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The thermocapillary motion of two bubbles along their line of centers in a uniform temperature gradient is investigated theoretically. The bubbles are moving in the direction of the temperature gradient. And the interaction between the leading bubble and the trailing one becomes significant as the separation distance between them is decreased greatly so that the bubble interaction is considered in this case. The appropriate equations of momentum and energy are solved using the method of reflections. In order to proceed analytically, sets of transformations between two coordinates are obtained. By using these transformations and the reflection process, accurate migration velocities of these two bubbles in the microgravity environment are derived for the limit of small Marangoni and Reynolds numbers. These results are employed to describe the thermocapillary motion of two bubbles and to estimate the effects of bubble size and the thermal gradient on the interaction between two bubbles. All of our results for the migration of the two bubbles demonstrate that the approach of the second bubble to the first one intensifies the mutual interaction between these two bubbles and yields some interesting thermocapillary motions.

We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus is linked to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), all of which are viewed as cytokine-driven malignancies. In particular, interleukin-6 (IL-6) has been found to promote the growth and proliferation of cells from KS and PEL. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]), which functions similarly to the cellular IL-6. Therefore, vIL-6 has been proposed to play an important role in tumor progression. Several groups have reported that vIL-6 is expressed from the HHV-8 genome at higher levels in PEL and MCD lesions than in KS lesions. However, it is not clear how vIL-6 expression is regulated. We characterized the transcription at the vIL-6 gene locus by Northern blot analysis and, in contrast to previous reports, we observed two distinct transcripts from induced PEL cell lines. This observation was confirmed by primer extension, as well as 5′ and 3′ rapid amplification of cDNA ends. Two transcription initiation sites and putative TATA boxes were mapped. A luciferase reporter system was used to show that each of the two putative TATA boxes contributed to vIL-6 promoter activity. Since virally encoded transcriptional activator Rta potently activates the viral lytic gene expression cascade, we examined the role of Rta in controlling vIL-6 gene expression and found that Rta activated the vIL-6 promoter. The Rta-responsive element was further mapped through a series of deletion constructs. Electrophoretic mobility shift assays demonstrated that Rta binds directly to the vIL-6 Rta-responsive element, and the core Rta-responsive element was mapped to a 26-bp region spanning from nucleotide 18315 to 18290 on the viral genome. We propose that the existence of two vIL-6 promoters offers opportunities for differential regulation of vIL-6 gene expression in different tissue types and may account for the variable vIL-6 levels observed in KS, PEL, and MCD.

Human immunodeficiency virus type-1 (HIV-1) infection dramatically increases the risk of development of Kaposi's sarcoma (KS) in individuals infected with Kaposi's sarcoma-associated herpesvirus (KSHV). In a primary effusion lymphoma (PEL) tissue culture model system, HIV-1 replication potently induced the lytic replication of KSHV and led to the secretion of soluble factors capable of inducing lytic KSHV replication in bystander cells. Here we demonstrate that HIV induces KSHV lytic replication through activation of the KSHV Rta. HIV gene expression activated the KSHV Rta promoter following viral infection or after transfection of proviral DNA. Although HIV-1 Tat has previously been implicated as an activator of KSHV lytic replication, Tat alone was unable to activate lytic replication and failed to activate the Rta promoter. We conclude that HIV activates KSHV lytic replication by inducing the KSHV Rta promoter and that factors other than HIV-1 Tat are required to mediate this effect.

Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an incidence of ~600 000 new cases per year and a 50% mortality rate for advanced disease. The mutational landscape of HNSCC has been recently elucidated, introducing the possibility for targeted therapeutic approaches. PIK3CA—which encodes the α-catalytic subunit of PI3K (PI3Kα)—is the most frequently altered actionable target in HNSCC, however it is not presently clear which patients benefit most from PI3Kα-inhibition. BYL719 is a leading PI3Kα inhibitor in clinical development for HNSCC. We previously examined the responses of a large panel of genetically-characterized HNSCC cell lines to BYL719 and identified activating HRAS G12V mutations as strong predictors for BYL719 resistance. Here we examine if this mutation is able to individually modulate BYL719 response through overexpression and knockdown studies. Methods: To determine if HRAS G12V was able to modulate BYL719 sensitivity, we knocked down HRAS in HRAS G12V and HRAS wild-type (WT) cell lines using RNA interference, and then treated cells over 10-point dose ranges with BYL719. Sensitivity was determined by calculating IC50 (half-maximal inhibitory concentration) values at 72 hours using PrestoBlue®. Constructs expressing wildtype (WT) HRAS or HRAS G12V were transfected into BYL719-sensitive cells for overexpression studies and cells were selected using G418 Sulfate for 1 month. Proliferation and BYL719 sensitivity (IC50 values) were measured as described. Immunoblotting was used to examine activity of the PI3K-AKT axis with and without BYL719 (1μM) for 24 hours. Results & Conclusions: HRAS knockdown sensitized HRAS G12V lines to BYL719 (lower IC50), but did not affect the response of WT HRAS cells. WT HRAS and HRAS G12V overexpression significantly increased cellular proliferation and promoted BYL719 resistance. In HRAS G12V cell lines, we observed high levels of active phosphorylated AKT (S473/T308), even in the presence of BYL719. Collectively these findings highlight the predictive role of HRAS G12V for innate BYL719 resistance and contribute to our understanding of which patients may respond best on BYL719 therapy. Citation Format: Kara M. Ruicci, Ren Sun, Morgan Black, Nicole Pinto, John Yoo, Kevin Fung, Danielle Macneil, Lauire Aillies, Joseph S. Mymryk, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. HRAS G12V predicts for innate resistance to PI3Kα inhibition in head and neck squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2017-3162

[This corrects the article DOI: 10.1371/journal.pgen.1005310.].

Objective To explore the effects of twist over-expression on the transdifferentiation of human peritoneal mesothelial cells(HPMCs).Methods The HPMCs were treated separately with 50mmol/L(high glucose group) and 5.6mmol/L(control group) D-glucose for 48h.The protein expression of twist,E-cadherin,Fsp1 and α-SMA were detected by Western blotting.The mRNA and protein expression of twist,E-cadherin,Fsp1 and α-SMA were tested by qRT-PCR and Western blotting after transfecting the plasmids pcDNA3.1-twist and empty vector pcDNA3.1 into HPMCs with Lipofectamine 2000 in vitro.The protein expression of twist,E-cadherin,Fsp1 and α-SMA were tested by Western blotting after silencing the twist expression by siRNA,empty vector pSlience and the parental cells.Results Compared with control group,the expression of twist,Fsp1 and α-SMA increased,and of E-cadherin decreased in high glucose group(P < 0.05).Compared with the empty vector group,after the over-expression of twist in HPMCs,the expression of E-cadherin decreased,and of Fsp1 and α-SMA increased in high glucose group(P < 0.05).Silencing the twist expression by siRNA,the expression of E-cadherin increased and of α-SMA and Fsp1 decreased in siTwist transfacted cells compared with pSlicencer-transfacted cell and parental cells(P < 0.05),and finally reversed the phenotype of MMT.Conclusion The over-expression of twist may promote the mesothelial-to-mesenchymal transition of HPMCs.

Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains., IMPORTANCE Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.

Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein–protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N-terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two-hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site-directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.

Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and β-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi’s sarcoma–associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. IMPORTANCE While KSHV can infect multiple cell types in vitro , only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK.In organellostudies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs.

The CALPUFF model was applied to simulate the ground-level atmospheric concentrations of Pb and Cd from municipal solid waste incineration (MSWI) plants, and the soil concentration model was used to estimate soil concentration increments after atmospheric deposition based on Monte Carlo simulation, then ecological risk assessment was conducted by the potential ecological risk index method. The results showed that the largest atmospheric concentrations of Pb and Cd were 5.59 x 109-3) microg x m(-3) and 5.57 x 10(-4) microg x m(-3), respectively, while the maxima of soil concentration incremental medium of Pb and Cd were 2.26 mg x kg(-1) and 0.21 mg x kg(-1), respectively; High risk areas were located next to the incinerators, Cd contributed the most to the ecological risk, and Pb was basically free of pollution risk; Higher ecological hazard level was predicted at the most polluted point in urban areas with a 55.30% probability, while in rural areas, the most polluted point was assessed to moderate ecological hazard level with a 72.92% probability. In addition, sensitivity analysis of calculation parameters in the soil concentration model was conducted, which showed the simulated results of urban and rural area were most sensitive to soil mix depth and dry deposition rate, respectively.

Capsid-associated tegument proteins have been identified in alpha- and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a β-sheet-rich triplex-binding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein. IMPORTANCE Several capsid-associated tegument proteins have been identified in the alpha- and betaherpesvirus subfamilies of the Herpesviridae . These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

Acute HIV-1 infection is characterized by the rapid generation of highly diverse genetic variants to adapt to the new host environment. Understanding the dynamics of viral genetic variation at this stage of infection is critical for vaccine design efforts and early drug treatment. Here, using a high-resolution deep sequencing approach targeting the HIV-1 gag region, we reveal very early immune pressure with dramatic subpopulation shifts in a single acutely infected participant providing further insight into the genetic dynamics of acute HIV-1 infection.

Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCE Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (∼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.

Combination chemotherapy with multiple drugs has been widely applied to cancer treatment owing to enhanced efficacy and reduced drug resistance. For drug combination experiment analysis, response surface modeling has been commonly adopted. In this paper, we introduce a Hill-based global response surface model and provide an application of the model to a 512-run drug combination experiment with three chemicals, namely AG490, U0126, and indirubin-3 ′ -monoxime (I-3-M), on lung cancer cells. The results demonstrate generally improved goodness of fit of our model from the traditional polynomial model, as well as the original Hill model on the basis of fixed-ratio drug combinations. We identify different dose–effect patterns between normal and cancer cells on the basis of our model, which indicates the potential effectiveness of the drug combination in cancer treatment. Meanwhile, drug interactions are analyzed both qualitatively and quantitatively. The distinct interaction patterns between U0126 and I-3-M on two types of cells uncovered by the model could be a further indicator of the efficacy of the drug combination. Copyright © 2014 John Wiley & Sons, Ltd.

Motivation: Next-generation sequencing technologies sequence viruses with ultra-deep coverage, thus promising to revolutionize our understanding of the underlying diversity of viral populations. While the sequencing coverage is high enough that even rare viral variants are sequenced, the presence of sequencing errors makes it difficult to distinguish between rare variants and sequencing errors. Results: In this article, we present a method to overcome the limitations of sequencing technologies and assemble a diverse viral population that allows for the detection of previously undiscovered rare variants. The proposed method consists of a high-fidelity sequencing protocol and an accurate viral population assembly method, referred to as Viral Genome Assembler (VGA). The proposed protocol is able to eliminate sequencing errors by using individual barcodes attached to the sequencing fragments. Highly accurate data in combination with deep coverage allow VGA to assemble rare variants. VGA uses an expectation–maximization algorithm to estimate abundances of the assembled viral variants in the population. Results on both synthetic and real datasets show that our method is able to accurately assemble an HIV viral population and detect rare variants previously undetectable due to sequencing errors. VGA outperforms state-of-the-art methods for genome-wide viral assembly. Furthermore, our method is the first viral assembly method that scales to millions of sequencing reads. Availability: Our tool VGA is freely available at http://genetics.cs.ucla.edu/vga/ Contact: serghei@cs.ucla.edu; eeskin@cs.ucla.edu

We present VGA, an accurate method for viral quasispecies assembly from ultra-deep sequencing data. The proposed method consists of a high-fidelity sequencing protocol and an accurate method for viral quasispecies assembly, referred to as Viral Genome Assembler (VGA). The proposed protocol is able to eliminate sequencing errors by using individual barcodes attached to the sequencing fragments. Results on both synthetic and real datasets show that our method able to accurately assemble HIV viral quasispecies and detect rare quasispecies previously undetectable due to sequencing errors. VGA outperforms state-of-the-art methods for the viral assembly. Furthermore, our method is the first viral assembly method which scales to millions of sequencing reads. Our tool VGA is freely available at http://genetics.cs.ucla.edu/vga/

A novel genome-wide genetics platform is presented in this study, which permits functional interrogation of all point mutations across a viral genome in parallel. Here we generated the first fitness profile of individual point mutations across the influenza virus genome. Critical residues on the viral genome were systematically identified, which provided a collection of subdomain data informative for structure-function studies and for effective rational drug and vaccine design. Our data was consistent with known, well-characterized structural features. In addition, we have achieved a validation rate of 68% for severely attenuated mutations and 94% for neutral mutations. The approach described in this study is applicable to other viral or microbial genomes where a means of genetic manipulation is available.

Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.

Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients., Author Summary The emergence of drug resistance during antiviral treatment limits treatment options and poses challenges to pharmaceutical development. Meanwhile, the search for novel antiviral compounds with chemical genetic screens has led to the identification of antiviral agents with undefined drug mechanisms. Daclatasvir, an effective NS5A inhibitor, is one such example. In traditional methods to identify critical residues governing drug-protein interactions, wild type virus is passaged under drug treatment pressure, enabling the identification of resistant mutations evolved after multiple viral passages. However, this method only characterizes a fraction of the positively selected variants. Here we have simultaneously quantified the relative change in replication fitness as well as the relative sensitivity to Daclatasvir for all possible single amino acid mutations in the NS5A domain IA, thereby identifying the entire panel of positions that interact with the drug. Using mathematical models, we predicted which mutations pose the greatest risk of causing emergence of resistance under different scenarios of treatment compliance. The mutant fitness and drug-sensitivity profiles obtained can also inform the patient-specific use of Daclatasvir and may facilitate the development of second-generation drugs with a higher genetic barrier to resistance.

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt , must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.

The use of a liquid culture system such as MGIT broth has greatly improved the sensitivity of isolating mycobacteria in clinical laboratories. Microscopic visualization of acid fast bacilli (AFB) in the culture positive MGIT broth remains the first routine step for rapidly indicating the presence of mycobacteria. We modified an ultraviolet (UV) light fixation process to increase AFB cells adherence to the slide. The retained haze proportion of a 1-cm circle marked area on the smear slide was quantified after the staining procedure indicating the adherence degree of AFB cells. More AFB cells were preserved on the slide after exposure to UV light of either germicidal lamp or UV crosslinker in a time-dependent manner. We demonstrated both the bovine serum albumin (BSA) in MGIT media and UV light exposure were required for enhancing fixation of AFB cells. While applying to AFB stains for 302 AFB positive MGIT broths in clinics, more AFB cells were retained and observed on smear slides prepared by the modified fixation procedure rather than by the conventional method. The modified fixation procedure was thus recommended for improving the sensitivity of microscopic diagnosis of AFB cells in culture positive MGIT broth.

The planar motion of a slightly distorted sphere around a fixed sphere in an unbounded fluid is investigated by a perturbation approach. An approximate velocity potential is derived in terms of sets of singularities by using the successive potential method. In a relative coordinate system moving with the uniform stream, the kinetic energy of the fluid is expressed as a function of 15 added masses. Approximate analytical solutions of added masses in series form are obtained and applied to determine the trajectories of the slightly distorted sphere around a fixed sphere. The hydrodynamic interaction between two bodies is computed based on the dynamical equations of motion. It is found that the presence of a sphere generates an effect on the planar motion of the slightly distorted sphere and the initial configuration of the slightly distorted sphere has a decisive influence on the development of its subsequent rotational motion.

The hydrodynamic interaction between two cylinders with rotational motion through an inviscid and incompressible fluid is investigated theoretically. The dynamical behaviors of an elliptic cylinder...

Herpesviruses are characterized as having two distinct life cycle phases: lytic replication and latency. The mechanisms of latency establishment and maintenance, as well as the switch from latency to lytic replication, are poorly understood. Human gammaherpesviruses, including Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with lymphoproliferative diseases and several human tumors. Unfortunately, the lack of cell lines to support efficient de novo productive infection and restricted host ranges of EBV and HHV-8 make it difficult to explore certain important biological questions. Murine gammaherpesvirus 68 (MHV-68, or γHV68) can establish de novo lytic infection in a variety of cell lines and is also able to infect laboratory mice, offering an ideal model with which to study various aspects of gammaherpesvirus infection. Here we describe in vitro studies of the mechanisms of the switch from latency to lytic replication of MHV-68. An MHV-68 gene, rta (replication and transcription activator), encoded primarily by open reading frame 50 (ORF50), is homologous to the rta genes of other gammaherpesviruses, including HHV-8 and EBV. HHV-8 and EBV Rta have been shown to play central roles in viral reactivation from latency. We first studied the kinetics of MHV-68 rta gene transcription during de novo lytic infection. MHV-68 rta was predominantly expressed as a 2-kb immediate-early transcript. Sequence analysis of MHV-68 rta cDNA revealed that an 866-nucleotide intron 5′ of ORF50 was removed to create the Rta ORF of 583 amino acids. To test the functions of MHV-68 Rta in reactivation, a plasmid expressing Rta was transfected into a latently infected cell line, S11E, which was established from a B-cell lymphoma in an MHV-68-infected mouse. Rta induced expression of viral early and late genes, lytic replication of viral DNA, and production of infectious viral particles. We conclude that Rta alone is able to disrupt latency, activate viral lytic replication, and drive the lytic cycle to completion. This study indicates that MHV-68 provides a valuable model for investigating regulation of the balance between latency and lytic replication in vitro and in vivo.

The general planar motion of an elliptic cylinder through an inviscid and incompressible fluid in the vicinity of a fixed circular cylinder is investigated analytically. The velocity potential is derived by using the successive-image method and the perturbation method, and it is used to determine the hydrodynamic interactions between them. In a relative coordinate system moving with the uniform flow, the kinetic energy of the fluid is expressed as a function of 15 generalized added masses due to the motion of these two cylinders. The generalized added masses are then employed to determine the translational and rotational motion of the elliptic cylinder based on the dynamical equations of motion that are obtained from Lagrange’s equations of motion. Numerical results show that the initial configuration of the elliptic cylinder greatly affects its subsequent motions.

Human herpesvirus 8 (HHV-8) infection has been implicated in the etiology of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD), three diseases that frequently develop in immunocompromised, human immunodeficiency virus-positive individuals. One hypothesis that would account for different pathological manifestations of infection by the same virus is that viral genes are differentially expressed in heterogeneous cell types. To test this hypothesis, we analyzed the localization and levels of expression of two viral genes expressed in latent and lytic infections and the viral homologue of interleukin-6 (vIL-6). We show that PEL parallels KS in the pattern of latent and lytic cycle viral gene expression but that the predominant infected cell type is a B cell. We also show that MCD differs from KS not only in the infected cell type (B-cell and T-cell lineage) but also in the pattern of viral gene expression. Only a few cells in the lesion are infected and all of these cells express lytic-cycle genes. Of possibly greater significance is the fact that in a comparison of KS, PEL, and MCD, we found dramatic differences in the levels of expression of vIL-6. Interleukin-6 is a B-cell growth and differentiation factor whose altered expression has been linked to plasma cell abnormalities, as well as myeloid and lymphoid malignancies. Our findings support the hypothesis that HHV-8 plays an important role in the pathogenesis of PEL and MCD, in which vIL-6 acts as an autocrine or paracrine factor in the lymphoproliferative processes common to both.

Antibody-fused interferons as an effective approach to enhance target specificity and antiviral efficacy of type I interferons

Polycrystal samples of La1-xSrxMnO3 (x=0.2, 0.3) have been synthesized using a sol-gel method. The effect of sintering process on the sample microstructure and Sr doping on the magnetoresistance for La1-xSrxMnO3 have been investigated. The results show that the increase of sintering temperature and time can promote the crystallization of samples and reduce the content of impurity. The additive of Sr makes Mn ion in materials relatively compact, and this causes the distorted deformation of the materials structure. The magnetoresistance (MR) changes with the Sr doping. When x=0.2, the maximum MR% of sample is about 43% and keep about 40% between 10~150K. At room temperature, the MR% will decrease to about 6.5%. When x=0.3, the order magnetoresistance (OMR) effect occures above the room temperature.

Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.

A survey for antibodies to a recombinant small viral capsid antigen (sVCA) of human herpesvirus type 8 (HHV‐8) was conducted in Sardinia, one of the world's highest incidence areas for classic Kaposi's sarcoma (KS). Prevalence of antibodies to HHV‐8 sVCA was greatest in patients with KS (95%), followed by family members (39%) and a Sardinian control population age‐ and sex‐matched to the relatives (11%). Within families, prevalence of antibodies was about equal among spouses, children, and siblings of KS patients, a finding that raises the possibilities of intrafamilial person‐to‐person or vertical transmission. Antibodies were detected 2–3 times more frequently in males than in females. The data show that prevalence of antibodies to HHV‐8 sVCA correlates with the distribution of classic KS in a high‐ incidence area. Clustering of seroprevalence within some families suggests the presence of familial risk factors for active HHV‐8 infection.

An overload was applied during a fatigue-crack-growth experiment, resulting in a temporary decrease in crack-growth rates, i.e., a retardation period. Neutron diffraction was used to investigate the retardation phenomenon by mapping the changes in the lattice-strain profiles around the fatigue-crack tip in a series of compact-tension (CT) specimens, which were fatigued to various stages through the retardation period after the overload. Following the overload, compressive-strain fields were observed along the loading direction close to the crack tip. As the crack grows out of the retardation period, the residual compressive strains decreased.

Interconversion between the linear genome of Epstein-Barr virus (EBV) present in virions and intracellular circular EBV DNA is a novel DNA recombination process. A previously characterized DNA binding activity called terminal repeat or tandem repeat binding protein (TRBP) was found to recognize several G-rich recombinogenic sequences in the EBV genome and in cellular DNA. TRBP was also found to be an autoantigen recognized by sera from certain patients with undifferentiated connective-tissue disorders. Here the transcription factor Sp1 has been identified as a component of TRBP and has been shown to be an autoantigen. Sp1 bound to recombination junctions of EBV DNA, such as those in the terminal repeats and in the large internal repeats, as well as to recombinogenic regions of cellular DNA, such as variable-number tandem repeats and switch regions of the immunoglobulin genes. We defined the ends of the linear EBV genome present in virions and showed that Sp1 binds to the sequence (GGGGTGGGGCATGGG) within EBV terminal repeats at the precise locus of interconversion of linear and circular viral DNA. Sp1 may be involved in DNA recombination.

Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein–protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interactions between 7 HCV and 94 human proteins, over 40% of which have been linked to HCV infection by other studies. These interactions develop a more complete picture of HCV infection, providing insight into HCV manipulation of pathways, such as lipid and cholesterol metabolism, that were previously linked to HCV infection and implicating novel targets within microtubule-organizing centers, the complement system and cell cycle regulatory machinery. In an effort to understand the relationship between HCV and related viruses, we compared the HCV 2a interactome to those of other HCV genotypes and to the related dengue virus. Greater overlap was observed between HCV and dengue virus targets than between HCV genotypes, demonstrating the value of parallel screening approaches when comparing virus–host cell interactomes. Using siRNAs to inhibit expression of cellular proteins, we found that five of the ten shared targets tested (CUL7, PCM1, RILPL2, RNASET2, and TCF7L2) were required for replication of both HCV and dengue virus. These shared interactions provide insight into common features of the viral life cycles of the family Flaviviridae.

Complement activation and inflammation have been suggested in the pathogenesis of stroke, mannose-binding lectin (MBL) were found to have roles during the process. The aim of this study was to investigate the relationship between acute ischemic stroke (AIS) and serum MBL levels in Chinese population. From January 1 to June 30 2013, all patients with first-ever AIS were recruited to participate in the study. Serum MBL levels and routine test were examined. The National Institutes of Health Stroke Scale (NIHSS) score was assessed on admission blinded to MBL levels. During the inclusion period, 148 patients with AIS were registered and completed study. The results indicated that the serum MBL levels were significantly (p

Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments.

Cellular contrast was demonstrated for the first time in lensfree on-chip holography by employing antibody-conjugated gold and silver nanoparticles (NPs) as contrast agents. Cytometric differentiation of CD4+ and CD8+ lymphocytes was achieved, after NP labeling, using machine learning algorithms.

Background Neck pain caused by cervical spondylosis (CS) has become one of the most common health problems around the world. Electroacupuncture (EA) has been employed to relieve CS neck pain, but there is limited clinical evidence for its effectiveness. Methods/Design This study consists of a randomized controlled trial (RCT) with two parallel arms: an acupuncture group and an EA group. Both groups will receive acupuncture at Jing-jiaji points for 30 minutes each time, for five sessions per week for a total of 20 sessions during this four-week period. In addition, the EA group will be connected with EA apparatus. The following outcome measurements will be used in examination of subjects: the Northwick Park Neck Pain Questionnaire (NPQ), McGill Pain Questionnaire (MPQ), and Short-Form 36 (SF-36) scale. All these outcomes will be examined at the start of the study, at the end of the second week, at four weeks after randomization, and one and three months after treatment cessation respectively. Discussion This study aims to assess the efficacy of EA, compared with acupuncture intervention at Jing-jiaji points for the CS neck pain. Trial registration Chinese Clinical Trials Register:ChiCTR-TRC-13003422.

Data on the epidemiology of hypertension in Chinese non-dialysis chronic kidney disease (CKD) patients are limited. The aim of the present study was to investigate the prevalence, awareness, treatment, and control of hypertension in the non-dialysis CKD patients through a nationwide, multicenter study in China.The survey was performed in 61 tertiary hospitals in 31 provinces, municipalities, and autonomous regions in China (except Hong Kong, Macao, and Taiwan). Trained physicians collected demographic and clinical data and measured blood pressure (BP) using a standardized protocol. Hypertension was defined as systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg, and/or use of antihypertensive medications. BP140/90 mmHg and130/80 mmHg were used as the 2 thresholds of hypertension control. In multivariate logistic regression with adjustment for sex and age, we analyzed the association between CKD stages and uncontrolled hypertension in non-dialysis CKD patients.The analysis included 8927 non-dialysis CKD patients. The prevalence, awareness, and treatment of hypertension in non-dialysis CKD patients were 67.3%, 85.8%, and 81.0%, respectively. Of hypertensive CKD patients, 33.1% and 14.1% had controlled BP to140/90 mmHg and130/80 mmHg, respectively. With successive CKD stages, the prevalence of hypertension in non-dialysis CKD patients increased, but the control of hypertension decreased (P0.001). When the threshold of BP130/80 mmHg was considered, the risk of uncontrolled hypertension in CKD 2, 3a, 3b, 4, and 5 stages increased 1.3, 1.4, 1.4, 2.5, and 4.0 times compared with CKD 1 stage, respectively (P0.05). Using the threshold of140/90 mmHg, the risk of uncontrolled hypertension increased in advanced stages (P0.05).The prevalence of hypertension Chinese non-dialysis CKD patients was high, and the hypertension control was suboptimal. With successive CKD stages, the risk of uncontrolled hypertension increased.

Background Acinetobacter baumannii hospital-acquired pneumonia (HAP) is associated with a high mortality worldwide. Non-ventilated patients with HAP (NVHAP) caused by nosocomial pathogens are reported to have a more favorable outcome than those with ventilator-associated pneumonia (VAP). The current study was designed to determine whether bacteremic patients with A. baumannii NVHAP also have a lower mortality than those receiving assisted ventilation. Methods This retrospective 10-year study was conducted at a 2900-bed teaching hospital located in Northern Taiwan. The population consisted of 144 patients with A. baumannii bacteremia and HAP. Of these 96 had VAP and 48 had NVHAP. Charts were reviewed for demographic characteristics, comorbidities, clinical manifestations, antimicrobial susceptibility, and 14-day mortality. Clonal relationships were determined by molecular typing. Results There were no significant differences between the two groups in comorbidities (Charlson scores). Patients with NVHAP were more likely to have developed bacteremia earlier, outside the ICU and undergone fewer invasive procedures. They had significantly lower APACHE II scores, fewer bilateral pneumonias and lower rates of antimicrobial resistance. No specific clones were identified in either group. The unadjusted (crude) 14-day mortality rates were not significantly different between the groups (NVHAP 43.8% vs. VAP 31.3%, p = 0.196). The adjusted 14-day mortality risk was significantly lower in ventilator-assisted patients (odds ratio = 0.201; 95% confidence interval = 0.075-0.538; p = 0.001). Conclusions Patients with bacteremic NVHAP and VAP caused by A. baumannii had similar crude mortality rates, but on logistic regression analysis those receiving ventilator assistance had a significantly lower mortality. This may have been due to better airway protection, more intensive monitoring with earlier diagnosis and treatment in patients with VAP, greater innate susceptibility to infection in those with NVHAP and differences in the virulence of A. baumannii.

Computational microscopy tools, in particular lensfree on-chip imaging, provide a large field-of-view along with a long depth-of-field, which makes it feasible to rapidly analyze large volumes of specimen using a compact and light-weight on-chip imaging architecture. To bring molecular specificity to this high-throughput platform, here we demonstrate the use of plasmon-resonant metallic nanoparticles to automatically recognize different cell types based on their plasmon-enhanced lensfree holograms, detected and reconstructed over a large field-of-view of e.g., ~24 mm².