Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response.

UNLABELLED: Small ubiquitin-like modifier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. IMPORTANCE: Posttranslational modifications with small ubiquitin-like modifier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection.

SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1.

UNLABELLED: Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE: Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1) as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signaling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signaling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.

Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states.

The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.

Prehospital Identification of Underlying Coronary Artery Disease by Community Paramedics.

There is a lack of definitive evidence that preventative, in-home medical care provided by highly trained community paramedics reduces acute health care utilization and improves the overall well-being of patients suffering from chronic diseases. The Expanding Paramedicine in the Community (EPIC) trial is a randomized controlled trial designed to investigate the use of community paramedics in chronic disease management (ClinicalTrials.gov ID: NCT02034045). This case of a patient randomized to the intervention arm of the EPIC study demonstrates how the added layer of frequent patient contact by community paramedics and real-time electronic medical record (EMR) correspondence between the paramedics, physicians and other involved practitioners prevented possible life-threatening complications. The visiting community paramedic deduced the need for an electrocardiogram, which prompted the primary care physician to order a stress test revealing abnormalities and thus a coronary artery bypass graft was performed without emergency procedures, unnecessary financial expenditure or further health degradation such as a myocardial infarction.

Depletion of intracellular zinc inhibits the ubiquitin ligase activity of viral regulatory protein ICP0 and restricts herpes simplex virus 1 replication in cell culture.

The viral ubiquitin ligase ICP0 stimulates the onset of HSV-1 lytic infection and productive reactivation of viral genomes from latency. In order to mediate these processes, it requires its C3HC4 RING finger domain, a tertiary structural fold that is coordinated by the binding of two zinc (Zn(2+)) atoms. Here we formally demonstrate that Zn(2+) binding and intracellular Zn(2+) levels are critical for ICP0's biochemical activity and that depletion of intracellular Zn(2+) severely attenuates HSV-1 replication.

SUMO pathway dependent recruitment of cellular repressors to herpes simplex virus type 1 genomes.

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2ß, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway.

Thoracic Surgeon Impressions of the Impact of the COVID-19 Pandemic on Lung Cancer Care-Lessons from the First Wave in Canada.

BACKGROUND: COVID-19 has invariably changed the way lung cancer surgical care is provided in Canada. Despite relevant management guidelines, the way in which cancer care has been affected has yet to be described for thoracic surgical populations. Routine lung cancer physiologic and staging assessments are unique in that they are droplet producing and aerosolizing procedures. Our objective was to quantify the effect of the COVID-19 pandemic on surgical lung cancer care as perceived by practicing thoracic surgeons during the first wave of the pandemic in Canada. METHODS: An electronic survey was distributed to members of the Canadian Association of Thoracic Surgeons. The survey was designed to determine surgeon perception of lung cancer preoperative care during the Canadian pandemic-instilled period of resource reallocation compared to standard care. Planned analyses were exploratory in nature; with count and frequency distributions of responses quantified. RESULTS: Fifty-three thoracic surgeons completed the survey. Responses were collected from all Canadian provinces. Little change in access to preoperative imaging was noted. However, a significant decrease in access to lung function and bronchoscopy testing occurred. Pulmonary surgery was perceived to be lengthier with reduced operating theater availability. Despite decreased OR access, only 40% of surgeons were aware of respective institutional mitigation strategies. SUMMARY: The COVID-19 pandemic has had an impact on standard lung cancer care preoperative workup. Further inquiry using institutional data is warranted to quantify its impact on cancer patient outcomes. Assessing the extent and effects of newly present barriers to standard lung cancer care is essential in forming appropriate mitigation strategies and planning for future pandemic waves.

High-Ambient Air Pollution Exposure Among Never Smokers Versus Ever Smokers With Lung Cancer.

INTRODUCTION: Air pollution may play an important role in the development of lung cancer in people who have never smoked, especially among East Asian women. The aim of this study was to compare cumulative ambient air pollution exposure between ever and never smokers with lung cancer. METHODS: A consecutive case series of never and ever smokers with newly diagnosed lung cancer were compared regarding their sex, race, and outdoor and household air pollution exposure. Using individual residential history, cumulative exposure to outdoor particulate matter (PM2.5) in a period of 20 years was quantified with a high-spatial resolution global exposure model. RESULTS: Of the 1005 patients with lung cancer, 56% were females and 33% were never smokers. Compared with ever smokers with lung cancer, never smokers with lung cancer were significantly younger, more frequently Asian, less likely to have chronic obstructive pulmonary disease or a family history of lung cancer, and had higher exposure to outdoor PM2.5 but lower exposure to secondhand smoke. Multivariable logistic regression analysis revealed a significant association with never-smoking patients with lung cancer and being female (OR = 4.01, 95% confidence interval [CI]: 2.76-5.82, p < 0.001), being Asian (ORAsian versus non-Asian = 6.48, 95% CI: 4.42-9.50, p < 0.001), and having greater exposure to air pollution (ORln_PM2.5 = 1.79, 95% CI: 1.10-7.2.90, p = 0.019). CONCLUSIONS: Compared with ever-smoking patients with lung cancer, never-smoking patients had strong associations with being female, being Asian, and having air pollution exposures. Our results suggest that incorporation of cumulative exposure to ambient air pollutants be considered when assessing lung cancer risk in combination with traditional risk factors.

The Diagnostic Accuracy and Sensitivity for Malignancy of Radial-Endobronchial Ultrasound and Electromagnetic Navigation Bronchoscopy for Sampling of Peripheral Pulmonary Lesions: Systematic Review and Meta-analysis.

BACKGROUND: Lung cancer screening with computed tomography chest is identifying peripheral pulmonary lesions (PPLs) suspicious for early-stage lung cancer at increasing rates. Radial-endobronchial ultrasound (R-EBUS) and electromagnetic navigation bronchoscopy (ENB) are 2 methods to sample PPLs to diagnose and treat early lung cancer. ENB has a higher operating financial cost, however, the rationale for its use is possible higher diagnostic accuracy versus R-EBUS. OBJECTIVE: The objective of this study was to determine the comparative diagnostic accuracy, sensitivity, and negative predictive value for R-EBUS and ENB in sampling PPLs. METHODS: A systematic review and meta-analysis were conducted. The Ovid Medline database was queried for original research reporting a diagnostic yield of R-EBUS or ENB for PPLs identified on computed tomography chest suspicious for malignancy. The I statistic assessed study heterogeneity. Random effects models produced pooled estimates of diagnostic accuracy and sensitivity for malignancy. Reasons for heterogeneity were explored with meta-regression. Publication bias and small study effects were assessed. RESULTS: A total of 41 studies involved 2988 lung nodules (R-EBUS 2102, ENB 886) in 3204 patients (R-EBUS 2097, ENB 1107). Overall sensitivity to detect cancer was 70.7% [95% confidence interval (CI): 67.2-74.0]; R-EBUS 70.5% (95% CI: 66.1-74.8), ENB 70.7% (95% CI: 64.7-76.8). Pooled overall diagnostic accuracy was 74.2% (95% CI: 71.0-77.3); R-EBUS 72.4% (95% CI: 68.7-76.1), ENB 76.4% (95% CI: 70.8-82.0). The localization modalities had comparative safety profiles of <2% complications. CONCLUSION: Both technologies have a high proportion of successful PPL localization with similar sensitivity for malignancy and accuracy. As such, both reasonable options for health care authorities to employ diagnostic algorithms.

Proteomic analysis of cells in the early stages of herpes simplex virus type-1 infection reveals widespread changes in the host cell proteome.

During infection by herpes simplex virus type-1 (HSV-1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV-1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV-1 replication, 2-DE and LC-MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock- and HSV-1-infected HEp-2 cells revealed a total of 103 protein spot changes. Of these, 63 were up-regulated and 40 down-regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV-1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV-1 infection.

Engineering cell lines for production of replication defective HSV-1 gene therapy vectors.

Herpes simplex virus type 1 (HSV-1) represents an attractive vehicle for a variety of gene therapy applications. To render this virus safe for clinical use, its cytotoxic genes must be removed without losing its ability to express transgenes efficiently. Our vectors are deleted for the essential immediate early genes ICP4 and ICP27. These genes are controlled by unique promoters having enhancer elements responsive to a viral structural protein VP16. The expression of these genes occurs prior to the activation of all other lytic functions and is thus required to initiate and complete the virus replication cycle. For large scale manufacture of clinical grade vectors, efficient cell lines must be generated that express the essential viral gene products in trans during vector propagation. Here we describe methods for engineering HSV-1 production cell lines that improve vector growth by altering the kinetics of complementing gene expression. We examined the ability of Vero cells independently transduced with ICP4 and ICP27 under transcriptional control of their respective promoters to support the growth of a replication defective vector (JDTOZHE), deleted for ICP4, ICP27 and approximately 20 kb of internal elements that are not required for virus growth in Vero cells. Vector yield on this cell line was 3 logs lower than wild-type virus grown on Vero cells. To understand the mechanism underlying poor vector yield, we examined the expression of ICP4 and ICP27 during virus complementation. While ICP27 was expressed immediately on vector infection, the expression of ICP4 was considerably delayed by 8-10 h, suggesting that the ICP4 promoter was not adequately activated by VP16 delivered by the infectious vector particle. Use of the ICP0 promoter to express ICP4 from the cellular genome resulted in higher induction levels and faster kinetics of ICP4 expression and a 10-fold improvement in vector yield. This study suggests that vector complementation is highly dependent on the kinetics of complementing gene expression and can lead to large differences in vector yield.

Computed tomography-guided platinum microcoil lung surgery: A cross-sectional study.

OBJECTIVE: The study objective was to provide a 5-year update on our tertiary-level institutional experience with computed tomography-guided platinum microcoil lung surgery. METHODS: A retrospective cross-sectional study was conducted. All patients admitted to the Thoracic Service at Vancouver General Hospital to undergo computed tomography-guided microcoil lung surgery were included. Key primary outcome variables were successful nodule localization and severity of adverse events associated with placement. Secondary outcomes included nodule characteristics on preoperative computed tomography chest and nodule surgical pathology. Continuous variables were reported as mean (± standard deviation), and counts were reported as proportions n (%). RESULTS: A total of 97 lung nodules were resected in 92 patients. Mean age was 65.3 (±10.6) years, and 59 (61%) were female. All 97 nodules (100%) were successfully localized using video-assisted thoracic surgery wedge resection. There were 59 cases (60.8%) of placement-related events noted on computed tomography of the chest. All were minor and self-limited in nature and did not require treatment: pneumothorax 45 (46.4%), lung hematoma 18 (18.6%), dislodgement 4 (4.1%), and hemoptysis 2 (2.1%). Mean nodule diameter was 13.2 mm (±6.7). Density was nonsolid in 27 (27.8%) and semi-solid in 27 (27.8%). There was a single case of positive surgical margin, and 4 (4.1%) went on to completion lobectomy. Non-small lung cancer was identified in 66 nodules. CONCLUSIONS: Computed tomography-guided platinum microcoil lung surgery is safe with a favorable clinical adverse event profile and is suitable for poor-risk patients. The method is efficient, yielding 100% diagnostic localization in our 5-year update. It eliminates the need for thoracotomy and palpation to localize worrisome subpleural tiny nodules. It is ideal for the management of changing nodules concerning for early lung cancer and diagnosis of small indeterminate lung nodules or metastases.

Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks.

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.

Preoperative computed tomography-guided microcoil localization of small peripheral pulmonary nodules: a prospective randomized controlled trial.

OBJECTIVES: Growing, small, peripheral, pulmonary nodules in patients at high risk for lung cancer lead to requests for video-assisted thoracoscopic (VATS) resection for pathologic diagnosis. The purpose of this randomized controlled trial was to determine if preoperative localization using percutaneously placed computed tomography (CT)-guided platinum microcoils decreases the need for thoracotomy or VATS anatomic resection (segmentectomy/lobectomy) for diagnosis. METHODS: Patients with undiagnosed nodules of 15 mm or less were randomized to either no localization or preoperative microcoil localization. Coils were placed with the distal end deep to the nodule and the superficial end coiled on the visceral pleural surface with subsequent visualization by intraoperative fluoroscopy and VATS. Nodules were removed by VATS wedge excision using endostaplers. The primary outcome was a VATS wedge excision for pathologic diagnosis of the nodule without the need for either thoracotomy or VATS anatomic resection. RESULTS: Sixty patients were randomized and 56 underwent surgery between March 2010 and June 2012. Twenty-nine underwent microcoil localization and 27 did not. The baseline characteristics (age, sex, forced expiratory volume in the first second of expiration, nodule size/depth) were similar. The coil group had a higher rate of successful diagnosis with VATS wedge resection alone (27/29 vs 13/27; P < .001), decreased operative time to nodule excision (37 ± 39 vs 100 ± 67 minutes; P < .001), and reduced stapler firings (3.7 ± 2.0 vs 5.9 ± 31; P = .003) with no difference in total costs. Pathologic diagnoses included 14 benign nodules, 32 primary lung malignancies, and 10 metastases. There were no clinically significant complications related to the coil placement or wedge resection. CONCLUSIONS: Preoperative CT-guided microcoil localization decreases the need for thoracotomy or VATS anatomic resection for the diagnosis of small peripheral pulmonary nodules.

Systemic perturbation of the ERK signaling pathway by the proteasome inhibitor, MG132.

Inhibition of the ubiquitin-proteasome protein degradation pathway has been identified as a viable strategy for anti-tumor therapy based on its broad effects on cell proliferation. By the same token, the variety of elicited effects confounds the interpretation of cell-based experiments using proteasome inhibitors such as MG132. It has been proposed that MG132 treatment reduces growth factor-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), at least in part through upregulation of dual specificity phosphatases (DUSPs). Here, we show that the effects of MG132 treatment on ERK signaling are more widespread, leading to a reduction in activation of the upstream kinase MEK. This suggests that MG132 systemically perturbs the intracellular phosphoproteome, impacting ERK signaling by reducing phosphorylation status at multiple levels of the kinase cascade.

Magnetic field-controlled microfluidic transport.

Several new forms of magnetohydrodynamic (MHD) flow occurring in the solution gap between two 250-microm-diameter Pt microdisk electrodes, oriented in a face-to-face geometry and immersed in a uniform magnetic field (1 T), are described. The MHD flow results from the Lorentz force generated by diffusion of electrochemically generated molecular ions through the magnetic field. Individual microscopic flow tubes ( approximately 50-microm radius) spanning the gap between the face-to-face electrodes are observed during the 1-e(-) reduction of nitrobenzene in acetonitrile solutions. The flow tubes extend up to approximately 2 cm in length and are stable for indefinite periods. Directional transport of the electrogenerated nitrobenzene radical anion over macroscopic distances within the flow tubes, with minimal diffusional broadening, is demonstrated using an ultramicroelectrode probe to map the convective flux of redox species. Pulsed MHD transport of small packets of molecules and the formation of large area (approximately 3 cm(2)), microscopically thin (25 microm) rotating sheets of solution are also demonstrated. The results suggest that electrochemical methods, in combination with magnetohydrodynamic principles, may be useful for external field-controlled microfluidic systems.