bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown
Authors: Galvis Alvaro E. , Fisher Hugh E. , Nitta Takayuki , Fan Hung , Camerini David 
Published: March 2014

ABSTRACT
Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different factors and occur in different intracellular locations.

IMPORTANCE
This study shows that HIV-1 reverse transcription starts in the cytoplasm but is completed in or on the surface of the nucleus. Moreover, we show that nuclear reverse transcription is dependent on the activity of the human RNA lariat debranchng enzyme (DBR1), while cytoplasmic reverse transcription is not. These findings may provide new avenues for inhibiting HIV-1 replication and therefore may lead to new medicines for treating HIV-1-infected individuals.
Identification of a Novel Rhabdovirus in Spodoptera frugiperda Cell Lines
Authors: Ma Hailun , Galvin Teresa A. , Glasner Dustin R. , Shaheduzzaman Syed , Khan Arifa S. 
Published: March 2014

ABSTRACT

The Sf9 cell line, derived from
Spodoptera frugiperda
, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order
Mononegavirales
in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the
S. frugiperda
rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the
Mononegavirales
from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus
Cytorhabdovirus
. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-
5
B
1-4
(Tn
5
; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses.

IMPORTANCE
The
Spodoptera frugiperda
Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms,
de novo
assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results.
The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity
Authors: Masante Cyril , El Najjar Farah , Chang Andres , Jones Angela , Moncman Carole L. , Dutch Rebecca Ellis 
Published: March 2014

ABSTRACTHuman metapneumovirus (HMPV) encodes three glycoproteins: the glycoprotein, which plays a role in glycosaminoglycan binding, the fusion (F) protein, which is necessary and sufficient for both viral binding to the target cell and fusion between the cellular plasma membrane and the viral membrane, and the small hydrophobic (SH) protein, whose function is unclear. The SH protein of the closely related respiratory syncytial virus has been suggested to function as a viroporin, as it forms oligomeric structures consistent with a pore and alters membrane permeability. Our analysis indicates that both the full-length HMPV SH protein and the isolated SH protein transmembrane domain can associate into higher-order oligomers. In addition, HMPV SH expression resulted in increases in permeability to hygromycin B and alteration of subcellular localization of a fluorescent dye, indicating that SH affects membrane permeability. These results suggest that the HMPV SH protein has several characteristics consistent with a putative viroporin. Interestingly, we also report that expression of the HMPV SH protein can significantly decrease HMPV F protein-promoted membrane fusion activity, with the SH extracellular domain and transmembrane domain playing a key role in this inhibition. These results suggest that the HMPV SH protein could regulate both membrane permeability and fusion protein function during viral infection.IMPORTANCEHuman metapneumovirus (HMPV), first identified in 2001, is a causative agent of severe respiratory tract disease worldwide. The small hydrophobic (SH) protein is one of three glycoproteins encoded by all strains of HMPV, but the function of the HMPV SH protein is unknown. We have determined that the HMPV SH protein can alter the permeability of cellular membranes, suggesting that HMPV SH is a member of a class of proteins termed viroporins, which modulate membrane permeability to facilitate critical steps in a viral life cycle. We also demonstrated that HMPV SH can inhibit the membrane fusion function of the HMPV fusion protein. This work suggests that the HMPV SH protein has several functions, though the steps in the HMPV life cycle impacted by these functions remain to be clarified.
Human Metapneumovirus SH and G Glycoproteins Inhibit Macropinocytosis-Mediated Entry into Human Dendritic Cells and Reduce CD4
+
T Cell Activation
Authors: Le Nouën Cyril , Hillyer Philippa , Brock Linda G. , Winter Christine C. , Rabin Ronald L. , Collins Peter L. , Buchholz Ursula J. 
Published: March 2014

ABSTRACT

Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC)
in vitro
and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4
+
T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4
+
T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4
+
T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4
+
T cells. This study describes a previously unknown mechanism of virus immune evasion.

IMPORTANCE
Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4
+
T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.
A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23
Authors: Richardson Lynn G. L. , Clendening Eric A. , Sheen Hyukho , Gidda Satinder K. , White K. Andrew , Mullen Robert T. 
Published: March 2014

ABSTRACT

Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus
Tomato bushy stunt virus
(TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of
Carnation Italian ringspot virus
(CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery.

IMPORTANCE
Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus
Carnation Italian ringspot virus
(CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants.
Feasibility of aerosol drug delivery to sleeping infants: a prospective observational study
Authors: Amirav Israel , Newhouse Michael T , Luder Anthony , Halamish Asaf , Omar Hamza , Gorenberg Miguel 
Published: March 2014

ObjectivesDelivery of inhaled medications to infants is usually very demanding and is often associated with crying and mask rejection. It has been suggested that aerosol administration during sleep may be an attractive alternative. Previous studies in sleeping children were disappointing as most of the children awoke and rejected the treatment. The SootherMask (SM) is a new, gentle and innovative approach for delivering inhaled medication to infants and toddlers. The present pilot study describes the feasibility of administering inhaled medications during sleep using the SM.DesignProspective observational study.SettingOut patients.Participants13 sleeping infants with recurrent wheezing who regularly used pacifiers and were <12 months old.InterventionParticipants inhaled technetium99mDTPA-labelled normal saline aerosol delivered via a Respimat Soft Mist Inhaler (SMI) (Boehringer-Ingelheim, Germany) and SM + InspiraChamber (IC; InspiRx Inc, New Jersey, USA).OutcomesThe two major outcomes were the acceptability of the treatment and the lung deposition (per cent of emitted dose).ResultsAll infants who fulfilled the inclusion criteria successfully received the SM treatment during sleep without difficulty. Mean lung deposition (±SD) averaged 1.6±0.5% in the right lung.ConclusionsThis study demonstrated that the combination of Respimat, IC and SM was able to administer aerosol therapy to all the sleeping infants who were regular pacifier users with good lung deposition. Administration of aerosols during sleep is advantageous since all the sleeping children accepted the mask and ensuing aerosol therapy under these conditions, in contrast to previous studies in which there was frequent mask rejection using currently available devices.Clinical Trial RegistryNCT01120938.
Identifying the key elements of an education package to up-skill multidisciplinary adult specialist palliative care teams caring for young adults with life-limiting conditions: an online Delphi study
Authors: Sivell Stephanie , Lidstone Victoria , Taubert Mark , Thompson Catherine , Nelson Annmarie 
Published: March 2014

ObjectivesTo collect the views of experts to inform the development of an education package for multidisciplinary adult specialist palliative care (SPC) teams caring for young people with life-limiting conditions.MethodsA modified online Delphi process collated expert opinion on format, delivery and content of an education package to up-skill adult SPC teams. Round 1 participants (n=44) answered free-text questions, generating items for Round 2. In Round 2, 68 participants rated the extent to which they agreed/disagreed with the items on 5-point Likert-type scales. Median and mean scores assessed the importance of each item. IQR scores assessed level of consensus for each item; items lacking consensus were rerated by 35 participants in Round 3.ResultsIn the Delphi, consensus was reached on a range of suggested formats, on who should deliver the training, and on several clinical, psychosocial and practical topics.ConclusionsDevelopment of a continuous/rolling programme of education, tailored for content and mode of delivery and incorporated into working practice is recommended. As a direct outcome of the results of this study, a series of six linked study days has been established, focusing specifically on the issues around caring for young adults with life-limiting conditions and palliative care needs.
A reconsideration of the correlation between veteran status and firearm suicide in the general population
Authors: Hoffmire Claire A , Bossarte Robert M 
Published: March 2014

BackgroundThe relationship between veteran status and firearm suicide has been evaluated previously, but multiple sources of bias limit conclusions. This study aimed to clarify the relationship between a history of military service and firearm suicide.MethodsData obtained from suicide death certificates from nine states (1999–2009) were analysed. History of military service was validated using data obtained from the Department of Veterans Affairs and Department of Defense. Modified Poisson Regression with robust SEs was used to compute adjusted prevalence ratios for the common outcome of firearm use among suicide decedents.ResultsMale veteran suicide decedents were 6% more likely to use firearms, and female veteran suicide decedents were 18% more likely to use firearms compared with their non-veteran peers.ConclusionsPrior estimates based on logistic regression and death certificate reporting of veteran status may have overestimated the relationship between veteran status and firearm use among suicide decedents.
How well do principal diagnosis classifications predict disability 12 months postinjury?
Authors: Gabbe Belinda J , Simpson Pam M , Lyons Ronan A , Polinder Suzanne , Rivara Frederick P , Ameratunga Shanthi , Derrett Sarah , Haagsma Juanita , Harrison James E 
Published: March 2014

BackgroundThe application of disability weights by nature of injury is central to the calculation of disability-adjusted life years (DALYs). Such weights should represent injury diagnosis groups that demonstrate homogeneity in disability outcomes. Existing classifications have not used empirical data in their development to inform groups that are homogeneous for disability outcomes, limiting the capacity to make informed recommendations for best practice in measuring injury burden.MethodsThe Validating and Improving injury Burden Estimates (Injury-VIBES) Study includes pooled data from over 30 000 injured participants recruited to six cohort studies. The International Classification of Disease 10th Revision (ICD-10) diagnosis codes were mapped to existing injury burden study groupings and prediction models were developed to measure the capacity of the injury groupings and ICD-10 diagnoses to predict disability outcomes at 12 months. Models were adjusted for age, gender and data source and investigated for discrimination using area under the receiver operating characteristic curve (AUC) and calibration using Hosmer–Lemeshow statistics and calibration curves.ResultsDiscrimination and calibration of models varied depending on the outcome measured. Models using full four-character ICD-10 diagnosis codes, rather than groupings of codes, demonstrated the highest discrimination ranging from an AUC (95% CI) of 0.627 (0.618 to 0.635) for the pain or discomfort item of the EQ-5D to 0.847 (0.841 to 0.853) for the extended Glasgow Outcome Scale independent living outcome. However, gain over other groupings was marginal.ConclusionsPrediction performance was best for measures of function such as independent living, mobility and self-care. The classifications were poorer predictors of anxiety/depression and pain/discomfort. There was no clearly superior classification.
Antitumor Activity of 7-Aminocarboxycoumarin Derivatives, a New Class of Potent Inhibitors of Lactate Influx but Not Efflux
Authors: Draoui Nihed , Schicke Olivier , Seront Emmanuel , Bouzin Caroline , Sonveaux Pierre , Riant Olivier , Feron Olivier 
Published: March 2014

Abstract
High lactate concentration in tumors is associated with bad prognosis. Lactate is released by glycolytic cells in tumors and recaptured by oxidative cancer cells to feed the tricarboxylic acid (TCA) cycle after conversion into pyruvate. Monocarboxylate transporters (MCT) mediate these fluxes of proton-linked lactate and represent attractive targets to interrupt lactate shuttle and to inhibit tumor growth. Here, we investigated the properties of 7-aminocarboxycoumarins (7ACC) developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment. The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models. Contrary to the reference MCT1 inhibitor AR-C155858, 7ACC unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters. 7ACC delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors. MCT target engagement was confirmed by the lack of activity of 7ACC on bladder UM-UC-3 carcinoma that does not express functional MCT. 7ACC also inhibited SiHa tumor relapse after treatment with cisplatin. Finally, we found that contrary to AR-C155858, 7ACC did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment. In conclusion, our results indicate that 7ACC selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects. Mol Cancer Ther; 13(6); 1410–8. ©2014 AACR.