bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stepwise Priming by Acidic pH and a High K
+
Concentration Is Required for Efficient Uncoating of Influenza A Virus Cores after Penetration
Authors: Stauffer Sarah , Feng Yuehan , Nebioglu Firat , Heilig Rosalie , Picotti Paola , Helenius Ari 
Published: August 2014

ABSTRACT

Influenza A virus (IAV) uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the prefusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 ion channel. The first weakens the interactions between the matrix protein, M1, and the viral ribonucleoprotein bundle. It involves a conformational change in a linker sequence and the C-terminal domain of M1 after exposure to a pH below 6.5. The second step is triggered by a pH of <6.0 and by the influx of K
+
ions. It causes additional changes in M1 as well as a loss of stability in the viral ribonucleoprotein bundle. Our results indicate that both the switch from Na
+
to K
+
in maturing endosomes and the decreasing pH are needed to prime IAV cores for efficient uncoating and infection of the host cell.

IMPORTANCE
The entry of IAV involves several steps, including endocytosis and fusion at late endosomes. Entry also includes disassembly of the viral core, which is composed of the viral ribonucleoproteins and the RNA genome. We have found that the uncoating process of IAV is initiated long before the core is delivered into the cytosol. M2, an ion channel in the viral membrane, is activated when the virus passes through early endosomes. Here, we show that protons entering the virus through M2 cause a conformational change in the matrix protein, M1. This weakens interactions between M1 and the viral ribonucleoproteins. A second change was found to occur when the virus enters late endosomes. The preacidified core is then exposed to a high concentration of K
+
, which affects the interactions between the ribonucleoproteins. Thus, when cores are finally delivered to the cytosol, they are already partially destabilized and, therefore, uncoating competent and infectious.
The Raccoon Polyomavirus Genome and Tumor Antigen Transcription Are Stable and Abundant in Neuroglial Tumors
Authors: Brostoff Terza , Dela Cruz Florante N. , Church Molly E. , Woolard Kevin D. , Pesavento Patricia A. 
Published: August 2014

ABSTRACT

Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by
in situ
hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells
in vitro
and in a mouse xenograft model. When tumor cells were cultured
in vitro
, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer.

IMPORTANCE
The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen genes, we cultured primary raccoon tumor cells and passaged them in mice, confirming the nonintegrated state of the virus and the maintenance of viral gene transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.
Activation of Intrinsic Immune Responses and Microglial Phagocytosis in an
Ex Vivo
Spinal Cord Slice Culture Model of West Nile Virus Infection
Authors: Quick Eamon D. , Leser J. Smith , Clarke Penny , Tyler Kenneth L. 
Published: August 2014

ABSTRACT

West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or spinal cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those that are dependent on infiltrating inflammatory cells has proven difficult. We used a murine
ex vivo
spinal cord slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days after
ex vivo
infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV, resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection, as exemplified by their amoeboid morphology, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. Our findings establish that CNS-resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses.

IMPORTANCE
There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a spinal cord slice culture (SCSC) model facilitates the study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses, including microglial activation and proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.
Distinct Mechanisms Regulate Exposure of Neutralizing Epitopes in the V2 and V3 Loops of HIV-1 Envelope
Authors: Upadhyay Chitra , Mayr Luzia M. , Zhang Jing , Kumar Rajnish , Gorny Miroslaw K. , Nádas Arthur , Zolla-Pazner Susan , Hioe Catarina E. 
Published: August 2014

ABSTRACT
Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (variable loop V2 integrin) epitopes compared to the accessibility of V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin α4β7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (MAbs) display extensive cross-reactivity with gp120 monomers from many subtypes but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similarly to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 MAbs but not to the V2i MAbs. Second, the contribution of N-glycans in masking V2i versus V3 epitopes was evaluated by testing the neutralization of pseudoviruses produced in the presence of a glycosidase inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i MAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-MAb interaction to 18 h before adding target cells increased virus neutralization by some V2i MAbs and all V3 MAbs tested. Consistent with this, V2i MAb binding to Env on the surface of transfected cells also increased in a time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic, but distinct factors modulate the antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites.

IMPORTANCE
Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 MAbs and some V2i MAbs display greater neutralization against relatively resistant HIV-1 isolates when the MAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines.
Epstein-Barr Virus Late Gene Transcription Depends on the Assembly of a Virus-Specific Preinitiation Complex
Authors: Aubry Valentin , Mure Fabrice , Mariamé Bernard , Deschamps Thibaut , Wyrwicz Lucjan S. , Manet Evelyne , Gruffat Henri 
Published: August 2014

ABSTRACT

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain
cis
-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique
cis
element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta- and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta- and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses.

IMPORTANCE
Control of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC).
Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication
Authors: Viskovska Maria , Anish Ramakrishnan , Hu Liya , Chow Dar-Chone , Hurwitz Amy M. , Brown Nicholas G. , Palzkill Timothy , Estes Mary K. , Prasad B. V. Venkataram 
Published: August 2014

ABSTRACT
Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated.

IMPORTANCE
Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs). A multifunctional RNA-binding NSP2 octamer with nucleotidyl phosphatase activity is central to viroplasm formation and RNA replication. Here we provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from peptide arrays are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1 and also point to NSP2's possible role in preventing the premature self-assembly of VP2 cores. Our findings lead us to propose that the NSP2 octamer with multiple enzymatic activities is a principal regulator of viroplasm formation, recruitment of viral proteins into the viroplasms, and possibly genome replication.
The long-term financial impact of electronic health record implementation
Authors: Howley Michael J , Chou Edgar Y , Hansen Nancy , Dalrymple Prudence W 
Published: August 2014

Abstract
Objective To examine the financial impact of electronic health record (EHR) implementation on ambulatory practices.
Methods We tracked the practice productivity (ie, number of patient visits) and reimbursement of 30 ambulatory practices for 2 years after EHR implementation and compared each practice to their pre-EHR implementation baseline.
Results Reimbursements significantly increased after EHR implementation even though practice productivity (ie, the number of patient visits) decreased over the 2-year observation period. We saw no evidence of upcoding or increased reimbursement rates to explain the increased revenues. Instead, they were associated with an increase in ancillary office procedures (eg, drawing blood, immunizations, wound care, ultrasounds).
Discussion The bottom line result—that EHR implementation is associated with increased revenues—is reassuring and offers a basis for further EHR investment. While the productivity losses are consistent with field reports, they also reflect a type of efficiency—the practices are receiving more reimbursement for fewer seeing patients. For the practices still seeing fewer patients after 2 years, the solution likely involves advancing their EHR functionality to include analytics. Although they may still see fewer patients, with EHR analytics, they can focus on seeing the right patients.
Conclusions Practice reimbursements increased after EHR implementation, but there was a long-term decrease in the number of patient visits seen in this ambulatory practice context.
Pancreatic enzyme replacement therapy (PERT) for malabsorption in patients with metastatic pancreatic cancer
Authors: Landers Amanda , Muircroft Wendy , Brown Helen 
Published: August 2014

PurposeThe diagnosis of metastatic pancreatic cancer (PC) carries a poor prognosis. PC is associated with weight loss and malabsorption in high rates secondary to pancreatic exocrine insufficiency. UK and USA guidelines exist recommending the empiric use of pancreatic enzyme replacement therapy (PERT) for quality of life in these patients. The aim of this study is to review the use of PERT in patients with metastatic PC referred to a specialist palliative care service.MethodsRetrospective observational study of patients referred to the service between January 2010 and July 2012 with a diagnosis of PC. Information about PERT use, tumour site and frequency of symptoms was collected.Results129 patients were referred, with a higher number in the eighth decade. Only 21% of this study group were prescribed PERT. Over 70% of patients had symptoms that could be attributable to malabsorption, mainly abdominal pain. Other symptoms such as bloating, wind and steatorrhoea were also common.ConclusionsGuidelines recommending empiric treatment of PERT in patients with metastatic PC are not currently being utilised.
Preliminary experience with the Pipeline Flex Embolization Device: technical note
Authors: Martínez-Galdámez M , Gil A , Caniego J L , Gonzalez E , Bárcena E , Perez S , Garcia-Bermejo P , Ortega-Gutierrez S 
Published: August 2014

BackgroundClinical experience with the Pipeline Embolization Device (PED) has been widely described in the literature since it obtained its European CE and FDA approvals in 2008 and 2011, respectively. The new generation of PED, the Pipeline Flex Embolization Device, received the CE mark of approval in March 2014. While the implant composition has not changed, its new delivery system has some differences. One of the main changes from the previous generation is a new delivery system that makes the device resheathable until deployed over 90% of its length. We present our preliminary experience using this device.MethodsBetween May and June 2014, six patients with six aneurysms were treated with the Pipeline Flex device.ResultsAll devices were placed properly, without technical difficulties. We successfully resheathed and repositioned the device in two cases. Minor and major intraprocedural or periprocedural events were noted.ConclusionsThe Pipeline Flex device allows more precise and controlled deployment than the current PED device. Although this preliminary experience seems positive, multicenter larger series will be needed to confirm the safety and durability of this new device.
Sudden Unexpected Death in North Carolina (SUDDEN): methodology review and screening results
Authors: Nanavati Parin P , Mounsey John Paul , Pursell Irion W , Simpson Ross J , Lewis Mary Elizabeth , Mehta Neil D , Williams Jefferson G , Bachman Michael W , Myers J Brent , Chung Eugene H 
Published: August 2014

ObjectivesThis paper describes the methodology for a prospective, community-based study of sudden unexpected death in Wake County, North Carolina.MethodsFrom 1 March to 29 June 2013, data of presumed cardiac arrest cases were captured from Wake County Emergency Medical Services. Participants were screened into the presumed sudden unexpected death group based on specific and sequential screening criteria, and medical and public records were collected for each participant in this group. A committee of independent cardiologists reviewed all data to determine final inclusion/exclusion of each participant into registry.ResultsWe received 398 presumed cardiac arrest referrals. Of these, 105 participants, age 18–65 years old, were identified as presumed sudden unexpected deaths. The primary reason for exclusion was survival to hospital (38%). Ninety-five per cent of participants in the presumed sudden unexpected death group experienced an unwitnessed death. Hypertension was present in almost 50%, while dyslipidaemia and diabetes mellitus were present in almost 25% of the same group. In addition, the presumed sudden unexpected death group includes 67.6% males (95% CI 58 to 76) whereas the control group only included 58.9% (95% CI 46 to 55) males.ConclusionsParticipant identification and data collection processes identify presumed sudden unexpected death cases and secure medical and public data for screening and final adjudication. The study infrastructure developed in Wake County will allow its expansion to other counties in North Carolina. Preliminary data indicate the study presently focuses on a population demographically representative of North Carolina.
Tectono-stratigraphic evolution of the Andean Orogen between 31 and 37°S (Chile and Western Argentina)
Authors: Charrier Reynaldo , Ramos Victor A. , Tapia Felipe , Sagripanti Lucía 
Published: August 2014

Abstract
In this classic segment, many tectonic processes, like flat-subduction, terrane accretion and steepening of the subduction, among others, provide a robust framework for their understanding. Five orogenic cycles, with variations in location and type of magmatism, tectonic regimes and development of different accretionary prisms, show a complex evolution. Accretion of a continental terrane in the Pampean cycle exhumed lower to middle crust in Early Cambrian. The Ordovician magmatic arc, associated metamorphism and foreland basin formation characterized the Famatinian cycle. In Late Devonian, the collision of Chilenia and associated high-pressure/low-temperature metamorphism contrasts with the late Palaeozoic accretionary prisms. Contractional deformation in Early to Middle Permian was followed by extension and rhyolitic (Choiyoi) magmatism. Triassic to earliest Jurassic rifting was followed by subduction and extension, dominated by Pacific marine ingressions, during Jurassic and Early Cretaceous. The Late Cretaceous was characterized by uplift and exhumation of the Andean Cordillera. An Atlantic ingression occurred in latest Cretaceous. Cenozoic contraction and uplift pulses alternate with Oligocene extension. Late Cenozoic subduction was characterized by the Pampean flat-subduction, the clockwise block tectonic rotations in the normal subduction segments and the magmatism in Payenia. These processes provide evidence that the Andean tectonic model is far from a straightforward geological evolution.