ResearchPad - biosynthetic-techniques https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase]]> https://www.researchpad.co/article/elastic_article_14506 The function of a protein is often dictated by a single defined fold, which in turn is determined by its amino acid sequences. However, multiple global conformations can be utilized by a protein to fulfill distinct functions under different circumstances. The flavivirus NS5 protein, a natural fusion of an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRP), may be such an example. Previously reported NS5 crystal structures exhibit two apparently different global conformations. In this work, we demonstrate that both conformations are conserved in the flaviviruses and important for virus proliferation, but only one of them is clearly relevant to RdRP catalysis, in particular at the early stages of the RNA synthesis.

]]>
<![CDATA[A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs]]> https://www.researchpad.co/article/5c897715d5eed0c4847d23fa

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of ‘superfolder’ green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 μg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens.

]]>
<![CDATA[Growth enhancement of porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein]]> https://www.researchpad.co/article/5c897777d5eed0c4847d2d6d

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.

]]>
<![CDATA[A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib]]> https://www.researchpad.co/article/5c8823f1d5eed0c4846393bf

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

]]>
<![CDATA[Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP]]> https://www.researchpad.co/article/5c61e917d5eed0c48496f7ea

Mutagenic translesion DNA polymerase V (UmuD′2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which onoff switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.

]]>
<![CDATA[Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription]]> https://www.researchpad.co/article/5c50c49ed5eed0c4845e8a43

Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.

]]>
<![CDATA[The transcriptome sequencing and functional analysis of eyestalk ganglions in Chinese mitten crab (Eriocheir sinensis) treated with different photoperiods]]> https://www.researchpad.co/article/5c478c7bd5eed0c484bd294c

Photoperiod plays an important role in individual growth, development, and metabolism in crustaceans. The growth and reproduction of crabs are closely related to the photoperiod. However, as of yet, there are still no transcriptomic reports of eyestalk ganglions treated under different photoperiods in the Chinese mitten crab (Eriocheir sinensis), which is a benthonic crab with high commercial value in Asia. In this study, we collected the eyestalk ganglions of crabs that were reared under different photoperiods, including a control group (L: D = 12 h: 12 h, named CC), a constant light group (L: D = 24 h: 0 h, named LL) and a constant darkness group (L: D = 0 h: 24 h, named DD). RNA sequencing was performed on these tissues in order to examine the effects of different photoperiods. The total numbers of clean reads from the CC, LL and DD groups were 48,772,584 bp, 53,943,281 bp and 53,815,178 bp, respectively. After de novo assembly, 161,380 unigenes were obtained and were matched with different databases. The DEGs were significantly enriched in phototransduction and energy metabolism pathways. Results from RT-qPCR showed that TRP channel protein (TRP) in the phototransduction pathway had a significantly higher level of expression in LL and DD groups than in the CC group. We found that the downregulation of the pyruvate dehydrogenase complex (PDC) gene and the upregulation phosphoenolpyruvate carboxykinase (PPC) gene were involved in energy metabolism processes in LL or DD. In addition, we also found that the upregulation of the expression level of the genes Gαq, pyruvate kinase (PK), NADH peroxidase (NADH) and ATPase is involved in phototransduction and energy metabolism. These results may shed some light on the molecular mechanism underlying the effect of photoperiod in physiological activity of E. sinensis.

]]>
<![CDATA[RNase H1 directs origin-specific initiation of DNA replication in human mitochondria]]> https://www.researchpad.co/article/5c37b7b0d5eed0c48449094a

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

]]>
<![CDATA[Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS]]> https://www.researchpad.co/article/5c3e505ed5eed0c484d80f2b

We investigated the effect of peroxisome proliferator-activated receptor δ (PPARδ) on angiotensin II (Ang II)-triggered hypertrophy of vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly inhibited Ang II-stimulated protein synthesis in a concentration-dependent manner, as determined by [3H]-leucine incorporation. GW501516-activated PPARδ also suppressed Ang II-induced generation of reactive oxygen species (ROS) in VSMCs. Transfection of small interfering RNA (siRNA) against PPARδ significantly reversed the effects of GW501516 on [3H]-leucine incorporation and ROS generation, indicating that PPARδ is involved in these effects. By contrast, these GW501516-mediated actions were potentiated in VSMCs transfected with siRNA against NADPH oxidase (NOX) 1 or 4, suggesting that ligand-activated PPARδ elicits these effects by modulating NOX-mediated ROS generation. The phosphatidylinositol 3-kinase inhibitor LY294002 also inhibited Ang II-stimulated [3H]-leucine incorporation and ROS generation by preventing membrane translocation of Rac1. These observations suggest that PPARδ is an endogenous modulator of Ang II-triggered hypertrophy of VSMCs, and is thus a potential target to treat vascular diseases associated with hypertrophic changes of VSMCs.

]]>
<![CDATA[Age threshold for recommending higher protein intake to prevent age-related muscle weakness: A cross-sectional study in Japan]]> https://www.researchpad.co/article/5c1ab85cd5eed0c484027ba4

Although insufficient dietary protein intake is a known risk factor for age-related muscle weakness, the optimal age at which higher protein intake is required to prevent muscle weakness is yet to be determined. Using a population-based panel survey of community-dwelling people aged 50–75 years, this cross-sectional study aimed to find the age threshold at which a higher protein intake is associated with higher muscle strength. We utilized a dataset from the Japanese Study of Aging and Retirement conducted between 2007 and 2011. Dietary protein intake was estimated using a validated dietary questionnaire and energy-adjusted via density method. Grip strength was measured using a Smedley-type handheld dynamometer. We calculated the marginal effect (and 95% confidence intervals) of protein intake on grip strength with stratification by age using multiple linear regression analyses with robust variance adjusting for potential confounders. There were 9,485 observations from 5,790 participants in the final analysis. Marginal effects of protein intake on grip strength increased with age, and it reached significance and had a positive impact only among men aged ≥75 years and women aged ≥65 years. With an additional 1% energy of protein intake, grip strength was increased by 0.10 kg and 0.19 kg for men and women aged ≥75 years, respectively. Our result indicated the possibility that women needed a high protein intake from a younger age compared with men. Further studies are needed to clarify from when a higher protein intake is recommended to prevent muscle weakness.

]]>
<![CDATA[Specificity and functional interplay between influenza virus PA-X and NS1 shutoff activity]]> https://www.researchpad.co/article/5c099405d5eed0c4842ae0b7

Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS1 proteins, the virus is capable of suppressing host protein synthesis, termed “host shutoff.” Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (pH1N1) viruses containing mutations affecting the expression of active PA-X and NS1. We analyzed viral growth, general shutoff activity, specificity of mRNA targets, and viral gene expressions. Our results showed that PA-X was the major contributor in reducing general host protein expression in the virus-infected cells. Intriguingly, our transcriptomic analysis from infected human airway A549 cells indicate that shutoff-active NS1 specifically targeted host mRNAs related to interferon (IFN) signaling pathways and cytokine release. Specificity of target mRNAs was less evident in PA-X, although it preferentially degraded genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of shutoff-active NS1, PA-X also degraded viral mRNAs, especially NS segments. The virus expressing shutoff-active NS1 with reduced amount of PA-X expression most efficiently suppressed antiviral and innate immune responses in human cells, indicating that influenza virus needs to optimize the contribution of these two shutoff proteins to circumvent host responses for its optimum growth.

]]>
<![CDATA[Frequent and biased odorant receptor (OR) re-selection in an olfactory placode-derived cell line]]> https://www.researchpad.co/article/5bb530db40307c24312bb0b4

We previously characterized a clonal olfactory placode-derived cell line (OP6) as a model system for studying odorant receptor (OR) choice, where individual OP6 cells, similar to olfactory sensory neurons in vivo, transcribe one allele (“monoallelic”) of one OR gene (“monogenic”). The OP6 cell line provides a unique opportunity to investigate intrinsic properties of OR regulation that cannot easily be investigated in vivo. First, whereas OR-expressing cells in vivo are post-mitotic, OP6 cells are immortalized, raising interesting questions about the stability of epigenetic states associated with OR selection/silencing as OP6 cells progress through the cell cycle. Second, OP6 cells have been isolated away from extrinsic developmental cues, and therefore, any long-term OR selection biases are likely to arise from intrinsic epigenetic states that persist in the absence of developmental context. In this study, we investigated OR re-selection frequency and selection biases within clonal OP6 cell populations. We found no evidence of OR stability through the cell cycle: our results were most consistent with OR re-selection events transpiring at least once per cell division, suggesting that chromatin states associated with OR selection in this system might not be maintained in the subsequent generation. In contrast, we found strong evidence for OR selection biases maintained over prolonged culturing across a diverse set of OP6 cell lineages, suggesting the persistence of intrinsic epigenetic states that advantage some OR loci over others. Together, our data suggest that in the absence of instructive cues, intrinsic epigenetic states influencing OR eligibility, but not those determining OR choice, might persist through the cell cycle.

]]>
<![CDATA[Role of altered proteostasis network in chronic hypobaric hypoxia induced skeletal muscle atrophy]]> https://www.researchpad.co/article/5bae98fc40307c0c23a1c155

Background

High altitude associated hypobaric hypoxia is one of the cellular and environmental perturbation that alters proteostasis network and push the healthy cell towards loss of muscle mass. The present study has elucidated the robust proteostasis network and signaling mechanism for skeletal muscle atrophy under chronic hypobaric hypoxia (CHH).

Methods

Male Sprague Dawley rats were exposed to simulated hypoxia equivalent to a pressure of 282 torr for different durations (1, 3, 7 and 14 days). After CHH exposure, skeletal muscle tissue was excised from the hind limb of rats for biochemical analysis.

Results

Chronic hypobaric hypoxia caused a substantial increase in protein oxidation and exhibited a greater activation of ER chaperones, glucose-regulated protein-78 (GRP-78) and protein disulphide isomerase (PDI) till 14d of CHH. Presence of oxidized proteins triggered the proteolytic systems, 20S proteasome and calpain pathway which were accompanied by a marked increase in [Ca2+]. Upregulated Akt pathway was observed upto 07d of CHH which was also linked with enhanced glycogen synthase kinase-3β (GSk-3β) expression, a negative regulator of Akt. Muscle-derived cytokines, tumor necrosis factor-α (TNF-α), interferon-ϒ (IFN-©) and interleukin-1β (IL-1β) levels significantly increased from 07d onwards. CHH exposure also upregulated the expression of nuclear factor kappa-B (NF-κB) and E3 ligase, muscle atrophy F-box-1 (Mafbx-1/Atrogin-1) and MuRF-1 (muscle ring finger-1) on 07d and 14d. Further, severe hypoxia also lead to increase expression of ER-associated degradation (ERAD) CHOP/ GADD153, Ub-proteasome and apoptosis pathway.

Conclusions

The disrupted proteostasis network was tightly coupled to degradative pathways, altered anabolic signaling, inflammation, and apoptosis under chronic hypoxia. Severe and prolonged hypoxia exposure affected the protein homeostasis which overwhelms the muscular system and tends towards skeletal muscle atrophy.

]]>
<![CDATA[Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo]]> https://www.researchpad.co/article/5989dab4ab0ee8fa60bac3a5

The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

]]>
<![CDATA[Effective Design of Multifunctional Peptides by Combining Compatible Functions]]> https://www.researchpad.co/article/5989da89ab0ee8fa60b9d6aa

Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf.

]]>
<![CDATA[Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells]]> https://www.researchpad.co/article/5989daebab0ee8fa60bbf540

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

]]>
<![CDATA[Spatial and Temporal Resolution of Global Protein Synthesis during HSV Infection Using Bioorthogonal Precursors and Click Chemistry]]> https://www.researchpad.co/article/5989daadab0ee8fa60baa15b

We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal new features from which we propose spatially linked platforms of newly synthesised protein processing after nuclear import.

]]>
<![CDATA[Comparative Evaluation of Real-Time PCR Methods for Human Noroviruses in Wastewater and Human Stool]]> https://www.researchpad.co/article/5989da23ab0ee8fa60b7f89d

Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently.

]]>
<![CDATA[Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein]]> https://www.researchpad.co/article/5989daeeab0ee8fa60bc0559

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.

]]>
<![CDATA[Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be1239

The intestinal epithelial barrier, composed of epithelial cells, tight junction proteins and intestinal secretions, prevents passage of luminal substances and antigens through the paracellular space. Dysfunction of the intestinal barrier integrity induced by toxins and pathogens is associated with a variety of gastrointestinal disorders and diseases. Although butyrate is known to enhance intestinal health, its role in the protection of intestinal barrier function is poorly characterized. Therefore, we investigated the effect of butyrate on intestinal epithelial integrity and tight junction permeability in a model of LPS-induced inflammation in IPEC-J2 cells. Butyrate dose-dependently reduced LPS impairment of intestinal barrier integrity and tight junction permeability, measured by trans-epithelial electrical resistance (TEER) and paracellular uptake of fluorescein isothiocyanate-dextran (FITC-dextran). Additionally, butyrate increased both mRNA expression and protein abundance of claudins-3 and 4, and influenced intracellular ATP concentration in a dose-dependent manner. Furthermore, butyrate prevented the downregulation of Akt and 4E-BP1 phosphorylation by LPS, indicating that butyrate might enhance tight junction protein abundance through mechanisms that included activation of Akt/mTOR mediated protein synthesis. The regulation of AMPK activity and intracellular ATP level by butyrate indicates that butyrate might regulate energy status of the cell, perhaps by serving as a nutrient substrate for ATP synthesis, to support intestinal epithelial barrier tight junction protein abundance. Our findings suggest that butyrate might protect epithelial cells from LPS-induced impairment of barrier integrity through an increase in the synthesis of tight junction proteins, and perhaps regulation of energy homeostasis.

]]>