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1.  A G-quadruplex-binding macrodomain within the “SARS-unique domain” is essential for the activity of the SARS-coronavirus replication-transcription complex 
Virology  2015;484:313-322.
The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the “SARS-unique domain”. The X domain was proposed to be an ADP-ribose-1″-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.
Graphical Abstract
doi:10.1016/j.virol.2015.06.016
PMCID: PMC4567502  PMID: 26149721
SARS-CoV replicon; SARS-unique domain; macrodomain; X-domain; reverse genetics; G-quadruplex; MERS-CoV
2.  A Cross-Sectional Comparison of Druggable Mutations in Primary Tumors, Metastatic Tissue, Circulating Tumor Cells, and Cell-Free Circulating DNA in Patients with Metastatic Breast Cancer: The MIRROR Study Protocol 
JMIR Research Protocols  2016;5(3):e167.
Background
Characterization of the driver mutations in an individual metastatic breast cancer (MBC) patient is critical to selecting effective targeted therapies. Currently, it is believed that the limited efficacy of many targeted drugs may be due to the expansion of drug resistant clones with different genotypes that were already present in the primary tumor. Identifying the genomic alterations of these clones, and introducing combined or sequential targeted drug regimens, could lead to a significant increase in the efficacy of currently available targeted therapies.
Objective
The primary objective of this study is to assess the concordance/discordance of mutations between the primary tumor and metastatic tissue in MBC patients. Secondary objectives include comparing the genomic profiles of circulating tumor cells (CTCs) and circulating free DNA (cfDNA) from peripheral blood with those of the primary tumor and metastatic tissue for each patient, evaluating these mutations in the signaling pathways that are relevant to the disease, and testing the feasibility of introducing liquid biopsy as a translational laboratory tool in clinical practice.
Methods
The multicenter, transversal, observational MIRROR study is currently ongoing in three participating hospitals. All consecutive patients with MBC confirmed by radiologic findings will be screened for eligibility, either at first relapse or if tumor regrowth occurs while on treatment for metastatic disease.
Results
Patient recruitment is currently ongoing. To date, 41 patients have a complete set of tissue samples available (plasma, CTCs, and formalin-fixed, paraffin-embedded primary tumor and metastatic tumor). However, none of these samples have undergone nucleic acids extraction or targeted deep sequencing.
Conclusions
The results of this study may have a significant influence on the practical management of patients with MBC, and may provide clues to clinicians that lead towards a better stratification of patients, resulting in more selective and less toxic treatments. Additionally, if genomic mutations found in metastatic tissues are similar to those detected in CTCs and/or cfDNA, liquid biopsies could prove to be a more convenient, non-invasive, and easily accessible source of genomic material for the analysis of mutations and other genomic aberrations in MBC.
Trial Registration
ClinicalTrials.gov NCT02626039; https://clinicaltrials.gov/ct2/show/NCT02626039 (Archived by WebCite at http://www.webcitation.org/6jlneVyoz)
doi:10.2196/resprot.6024
PMCID: PMC5005674  PMID: 27531554
breast neoplasm; drug therapy; genetics; molecular targeted therapy; sequence analysis.
3.  Proinflammatory cytokines and matrix metalloproteinases in CSF of patients with VZV vasculopathy 
Objective:
To determine the levels of proinflammatory cytokines and matrix metalloproteinases (MMPs) in the CSF of patients with virologically verified varicella zoster virus (VZV) vasculopathy.
Methods:
CSF from 30 patients with virologically verified VZV vasculopathy was analyzed for levels of proinflammatory cytokines and MMPs using the Meso Scale Discovery multiplex ELISA platform. Positive CNS inflammatory disease controls were provided by CSF from 30 patients with multiple sclerosis. Negative controls were provided by CSF from 20 healthy controls.
Results:
Compared to multiple sclerosis CSF and CSF from healthy controls, levels of interleukin (IL)-8, IL-6, and MMP-2 were significantly elevated in VZV vasculopathy CSF.
Conclusions:
CSF of patients with VZV vasculopathy revealed a unique profile of elevated proinflammatory cytokines, IL-8 and IL-6, along with elevated MMP-2. The relevance of these cytokines to the pathogenesis of VZV vasculopathy requires further study.
doi:10.1212/NXI.0000000000000246
PMCID: PMC4907802  PMID: 27340684
4.  Impact of Vesicular Stomatitis Virus M Proteins on Different Cellular Functions 
PLoS ONE  2015;10(6):e0131137.
Three different matrix (M) proteins termed M1, M2 and M3 have been described in cells infected with vesicular stomatitis virus (VSV). Individual expression of VSV M proteins induces an evident cytopathic effect including cell rounding and detachment, in addition to a partial inhibition of cellular protein synthesis, likely mediated by an indirect mechanism. Analogous to viroporins, M1 promotes the budding of new virus particles; however, this process does not produce an increase in plasma membrane permeability. In contrast to M1, M2 and M3 neither interact with the cellular membrane nor promote the budding of double membrane vesicles at the cell surface. Nonetheless, all three species of M protein interfere with the transport of cellular mRNAs from the nucleus to the cytoplasm and also modulate the redistribution of the splicing factor. The present findings indicate that all three VSV M proteins share some activities that interfere with host cell functions.
doi:10.1371/journal.pone.0131137
PMCID: PMC4474437  PMID: 26091335
6.  CXCL13 is a Biomarker of Inflammation in Multiple Sclerosis, Neuromyelitis Optica, and Other Neurological Conditions 
CXCXL13, a B-cell chemokine, has been proposed as a biomarker in a variety of conditions, some of which can mimic multiple sclerosis (MS) and can have very high levels. In this case-control study, CSF CXCL13 was elevated in MS, neuromyelitis optica (NMO), and other inflammatory neurological controls compared to noninflammatory controls. Levels did not differentiate disease groups. For all subjects taken together, CSF CXCL13 correlated with CSF WBC, oligoclonal band numbers, CSF protein, EDSS, and neurofilament levels. In subgroup analyses, CSF CXCL13 correlated with CSF WBC in NMO and IgG index in MS. Additionally, serum CXCL13 was elevated in NMO.
doi:10.1177/1352458512473362
PMCID: PMC3959125  PMID: 23322500
Multiple sclerosis; neuromyelitis optica; CXCL13; neuroinflammation; neurofilament; myelin basic protein; biomarker
7.  Role of Magnetic Resonance Imaging, Cerebrospinal Fluid, and Electroencephalogram in Diagnosis of Sporadic Creutzfeldt-Jakob Disease 
Journal of neurology  2012;260(2):498-506.
Sporadic Creutzfeldt-Jakob disease (sCJD) is a rapidly progressive dementia (RPD) that can be difficult to identify ante-mortem, with definitive diagnosis requiring tissue confirmation. We describe the clinical, magnetic resonance imaging (MRI), cerebrospinal fluid (CSF), and electroencephalogram (EEG) measures of a small cohort of 30 patients evaluated for RPD. Clinical and diagnostic measures were cross-sectionally obtained from 17 sCJD patients (15 definite, 2 probable), 13 non-prion rapidly progressive dementia patients (npRPD), and 18 unimpaired controls. In a subset of patients (9 sCJD and 9 npRPD) diffusion tensor imaging (DTI) measures [fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD)] were also obtained for the caudate, corpus callosum, posterior limb of the internal capsule, pulvinar, precuneus, and frontal lobe. Differences among groups were assessed by an analysis of variance. Compared to npRPD individuals, sCJD patients had cerebellar dysfunction, significantly higher CSF tau, “positive” CSF 14-3-3, and hyperintensities on diffusion weighted imaging (DWI) that met previously established imaging criteria for sCJD. EEG changes were similar for the two groups. In addition, sCJD patients had significant decreases in DTI measures (MD, AD, RD but not FA) within the caudate and pulvinar compared to either npRPD patients or unimpaired controls. Our results confirm that CSF abnormalities and MRI (especially DWI) can assist in distinguishing sCJD patients from npRPD patients. Future longitudinal studies using multiple measures (including CSF and MRI) are needed for evaluating pathological changes seen in sCJD patients.
doi:10.1007/s00415-012-6664-6
PMCID: PMC3651672  PMID: 22968768
Creutzfeldt-Jakob disease; diffusion magnetic resonance imaging; cerebrospinal fluid
8.  Association of Neuromyelitis Optica With Severe and Intractable Pain 
Archives of neurology  2012;69(11):1482-1487.
Objective
To contrast differences in pain and treatment outcomes between neuromyelitis optica (NMO) and multiple sclerosis (MS).
Design
Retrospective, cross-sectional cohort study.
Setting
Academic MS center.
Patients
Complete ascertainment of an academic MS center cohort of NMO and an MS comparison sample cohort.
Main Outcome Measures
Current pain was quantified by a 10-point scale and the McGill Pain Questionnaire. Expanded Disability Status Scale score and number of involved spinal cord levels were collected in addition to testing for cognition, fatigue, depression, and quality of life. Number and types of pain medications were tabulated.
Results
Current pain was more common in subjects with NMO (n=29) vs MS (n=66) (86.2% vs 40.9%; P<.001) and more severe on a 10-point scale (5.38 vs 1.85; P <.001). Pain remained more common after controlling for disability and number of spinal cord segments (P=.03). Prescription pain medication was used more frequently in subjects with NMO compared with subjects with MS (75.9% vs 37.8%; P<.001), often requiring more than 1 medication (65.5% vs 15.2%; P<.001). No subject with NMO taking pain medication (22 of 29) rated their current pain as 0 of 10, whereas almost half of those taking pain medication with MS were currently free of pain (0% vs 48%; P=.006).
Conclusions
Neuromyelitis optica is frequently associated with severe pain that appears insufficiently controlled by pharmacologic interventions. Future studies should evaluate the efficacy of a multidisciplinary and multimodal approach to pain management.
doi:10.1001/archneurol.2012.768
PMCID: PMC3561507  PMID: 22926050
9.  Rituximab-Associated Progressive Multifocal Leukoencephalopathy in Rheumatoid Arthritis 
Archives of neurology  2011;68(9):1156-1164.
Objective
To describe the development of progressive multifocal leukoencephalopathy (PML) in patients with rheumatoid arthritis (RA) treated with rituximab.
Design
Case study.
Setting
Clinical care for patients with rheumatologic diseases. Most were referred to academic centers for care after diagnosis (Washington University, St Louis, Missouri; Karolinska Insitute, Stockholm, Sweden; and Royal Melbourne Hospital, Melbourne, Australia) while one was cared for in a neurology practice in Dallas, Texas, with consultation by an academic neurovirologist from the University of Colorado in Denver.
Patients
Four patients developing PML in the setting of rituximab therapy for RA.
Intervention
Rituximab therapy.
Main Outcome Measures
Clinical and pathological observations.
Results
Four patients from an estimated population of 129 000 exposed to rituximab therapy for RA are reported in whom PML developed after administration of this drug. All were women older than 50 years, commonly with Sjögren syndrome and a history of treatment for joint disease ranging from 3 to 14 years. One case had no prior biologic and minimal immunosuppressive therapy. Progressive multifocal leukoencephalopathy presented as a progressive neurological disorder, with diagnosis confirmed by detection of JC virus DNA in the cerebrospinal fluid or brain biopsy specimen. Two patients died in less than 1 year from PML diagnosis, while 2 remain alive after treatment withdrawal. Magnetic resonance scans and tissue evaluation confirmed the frequent development of inflammatory PML during the course of the disease.
Conclusion
These cases suggest an increased risk, about 1 case per 25 000 individuals, of PML in patients with RA being treated with rituximab. Inflammatory PML may occur in this setting even while CD20 counts remain low.
doi:10.1001/archneurol.2011.103
PMCID: PMC3428054  PMID: 21555606
10.  The immuno-pathophysiology of multiple sclerosis 
Neurologic clinics  2011;29(2):257-278.
doi:10.1016/j.ncl.2010.12.009
PMCID: PMC3080042  PMID: 21439440
multiple sclerosis; neuro-immunology; demyelination; pathogenesis
11.  PATHOGENICITY OF SEVERE ACUTE RESPIRATORY CORONAVIRUS DELETION MUTANTS IN hACE-2 TRANSGENIC MICE 
Virology  2008;376(2):379-389.
Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-Δ[6-9b]), the structural gene E (rSARS-CoV-ΔE), and a combination of both sets of genes (rSARS-CoV-Δ[E,6-9b]) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.1 and CaCo-2) cell lines and for transgenic (Tg) mice expressing the SARS-CoV receptor human angiotensin converting enzyme-2 (hACE-2), indicating that none of these proteins is essential for the viral cycle. Furthermore, in Vero E6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the final morphology of the virions. Nevertheless, in the absence of E protein, release of virus particles efficacy was reduced. Viruses lacking E protein grew about 100-fold lower than the wt virus in lungs of Tg infected mice but did not grow in the brains of the same animals, in contrast to the rSARS-CoV-Δ[6-9b] virus, which grew almost as well as the wt in both tissues. Viruses lacking E protein were highly attenuated in the highly sensitive hACE-2 Tg mice, in contrast to the minimal rSARS-CoV-Δ[6-9b] and wt viruses. These data indicate that E gene might be a virulence factor influencing replication level, tissue tropism and pathogenicity of SARS-CoV, suggesting that ΔE attenuated viruses are promising vaccine candidates.
doi:10.1016/j.virol.2008.03.005
PMCID: PMC2810402  PMID: 18452964
12.  A Live Attenuated Severe Acute Respiratory Syndrome Coronavirus Is Immunogenic and Efficacious in Golden Syrian Hamsters▿  
Journal of Virology  2008;82(15):7721-7724.
The immunogenicity and protective efficacy of a live attenuated vaccine consisting of a recombinant severe acute respiratory syndrome (SARS) coronavirus lacking the E gene (rSARS-CoV-ΔE) were studied using hamsters. Hamsters immunized with rSARS-CoV-ΔE developed high serum-neutralizing antibody titers and were protected from replication of homologous (SARS-CoV Urbani) and heterologous (GD03) SARS-CoV in the upper and lower respiratory tract. rSARS-CoV-ΔE-immunized hamsters remained active following wild-type virus challenge, while mock-immunized hamsters displayed decreased activity. Despite being attenuated in replication in the respiratory tract, rSARS-CoV-ΔE is an immunogenic and efficacious vaccine in hamsters.
doi:10.1128/JVI.00304-08
PMCID: PMC2493341  PMID: 18463152

Results 1-12 (12)