bluuebunny/crossref_metadata_2025_split huggingface dataset:
Higher-Order Structure of the Rous Sarcoma Virus SP Assembly Domain
Authors: Bush Di L. , Monroe Eric B. , Bedwell Gregory J. , Prevelige Peter E. , Phillips Judith M. , Vogt Volker M. 
Published: March 2014

ABSTRACT

Purified retroviral Gag proteins can assemble
in vitro
to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using
in vitro
assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly.

IMPORTANCE
The structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.
The Host Protease TMPRSS2 Plays a Major Role in
In Vivo
Replication of Emerging H7N9 and Seasonal Influenza Viruses
Authors: Sakai Kouji , Ami Yasushi , Tahara Maino , Kubota Toru , Anraku Masaki , Abe Masako , Nakajima Noriko , Sekizuka Tsuyoshi , Shirato Kazuya , Suzaki Yuriko , Ainai Akira , Nakatsu Yuichiro , Kanou Kazuhiko , Nakamura Kazuya , Suzuki Tadaki , Komase Katsuhiro , Nobusawa Eri , Maenaka Katsumi , Kuroda Makoto , Hasegawa Hideki , Kawaoka Yoshihiro , Tashiro Masato , Takeda Makoto 
Published: March 2014

ABSTRACT

Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s)
in vivo
. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype,
in vivo
. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2
+/+
wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ≥1,000 50% lethal doses (LD
50
) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage
in vivo
and, thus, that TMPRSS2 expression is essential for IAV replication
in vivo
.

IMPORTANCE
Influenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs
in vivo
through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2
in vitro
. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health.
Template Role of Double-Stranded RNA in Tombusvirus Replication
Authors: Kovalev Nikolay , Pogany Judit , Nagy Peter D. 
Published: March 2014

ABSTRACT

Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(−)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (−)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of
Tomato bushy stunt virus
(TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (−)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny.

IMPORTANCE
Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (−)RNA in the membrane-bound viral replicase, we performed complete RNA replication of
Tomato bushy stunt virus
(TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (−)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.
The Spectrum of Children's Palliative Care Needs: a classification framework for children with life-limiting or life-threatening conditions
Authors: Shaw Karen L , Brook Lynda , Mpundu-Kaambwa Christine , Harris Nicky , Lapwood Susie , Randall Duncan 
Published: March 2014

ObjectivesThis paper examined the potential of a new classification framework,The Spectrum of Children's Palliative Care Needs, to facilitate identification of children with palliative care needs for the purposes of minimum data set collection and population needs assessment.MethodsHealth and social care professionals (n=50) in a range of paediatric palliative care settings applied The Spectrum to (i) clinical vignettes and (ii) consecutive children on their caseloads. They also provided confidence ratings and written comments about their experiences. Inter-rater reliability, conceptual validity, acceptability, feasibility and sustainability were examined. A subset of professionals (n=9) also participated in semistructured telephone interviews to provide further insight.ResultsInter-rater reliability for the vignettes (κ=0.255) was fair. However, professionals were more confident applying The Spectrum to their caseloads, which included children (n=74) with a range of life-limiting/life-threatening conditions. The Spectrum made conceptual sense in relation to these children and was considered to offer a meaningful way to define the eligible population in service mapping. Benefits for clinical work (eg, facilitating patient review, workload management, clinical audit) and research were also identified. However, important threats to reliability were highlighted.ConclusionsPreliminary assessment of The Spectrum confirms its potential to promote consistent data set collection in children's palliative care. The results have been used to produce a revised version and user guidelines to address issues raised by participants. However, further research is required to further validate the framework and establish its relevance to families’ self-defined needs.
Parents and end-of-life decision-making for their child: roles and responsibilities
Authors: Sullivan Jane , Gillam Lynn , Monagle Paul 
Published: March 2014

BackgroundWhether parents want to be and should be the decision-maker for their child in end-of-life matters are contested clinical and ethical questions. Previous research outcomes are equivocal.MethodA qualitative interview method was used to examine the views and experiences of 25 bereaved parents in end-of-life decision-making for their child. Data were analysed thematically.ResultsThree types of decision-making roles were identified: self-determined, guided (both involving active decision-making) and acquiescent (passive).The majority of parents had been active in the decision-making process for their child. They perceived themselves as the ultimate end-of-life decision-maker. This was perceived as part of their parental responsibility. A minority of parents did not consider that they had been an active, ultimate decision-maker. Generally, parents in the self-determined and guided groups reported no negative consequences from their decision-making involvement. Importantly, parents in the acquiescent group described their experience as difficult at the time and subsequently, although not all difficulties related directly to decision-making. Parents considered that in principle parents should be the end-of-life decision-maker for their child, but understood personal characteristics and preference could prevent some parents from taking this role.ConclusionsThis study unequivocally supports parents’ desire to fulfil the end-of-life decision-making role. It provides a nuanced understanding of parents’ roles and contributes evidence for the ethical position that parents should be the end-of-life decision-makers for their child, unless not in the child's best interests. On the whole, parents want this role and can manage its consequences. Indeed, not being the end-of-life decision-maker could be detrimental to parents’ well-being.
Service user perspectives on palliative care education for health and social care professionals supporting people with learning disabilities
Authors: McLaughlin Dorry , Barr Owen , McIlfatrick Sonja , McConkey Roy 
Published: March 2014

Background and objectivesEvidence from European and American studies indicates limited referrals of people with learning (intellectual) disabilities to palliative care services. Although professionals’ perceptions of their training needs in this area have been studied, the perceptions of people with learning disabilities and family carers are not known. This study aimed to elicit the views of people with learning disabilities, and their family carers concerning palliative care, to inform healthcare professional education and training.MethodsA qualitative, exploratory design was used. A total of 17 people with learning disabilities were recruited to two focus groups which took place within an advocacy network. Additionally, three family carers of someone with a learning disability, requiring palliative care, and two family carers who had been bereaved recently were also interviewed.ResultsCombined data identified the perceived learning needs for healthcare professionals. Three subthemes emerged: ‘information and preparation’, ‘provision of care’ and ‘family-centred care’.ConclusionsThis study shows that people with learning disabilities can have conversations about death and dying, and their preferred end-of-life care, but require information that they can understand. They also need to have people around familiar to them and with them. Healthcare professionals require skills and knowledge to effectively provide palliative care for people with learning disabilities and should also work in partnership with their family carers who have expertise from their long-term caring role. These findings have implications for educators and clinicians.
The uncertainties associated with the application of through-diffusion, the steady-state method: a case study of strontium diffusion
Authors: Gondolli J. , Večerník P. 
Published: March 2014

Abstract

In this study, the uncertainties associated with through-diffusion, the steady-state method used for determination of the pore diffusion coefficient (
D
p
), were analysed and evaluated. The diffusion of strontium through the sample of compacted bentonite was studied to evaluate various sources of uncertainties and compare their contribution with uncertainty of
D
p
. Different diffusion experiment arrangements were used to test the effect of solution accessibility to the filter membranes. Three main general factors influencing the uncertainty of
D
p
at given conditions were identified: (a) the compacted sample preparation and its physical properties; (b) the properties of the diffusion cell; and (c) the strontium concentration analyses. Some of the identified sources can be treated as fixed with limited extent (e.g. the deformation of components of the diffusion cell), but some sources must be treated as variable with wide extent depending on the experimental conditions (e.g. the physical properties of the compacted bentonite sample – moisture, density, porosity). The variable uncertainty sources have to be taken into account, especially in repeatability and reproducibility tests. It was concluded that each of the diffusion cells with compacted bentonite sample represents a unique diffusion system. It is preferable to present the results of diffusion experiments as a range of values rather than as one average value with an uncertainty.
Contributions of the Host Microenvironment to Cancer-Induced Bone Disease
Authors: Olechnowicz Sam W.Z. , Edwards Claire M. 
Published: March 2014

Abstract
The bone marrow provides a specialized and highly supportive microenvironment for tumor growth and development of the associated bone disease. It is a preferred site for breast and prostate cancer bone metastasis and the hematologic malignancy, multiple myeloma. For many years, researchers have focused upon the interactions between tumor cells and the cells directly responsible for bone remodeling, namely osteoclasts and osteoblasts. However, there is ever-increasing evidence for a multitude of ways in which the bone marrow microenvironment can promote disease pathogenesis, including via cancer-associated fibroblasts, the hematopoietic stem cell niche, myeloid-derived suppressor cells, and the sympathetic nervous system. This review discusses the recent advances in our understanding of the contribution of the host microenvironment to the development of cancer-induced bone disease. Cancer Res; 74(6); 1625–31. ©2014 AACR.
Hypoxia-Inducible Factor-1 Promotes Pancreatic Ductal Adenocarcinoma Invasion and Metastasis by Activating Transcription of the Actin-Bundling Protein Fascin
Authors: Zhao Xiao , Gao Song , Ren He , Sun Wei , Zhang Huan , Sun Jianwei , Yang Shengyu , Hao Jihui 
Published: March 2014

Abstract
Because of the early onset of local invasion and distant metastasis, pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignant tumor, with a 5-year survival rate of less than 5%. In this study, we investigated the role of fascin, a prometastasis actin-bundling protein, in PDAC progression, invasion, and the molecular mechanisms underlying fascin overexpression in PDAC. Our data showed that the expression levels of fascin were higher in cancer tissues than in normal tissues, and fascin overexpression correlated with the PDAC differentiation and prognosis. Fascin overexpression promoted PDAC cell migration and invasion by elevating matrix metalloproteinase-2 (MMP-2) expression. Fascin regulated MMP-2 expression through protein kinase C and extracellular signal—regulated kinase. Importantly, our data showed that hypoxia induced fascin overexpression in PDAC cells by promoting the binding of hypoxia-inducible factor-1 (HIF-1) to a hypoxia response element on the fascin promoter and transactivating fascin mRNA transcription. Intriguingly, HIF-1α expression levels in PDAC patient specimens significantly correlated with fascin expression. Moreover, immunohistochemistry staining of consecutive sections demonstrated colocalization between HIF-1α and fascin in PDAC specimens, suggesting that hypoxia and HIF-1α were responsible for fascin overexpression in PDAC. When ectopically expressed, fascin was able to rescue PDAC cell invasion after HIF-1α knockdown. Our results demonstrated that fascin is a direct target gene of HIF-1. Our data suggested that the hypoxic tumor microenvironment in PDAC might promote invasion and metastasis by inducing fascin overexpression, and fascin might be targeted to block PDAC progression. Cancer Res; 74(9); 2455–64. ©2014 AACR.
Reverse-Phase Protein Array Profiling of Oropharyngeal Cancer and Significance of PIK3CA Mutations in HPV-Associated Head and Neck Cancer
Authors: Sewell Andrew , Brown Brandee , Biktasova Asel , Mills Gordon B. , Lu Yiling , Tyson Darren R. , Issaeva Natalia , Yarbrough Wendell G. 
Published: March 2014

Abstract
Purpose: Human papilloma virus (HPV)–associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV−) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(−) OPSCC and between tumors with and without PIK3CA mutations.
Experimental Design: Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(−) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA.
Results: Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(−) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(−) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor.
Conclusions: Protein expression analyses suggest that HPV(+) and HPV(−) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers. Clin Cancer Res; 20(9); 2300–11. ©2014 AACR.
Therapeutic Efficacy of p53 Restoration in Mdm2-Overexpressing Tumors
Authors: Li Qin , Zhang Yun , El-Naggar Adel K. , Xiong Shunbin , Yang Peirong , Jackson James G. , Chau Gilda , Lozano Guillermina 
Published: March 2014

Abstract
The p53 (TP53) tumor suppressor is the most frequently mutated gene in human cancers. Restoring expression of wild-type p53 has led to tumor growth suppression in a variety of tumor models that are p53 deficient. Other mechanisms, for example, upregulation of Mdm2, exist in tumors to inactivate the p53 pathway. Mdm2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation, is present at high levels in many tumors with wild-type p53. In this study, the effects of restoring p53 activity were probed in Mdm2-overexpressing tumors genetically using animal models. Here, it was demonstrated that elevated levels of Mdm2 and decreased levels of p53 act additively to dampen p53 activity in DNA damage response and tumor development. Our data further indicate that restoration of wild-type p53 expression in Mdm2-overexpressing angiosarcomas results in tumor stasis and regression in some cases. Finally, it was determined that restored p53 suppressed cell proliferation but did not elicit apoptosis in the Mdm2-overexpressing angiosarcomas.
Implications: Restoration of wild-type p53 expression in Mdm2-overexpressing tumors suppresses tumor growth, which represents a potential clinical strategy to treat tumors with high levels of Mdm2.
Visual Overview: http://mcr.aacrjournals.org/content/12/6/901/F1.large.jpg.
Mol Cancer Res; 12(6); 901–11. ©2014 AACR.