located in the env gene through their cis-acting targets. We have identified and characterized a 72 bp TBS (Borf-1) responsive element located in BFV3026, internal promoter (TREIP) by deletion mutant and transient expression assay. This cis-acting target element in the internal promoter has the properties of a transcriptional enhancer which functions independently of its orientation, position and also in heterologous promoters (BFV LTR and bovine immunodeficiency virus, BIV LTR). Alignments reveal that there are positional similarity and sequence homology among BFV TREIP, SFV-1 TREIP proximal element and SFV-3 TREIPH, which suggests that this kind of cis-acting elements possesses some common functional character.

Lnc-RP5 Regulates the mi R-129-5p/Notch1/PFV Internal Promoter Axis to Promote the Expression of the Prototype Foamy Virus Transactivator Tas

Prototype foamy virus(PFV)is a unique retrovirus that infects animals and humans and does not cause clinical symptoms.Long noncoding RNAs(lncRNAs)are believed to exert multiple regulatory functions during viral infections.Previously,we utilized RNA sequencing(RNA-seq)to characterize and identify the lncRNA lnc-RP5-1086 D14.3.1-1:1(lnc-RP5),which is markedly decreased in PFV-infected cells.However,little is known about the function of lnc-RP5 during PFV infection.In this study,we identified lnc-RP5 as a regulator of the PFV transcriptional transactivator(Tas).Lnc-RP5 enhanced the activity of the PFV internal promoter(IP).The expression of PFV Tas was found to be promoted by lnc-RP5.Moreover,mi R-129-5 p was found to be involved in the lnc-RP5-mediated promotion of PFV IP activity,while the Notch1 protein suppressed the activity of PFV IP and the expression of Tas.Our results demonstrate that lnc-RP5 promotes the expression of PFV Tas through the miR-129-5 p/Notch1/PFV IP axis.This work provides evidence that host lnc RNAs can manipulate PFV replication by employing mi RNAs and proteins during an early viral infection.

Membrane-spanning domain of bovine foamy virus transmembrane protein having cytotoxicity

Foamy viruses(FVs)have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane(TM)proteins include a region of hydrophobic domain called membrane-spanning domain(MSD),which contains several stretches of hydrophobic amino acids.To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli,a series of mutants were constructed and expressed in the E.coli BL21(DE3)using pET-32a(+)as expressing vector.The results showed that only TM3 without MSD was expressed in E.coli,whereas the other two containing full or part of the MSD(TM1 and TM2)could not be expressed.Furthermore,the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD,especially on the stretches of hydrophobic amino acids.Western blotting analysis showed that TM3 protein was purified with affinity purification.

Pediatric non-alcoholic steatohepatitis:The first report on the ultrastructure of hepatocyte mitochondria

AIM: To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH).

The pathology of unusual subtypes of prostate cancer

There are some current literatures describing the morphologic change of prostate carcinoma variants. Some subtypes do not respond to hormone deprivation therapy, for example adenosquamous and squamous cell carcinoma （SQCC）, basaloid and adenoid cystic carcinoma （ACC）, small cell carcinoma （SmCC）, sarcomatoid carcinoma, urothelial carcinoma; some are defined in special Gleason grade, some develop different prognosis. So, it is very important to identify these rare subtypes to avoid misdiagnosis. In this review, we aim to describe the typical clinicopathological features of the rare variants of prostate cancer, including prostate acinar adenocarcinoma morphologic variants.

Preparation of BFV Gag Antiserum and Preliminary Study on Cellular Distribution of BFV

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Retroviral integrase protein and intasome nucleoprotein complex structures

Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral integrase(IN) protein. IN catalyzes two distinct chemical reactions: 3'-processing, whereby the viral DNA is recessed by a di- or trinucleotide at its 3'-ends, and strand transfer, in which the processed viral DNA ends are inserted into host chromosomal DNA. Although IN has been studied as a recombinant protein since the 1980 s, detailed structural understanding of its catalytic functions awaited high resolution structures of functional IN-DNA complexes or intasomes, initially obtained in 2010 for the spumavirus prototype foamy virus(PFV). Since then, two additional retroviral intasome structures, from the α-retrovirus Rous sarcoma virus(RSV) and β-retrovirus mouse mammary tumor virus(MMTV), have emerged. Here, we briefly review the history of IN structural biology prior to the intasome era, and then compare the intasome structures of PFV, MMTV and RSV in detail. Whereas the PFV intasome is characterized by a tetrameric assembly of IN around the viral DNA ends, the newer structures harbor octameric IN assemblies. Although the higher order architectures of MMTV and RSV intasomes differ from that of the PFV intasome, they possess remarkably similar intasomal core structures. Thus, retroviral integration machineries have adapted evolutionarily to utilize disparate IN elements to construct convergent intasome core structures for catalytic function.

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

Foaminess of Refining Slags System

The experiments on the foaminess of the CaO-MgO-Al2O3 -SiO2 -CaF2 refining slag system have been carried out. The obtained results are as followst (1) relative foaming height linearly increases with the increasing of the flowrate of blowing gas; (2) the proper content of MgO is about 11% when slag basicity B<2.5 and it should lower when slag basicity will be higher; (3) the better content of Al2O3, (MgO)+(Al2O3 ) is 15% and 20 % ～ 26 % respectively at the range of lower basicities; (4) the effect of slag basicity on the foaminess is complex and its optimizing vaiue is 1.9 at the specific contents of MgO, Al2O3 and CaF2.

CO2 flooding strategy to enhance heavy oil recovery

CO_(2) flooding is one of the most promising techniques to enhance both light and heavy oil recovery.In light oil recovery,the production pressure in CO_(2) flooding in general keeps constant in order to maintain the miscibility of injected CO_(2) and crude oil;while in heavy oil recovery,a depleting pressure scheme may be able to induce foamy oil flow,thus the oil recovery could be further enhanced.In this study,different pressure control schemes were tested by 1-D core-flooding experiments to obtain an optimized one.Numerical simulations were conducted to history match all experimental data to understand the mechanisms and characteristics of different CO_(2) flooding strategies.For the core-flooding experiments,1500 mD sandstone cores,formation brine and a heavy oil sample with a viscosity of about 869.3 cp at reservoir condition(55℃ and 11 MPa)were used.Before each CO_(2) flooding test,early stage water-flooding was conducted until the water cut reached 90%.Different CO_(2) injection rates and production pressure control strategies were tested through core-flooding experiments.Experimental results indicated that a slower CO_(2) injection rate(2 ml/min)led to a higher recovery factor from 31.1%to 36.7%,compared with a high CO_(2) injection rate of 7 ml/min;for the effects of different production strategies,a constant production pressure at the production port yielded a recovery factor of 31.1%;while a pressure depletion with 47.2 kPa/min at the production port yielded 7%more oil recovery;and the best pressure control scheme in which the production pressure keeping constant during CO_(2) injection period,then depleting the model pressure with the injector shut-in yielded a recovery factor of 42.5%of the initial OOIP.For the numerical simulations study,the same oil relative permeability curve was applied to match the experimental results to all tests.Different gas relative permeability curves were obtained when the production pressure schemes are different.A much lower gas relative permeability curve and a higher critical gas saturation were achieved in the best pressure control scheme case compared to other cases.The lower gas relative permeability curve indicates that foamy oil was formed in the pressure depletion processes.Through this study,it is suggested that the pressure control scheme can be optimized in order to maximize the CO_(2) injection performance for enhanced heavy oil recovery.