Cystathionine gamma-lyase/H2 S signaling facilitates myogenesis under aging and injury condition.

Hydrogen sulfide (H2 S) can be endogenously produced and belongs to the class of signaling molecules known as gasotransmitters. Cystathionine gamma-lyase (CSE)-derived H2 S is implicated in the regulation of cell differentiation and the aging process, but the involvements of the CSE/H2 S system in myogenesis upon aging and injury have not been explored. In this study, we demonstrated that CSE acts as a major H2 S-generating enzyme in skeletal muscles and is significantly down-regulated in aged skeletal muscles in mice. CSE deficiency exacerbated the age-dependent sarcopenia and cardiotoxin-induced injury/regeneration in mouse skeletal muscle, possibly attributed to inefficient myogenesis. In contrast, supplement of NaHS (an H2 S donor) induced the expressions of myogenic genes and promoted muscle regeneration in mice. In vitro, incubation of myoblast cells (C2C12) with H2 S promoted myogenesis, as evidenced by the inhibition of cell cycle progression and migration, altered expressions of myogenic markers, elongation of myoblasts, and formation of multinucleated myotubes. Myogenesis was also found to upregulate CSE expression, while blockage of CSE/H2 S signaling resulted in a suppression of myogenesis. Mechanically, H2 S significantly induced the heterodimer formation between MEF2c and MRF4 and promoted the binding of MEF2c/MRF4 to myogenin promoter. MEF2c was S-sulfhydrated at both cysteine 361 and 420 in the C-terminal transactivation domain, and blockage of MEF2c S-sulfhydration abolished the stimulatory role of H2 S on MEF2c/MRF4 heterodimer formation. These findings support an essential role for H2 S in maintaining myogenesis, presenting it as a potential candidate for the prevention of age-related sarcopenia and treatment of muscle injury.

Creation of superposition of arbitrary states encoded in two high-Q cavities.

The principle of superposition is a key ingredient for quantum mechanics. A recent work [Phys. Rev. Lett.116, 110403 (2016)10.1103/PhysRevLett.116.110403] has shown that a quantum adder that deterministically generates a superposition of two unknown states is forbidden. Here we consider the implementation of the probabilistic quantum adder in the 3D cavity-transmon system. Our implementation is based on a three-level superconducting transmon qubit dispersively coupled to two cavities. Numerical simulations show that high-fidelity generation of the superposition of two coherent states is feasible with current circuit QED technology. Our method also works for other physical systems such as two optical cavities coupled to a three-level atom or two nitrogen-vacancy center ensembles interacted with one three-level superconducting flux qubit.

RNA disruption indicates CHOP therapy efficacy in canine lymphoma.

BACKGROUND: Assessment of the efficacy of a multi-agent chemotherapy protocol in which cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are administered in canine lymphoma is generally performed by physical measurement of lymph node diameter. However, no consistent correlation has been made with prognostic indicators and the length or absence of clinical remission based on lymph node size. RNA disruption measured mid-therapy has been correlated with increased disease-free survival in recent studies of human cancer and was assessed in this study of canine lymphoma patients. Fine needle aspirate samples were taken before treatment and at weeks 3, 6, and 11 of CHOP therapy. RNA was isolated from these samples and assessed using an Agilent Bioanalyzer. RNA disruption assay (RDA) analysis was performed on the data from the resulting electropherograms. RESULTS: An increased RNA disruption index (RDI) score was significantly associated with improved progression-free survival. CONCLUSIONS: Predicting the risk of early relapse during chemotherapy could benefit veterinary patients by reducing ineffective treatment and could allow veterinary oncologists to switch earlier to a more effective drug regimen.

An Adaptive Track Segmentation Algorithm for a Railway Intrusion Detection System.

Video surveillance-based intrusion detection has been widely used in modern railway systems. Objects inside the alarm region, or the track area, can be detected by image processing algorithms. With the increasing number of surveillance cameras, manual labeling of alarm regions for each camera has become time-consuming and is sometimes not feasible at all, especially for pan-tilt-zoom (PTZ) cameras which may change their monitoring area at any time. To automatically label the track area for all cameras, video surveillance system requires an accurate track segmentation algorithm with small memory footprint and short inference delay. In this paper, we propose an adaptive segmentation algorithm to delineate the boundary of the track area with very light computation burden. The proposed algorithm includes three steps. Firstly, the image is segmented into fragmented regions. To reduce the redundant calculation in the evaluation of the boundary weight for generating the fragmented regions, an optimal set of Gaussian kernels with adaptive directions for each specific scene is calculated using Hough transformation. Secondly, the fragmented regions are combined into local areas by using a new clustering rule, based on the region's boundary weight and size. Finally, a classification network is used to recognize the track area among all local areas. To achieve a fast and accurate classification, a simplified CNN network is designed by using pre-trained convolution kernels and a loss function that can enhance the diversity of the feature maps. Experimental results show that the proposed method finds an effective balance between the segmentation precision, calculation time, and hardware cost of the system.

High-Speed Railway Intruding Object Image Generating with Generative Adversarial Networks.

Foreign object intrusion is a great threat to high-speed railway safety operations. Accurate foreign object intrusion detection is particularly important. As a result of the lack of intruding foreign object samples during the operational period, artificially generated ones will greatly benefit the development of the detection methods. In this paper, we propose a novel method to generate railway intruding object images based on an improved conditional deep convolutional generative adversarial network (C-DCGAN). It consists of a generator and multi-scale discriminators. Loss function is also improved so as to generate samples with a high quality and authenticity. The generator is extracted in order to generate foreign object images from input semantic labels. We synthesize the generated objects to the railway scene. To make the generated objects more similar to real objects, on scale in different positions of a railway scene, a scale estimation algorithm based on the gauge constant is proposed. The experimental results on the railway intruding object dataset show that the proposed C-DCGAN model outperforms several state-of-the-art methods and achieves a higher quality (the pixel-wise accuracy, mean intersection-over-union (mIoU), and mean average precision (mAP) are 80.46%, 0.65, and 0.69, respectively) and diversity (the Fréchet-Inception Distance (FID) score is 26.87) of generated samples. The mIoU of the real-generated pedestrian pairs reaches 0.85, and indicates a higher scale of accuracy for the generated intruding objects in the railway scene.

One-step implementation of a hybrid Fredkin gate with quantum memories and single superconducting qubit in circuit QED and its applications.

In a recent remarkable experiment [Sci. Adv. 2, e1501531 (2016)], a 3-qubit quantum Fredkin (i.e., controlled-SWAP) gate was demonstrated by using linear optics. Here we propose a simple experimental scheme by utilizing the dispersive interaction in superconducting quantum circuit to implement a hybrid Fredkin gate with a superconducting flux qubit as the control qubit and two separated quantum memories as the target qudits. The quantum memories considered here are prepared by the superconducting coplanar waveguide resonators or nitrogen-vacancy center ensembles. In particular, it is shown that this Fredkin gate can be realized using a single-step operation and more importantly, each target qudit can be in an arbitrary state with arbitrary degrees of freedom. Furthermore, we show that this experimental scheme has many potential applications in quantum computation and quantum information processing such as generating arbitrary entangled states (discrete-variable states or continuous-variable states) of the two memories, measuring the fidelity and the entanglement between the two memories. With state-of-the-art circuit QED technology, the numerical simulation is performed to demonstrate that two-memory NOON states, entangled coherent states, and entangled cat states can be efficiently synthesized.

Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States.

BACKGROUND: Emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) in North America, Asia and Europe has caused severe economic loss to the global swine industry. However, the virome of PEDV infected pigs and its effect on disease severity remains unknown. The advancements of sequencing technology have made it possible to characterize the entire microbiome of different body sites for any host. METHODS: The objective of this study was to characterize the RNA virome in PEDV-positive pigs using the hypothesis-free metagenomics approach based on next-generation sequencing. Specifically, 217 PEDV-positive swine fecal swab samples collected from diarrheic piglets over 17 US states during 2015-2016 were analyzed. RESULTS: A Kraken algorithm-based bioinformatics analysis revealed the presence of up to 9 different RNA genera besides PEDV (Alphacoronavirus genus), including Mamastrovirus (52%, 113/217), Enterovirus (39%, 85/217), Sapelovirus (31%, 67/217), Posavirus (30%, 66/217), Kobuvirus (23%, 49/217), Sapovirus (13%, 28/217), Teschovirus (10%, 22/217), Pasivirus (9%, 20/217), and Deltacoronavirus (3%, 6/217). There were 58 out of 217 piglets (27%) have PEDV infection alone whereas the remaining 159 (73%) shed 2 up to 9 different viruses. CONCLUSION: These findings demonstrated that PEDV infected diarrheic pigs had an extensive RNA viral flora consisting of four different families: Astroviridae, Picornaviridae, Caliciviridae, and Coronaviridae.

Dexamethasone treatment did not exacerbate Seneca Valley virus infection in nursery-age pigs.

BACKGROUND: Senecavirus A, commonly known as Seneca Valley virus (SVV), is a picornavirus that has been infrequently associated with porcine idiopathic vesicular disease (PIVD). In late 2014 there were multiple PIVD outbreaks in several states in Brazil and samples from those cases tested positive for SVV. Beginning in July of 2015, multiple cases of PIVD were reported in the United States in which a genetically similar SVV was also detected. These events suggested SVV could induce vesicular disease, which was recently demonstrated with contemporary US isolates that produced mild disease in pigs. It was hypothesized that stressful conditions may exacerbate the expression of clinical disease and the following experiment was performed. Two groups of 9-week-old pigs were given an intranasal SVV challenge with one group receiving an immunosuppressive dose of dexamethasone prior to challenge. After challenge animals were observed for the development of clinical signs and serum and swabs were collected to study viral shedding and antibody production. In addition, pigs were euthanized 2, 4, 6, 8, and 12 days post inoculation (dpi) to demonstrate tissue distribution of virus during acute infection. RESULTS: Vesicular disease was experimentally induced in both groups with the duration and magnitude of clinical signs similar between groups. During acute infection [0-14 days post infection (dpi)], SVV was detected by PCR in serum, nasal swabs, rectal swabs, various tissues, and in swabs from ruptured vesicles. From 15 to 30 dpi, virus was less consistently detected in nasal and rectal swabs, and absent from most serum samples. Virus neutralizing antibody was detected by 5 dpi and lasted until the end of the study. CONCLUSION: Treatment with an immunosuppressive dose of dexamethasone did not drastically alter the clinical disease course of SVV in experimentally infected nursery aged swine. A greater understanding of SVV pathogenesis and factors that could exacerbate disease can help the swine industry with control and prevention strategies directed against this virus.

Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A.

Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. We report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions.

Role of autophagy and lysosomal drug sequestration in acquired resistance to doxorubicin in MCF-7 cells.

BACKGROUND: The roles and mechanisms involved in starvation-induced autophagy in mammalian cells have been extensively studied. However, less is known about the potential role for autophagy as a survival pathway in acquired drug resistance in cancer cells under nutrient-rich conditions. METHODS: We selected MCF-7 breast tumor cells for survival in increasing concentrations of doxorubicin and assessed whether the acquisition of doxorubicin resistance was accompanied by changes in doxorubicin and lysosome localization and the activation of autophagy, as assessed by laser scanning confocal microscopy with or without immunohistochemical approaches. The ultrastructure of cells was also viewed using transmission electron microscopy. Cellular levels of autophagy and apoptosis-related proteins were assessed by immunoblotting techniques, while protein turnover was quantified using a flux assay. RESULTS: As cells acquired resistance to doxorubicin, the subcellular location of the drug moved from the nucleus to the perinuclear region. The location of lysosomes and autophagosomes also changed from being equally distributed throughout the cytoplasm to co-localizing with doxorubicin in the perinuclear region. There was an apparent temporal correlation between the acquisition of doxorubicin resistance and autophagy induction, as measured by increases in monodansylcadaverine staining, LC3-II production, and co-localization of LAMP1 and LC3-II immunofluorescence. Electron microscopy revealed an increase in cytoplasmic vacuoles containing mitochondria and other cellular organelles, also suggestive of autophagy. Consistent with this view, a known autophagy inhibitor (chloroquine) was highly effective in restoring doxorubicin sensitivity in doxorubicin-resistant cells. Moreover, this induction of autophagy correlated temporally with increased expression of the selective cargo receptor p62, which facilitates the delivery of doxorubicin-damaged mitochondria and other organelles to autophagosomes. Finally, we suggest that autophagy associated with doxorubicin resistance may be distinct from classical starvation-induced autophagy, since Beclin 1 and Atg7 expression did not change upon acquisition of doxorubicin resistance, nor did recombinant Bcl2 overexpression or an Atg7 knockdown alter doxorubicin cytotoxicity. CONCLUSION: Taken together, our findings suggest that doxorubicin resistance in MCF-7 breast cancer cells is mediated, at least in part, by the activation of autophagy, which may be distinct from starvation-induced autophagy.

RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.

BACKGROUND: Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced "RNA disruption" is, the extent to which it is associated with drug response or what the underlying mechanisms are. METHODS: Ovarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor. RESULTS: All chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3'-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption. CONCLUSIONS: Supporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.

Tumor RNA disruption predicts survival benefit from breast cancer chemotherapy.

In a prior substudy of the CAN-NCIC-MA.22 clinical trial (ClinicalTrials.gov identifier NCT00066443), we observed that neoadjuvant chemotherapy reduced tumor RNA integrity in breast cancer patients, a phenomenon we term "RNA disruption." The purpose of the current study was to assess in the full patient cohort the relationship between mid-treatment tumor RNA disruption and both pCR post-treatment and, subsequently, disease-free survival (DFS) up to 108 months post-treatment. To meet these objectives, we developed the RNA disruption assay (RDA) to quantify RNA disruption and stratify it into 3 response zones of clinical importance. Zone 1 is a level of RNA disruption inadequate for pathologic complete response (pCR); Zone 2 is an intermediate level, while Zone 3 has high RNA disruption. The same RNA disruption cut points developed for pCR response were then utilized for DFS. Tumor RDA identified >fourfold more chemotherapy non-responders than did clinical response by calipers. pCR responders were clustered in RDA Zone 3, irrespective of tumor subtype. DFS was about 2-fold greater for patients with tumors in Zone 3 compared to Zone 1 patients. Kaplan-Meier survival curves corroborated these findings that high tumor RNA disruption was associated with increased DFS. DFS values for patients in zone 3 that did not achieve a pCR were similar to that of pCR recipients across tumor subtypes, including patients with hormone receptor positive tumors that seldom achieve a pCR. RDA appears superior to pCR as a chemotherapy response biomarker, supporting the prospect of its use in response-guided chemotherapy.

Experimental Seneca Valley virus infection in sows and their offspring.

Neonatal mortality has been increasingly reported on swine breeding farms experiencing swine idiopathic vesicular disease (SIVD) outbreaks, which can be accompanied by lethargy, diarrhea, and neurologic signs in neonates. Seneca Valley Virus (SVV), or Senecavirus A, has been detected in clinical samples taken from pigs with SIVD. Experimental SVV inoculation has caused vesicular disease in pigs, particularly during the stages from weaning to finishing. However, it remains crucial to investigate whether SVV directly contributes to the increase in neonatal mortality rates. The following study was conducted to chronicle the pathogenesis of SVV infection in sows and their offspring. Ten sows were intranasally inoculated with 4.75 × 107 plaque-forming units of the virus per sow either late in gestation (n = 5) or within fourteen days of farrowing (n = 5). Each sow replicated SVV following intranasal inoculation, but only one out of ten sows developed a vesicular lesion on the snout. Evidence of transplacental infection was observed in two litters, and an additional two litters became infected following parturition out of five litters from sows inoculated in late gestation. No clinical signs were observed in the infected neonates. Likewise, no clinical signs were observed in the other five litters inoculated after farrowing, although each piglet did replicate the challenge virus. In this study, the experimental challenge of SVV did not result in neonatal mortality in contrast to observations in the field; however, it has shed light on the pathogenesis of the virus, the transmission of SVV between sows and their offspring, and host immune response that can help shape control measures in the field.

Utility of Feathers for Avian Influenza Virus Detection in Commercial Poultry.

The present study evaluated the potential utility of feather samples for the convenient and accurate detection of avian influenza virus (AIV) in commercial poultry. Feather samples were obtained from AIV-negative commercial layer facilities in Iowa, USA. The feathers were spiked with various concentrations (106 to 100) of a low pathogenic strain of H5N2 AIV using a nebulizing device and were evaluated for the detection of viral RNA using a real-time RT-PCR assay immediately or after incubation at -20, 4, 22, or 37 °C for 24, 48, or 72 h. Likewise, cell culture medium samples with and without the virus were prepared and used for comparison. In the spiked feathers, the PCR reliably (i.e., 100% probability of detection) detected AIV RNA in eluates from samples sprayed with 103 EID50/mL or more of the virus. Based on half-life estimates, the feathers performed better than the corresponding media samples (p < 0.05), particularly when the samples were stored at 22 or 37 °C. In conclusion, feather samples can be routinely collected from a poultry barn as a non-invasive alternative to blood or oropharyngeal-cloacal swab samples for monitoring AIV.

RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells.

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed "RNA disruption". Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells.

INTRODUCTION: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance. METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR). RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways. CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.

Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization.

BACKGROUND: Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. METHODS: To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. RESULTS: Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the "hit list" was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. CONCLUSIONS: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug's properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization.

Sequence analysis of the NSP2, ORF5, and ORF7 genes of 11 PRRS virus isolates from China.

During a tracing investigation of blue ear disease in China conducted from January to November 2008, 11 porcine reproductive and respiratory syndrome virus (PRRSV) isolates were collected from eight provinces including Liaoning, Jilin, Hebei, Shandong, Henan, Shanghai, Zhejiang, and Guangxi. The complete gene sequences of the NSP2, ORF5, and ORF7 genes from these 11 PRRSV isolates were amplified, cloned and detected by RT-PCR and then compared with the published sequences of other strains. The results showed that all of the isolates genotypically belonged to an American strain, but shared high homology with JXA1, the highly pathogenic strain endemic to China. All of the 11 PRRSV isolates demonstrated a 90-nucleotide deletion in the NSP2 gene, suggesting that the main epidemic of PRRSV in 2008 was due to this gene deletion isolate. More consistent mutations were found in specific regions of the ORF5 and ORF7 genes of the 11 PRRSV isolates, such as the signal peptide and transmembrane regions of GP5 and the Pat 7 motif of the N protein. Whether these mutations influence nuclear localization of PRRSV requires confirmation by future studies.

Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library. RESULTS: Major changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold. CONCLUSIONS: The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

Evaluation of PCR-based hemagglutinin subtyping as a tool to aid in surveillance of avian influenza viruses in migratory wild birds.

The surveillance of migratory wild birds (MWBs) for avian influenza virus (AIV) allows detecting the emergence of highly pathogenic AIV that can infect domestic poultry and mammals, new subtypes, and antigenic/genetic variants. The current AIV surveillance system for MWBs in the United States is based on virus isolation (VI) followed by sequencing isolates. This system primarily focuses on the early detection of H5 and H7 AIVs. However, it is suboptimal in assessing diverse AIV subtypes at any given time because of the low VI success rate. To improve such a shortfall, a SYBR® Green-based real-time reverse transcription-polymerase chain reaction (rtRT-PCR) panel was developed for direct HA subtyping of AIVs in oropharyngeal-cloacal (OPC) swabs from MWBs. Under optimal conditions, the PCR panel detected AIVs of all 16 different HA subtypes with an average limit of detection of 102.6 copies/reaction (2 µl of extract). In testing 90 OPC swabs from 13 MWB species, the PCR provided a significantly faster turnaround of results and demonstrated the presence of more subtypes and concurrent infection among MWBs compared to what the current surveillance testing algorithm showed. In conclusion, newly developed SYBR® Green rtRT-PCR panel can be a useful tool for monitoring MWBs for AIVs.