Efficient Sorting of Semiconducting Single-Walled Carbon Nanotubes in Bio-Renewable Solvents Through Main-Chain Engineering of Conjugated Polymers.

Conjugated polymer sorting is recognized as an efficient and scalable method for the selective extraction of semiconducting single-walled carbon nanotubes (s-SWCNTs). However, this process typically requires the use of nonpolar and aromatic solvents as the dispersion medium, which are petroleum-based and carry significant production hazards. Moreover, there is still potential for improving the efficiency of batch purification. Here, this study presents fluorene-based conjugated polymer that integrates diamines containing ethylene glycol chains (ODA) as linkers within the main chain, to effectively extract s-SWCNTs in bio-renewable solvents. The introduction of ODA segments enhances the solubility in bio-renewable solvents, facilitating effective wrapping of s-SWCNTs in polar environments. Additionally, the ODA within the main chain enhances affinity to s-SWCNTs, thereby contributing to increased yields and purity. The polymer achieves a high sorting yield of 55% and a purity of 99.6% in dispersion of s-SWCNTs in 2-Methyltetrahydrofuran. Thin-film transistor arrays fabricated with sorted s-SWCNTs solution through slot-die coating exhibit average charge carrier mobilities of 20-23 cm2 V⁻¹ s⁻¹ and high on/off current ratios exceeding 105 together with high spatial uniformity. This study highlights the viability of bio-renewable solvents in the sorting process, paving the way for the eco-friendly approach to the purification of SWCNTs.

Targeting NLRP3 signaling reduces myocarditis-induced arrhythmogenesis and cardiac remodeling.

BACKGROUND: Myocarditis substantially increases the risk of ventricular arrhythmia. Approximately 30% of all ventricular arrhythmia cases in patients with myocarditis originate from the right ventricular outflow tract (RVOT). However, the role of NLRP3 signaling in RVOT arrhythmogenesis remains unclear. METHODS: Rats with myosin peptide-induced myocarditis (experimental group) were treated with an NLRP3 inhibitor (MCC950; 10 mg/kg, daily for 14 days) or left untreated. Then, they were subjected to electrocardiography and echocardiography. Ventricular tissue samples were collected from each rat's RVOT, right ventricular apex (RVA), and left ventricle (LV) and examined through conventional microelectrode and histopathologic analyses. In addition, whole-cell patch-clamp recording, confocal fluorescence microscopy, and Western blotting were performed to evaluate ionic currents, intracellular Ca2+ transients, and Ca2+-modulated protein expression in individual myocytes isolated from the RVOTs. RESULTS: The LV ejection fraction was lower and premature ventricular contraction frequency was higher in the experimental group than in the control group (rats not exposed to myosin peptide). Myocarditis increased the infiltration of inflammatory cells into cardiac tissue and upregulated the expression of NLRP3; these observations were more prominent in the RVOT and RVA than in the LV. Furthermore, experimental rats treated with MCC950 (treatment group) improved their LV ejection fraction and reduced the frequency of premature ventricular contraction. Histopathological analysis revealed higher incidence of abnormal automaticity and pacing-induced ventricular tachycardia in the RVOTs of the experimental group than in those of the control and treatment groups. However, the incidences of these conditions in the RVA and LV were similar across the groups. The RVOT myocytes of the experimental group exhibited lower Ca2+ levels in the sarcoplasmic reticulum, smaller intracellular Ca2+ transients, lower L-type Ca2+ currents, larger late Na+ currents, larger Na+-Ca2+ exchanger currents, higher reactive oxygen species levels, and higher Ca2+/calmodulin-dependent protein kinase II levels than did those of the control and treatment groups. CONCLUSION: Myocarditis may increase the rate of RVOT arrhythmogenesis, possibly through electrical and structural remodeling. These changes may be mitigated by inhibiting NLRP3 signaling.

Chronic Partial Sleep Deprivation Increased the Incidence of Atrial Fibrillation by Promoting Pulmonary Vein and Atrial Arrhythmogenesis in a Rodent Model.

Sleep deprivation (SD) is a recognized risk factor for atrial fibrillation (AF), yet the precise molecular and electrophysiological mechanisms behind SD-induced AF are unclear. This study explores the electrical and structural changes that contribute to AF in chronic partial SD. We induced chronic partial SD in Wistar rats using a modified multiple-platform method. Echocardiography demonstrated impaired systolic and diastolic function in the left ventricle (LV) of the SD rats. The SD rats exhibited an elevated heart rate and a higher low-frequency to high-frequency ratio in a heart-rate variability analysis. Rapid transesophageal atrial pacing led to a higher incidence of AF and longer mean AF durations in the SD rats. Conventional microelectrode recordings showed accelerated pulmonary vein (PV) spontaneous activity in SD rats, along with a heightened occurrence of delayed after-depolarizations in the PV and left atrium (LA) induced by tachypacing and isoproterenol. A Western blot analysis showed reduced expression of G protein-coupled receptor kinase 2 (GRK2) in the LA of the SD rats. Chronic partial SD impairs LV function, promotes AF genesis, and increases PV and LA arrhythmogenesis, potentially attributed to sympathetic overactivity and reduced GRK2 expression. Targeting GRK2 signaling may offer promising therapeutic avenues for managing chronic partial SD-induced AF. Future investigations are mandatory to investigate the dose-response relationship between SD and AF genesis.

Deletion of gene OV132 attenuates Orf virus more effectively than gene OV112.

Orf virus (ORFV), a Parapoxvirus in Poxviridae, infects sheep and goats resulting in contagious pustular dermatitis. ORFV is regarded as a promising viral vector candidate for vaccine development and oncolytic virotherapy. Owing to their potential clinical application, safety concerns have become increasingly important. Deletion of either the OV132 (encoding vascular endothelial growth factor, VEGF) or OV112 (encoding the chemokine binding protein, CBP) genes reduced ORFV infectivity, which has been independently demonstrated in the NZ2 and NZ7 strains, respectively. This study revealed that the VEGF and CBP gene sequences of the local strain (TW/Hoping) shared a similarity of 47.01% with NZ2 and 90.56% with NZ7. Due to the high sequence divergence of these two immunoregulatory genes among orf viral strains, their contribution to the pathogenicity of Taiwanese ORFV isolates was comparatively characterized. Initially, two ORFV recombinants were generated, in which either the VEGF or CBP gene was deleted and replaced with the reporter gene EGFP. In vitro assays indicated that both the VEGF-deletion mutant ORFV-VEGFΔ-EGFP and the CBP deletion mutant ORFV-CBPΔ-EGFP were attenuated in cells. In particular, ORFV-VEGFΔ-EGFP significantly reduced plaque size and virus yield compared to ORFV-CBPΔ-EGFP and the wild-type control. Similarly, in vivo analysis revealed no virus yield in the goat skin biopsy infected by ORFV-VEGFΔ-EGFP, and significantly reduced the virus yield of ORFV-CBPΔ-EGFP relative to the wild-type control. These results confirmed the loss of virulence of both deletion mutants in the Hoping strain, whereas the VEGF-deletion mutant was more attenuated than the CBP deletion strain in both cell and goat models. KEY POINTS: • VEGF and CBP genes are crucial in ORFV pathogenesis in the TW/Hoping strain • The VEGF-deletion mutant virus was severely attenuated in both cell culture and animal models • Deletion mutant viruses are advantageous vectors for the development of vaccines and therapeutic regimens.

Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting.

PURPOSE: A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection. METHODS: Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60 min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR). RESULTS: Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30 min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05). CONCLUSION: CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

Glucagon-like Peptide-1 Receptor Activation Reduces Pulmonary Vein Arrhythmogenesis and Regulates Calcium Homeostasis.

Glucagon-like peptide-1 (GLP-1) receptor agonists are associated with reduced atrial fibrillation risk, but the mechanisms underlying this association remain unclear. The GLP-1 receptor agonist directly impacts cardiac Ca2+ homeostasis, which is crucial in pulmonary vein (PV, the initiator of atrial fibrillation) arrhythmogenesis. This study investigated the effects of the GLP-1 receptor agonist on PV electrophysiology and Ca2+ homeostasis and elucidated the potential underlying mechanisms. Conventional microelectrodes and whole-cell patch clamp techniques were employed in rabbit PV tissues and single PV cardiomyocytes before and after GLP-1 (7-36) amide, a GLP-1 receptor agonist. Evaluations were conducted both with and without pretreatment with H89 (10 µM, an inhibitor of protein kinase A, PKA), KN93 (1 µM, an inhibitor of Ca2+/calmodulin-dependent protein kinase II, CaMKII), and KB-R7943 (10 µM, an inhibitor of Na+/Ca2+ exchanger, NCX). Results showed that GLP-1 (7-36) amide (at concentrations of 1, 10, and 100 nM) reduced PV spontaneous activity in a concentration-dependent manner without affecting sinoatrial node electrical activity. In single-cell experiments, GLP-1 (7-36) amide (at 10 nM) reduced L-type Ca2+ current, NCX current, and late Na+ current in PV cardiomyocytes without altering Na+ current. Additionally, GLP-1 (7-36) amide (at 10 nM) increased sarcoplasmic reticulum Ca2+ content in PV cardiomyocytes. Furthermore, the antiarrhythmic effects of GLP-1 (7-36) amide on PV automaticity were diminished when pretreated with H89, KN93, or KB-R7943. This suggests that the GLP-1 receptor agonist may exert its antiarrhythmic potential by regulating PKA, CaMKII, and NCX activity, as well as modulating intracellular Ca2+ homeostasis, thereby reducing PV arrhythmogenesis.

Role of Endothelin-1 in Right Atrial Arrhythmogenesis in Rabbits with Monocrotaline-Induced Pulmonary Arterial Hypertension.

Atrial arrhythmias are considered prominent phenomena in pulmonary arterial hypertension (PAH) resulting from atrial electrical and structural remodeling. Endothelin (ET)-1 levels correlate with PAH severity and are associated with atrial remodeling and arrhythmia. In this study, hemodynamic measurement, western blot analysis, and histopathology were performed in the control and monocrotaline (MCT, 60 mg/kg)-induced PAH rabbits. Conventional microelectrodes were used to simultaneously record the electrical activity in the isolated sinoatrial node (SAN) and right atrium (RA) tissue preparations before and after ET-1 (10 nM) or BQ-485 (an ET-A receptor antagonist, 100 nM) perfusion. MCT-treated rabbits showed an increased relative wall thickness in the pulmonary arterioles, mean cell width, cross-sectional area of RV myocytes, and higher right ventricular systolic pressure, which were deemed to have PAH. Compared to the control, the spontaneous beating rate of SAN-RA preparations was faster in the MCT-induced PAH group, which can be slowed down by ET-1. MCT-induced PAH rabbits had a higher incidence of sinoatrial conduction blocks, and ET-1 can induce atrial premature beats or short runs of intra-atrial reentrant tachycardia. BQ 485 administration can mitigate ET-1-induced RA arrhythmogenesis in MCT-induced PAH. The RA specimens from MCT-induced PAH rabbits had a smaller connexin 43 and larger ROCK1 and phosphorylated Akt than the control, and similar PKG and Akt to the control. In conclusion, ET-1 acts as a trigger factor to interact with the arrhythmogenic substrate to initiate and maintain atrial arrhythmias in PAH. ET-1/ET-A receptor/ROCK signaling may be a target for therapeutic interventions to treat PAH-induced atrial arrhythmias.

Effects of Adrenomedullin on Atrial Electrophysiology and Pulmonary Vein Arrhythmogenesis.

Adrenomedullin, a peptide with vasodilatory, natriuretic, and diuretic effects, may be a novel agent for treating heart failure. Heart failure is associated with an increased risk of atrial fibrillation (AF), but the effects of adrenomedullin on atrial arrhythmogenesis remain unclear. This study investigated whether adrenomedullin modulates the electrophysiology of the atria (AF substrate) or pulmonary vein (PV; AF trigger) arrhythmogenesis. Conventional microelectrode or whole-cell patch clamps were used to study the effects of adrenomedullin (10, 30, and 100 pg/mL) on the electrical activity, mechanical response, and ionic currents of isolated rabbit PV and sinoatrial node tissue preparations and single PV cardiomyocytes. At 30 and 100 pg/mL, adrenomedullin significantly reduced the spontaneous beating rate of the PVs from 2.0 ± 0.4 to 1.3 ± 0.5 and 1.1 ± 0.5 Hz (reductions of 32.9% ± 7.1% and 44.9 ± 8.4%), respectively, and reduced PV diastolic tension by 12.8% ± 4.1% and 14.5% ± 4.1%, respectively. By contrast, adrenomedullin did not affect sinoatrial node beating. In the presence of L-NAME (a nitric oxide synthesis inhibitor, 100 µM), adrenomedullin (30 pg/mL) did not affect the spontaneous beating rate or diastolic tension of the PVs. In the single-cell experiments, adrenomedullin (30 pg/mL) significantly reduced the L-type calcium current (ICa-L) and reverse-mode current of the sodium-calcium exchanger (NCX). Adrenomedullin reduces spontaneous PV activity and PV diastolic tension by reducing ICa-L and NCX current and thus may be useful for treating atrial tachyarrhythmia.

Toll-like receptor 4 activation modulates pericardium-myocardium interactions in lipopolysaccharide-induced atrial arrhythmogenesis.

AIMS: Inflammation plays a role in the pathogenesis of atrial fibrillation (AF). Pericarditis enhanced atrial arrhythmogenesis, but the role of the pericardium remains unclear in AF. Activation of the toll-like receptor 4 (TLR4) by binding to lipopolysaccharide (LPS) promotes cardiac electrical remodelling. In this study, we hypothesized that pericarditis may induce atrial arrhythmogenesis via pericardium-myocardium interactions by TLR4 signalling. METHODS AND RESULTS: Pericarditis was induced in rabbits by injecting LPS (1-2 mg/kg) into the pericardium. Conventional microelectrodes were used to record the action potentials of left atrial (LA) posterior walls (LAPWs) and LA appendages (LAAs) with and without attached pericardium in the control or pericarditis-induced rabbits. Cytokine array was used to measure the expression levels of proinflammatory cytokines in control and LPS-treated pericardium. Compared with the controls, the LPS-treated pericardium had higher expressions of IL-1α, IL-8, and MIP-1ß. Rapid atrial pacing-induced burst firing in LPS-treated LAPWs and LAAs, and in control LAPWs (but not in LAAs). The incidence of pacing-induced spontaneous activity and burst firing was increased by LPS-treated pericardium but was attenuated by the control pericardium. Moreover, burst firing induced by LPS-treated pericardium was blocked upon administration of the TLR4 inhibitor, TAK-242 (100 ng/mL), ryanodine receptor inhibitor (ryanodine, 3 µM), or calmodulin kinase II inhibitor (KN-93, 1 µM). CONCLUSIONS: Healthy and inflamed pericardium differently modulate LPS-induced atrial arrhythmogenesis. Targeting pericardium via TLR4 signalling may be a novel therapeutic strategy for AF.

Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication.

Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine-to-inosine (A-to-I)-editing activity. Finally, the association with OV20.0 suppressed the A-to-I-editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.IMPORTANCE Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection.

What does cell therapy manufacturing cost? A framework and methodology to facilitate academic and other small-scale cell therapy manufacturing costings.

BACKGROUND AIMS: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies. METHODS: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€). RESULTS: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices. CONCLUSIONS: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs.

Web-Based Dashboard for the Interactive Visualization and Analysis of National Risk-Standardized Mortality Rates of Sepsis in the US.

Sepsis mortality is heavily influenced by the quality of care in hospitals. Comparing risk-standardized mortality rate (RSMR) of sepsis patients in different states in the United States has potentially important clinical and policy implications. In the current study, we aimed to compare national sepsis RSMR using an interactive web-based dashboard. We analyzed sepsis mortality using the National Inpatient Sample Database of the US. The RSMR was calculated by the hierarchical logistic regression model. We wrote the interactive web-based dashboard using the Shiny framework, an R package that integrates R-based statistics computation and graphics generation. Visual summarizations (e.g., heat map, and time series chart), and interactive tools (e.g., year selection, automatic year play, map zoom, copy or print data, ranking data by name or value, and data search) were implemented to enhance user experience. The web-based dashboard (https://sepsismap.shinyapps.io/index2/) is cross-platform and publicly available to anyone with interest in sepsis outcomes, health inequality, and administration of state/federal healthcare. After extrapolation to the national level, approximately 35 million hospitalizations were analyzed for sepsis mortality each year. Eight years of sepsis mortality data were summarized into four easy to understand dimensions: Sepsis Identification Criteria; Sepsis Mortality Predictors; RSMR Map; RSMR Trend. Substantial variation in RSMR was observed for different states in the US. This web-based dashboard allows anyone to visualize the substantial variation in RSMR across the whole US. Our work has the potential to support healthcare transparency, information diffusion, health decision-making, and the formulation of new public policies.

Use Microfluidic Chips to Study the Phototaxis of Lung Cancer Cells.

Cell migration is an important process involved in wound healing, tissue development, and so on. Many studies have been conducted to explore how certain chemicals and electric fields induce cell movements in specific directions, which are phenomena termed chemotaxis and electrotaxis, respectively. However, phototaxis, the directional migration of cells or organisms toward or away from light, is rarely investigated due to the difficulty of generating a precise and controllable light gradient. In this study, we designed and fabricated a microfluidic chip for simultaneously culturing cells and generating a blue light gradient for guiding cell migration. A concentration gradient was first established inside this chip, and by illuminating it with a blue light-emitting diode (LED), a blue light gradient was generated underneath. Cell migration in response to this light stimulus was observed. It was found that lung cancer cells migrated to the dark side of the gradient, and the intracellular reactive oxygen species (ROS) was proportional to the intensity of the blue light.

Identifying Unmet Treatment Needs for Patients With Osteoporotic Fracture: Feasibility Study for an Electronic Clinical Surveillance System.

BACKGROUND: Traditional clinical surveillance relied on the results from clinical trials and observational studies of administrative databases. However, these studies not only required many valuable resources but also faced a very long time lag. OBJECTIVE: This study aimed to illustrate a practical application of the National Taiwan University Hospital Clinical Surveillance System (NCSS) in the identification of patients with an osteoporotic fracture and to provide a high reusability infrastructure for longitudinal clinical data. METHODS: The NCSS integrates electronic medical records in the National Taiwan University Hospital (NTUH) with a data warehouse and is equipped with a user-friendly interface. The NCSS was developed using professional insight from multidisciplinary experts, including clinical practitioners, epidemiologists, and biomedical engineers. The practical example identifying the unmet treatment needs for patients encountering major osteoporotic fractures described herein was mainly achieved by adopting the computerized workflow in the NCSS. RESULTS: We developed the infrastructure of the NCSS, including an integrated data warehouse and an automatic surveillance workflow. By applying the NCSS, we efficiently identified 2193 patients who were newly diagnosed with a hip or vertebral fracture between 2010 and 2014 at NTUH. By adopting the filter function, we identified 1808 (1808/2193, 82.44%) patients who continued their follow-up at NTUH, and 464 (464/2193, 21.16%) patients who were prescribed anti-osteoporosis medications, within 3 and 12 months post the index date of their fracture, respectively. CONCLUSIONS: The NCSS systems can integrate the workflow of cohort identification to accelerate the survey process of clinically relevant problems and provide decision support in the daily practice of clinical physicians, thereby making the benefit of evidence-based medicine a reality.

Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus.

Double-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from the Poxviridae family, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKR in vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.IMPORTANCE Our previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol 89:4966-4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds directly to the RNA binding domains (RBDs) of PKR, and this interaction does not require dsRNA. Moreover, OV20.0 interacts with or occupies the RBD2 and the kinase domain of PKR, which then prevents PACT binding to PKR. Finally, OV20.0 associates with PACT via the RBDs, which may reduce the ability of PACT to induce PKR activation. The findings in this study provide new concepts in relation to how ORFV modulates PKR activation.

Functional analysis of the short isoform of orf virus protein OV20.0.

UNLABELLED: Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE: The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant.

Goatpoxvirus ATPase activity is increased by dsDNA and decreased by zinc ion.

Viral-encoded ATPase can act as a part of molecular motor in genome packaging of DNA viruses, such as vaccinia virus and adenovirus, by ATP hydrolysis and interaction with DNA. Poxviral ATPase (also called A32) is involved in genomic double-stranded DNA (dsDNA) encapsidation, and inhibition of the expression of A32 causes formation of immature virions lacking viral DNA. However, the role of A32 in goatpoxvirus genome packaging and its dsDNA binding property are not known. In this study, purified recombinant goatpoxvirus A32 protein (rA32) was examined for its dsDNA binding property as well as the effect of dsDNA on ATP hydrolysis. We found that rA32 could bind dsDNA, and its ATPase activity was significant increased with dsDNA binding. Effects of magnesium and calcium ions on ATP hydrolysis were investigated also. The ATPase activity was dramatically enhanced by dsDNA in the presence of Mg(2+); in contrast, ATPase function was not altered by Ca(2+). Furthermore, the enzyme activity of rA32 was completely blocked by Zn(2+). Regarding DNA-protein interaction, the rA32-ATP-Mg(2+) showed lower dsDNA binding affinity than that of rA32-ATP-Ca(2+). The DNA-protein binding was stronger in the presence of zinc ion. Our results implied that A32 may play a role in viral genome encapsidation and DNA condensation.

Establishment of a trimodality analytical platform for tracing, imaging and quantification of gold nanoparticles in animals by radiotracer techniques.

This study aims to establish a (198)Au-radiotracer technique for in vivo tracing, rapid quantification, and ex vivo visualization of PEGylated gold nanoparticles (GNPs) in animals, organs and tissue dissections. The advantages of GNPs lie in its superior optical property, biocompatibility and versatile conjugation chemistry, which are promising to develop diagnostic probes and drug delivery systems. (198)Au is used as a radiotracer because it simultaneously emits beta and gamma radiations with proper energy and half-life; therefore, (198)Au can be used for bioanalytical purposes. The (198)Au-tagged radioactive gold nanoparticles ((198)Au-GNPs) were prepared simply by irradiating the GNPs in a nuclear reactor through the (197)Au(n,γ)(198)Au reaction and subsequently the (198)Au-GNPs were subjected to surface modification with polyethylene glycol to form PEGylated (198)Au-GNPs. The (198)Au-GNPs retained physicochemical properties that were the same as those of GNP before neutron irradiation. Pharmacokinetic and biodisposition studies were performed by intravenously injecting three types of (198)Au-GNPs with or without PEGylation into mice; the γ radiation in blood specimens and dissected organs was then measured. The (198)Au-radiotracer technique enables rapid quantification freed from tedious sample preparation and shows more than 95% recovery of injected GNPs. Clinical gamma scintigraphy was proved feasible to explore spatial- and temporal-resolved biodisposition of (198)Au-GNPs in living animals. Moreover, autoradiography, which recorded beta particles from (198)Au, enabled visualizing the heterogeneous biodisposition of (198)Au-GNPs in different microenvironments and tissues. In this study, the (198)Au-radiotracer technique facilitated creating a trimodality analytical platform for tracing, quantifying and imaging GNPs in animals.

Complexity Analysis of Beat-to-Beat Skin-Surface Laser-Doppler Flowmetry Signals in Stroke Patients.

OBJECTIVES: This study performed skin-surface LDF measurements and SampEn analysis with the aims of (i) capturing the temporal complexity of cerebral hemodynamics in stroke patients and (ii) discriminating stroke patients from healthy control subjects. We also investigated the response induced by AS in beat-to-beat SampEn indexes of LDF signals. METHODS: LDF signals were obtained at bilateral TaiYang acupoints in 52 stroke patients. Each assessment involved a 20-minute baseline recording, a 20-minute AS, and a subsequent 20-minute recording. The FDT, FRT, and PW were calculated for each pulse of the LDF signals, and then their SampEn values were calculated. RESULTS: The SampEn values of FRT were significantly larger in the stroke group (1.064 ± 0.052 and p = 0.013 at the stroke side; 1.059 ± 0.055 and p = 0.017 at the controlateral side) than in the control group (0.975 ± 0.120). On the stroke side, the SampEn of value of FRT was significantly decreased following AS (1.064 ± 0.052 to 1.008 ± 0.060; p = 0.027). CONCLUSION: Larger SampEn values of FRT can be partly attributed to the local regulatory activities that are present in the stroke subjects when facing the induced abnormal vascular conditions and blood flow perfusion resistance. The present findings could aid the development of a noninvasive monitoring technique that will enable discrimination of the different microcirculatory responses in stroke patients.

Roles of nucleic acid substrates and cofactors in the vhs protein activity of pseudorabies virus.

Pseudorabies virus (PrV) belongs to the α-herpesvirinae of which human simplex virus (HSV) is the prototype virus. One of the hallmarks of HSV infection is shutoff of protein synthesis that is mediated by various viral proteins including vhs (virion host shutoff), which is encoded by the UL41 gene. However, the function of PrV vhs is poorly understood. Due to the low sequence similarity (39.3%) between the HSV and PrV UL41 proteins, vhs might not share the same biochemistry characteristics. The purpose of this study was to characterize the nuclease activity of the PrV vhs protein with respect to substrate specificity, its requirements in terms of cofactors, and the protein regions, as well as key amino acids, which contribute to vhs activity. Our results indicated that, similar to HSV vhs, PrV vhs is able to degrade ssRNA and mRNA. However, PrV vhs also targeted rRNA for degradation, which is novel compared to the HSV-1 vhs. Activity assays indicated that Mg(2+) alone enhances RNA degradation mediated by PrV vhs, while K(+) and ATP are not sufficient to induce activity. Finally, we demonstrated that each of the four highly conserved functional boxes of PrV vhs contributes to RNA degradation and that, in particular, residues 152, 169, 171, 172, 173 343, 345, 352 and 356, which are conserved among α-herpesviruses, are key amino acids needed for PrV vhs ribonuclease activity.