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Previous studies have shown that West Nile (Sarafend) virus matured by budding at the plasma membrane, which differs from the usual intracellular maturation of other flaviviruses. The present study investigated the trafficking mechanism of the envelope (E) and capsid (C) proteins of West Nile (Sarafend) virus during the replication cycle. The use of time-based double-immunofluorescence labelling coupled with the Triton X-100 extraction procedure revealed that both the E and C proteins were transported from the perinuclear region towards the plasma membrane along the microtubules simultaneously. The strong association of these virus proteins with the microtubules was demonstrated further with Triton X-100 extraction procedure coupled with double immunogold-labelling. Extraction of infected cells with Triton X-100 in high salt also revealed that virus E proteins were associated with the microtubules via protein-protein interaction. The disruption of microtubules with vinblastine sulphate inhibited the trafficking of both the virus E and C proteins. Both virus structural proteins were observed to co-localise and retained within vinblastine sulphate-induced microtubulin paracrystals. Extracellular virus production was also reduced drastically by vinblastine sulphate at non-cytotoxic concentration. Subsequent studies revealed that the transportation of virus E protein was associated with the microtubules-based motor protein, kinesin.

We report a technique for generation of multiple energy bandgaps using a combination of one-step gray mask lithography and low-energy arsenic ion implantation induced disordering. Using this technique, we have successfully integrated 12-section with variable energy bandgaps on a single InGaAs/InGaAsP laser heterostructure. When compared to conventional processes, this novel technique is simple, promising and cost effective.

In this work, we study the regional dependence of transport behavior of microalgae Chlorella vulgaris inside microfluidic channel on applied fluid flow rate. The microalgae are treated as spherical naturally buoyant particles. Deviation from the normal diffusion or Brownian transport is characterized based on the scaling behavior of the mean square displacement (MSD) of the particle trajectories by resolving the displacements in the streamwise (flow) and perpendicular directions. The channel is divided into three different flow regions, namely center region of the channel and two near-wall boundaries and the particle motions are analyzed at different flow rates. We use the scaled Brownian motion to model the transitional characteristics in the scaling behavior of the MSDs. We find that there exist anisotropic anomalous transports in all the three flow regions with mixed sub-diffusive, normal and super-diffusive behavior in both longitudinal and transverse directions.

Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC). NK cells responded by cytokine production and the lysis of target cells. Notably, in the absence of significant monokine production by DenV-infected DC, it was the combination of type I IFNs and TNF-α produced by DenV-infected DC that was important for stimulating the IFN-γ and cytotoxic responses of NK cells. Cell-bound factors enhanced NK cell IFN-γ production. In particular, reduced HLA class I expression was observed on DenV-infected DC, and IFN-γ production was enhanced in licensed/educated NK cell subsets. NK–DC cell contact was also identified as a requirement for a cytotoxic response, and there was evidence for both perforin/granzyme as well as Fas/Fas ligand–dependent pathways of killing by NK cells. In summary, our results have uncovered a previously unappreciated role for the combined effect of type I IFNs, TNF-α, and cell surface receptor–ligand interactions in triggering the antidengue response of primary human NK cells.

Background: Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, ∼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort. Methods: PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response. Results: PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1β, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P

Objectives:Continuous positive airway pressure (CPAP) has become the standard of care for obstructive sleep apnea (OSA). However, CPAP compliance has been shown to be poor and surgical correction of upper airway may be an option for selected patients. The study aimed to assess the outcomes of patients presenting to sleep disorder clinic with OSA.Methods:Subjects were identified by reviewing sleep laboratory records of patients presented for full polysomnography from 2010 to 2014. Case notes were traced and data collected retrospectively. The treatment prescribed include weight reduction, CPAP, surgery and oral appliance. The outcomes of treatment options offered were analyzed and full polysomnography parameters before and after treatment were compared.Results:Less than 20% of patients underwent surgical correction of the upper airway. Less than 50% of the patients used an oral appliance. More than 60% of patients showed improvement in the respiratory disturbance index (RDI) after the surgery. About 80% of...

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) modulates food intake and body weight under physiological and pathological conditions by acting on the hypothalamus and brainstem. When overexpressed in disease, such as in advanced cancer, elevated serum MIC-1/GDF15 levels lead to an anorexia/cachexia syndrome. To gain a better understanding of its actions in the brainstem we studied MIC-1/GDF15 induced neuronal activation identified by induction of Fos protein. Intraperitoneal injection of human MIC-1/GDF15 in mice activated brainstem neurons in the area postrema (AP) and the medial (m) portion of the nucleus of the solitary tract (NTS), which did not stain with tyrosine hydroxylase (TH). To determine the importance of these brainstem nuclei in the anorexigenic effect of MIC-1/GDF15, we ablated the AP alone or the AP and the NTS. The latter combined lesion completely reversed the anorexigenic effects of MIC-1/GDF15. Altogether, this study identified neurons in the AP and/or NTS, as being critical for the regulation of food intake and body weight by MIC-1/GDF15.

The liver is a multifunctional organ that is involved in a number of critical excretory, synthetic, and metabolic functions. Biochemical assessment of these functions in children involves a number of tests performed in clinical laboratories. Many of the most commonly utilized serum chemistry tests, such as the aminotransferase and alkaline phosphatase (AP) levels, are often referred to as liver function tests, which is a misnomer as these do not actually measure or indicate liver function. Rather, these tests should be referred to as liver enzyme tests, with the term liver function tests reserved for true measures of hepatocyte synthetic function such as serum albumin levels and the prothrombin time (PT) or international normalized ratio. Any single biochemical test provides limited information that must be placed in the context of the entire clinical and historical picture. Currently available laboratory evaluative tests of the liver are used to: (1) screen for and document liver injury; (2) identify the type or pattern of liver disorder and the site of injury; (3) make a prognosis and follow-up children with chronic liver disease; and (4) serially monitor the course of liver disease, evaluate the response to treatment, and adjust a treatment regimen, when appropriate. The widespread availability and frequent use of serum chemistry tests in children have resulted in an increase in the number of both normal and abnormal liver chemistry test values that must be evaluated by physicians. However, some limitations of liver biochemical tests must be recognized. First, screening laboratory tests may lack sensitivity. That is, if a liver chemistry test is normal, it does not ensure that the patient is free of liver disease. Children with chronic liver disease can have normal serum aminotransferase levels. Second, these tests are not specific for liver dysfunction. For example, serum aminotransferases may be elevated in patients with a non-hepatic disorder such as a musculoskeletal condition or cardiomyopathy. Finally, liver chemistry tests rarely provide a specific diagnosis; rather, they suggest a general category of liver disorder. For example, abnormal liver biochemical tests do not distinguish viral hepatitis from autoimmune hepatitis, or delineate intrahepatic from extrahepatic etiologies of cholestasis.

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the syntheticβ-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can developin vivoduring treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81B. pseudomalleiisolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility inB. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81B. pseudomallei, six multidrug resistant (MDR) isolates carriedbpeB,amrB, andBPSS1119andpenAgenes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B).

Background / Aims : Plasminogen directs matrix proteolysis during liver repair. Based on the role of hepatic stellate cells (HSCs) on matrix production, we investigated whether plasminogen-driven matrix proteolysis modulates the phenotype of HSCs. Methods : Carbon tetrachloride was injected intraperitoneally into mice deficient in plasminogen, fibrinogen, or both, and to normal littermates, followed by determination of the phenotype of HSCs, matrix deposition, and apoptosis. Results : Activation of HSCs was restricted to the zone of injury and increased >ten-fold above baseline regardless of the plasminogen status 2 days after toxin. Thereafter, the number of activated HSCs decreased to baseline levels between 7 and 14 days in normal mice, but remained elevated in plasminogen-deficient livers ∼ten-fold above non-targeted littermates. Despite the zonal increase in activated HSCs, the total number of desmin-stained HSCs was similar along the lobule in both genotypes. No appreciable difference in apoptosis of perisinusoidal cells was found between genotypes; however, fibrillary material was present in the subsinusoidal space of Plg 0 livers. This fibrillary material was not fibrin, as demonstrated by similar findings in Plg 0 /Fib 0 mice, which accumulated fibronectin in injured areas. Conclusions : Proteolytic clearance of non-fibrin matrix components by plasminogen must occur for HSCs to restore the quiescent phenotype during liver repair.

Two dengue 2-specific IgM monoclonal antibodies (MAb) recognised spatially unrelated epitopes on the envelope (E) protein of dengue 2 virus, which were also recognised by serum from 20 and 50%, respectively, of patients with a primary dengue 2 infection. Dengue 2 virus populations escaping neutralisation by MAb 6B2 (representing the majority population of dengue 2-specific IgM MAbs ) had a deduced amino acid change (G-S) in the pre-Membrane (prM) protein at position 15 and a second in the E protein at E311 (E-G). The change in the E protein was adjacent to the only other epitopes on dengue 2 virus (E307, E383-385) involved in neutralisation that have been identified but that were recognised by IgG antibodies. Dengue 2 virus escaping neutralisation by IgM MAb 10F2, representing the minority population of dengue 2-specific IgM MAbs, had the same deduced amino acid change (G-S) at prM15 as the 6B2 neutralisation escape mutant dengue 2 virus population and four deduced amino acid changes in the E protein (E69, T-I, in the glycosylation motif; E71, E-D; E112, S-G; E124, I-N), which may be close enough to each other to form a single epitope and a fifth at E402 (F-L) in a region of the E protein of TBE virus essential for the low pH-induced E protein dimer-trimer transition. The 10F2 neutralisation escape mutant, but not the 6B2 one, had lost its ability to cause fusion from within Aedes albopictus mosquito cells and was inactivated more rapidly than the 6B2 neutralisation escape mutant and wild type viruses at 42°C. Dengue 2 viruses passaged in BHK cells in the absence of a selecting antibody, shared a common amino acid (S) at E53, which differed from both wild type and neutralisation escape mutant virus populations at this position (P) and may have been responsible for a significant reduction in the ability of these “passage control” virus populations to be neutralised by both 6B2 and 10F2 antibodies. J. Med. Virol. 65:315–323, 2001. © 2001 Wiley-Liss, Inc.

The study reported here employed a 2 (wheelchair-bound versus non-wheelchair-bound) × 2 (Caucasian versus Chinese) × 2 (programmer versus computer operator) full factorial experiment design to examine the effects of evaluator biases on the perceived employability of job applicants. Results based on 640 evaluation reports showed that evaluators tended to accord higher employability ratings to job applicants with apparent physical disabilities and those with a higher level of job skill. The positive bias in favor of applicants with disabilities in terms of perceived employability was greater for Caucasian than for Chinese job applicants. These results suggested that there might be race-disability interactions in the evaluation process. Implications for research on employment discrimination were discussed.

It has been difficult to detect and visualize the physical nucleocapsid particles during the replication process of the flaviviruses. The use of cryo-immunoelectron microscopy has clearly revealed the capsid proteins and nucleocapsid particles of West Nile (Sarafend) virus (a flavivirus) for the first time. Physical nucleocapsid particles accumulated in large numbers from 8 hr postinfection. Double immunolabeling of the envelope and capsid proteins showed a close association of these structural proteins for most of the replication cycle. By 10 hr postinfection, budding of nucelocapsids from the plasma membrane was very obvious. Although maturation at the plasma membrane was the dominant mode, during late infection, intracellular maturation into large vacuoles was also observed. J. Med. Virol. 65:758–764, 2001. © 2001 Wiley-Liss, Inc.

In an attempt to identify novel chemokine receptor genes, cDNA expressed sequence tags (EST) were analyzed for a significant homology with mammalian chemokine receptors. The sequence from one of the selected EST clones was used to generate a full-length cDNA encoding a putative seven transmembrane receptor, CCRL1-CC chemokine receptor like 1. The full-length receptor encodes a polypeptide of 350 amino acids and has about 35% homology to the chemokine receptors CCR6 and CCR7. Northern blot analysis indicates predominant expression of about 5.0, 2.0 and 1.3 kb mRNA forms in human heart tissue, while low-level expression of the 2.0 and 1.3 kb forms was observed in lung, pancreas and spleen and in fetal tissues. In-situ hybridization confirmed the presence of CCRL1 mRNA in cardiac muscle cells. Similar to the chemokine receptor CCR6, CCRL1 maps to chromosome 6 and has one intron in the 5′ untranslated region. Coupled transcription–translation of CCRL1 cDNA yielded a glycosylated polypeptide of about 45 kDa.

We report the results of the time-resolved emission spectra and the decay curves, concerning the important green emission transition at 544 nm ( 5 D 4 → 7 F 5 ). Five other transitions at 489 nm ( 5 D 4 → 7 F 6 ), 585 nm ( 5 D 4 → 7 F 4 ), 620 nm ( 5 D 4 → 7 F 3 ), 674 nm ( 5 D 4 → 7 F 2 ) and 648 nm ( 5 D 4 → 7 F 1 ) of Tb 3+ : (La,Y)PO 4 and (La,Gd)PO 4 were measured to understand the influences of the wavelengths of excitation, in emitting green color from these two novel phosphor materials. It was observed that the spectral features possess sharp and bright emission for potential applications on the monitors of the television and some other related electronic systems, in observing the images in green color.

Polyclonal antibodies derived from dengue virus immune sera and 3H5 monoclonal antibody showed potent neutralisation effect on dengue-2 virus in the plaque reduction neutralisation assay. This study demonstrated that antibodies present in immune human sera and 3H5 monoclonal antibody neutralised dengue-2 virus by altering the virus entry pathway into cells. In the presence of neutralising antibodies, dengue-2 virus was endocytosed by LLC-MK2 cells. The endocytosis process involved ruffling of antibody-coated virions by cellular pseudopodia and invagination of cell membrane. This mode of entry is atypical as compared to direct fusion of dengue-2 virus with cell membrane in the absence of antibody. The virions were internalised in the form of virion-antibody complexes consisting of single or clumps of virions. After 3 minutes of incubation, neutralised virions were detected in cellular vesicles, and signs of intra-endosomal penetration into cytoplasm were not evident even after a prolonged incubation of 10 minutes, suggesting that viral uncoating was compromised. Vesicle-bound virions were no longer detected after 20 minutes of incubation. In addition, no sign of viral replication was detected in cells infected with "neutralised" virions by immunofluorescence assay. This indicated that internalised virions had been degraded leading to abortive infection. In conclusion, antibodies present in 3H5 monoclonal antibody and human immune sera rendered dengue-2 virus non-infective by neutralising the viral fusion site and causing alteration of viral entry mode. Antibodies in immune sera but not 3H5 monoclonal antibody also exerted minimal inhibitory effect on virus binding and internalisation.

The Western blot (WB) assay was used to determine dengue virus antibodies present in human immune sera arising from recent primary and secondary dengue virus infections in Singapore. Cell lysates of dengue-2 virus-infected C6/36 and Vero cells were used. Antibodies directed against structural proteins of dengue-2 virus including envelope (E, gp60/50), capsid-premembrane (C-PrM, gp35), and premembrane (PrM, gp20) were detected, with antibody against envelope protein being most dominant. Similar WB profiles were detected in both primary and secondary dengue virus infections. The reactivity rate of antibodies to dengue-2 virus proteins was higher in infected Vero cell lysate than in infected C6/36 cell lysate, with the exception of antibodies to nonstructural proteins of NS1 and NS3, which were detected predominantly in infected C6/36 cell lysate. More than 75% of "normal" individuals (with no complaint of recent dengue virus infection) examined had low levels of dengue virus antibodies, but all presented with similar WB profiles as patients with recent dengue virus infections. This finding reflects a high seroprevalence of dengue virus infections and the long lasting nature of E, C-PrM, and PrM antibodies. Results from this study indicate that in natural dengue virus infections, native E, C-PrM, and PrM antigens of dengue virus are immunogenic and elicit long-lasting antibodies.

Blood resistivity is an important quantity which value influences the results of various methods used in the study of heart and circulation. In this paper, the relationship between blood resistivity and velocity of blood flow was evaluated and analyzed based upon a probe using six-ring electrodes and a circulatory model. The experimental results indicated that the change in blood resistivity was only /spl plusmn/1.1% when the velocity of blood flow changed from 2.83 cm/s to 40 cm/s and it rose to 23% when the velocity was lower than 2.83 cm/s.

The present paper reviews the applications and recent developments of microwave digestion or extraction as a sample preparation technique. This technique has been applied successfully in both organic and inorganic determinations for various sample matrices. It offers the benefits of rapid sample preparation, ease of automation and reduction in background contamination. Good recoveries are generally obtained.

Initial experiments using whole cells have shown that there were specific and saturable interactions between kunjin (KUN) virus and receptor molecules on the Vero cell surfaces. Solubilisation of Vero cell plasma membranes with octyl-D-glucoside (OG) yielded an extract which also interacted specifically with KUN virus. This was proven using electron microscopy. When the virus-OG-extract complex was exposed onto Vero cell monolayers, no KUN virus was observed to enter into the whole cells. This would imply that there was virus-receptor interaction with the OG-extract leaving no free virus to attach to the whole cells. The attachment kinetics of KUN virus was studied further using the Scatchard analysis which indicated the involvement of more than one interactive macromolecule in the attachment event.

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus–host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.

BackgroundIn 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens.MethodsOf the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted.ResultsElectron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the familyPicornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range.ConclusionsThe etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.

Rheumatoid Arthritis (RA) has been associated with anemia. The treatment of anemia in RA includes blood transfusions, erythropoietic agents and iron supplements. Our observations suggest that the treatment of the inflammatory disease is associated with an improvement of hemoglobin (Hgb) levels in RA patients.Record review, analyzing anemic RA patients and hemoglobin level changes after intensification of RA therapy. Three initial regimes were identified: corticosteroid (Prednisone), Disease Modifying Anti-rheumatic Drugs (DMARD's) plus Prednisone and for patients with no initial treatment with the initiation of Prednisone and or TNF a inhibitors (Etanercept).8 female and 2 male patients with average age 59.7 + 14 years were selected. The results for the group treated with Prednisone initially N=4; showed a 0.9 g/dL increase in Hgb and Hct increase of 3% after addition of Etanercept. Those with DMARDs and corticosteroid regime had an Hgb and Hct increase of 1.2 g/dL and 3.5% after addition of Etanercept. The group with no initial treatment was further divided in two groups. The first was treated with Etanercept and the second was treated with Prednisone, resulting in average Hgb increases of 1.9 g/dl Hgb and 1.8 g/dL, and HCT increase 4% and 3.9% respectively. The global average change in Hgb was 1.3g/dL and HCT increase 3.2%.The intensity of treatment of RA including Etanercept and Prednisone improved cases of mild anemia in rheumatoid arthritis.

Flavivirus capsid (C) protein is a key structural component of virus particles. The non-structural role of C protein in the pathogenesis of arthropod-borne flaviviruses is not clearly deciphered. This study showed that West Nile virus (WNV) and dengue virus (DENV) utilized C protein to reduce human Sec3p (hSec3p) levels at post-transcriptional level through activation of chymotrypsin-like proteolytic function of 20S proteasome. Mutagenesis studies confirmed amino acids 14, 109-114 of WNV C protein and 13, 102-107 of DENV C protein played an important role in activating the proteolytic function of 20S proteasome. Amino acid residues at 14 (WNV) and 13 (DENV) of C protein were important for C protein-hSec3p binding and physical interaction between C protein and hSec3p was essential to execute hSec3p degradation. Degradation motif required to degrade hSec3p resided between amino acid residues 109-114 of WNV C protein and 102-107 of DENV C protein. Proteasomes, hSec3p binding motif and degradation motif on C protein must be intact for efficient flavivirus production. Clinical isolates of DENV showed more pronounced effect in manipulating the proteasomes and reducing hSec3p levels. This study portrayed the non-structural function of C protein that helped the flavivirus to nullify the antiviral activity of hSec3p by accelerating its degradation and facilitating efficient binding of elongation factor 1α with flaviviral RNA genome.

Dengue Virus (DENV), a flavivirus spread by mosquito vectors, can cause vascular leakage and hemorrhaging. However, the processes that underlie increased vascular permeability and pathological plasma leakage during viral hemorrhagic fevers are largely unknown. Mast cells (MCs) are activated in vivo during DENV infection, and we show that this elevates systemic levels of their vasoactive products, including chymase, and promotes vascular leakage. Treatment of infected animals with MC-stabilizing drugs or a leukotriene receptor antagonist restores vascular integrity during experimental DENV infection. Validation of these findings using human clinical samples revealed a direct correlation between MC activation and DENV disease severity. In humans, the MC-specific product, chymase, is a predictive biomarker distinguishing dengue fever (DF) and dengue hemorrhagic fever (DHF). Additionally, our findings reveal MCs as potential therapeutic targets to prevent DENV-induced vasculopathy, suggesting MC-stabilizing drugs should be evaluated for their effectiveness in improving disease outcomes during viral hemorrhagic fevers. DOI: http://dx.doi.org/10.7554/eLife.00481.001, eLife digest Dengue fever is an infectious tropical disease that is transmitted by mosquitoes carrying dengue virus. Almost half of the world’s population lives in dengue-plagued regions and it is estimated that between 50 and 100 million people are infected annually. However, there is no effective vaccine and researchers have only a limited understanding of the mechanisms behind the disease. While most infected individuals experience fever, headache, muscle and joint pain, and a skin rash, a small percentage go on to develop a life-threatening condition known as dengue hemorrhagic fever (DHF). This is marked by internal bleeding, and by the leakage of water and salts from blood vessels (vascular leakage). At present, it is difficult to tell which patients will develop this complication, making it hard to tailor treatment appropriately. Here, St John et al. reveal that the vascular leakage that occurs in DHF is triggered by mast cells, which line blood vessels and regulate their permeability through the release of molecules such as histamines and leukotrienes. They also found that mice deficient in mast cells did not show dengue-induced vascular leakage, and that wild-type animals treated with drugs that block the actions of proteins produced by these cells, showed less vascular leakage than controls. Moreover, levels of an enzyme called chymase, another mast cell product, are higher in human patients with DHF than in those with dengue fever. Since chymase release occurs early in infection, tests for the presence of this enzyme could be used to predict which patients are likely to develop DHF. The work of St John et al. suggests new lines of inquiry into the mechanisms that lead some individuals infected with dengue fever to develop DHF, and indicates that drugs that target mast cells could offer an effective treatment. DOI: http://dx.doi.org/10.7554/eLife.00481.002

Dengue virus (DENV) capsid (C) protein is one of the three structural proteins that form a mature virus. The main challenge impeding the study of this protein is to generate pure non-truncated, full-length C proteins for structural and functional studies. This is mainly due to its small molecular weight, highly positively charged, stability and solubility properties. Here, we report a strategy to construct, express, biotinylate and purify non-truncated, full-length DENV C protein. A 6× His tag and a biotin acceptor peptide (BAP) were cloned at the N-terminus of C protein using overlapping extension-polymerase chain reaction method for site-specific biotinylation. The final construct was inserted into pET28a plasmid and BL-21 (CodonPlus) expression competent cell strain was selected as there are 12% rare codons in the C protein sequence. Strikingly, we found that our recombinant proteins with BAP were biotinylated endogenously with high efficiency in Escherichia coli BL-21 strains. To purify this His-tagged C protein, nickel-nitriloacetic acid affinity chromatography was first carried out under denaturing condition. After stepwise dialysis and concurrent refolding, ion exchange-fast protein liquid chromatography was performed to further separate the residual contaminants. To obtain C protein with high purity, a final round of purification with size exclusion chromatography was carried out and a single peak corresponding to C protein was attained. With this optimized sequential purification protocol, we successfully generated pure biotinylated full-length DENV C protein. The functionality of this purified non-truncated DENV C protein was examined and it was suitable for structural and molecular studies.

CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55GagHIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun). The two resulting chimeric proteins, DIII-DENV1-RIgE and DIII-WNVKun-RIgE, were addressed to the plasma membrane, exposed at the surface of human and insect cells, and incorporated into extracellular VLPs when coexpressed with Pr55GagHIV in insect cells. The DIII domains were accessible at the surface of retroviral VLPs, as shown by their reactivity with specific antibodies, and notably antibodies from patient sera. The DIII-RIgE proteins were found to be incorporated in VLPs made of SIV, MLV, or chimeric MLV-HIV Gag precursors, indicating that DIII-RIgE could pseudotype a wide variety of retroviral VLPs. VLP-displayed DIII were capable of inducing specific neutralizing antibodies against DENV and WNV in mice. Although the neutralization response was modest, our data confirmed the capability of DIII to induce a flavivirus neutralization response, and suggested that our VLP-displayed CD16-RIgE-based platform could be developed as a vaccine vector against different flaviviruses and other viral pathogens.

Homeostatic pressure-driven compaction is a ubiquitous mechanical force in multicellular organisms and is proposed to be important in the maintenance of multicellular tissue integrity and function. Previous cell-free biochemical models have demonstrated that there are cross-talks between compaction forces and tissue structural functions, such as cell-cell adhesion. However, its involvement in physiological tissue function has yet to be directly demonstrated. Here, we use the bile canaliculus (BC) as a physiological example of a multicellular functional structure in the liver, and employ a novel 3D microfluidic hepatocyte culture system to provide an unprecedented opportunity to experimentally modulate the compaction states of primary hepatocyte aggregates in a 3D physiological-mimicking environment. Mechanical compaction alters the physical attributes of the hepatocyte aggregates, including cell shape, cell packing density and cell-cell contact area, but does not impair the hepatocytes' remodeling and functional capabilities. Characterization of structural and functional polarity shows that BC formation in compact hepatocyte aggregates is accelerated to as early as 12 hours post-seeding; whereas non-compact control requires 48 hours for functional BC formation. Further dynamic immunofluorescence imaging and gene expression profiling reveal that compaction accelerated BC formation is accompanied by changes in actin cytoskeleton remodeling dynamics and transcriptional levels of hepatic nuclear factor 4α and Annexin A2. Our report not only provides a novel strategy of modeling BC formation for in vitro hepatology research, but also shows a first instance that homeostatic pressure-driven compaction force is directly coupled to the higher-order multicellular functions.

Anorexia/cachexia is a common and currently mostly untreatable complication of advanced cancer. It is also a feature of a number of chronic diseases and can also occur as part of the normal ageing process. Over recent years, two different, but sometimes overlapping, processes have been identified to mediate anorexia/cachexia: those that act primarily on muscle reducing its mass and function, and processes that decrease nutrition leading to loss of both fat and muscle. In the case of at least some cancers, the latter process is sometimes driven by marked overexpression of macrophage inhibitory cytokine-1/growth differentiation factor 15 (MIC-1/GDF15). MIC-1/GDF15 is a transforming growth factor beta (TGF-β) family cytokine that is found in the serum of all normal individuals at an average concentration of about 0.6 ng/ml. Its increased expression in both cancers and other diseases can result in 10–100-fold or more elevation of its serum levels. In experimental animals, serum MIC-1/GDF15 levels at the lower end of this range induce anorexia by direct actions of the circulating cytokine on feeding centres in the brain. Mice with tumours overexpressing MIC-1/GDF15 display decreased food intake, loss of lean and fat mass and cachexia. That this process also mediates anorexia/cachexia in humans is suggested by the fact that there is a direct correlation between the degree of serum MIC-1/GDF15 elevation and the amount of cancer-related weight loss, the first such relationship demonstrated. Further, in experimental animals, weight loss can be reversed by neutralisation of tumour-produced MIC-1/GDF15 with a specific monoclonal antibody, suggesting the possibility of effective therapy of patients with the devastating complication of anorexia/cachexia.

5039Background: DTX improves symptoms and survival in mCRPC; however, ~ 50% of patients have DTX resistant disease. Early markers of DTX resistance will allow rapid cessation of ineffective therapy...

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).

Cryotechniques in combination with electron microscopy were used in an attempt to obtain more precise morphological details of flavivirus-induced structures. From conventional chemical fixation procedure, proliferation of endoplasmic reticulum, formation of microtubule paracrystals and clusters of smooth membrane vesicles (with ‘thread-like’ enclosures) were observed. These induced changes are typical for flavivirus infections. The images obtained from cryosections were disappointing as the structures were not well preserved. On the other hand, cryosubstituted-infected cells gave revealing images of the virus-induced structures. The most obvious difference between the cryosubstituted and chemical fixed processes was on the morphology of the ‘thread-like’ structure. The ‘thread-like’ structures instead appeared as dense cores. The morphology of the virus particles was also better defined. The envelope of the virus appeared clearly differentiated from the nucleocapsid. The most important finding was that the cryosubstituted technique was able to preserve the structures of the flavivirus nucleocapsids which so far has not been convincing reported with chemical processing.

West Nile (Sarafend) virus was distinctly observed to bud from the plasma membrane rather than mature intracellularly. This has been observed with transmission electron microscopy. Using conventional scanning electron microscopy, budding at the plasma membrane especially at the filopodia was clearly illustrated. Immunogold labelling against the virus envelope protein was also performed to confirm this mode of exit. The gold particles were observed to be located at the sites where virus budding was seen under the field emission scanning electron microscope.