Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

[GRADE guidelines 20: Assessing the certainty of evidence in the importance of outcomes or values and preferences - inconsistency, imprecision, and other domains]. / GRADE Leitlinien: 20. Untersuchung der Vertrauenswürdigkeit in die Evidenz zur Beurteilung der Wichtigkeit von Endpunkten oder Werten und Präferenzen ­ Inkonsistenz, unzureichende Präzision und andere Domänen.

OBJECTIVE: To provide Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) guidance for assessing inconsistency, imprecision, and other domains for the certainty of evidence about the relative importance of outcomes. STUDY DESIGN AND SETTING: We applied the GRADE domains to rate the certainty of evidence in the importance of outcomes to several systematic reviews, iteratively reviewed draft guidance, and consulted GRADE members and other stakeholders for feedback. RESULTS: We describe the rationale for considering the remaining GRADE domains when rating the certainty in a body of evidence for the relative importance of outcomes. As meta-analyses are not common in this context, inconsistency and imprecision assessments are challenging. Furthermore, confusion exists about inconsistency, imprecision, and true variability in the relative importance of outcomes. To clarify this issue, we suggest that the true variability is neither equivalent to inconsistency nor imprecision. Specifically, inconsistency arises from population, intervention, comparison and outcome and methodological elements that should be explored and, if possible, explained. The width of the confidence interval and sample size inform judgments about imprecision. We also provide suggestions on how to detect publication bias and discuss the domains to rate up the certainty. CONCLUSION: We provide guidance and examples for rating inconsistency, imprecision, and other domains for a body of evidence describing the relative importance of outcomes.

Effect of an emergency department education and empowerment intervention on uncontrolled hypertension in a predominately minority population: The AHEAD2 randomized clinical pilot trial.

OBJECTIVE: To determine whether an emergency department (ED) education and empowerment intervention coupled with early risk assessment can help improve blood pressure (BP) in a high-risk population. METHODS: A hypertension emergency department intervention aimed at decreasing disparities (AHEAD2) is a 3-arm, single-site randomized pilot trial for feasibility in an urban academic ED. A total of 150 predominantly ethnic minorities with no primary care provider and severely elevated blood pressure (BP) (≥160/100 mm Hg) were enrolled over 10 months. Participants were randomized into 1 of 3 study arms: (1) enhanced usual care (EUC), (2) ED-initiated screening, brief intervention, and referral for treatment (ED-SBIRT), or (3) ED- SBIRT plus a 48-72 hours post-acute care hypertension transition clinic (ED-SBIRT+PACHT-c). Primary outcomes were change in systolic and diastolic BP (SBP and DBP) from baseline to 9 months. Secondary outcomes were BP control (BP <140/90 mm Hg), changes in hypertension knowledge, medication adherence, and limited bedside echocardiogram (LBE) findings. RESULTS: SBP reduction from baseline to month 9 was -26.8 (95% confidence interval [CI]: -32.8, -20.7) mm Hg for ED-SBIRT, -23.4 (95% CI: -29.5, -17.3) mm Hg for ED-SBIRT+PACHT-c, and -18.9 (95% CI: -24.9, -12.9) mm Hg for EUC. DBP decreased by -12.5 (95% CI: -16.1, -9.0) mm Hg for ED-SBIRT, -11.3 (95% CI: -14.8, -7.7) mm Hg for ED-SBIRT+PACHT-c, and -8.4 (95% CI: -11.9, -4.9) mm Hg for EUC. A multicomponent intervention compared with EUC resulted in SBP decrease of -7.9 mm Hg (95% CI: -16.4, 0.6). At 9 months, hypertension was controlled for 29.3% (95% CI: 20.3, 38.3) of intervention and 23.5% (95% CI: 11.9, 35.2) of EUC participants. All groups saw improvements in hypertension knowledge, medication adherence, and LBEs, with greater improvements in intervention groups. CONCLUSIONS: The study findings suggest that a multicomponent intervention comprising of ED education and empowerment coupled with early risk assessment may help improve BP in a high-risk population.

[GRADE Guidelines: 19. Assessing the certainty of evidence in the importance of outcomes or values and preferences: Risk of bias and indirectness]. / GRADE-Leitlinien: 19. Bewertung der Vertrauenswürdigkeit der Evidenz für die Bedeutung von Endpunkten oder Werten und Präferenzen ­ Risiko für Bias und Indirektheit.

OBJECTIVES: The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) working group defines patient values and preferences as the relative importance patients place on the main health outcomes. We provide GRADE guidance for assessing the risk of bias and indirectness domains for certainty of evidence about the relative importance of outcomes. STUDY DESIGN AND SETTING: We applied the GRADE domains to rate the certainty of evidence in the importance of outcomes to several systematic reviews, iteratively reviewed draft guidance and consulted GRADE members and other stakeholders for feedback. RESULTS: This is the first of two articles. A body of evidence addressing the importance of outcomes starts at "high certainty"; concerns with risk of bias, indirectness, inconsistency, imprecision, and publication bias lead to downgrading to moderate, low, or very low certainty. We propose the following subdomains of risk of bias: selection of the study population, missing data, the type of measurement instrument, and confounding; we have developed items for each subdomain. The population, intervention, comparison, and outcome elements associated with the evidence determine the degree of indirectness. CONCLUSION: This article provides guidance and examples for rating the risk of bias and indirectness for a body of evidence summarizing the importance of outcomes.

Community Targeting of Uncontrolled Hypertension: Results of a Hypertension Screening and Education Intervention in Community Churches Serving Predominantly Racial/Ethnic Minority Populations.

Background. Uncontrolled hypertension is the primary risk factor for the development of cardiovascular complications and particularly burdens racial/ethnic minority populations. Aim. To determine the effectiveness of a community hypertension screening, education, and empowerment intervention on blood pressure (BP) improvement. Method. We screened 152 participants across four churches in predominantly racial/ethnic minority neighborhoods for elevated BP. During this visit, those with BP ≥ 140/90 mmHg were enrolled in the study and completed interventions. Individuals with moderately elevated BP (≥140/90 and <160/100 mmHg; Group 1) viewed a 3-minute hypertension education video. Individuals with severely elevated BP (≥160/100 mmHg; Group 2) additionally viewed echocardiograms images with subclinical changes from uncontrolled hypertension and had a brief on-site medication review with a pharmacist. Both groups received automated BP monitors and information on neighborhood federally qualified health centers for primary care. Participants returned to each church for follow-up 3 months later. We analyzed BP difference at 3 months and percentage with controlled BP for each group. Results. For Group 1, mean baseline and follow-up BPs were 143.5/88.0 mmHg and 138.5/85.8 mmHg, respectively. For Group 2, BPs significantly decreased from 165.4/98.3 mmHg to 150.4/90.8 mmHg. After the intervention, participants with controlled BP for Groups 1 and 2 were 35.5% and 55.2%, respectively. Discussion. Participants in both groups had BP improvements. Greater improvements were in individuals at higher cardiovascular risk due to severely elevated BPs (≥160/100 mmHg). Conclusion. This pilot highlights the impact that streamlined empowerment interventions with dedicated health personnel can have in high-risk communities with elevated BPs.

Direct Automated MALDI Mass Spectrometry Analysis of Cellular Transporter Function: Inhibition of OATP2B1 Uptake by 294 Drugs.

OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.

Barriers and Facilitators to Hypertension Control Following Participation in a Church-Based Hypertension Intervention Study.

PURPOSE: Hypertension is the primary risk factor for development of cardiovascular complications. Community-initiated interventions have proven effective in reducing cardiovascular disease risk among individuals who might otherwise face barriers to care. The purposes of this study were to gain feedback on a church-based hypertension intervention study and assess barriers and facilitators to hypertension control after participation in the study. DESIGN: Qualitative study of 4 focus groups. SETTING: Focus groups took place at 4 churches in primarily minority neighborhoods of Chicago, Illinois, in summer 2017. PARTICIPANTS: Thirty-one community members participated in the focus groups. METHOD: The Community Targeting of Uncontrolled Hypertension (CTOUCH) study was a church-based screening, brief intervention, and referral for treatment program for hypertension. Following the study completion, participants were invited to join a focus group to provide feedback on the study and discuss barriers and facilitators to hypertension control. The authors used the Framework Method to analyze the data. RESULTS: Community Targeting of Uncontrolled Hypertension was well received by participants, particularly the awareness of their individual blood pressure and subsequent education on risk modification. The most common facilitators for hypertension control were social support, knowing how to control hypertension, and community resources. The most common barriers to hypertension control were lack of hypertension knowledge, negative primary care experiences, and lack of disease awareness. CONCLUSION: Knowledge of barriers and facilitators can inform areas of success and opportunities for improvement in community-based hypertension programs including future renditions in CTOUCH.

Quantitative Mass Spectrometry Imaging Reveals Mutation Status-independent Lack of Imatinib in Liver Metastases of Gastrointestinal Stromal Tumors.

Mass spectrometry imaging (MSI) is an enabling technology for label-free drug disposition studies at high spatial resolution in life science- and pharmaceutical research. We present the first extensive clinical matrix-assisted laser desorption/ionization (MALDI) quantitative mass spectrometry imaging (qMSI) study of drug uptake and distribution in clinical specimen, analyzing 56 specimens of tumor and corresponding non-tumor tissues from 27 imatinib-treated patients with the biopsy-proven rare disease gastrointestinal stromal tumors (GIST). For validation, we compared MALDI-TOF-qMSI with conventional UPLC-ESI-QTOF-MS-based quantification from tissue extracts and with ultra-high resolution MALDI-FTICR-qMSI. We introduced a novel generalized nonlinear calibration model of drug quantities based on computational evaluation of drug-containing areas that enabled better data fitting and assessment of the inherent method nonlinearities. Imatinib tissue spatial maps revealed striking inefficiency in drug penetration into GIST liver metastases even though the corresponding healthy liver tissues in the vicinity showed abundant imatinib levels beyond the limit of quantification (LOQ), thus providing evidence for secondary drug resistance independent of mutation status. Taken together, these findings underscore the important application of MALDI-qMSI in studying the spatial distribution of molecularly targeted therapeutics in oncology, namely to serve as orthogonal post-surgical approach to evaluate the contribution of anticancer drug disposition to resistance against treatment.

Advanced MALDI mass spectrometry imaging in pharmaceutical research and drug development.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has emerged as a key technology for label-free bioanalysis of the spatial distribution of biomolecules, pharmaceuticals and other xenobiotics in tissue sections. Recent advances in instrumentation, sample preparation, multimodal workflows, quantification, analytical standardization and 'big data' processing have led to widespread utilization of MALDI MSI in pharmaceutical research. These developments have led to applications of the technology in drug discovery beyond drug disposition analysis, most notably in pharmacodynamic biomarker research and in toxicology.

Author Correction: Fourier Transform Infrared Microscopy Enables Guidance of Automated Mass Spectrometry Imaging to Predefined Tissue Morphologies.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Analyzing the antiseptic capacity of silver-functionalized poly(ethylene glycol)-heparin hydrogels after human whole blood exposure.

Advanced blood contacting biomaterials are designed to combine antiseptic and anticoagulant functionalities. Here, we present a new in vitro methodology for the analysis of bacterial adhesion and growth after the preceding human whole blood incubation of the tested materials. Poly(styrene) surfaces as well as thrombin-responsive and non-responsive poly(ethylene glycol)-heparin hydrogel coatings, with and without silver functionalization, were analyzed with this approach using freshly drawn human whole blood and various human pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). Adsorbed blood proteins and adherent immune cells were observed to suppress bacterial colonization on poly(styrene) surfaces. Silver functionalization of responsive and non-responsive poly(ethylene glycol)-heparin hydrogels had no influence on microbial attachment but decreased bacterial proliferation and viability. Whole blood pre-incubation did not affect the antimicrobial properties of the tested silver-modified hydrogels. In sum, our introduced multistage incubation test revealed the antibacterial effects as well as antiseptic-permissive characteristics of blood-borne interfacial layers on polymeric biomaterials.

Fourier Transform Infrared Microscopy Enables Guidance of Automated Mass Spectrometry Imaging to Predefined Tissue Morphologies.

Multimodal imaging combines complementary platforms for spatially resolved tissue analysis that are poised for application in life science and personalized medicine. Unlike established clinical in vivo multimodality imaging, automated workflows for in-depth multimodal molecular ex vivo tissue analysis that combine the speed and ease of spectroscopic imaging with molecular details provided by mass spectrometry imaging (MSI) are lagging behind. Here, we present an integrated approach that utilizes non-destructive Fourier transform infrared (FTIR) microscopy and matrix assisted laser desorption/ionization (MALDI) MSI for analysing single-slide tissue specimen. We show that FTIR microscopy can automatically guide high-resolution MSI data acquisition and interpretation without requiring prior histopathological tissue annotation, thus circumventing potential human-annotation-bias while achieving >90% reductions of data load and acquisition time. We apply FTIR imaging as an upstream modality to improve accuracy of tissue-morphology detection and to retrieve diagnostic molecular signatures in an automated, unbiased and spatially aware manner. We show the general applicability of multimodal FTIR-guided MALDI-MSI by demonstrating precise tumor localization in mouse brain bearing glioma xenografts and in human primary gastrointestinal stromal tumors. Finally, the presented multimodal tissue analysis method allows for morphology-sensitive lipid signature retrieval from brains of mice suffering from lipidosis caused by Niemann-Pick type C disease.

A hypertension emergency department intervention aimed at decreasing disparities: Design of a randomized clinical trial.

Effective interventions to identify and treat uncontrolled hypertension (HTN), particularly in underrepresented populations that use the emergency department (ED) for primary care, are critically needed. Uncontrolled HTN contributes significantly to cardiovascular morbidity and mortality and is more frequently encountered among patients presenting to the ED as compared to the primary care setting. EDs serve as the point of entry into the health care system for high-risk patient populations, including minority and low-income patients. Previous studies have demonstrated that the prevalence of uncontrolled/undiagnosed HTN in patients presenting to the ED is alarmingly high. Thus ED engagement and early risk assessment/stratification is a feasible innovation to help close health disparity gaps in HTN. A Hypertension Emergency Department Intervention Aimed at Decreasing Disparities (AHEAD2) trial, funded by the National Heart, Lung, and Blood Institute (NHLBI) is a three-arm single site randomized clinical pilot trial of adults presenting to the ED with Stage 2 hypertension (blood pressure [BP]>160/100) comparing (1) an ED-initiated Screening, Brief Intervention, and Referral for Treatment (SBIRT) focused on HTN, (2) the same ED-initiated SBIRT coupled with a Post-Acute Care Hypertension Transition Consultation by ED Clinical Pharmacists, and (3) usual care. The primary outcome is mean BP differences between study arms. Secondary outcomes are proportion of participants with BP control (BP<140/90mmHg), and improvements in HTN knowledge and medication adherence scores between study arms. The objective of this report is to describe the development of the AHEAD2 trial, including the methods, research infrastructure, and other features of the randomized clinical trial design.

QUALITY OF SAMPLE SIZE ESTIMATION IN TRIALS OF MEDICAL DEVICES: HIGH-RISK DEVICES FOR NEUROLOGICAL CONDITIONS AS EXAMPLE.

BACKGROUND: The aim of this study was to assess the quality of reporting sample size calculation and underlying design assumptions in pivotal trials of high-risk medical devices (MDs) for neurological conditions. METHODS: Systematic review of research protocols for publicly registered randomized controlled trials (RCTs). In the absence of a published protocol, principal investigators were contacted for additional data. To be included, trials had to investigate a high-risk MD, registered between 2005 and 2015, with indications stroke, headache disorders, and epilepsy as case samples within central nervous system diseases. Extraction of key methodological parameters for sample size calculation was performed independently and peer-reviewed. RESULTS: In a final sample of seventy-one eligible trials, we collected data from thirty-one trials. Eighteen protocols were obtained from the public domain or principal investigators. Data availability decreased during the extraction process, with almost all data available for stroke-related trials. Of the thirty-one trials with sample size information available, twenty-six reported a predefined calculation and underlying assumptions. Justification was given in twenty and evidence for parameter estimation in sixteen trials. Estimates were most often based on previous research, including RCTs and observational data. Observational data were predominantly represented by retrospective designs. Other references for parameter estimation indicated a lower level of evidence. CONCLUSIONS: Our systematic review of trials on high-risk MDs confirms previous research, which has documented deficiencies regarding data availability and a lack of reporting on sample size calculation. More effort is needed to ensure both relevant sources, that is, original research protocols, to be publicly available and reporting requirements to be standardized.

Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer.

Gastric cancer (GC) remains a malignant disease with high mortality. Patients are frequently diagnosed in advanced stages where survival prognosis is poor. Thus, there is high medical need to find novel drug targets and treatment strategies. Recently, the comprehensive molecular characterization of GC subtypes revealed mutations in the small GTPase RHOA as a hallmark of diffuse-type GC. RHOA activates RHO-associated protein kinases (ROCK1/2) which regulate cell contractility, migration and growth and thus may play a role in cancer. However, therapeutic benefit of RHO-pathway inhibition in GC has not been shown so far. The ROCK1/2 inhibitor 1-(5-isoquinoline sulfonyl)-homopiperazine (HA-1077, fasudil) is approved for cerebrovascular bleeding in patients. We therefore investigated whether fasudil (i.p., 10 mg/kg per day, 4 times per week, 4 weeks) inhibits tumor growth in a preclinical model of GC. Fasudil evoked cell death in human GC cells and reduced the tumor size in the stomach of CEA424-SV40 TAg transgenic mice. Small animal PET/CT confirmed preclinical efficacy. Mass spectrometry imaging identified a translatable biomarker for mouse GC and suggested rapid but incomplete in situ distribution of the drug to gastric tumor tissue. RHOA expression was increased in the neoplastic murine stomach compared with normal non-malignant gastric tissue, and fasudil reduced (auto) phosphorylation of ROCK2 at THR249 in vivo and in human GC cells in vitro. In sum, our data suggest that RHO-pathway inhibition may constitute a novel strategy for treatment of GC and that enhanced distribution of future ROCK inhibitors into tumor tissue may further improve efficacy.

Clinical Presentation and Multi-Parametric Screening Surrogates of Ischemic Stroke Patients Suffering from Infective Endocarditis.

BACKGROUND: Infective endocarditis (IE) represents a life-threatening condition due to complications like cardiac failure and thromboembolism. In ischemic stroke, IE formally excludes patients from approaches addressing the recanalization of occluded vessels, challenging decision-making in the early phase of hospitalization. This study aimed at the rate and clinical course of stroke patients with IE and explored clinical, imaging-based and serum parameters, which would allow early identification. METHODS: A hospital-based registry containing 1,531 ischemic stroke patients was screened for IE identified by echocardiography. In addition to clinical parameters, patterns of cerebral manifestation as well as a variety of inflammatory serum and myocardial markers were analyzed concerning their predictive impact for identifying affected patients. RESULTS: IE was found in 26 patients (1.7%) and was associated with an increased body temperature and cardiac murmurs. Patients suffering from IE demonstrated a more severe clinical affection at hospital discharge and an impaired symptom decline during hospitalization, further deteriorated by the use of systemic thrombolysis. Distribution of cerebral infarction patterns did not differ between the groups. C-reactive protein (CRP) and leukocyte count as well as troponin and myoglobin, taken at hospital admission, were found to be significantly associated with IE. CONCLUSIONS: IE in stroke patients is associated with worse clinical outcome, complicated by intravenously applied thrombolysis, and therefore needs to be screened during the early phase of hospitalization. Increased serum levels of CRP and leukocyte count in combination with an increased body temperature or abnormal cardiac murmurs should entail rapid initiation of further diagnostics, that is, transoesophageal echocardiography.

The stress kinase GCN2 does not mediate suppression of antitumor T cell responses by tryptophan catabolism in experimental melanomas.

Tryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific Gcn2 knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of Tdo did not differentially affect GCN2-proficient vs. GCN2-deficient T cells in vivo. Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Interaction of VP8 with mRNAs of bovine herpesvirus-1.

The UL47 gene product, VP8, is one of the most abundant tegument proteins of bovine herpesvirus-1 (BoHV-1). Deletion of VP8 leads to impaired growth in tissue culture, and VP8 is indispensable for BHV-1 replication in cattle. To elucidate the biological functions of VP8, we explored its interaction with mRNAs of immediate early (bICP0), early (gB, gD) and late (gC) genes of BoHV-1. FLAG-tagged VP8 was pulled down from COS-7 cells co-transfected with plasmids encoding VP8 and either gB, gC, gD or bICP0. This was followed by RNA extraction, cDNA synthesis and qPCR, which demonstrated binding of VP8 to bICP0, gB, gC and gD mRNAs in the cytoplasm and nucleus. These results were supported by co-localization of VP8 with bICP0, gB, gC and gD mRNAs in the nucleus as determined by confocal microscopy. Amino acids 259-342, located in the conserved portion of UL47 homologues, were found to contain the RNA binding region on VP8. To further characterize these interactions, Northwestern blotting was performed by immobilizing VP8 on a nitrocellulose membrane followed by hybridization with in vitro transcribed bICP0 mRNA. The results demonstrated binding of VP8 to intron-less mRNA but not intron-containing mRNA of bICP0. In addition, the interaction of VP8 with bICP0 mRNA was confirmed in vitro by RNA electrophoretic mobility shift assay, which also showed that the zinc finger and acidic domains both interact with VP8. Based on these results, we concluded that VP8 binds to intron-less mRNAs of bICP0, gB, gC and gD.