bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation
Authors: Orabi Ahmed , Bieringer Maria , Geerlof Arie , Bruss Volker 
Published: July 2015

ABSTRACT

The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain [MD]) in the large surface protein L and a narrow region (matrix binding domain [MBD]) including isoleucine 126 on the capsid surface have been mapped, in which point mutations such as core I126A specifically blocked nucleocapsid envelopment. It is possible that the two domains interact with each other during virion morphogenesis. By the systematic evolution of ligands by exponential enrichment (SELEX) method, we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications, 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50% of the clones. The main aptamer, AO-01 (13 clones, 18%), showed the lowest dissociation constant (

K
d

) of 180 ± 82 nM for capsid binding among the four molecules. Its

K
d

for I126A capsids was 1,306 ± 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47% inhibition of virion production at 3 days posttransfection, but there was no inhibition by cotransfection of an aptamer with a random sequence. The half-life of AO-01 in cells was 2 h, which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction with a low-molecular-weight substance might represent a new approach for an antiviral therapy.

IMPORTANCE
Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55-base-long single-stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The results substantiate the current model for HBV morphogenesis and show that the capsid envelope interaction is a potential antiviral target.
Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus
Authors: Adu-Gyamfi Emmanuel , Johnson Kristen A. , Fraser Mark E. , Scott Jordan L. , Soni Smita P. , Jones Keaton R. , Digman Michelle A. , Gratton Enrico , Tessier Charles R. , Stahelin Robert V. 
Published: July 2015

ABSTRACT
Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles.

IMPORTANCE
The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.
Longitudinal Detection of Prion Shedding in Saliva and Urine by Chronic Wasting Disease-Infected Deer by Real-Time Quaking-Induced Conversion
Authors: Henderson Davin M. , Denkers Nathaniel D. , Hoover Clare E. , Garbino Nina , Mathiason Candace K. , Hoover Edward A. 
Published: July 2015

ABSTRACT

Chronic wasting disease (CWD) is an emergent, rapidly spreading prion disease of cervids. Shedding of infectious prions in saliva and urine is thought to be an important factor in CWD transmission. To help to elucidate this issue, we applied an
in vitro
amplification assay to determine the onset, duration, and magnitude of prion shedding in longitudinally collected saliva and urine samples from CWD-exposed white-tailed deer. We detected prion shedding as early as 3 months after CWD exposure and sustained shedding throughout the disease course. We estimated that the 50% lethal dose (LD
50
) for cervidized transgenic mice would be contained in 1 ml of infected deer saliva or 10 ml of urine. Given the average course of infection and daily production of these body fluids, an infected deer would shed thousands of prion infectious doses over the course of CWD infection. The direct and indirect environmental impacts of this magnitude of prion shedding on cervid and noncervid species are surely significant.

IMPORTANCE
Chronic wasting disease (CWD) is an emerging and uniformly fatal prion disease affecting free-ranging deer and elk and is now recognized in 22 U.S. states and 2 Canadian provinces. It is unique among prion diseases in that it is transmitted naturally through wild populations. A major hypothesis to explain CWD's florid spread is that prions are shed in excreta and transmitted via direct or indirect environmental contact. Here we use a rapid
in vitro
assay to show that infectious doses of CWD prions are in fact shed throughout the multiyear disease course in deer. This finding is an important advance in assessing the risks posed by shed CWD prions to animals as well as humans.
The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry
Authors: Loustalot Fabien , Kremer Eric J. , Salinas Sara 
Published: July 2015

ABSTRACT

The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FK
CAV
), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FK
CAV
entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry.

IMPORTANCE
Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family.
Plasmid-Mediated Quinolone Resistance
Authors: Jacoby George A. , Strahilevitz Jacob , Hooper David C. 
Published: July 2015

ABSTRACTThree mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genesqnrA, qnrB, qnrC, qnrD, qnrS,andqnrVCcode for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. Theqnrgenes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.
Conditional Activation of Toxin-Antitoxin Systems: Postsegregational Killing and Beyond
Authors: Hernández-Arriaga Ana María , Chan Wai Ting , Espinosa Manuel , Díaz-Orejas Ramón 
Published: July 2015

ABSTRACT

Toxin-antitoxin (TA) systems are small genetic modules formed by a stable toxin and an unstable antitoxin that are widely present in plasmids and in chromosomes of Bacteria and Archaea. Toxins can interfere with cell growth or viability, targeting a variety of key processes. Antitoxin inhibits expression of the toxin, interacts with it, and neutralizes its effect. In a plasmid context, toxins are kept silent by the continuous synthesis of the unstable antitoxins; in plasmid-free cells (segregants), toxins can be activated owing to the faster decay of the antitoxin, and this results in the elimination of these cells from the population (
p
ost
s
egregational
k
illing [PSK]) and in an increase of plasmid-containing cells in a growing culture. Chromosomal TA systems can also be activated in particular circumstances, and the interference with cell growth and viability that ensues contributes in different ways to the physiology of the cell. In this article, we review the conditional activation of TAs in selected plasmidic and chromosomal TA pairs and the implications of this activation. On the whole, the analysis underscores TA interactions involved in PSK and points to the effective contribution of TA systems to the physiology of the cell.
DNA Assembly Tools and Strategies for the Generation of Plasmids
Authors: Baek Chang-Ho , Liss Michael , Clancy Kevin , Chesnut Jonathan , Katzen Federico 
Published: July 2015

ABSTRACT

Since the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago, molecular biology has evolved into a multidisciplinary field that has democratized the conversion of a digitized DNA sequence stored in a computer into its biological counterpart, usually as a plasmid, stored in a living cell. In this article, we summarize the most relevant tools that allow the swift assembly of DNA sequences into useful plasmids for biotechnological purposes. We cover the main components and stages in a typical DNA assembly workflow, namely
in silico
design,
de novo
gene synthesis, and
in vitro
and
in vivo
sequence assembly methodologies.
Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other
Firmicutes
Authors: Schwarz Stefan , Shen Jianzhong , Wendlandt Sarah , Feßler Andrea T. , Wang Yang , Kadlec Kristina , Wu Cong-Ming 
Published: July 2015

ABSTRACT

In staphylococci and other
Firmicutes
, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or
in toto
into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other
Firmicutes
have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other
Firmicutes
.
Resolution of Multimeric Forms of Circular Plasmids and Chromosomes
Authors: Crozat Estelle , Fournes Florian , Cornet François , Hallet Bernard , Rousseau Philippe 
Published: July 2015

ABSTRACT
One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes.
Using Plasmids as DNA Vaccines for Infectious Diseases
Authors: Tregoning John S. , Kinnear Ekaterina 
Published: July 2015

ABSTRACTDNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.
Rationale and design of three observational, prospective cohort studies including biobanking to evaluate and improve diagnostics, management strategies and risk stratification in venous thromboembolism: theVTEval Project
Authors: Frank Bernd , Ariza Liana , Lamparter Heidrun , Grossmann Vera , Prochaska Jürgen H , Ullmann Alexander , Kindler Florentina , Weisser Gerhard , Walter Ulrich , Lackner Karl J , Espinola-Klein Christine , Münzel Thomas , Konstantinides Stavros V , Wild Philipp S 
Published: July 2015

IntroductionVenous thromboembolism (VTE) with its two manifestations deep vein thrombosis (DVT) and pulmonary embolism (PE) is a major public health problem. TheVTEval Projectaims to investigate numerous research questions on diagnosis, clinical management, treatment and prognosis of VTE, which have remained uncertain to date.Methods and analysisTheVTEval Projectconsists of three observational, prospective cohort studies on VTE comprising cohorts of individuals with a clinical suspicion of acute PE (with or without DVT), with a clinical suspicion of acute DVT (without symptomatic PE) and with an incidental diagnosis of VTE (PE or DVT). TheVTEval Projectexpects to enrol a total of approximately 2000 individuals with subsequent active and passive follow-up investigations over a time period of 5 years per participant. Time points for active follow-up investigations are at months 3, 6, 12, 24 and 36 after diagnosis (depending on the disease cohort); passive follow-up investigations via registry offices and the cancer registry are performed 48 and 60 months after diagnosis for all participants. Primary short-term outcome is defined by overall mortality (PE-related death and all other causes of death), primary long-term outcome by symptomatic VTE (PE-related death, recurrence of non-fatal PE or DVT). TheVTEval Projectincludes three ‘all-comer’ studies and involves the standardised acquisition of high-quality data, covering the systematic assessment of VTE including symptoms, risk profile, psychosocial, environmental and lifestyle factors as well as clinical and subclinical disease, and it builds up a large state-of-the-art biorepository containing various materials from serial blood samplings.Ethics and disseminationTheVTEval Projecthas been approved by the local data safety commissioner and the responsible ethics committee (reference no. 837.320.12 (8421-F)). Trial results will be published in peer-reviewed journals and presented at national and international scientific meetings.Trial registration numberNCT02156401.
Reliable EGFR mutation testing in ultrasound-guided supraclavicular lymph node fine-needle aspirates: a cohort study with diagnostic performance analysis
Authors: Awwad Amir , Tiwari Sandeep , Sovani Vishakha , Baldwin David R , Kumaran Maruti 
Published: July 2015

Introduction15–30% of patients with lung cancer will have supraclavicular and cervical lymphadenopathy (SCLN). Ultrasound (US)-guided fine-needle aspiration (FNA) cytology is regarded as an effective diagnostic tool in small size lymph nodes (LNs) and impalpable positron emission tomography detected nodes. We evaluated our diagnostic service performance in relation to the adequacy of samples for epidermal growth factor receptor (EGFR) mutation.MethodsRetrospective data analysis from electronic records, searching for all suspected lung cancer referrals that underwent US of the neck±FNA, over a continuous period of 4 years.ResultsOf 306 cases with suspected lung cancer referred to our department for US FNA of SCLN, 228 patients underwent the procedure. Of the remaining 78 patients, LNs were not detected in 52 cases and appeared benign in 26. Cytological diagnosis was established in 171 patients (75%) for treatment decisions without further investigations. The remaining 57 patients had further investigations; 45 reconfirmed the US-guided FNA diagnosis. The average LN size was 12.9 mm, and positive cytology was obtained in LNs ranging from 3 to 45 mm. Of 57 adenocarcinoma cases, 34 were tested for EGFR confirming 4 positive, 25 negative and 5 insufficient samples. No complications were recorded.ConclusionsUS-guided FNA of SCLN remains an important diagnostic tool in lung cancer. Adequate tissue can be obtained for reliable diagnosis from LNs and for EGFR mutational analysis, without the need for more invasive and expensive investigations in more than 80% of cases.
Subcellular localisation of anillin is associated with different survival outcomes in upper urinary tract urothelial carcinoma
Authors: Liang Peir-In , Chen Wan Tzu , Li Chien-Feng , Li Ching-Chia , Li Wei-Ming , Huang Chun-Nung , Yeh Hsin-Chih , Ke Hung-Lung , Wu Wen-Jeng , Chai Chee-Yin 
Published: July 2015

BackgroundThe protein anillin (ANLN) has important roles in cell cytokinesis. Until now, no studies have evaluated the role of ANLN expression in a large cohort of patients with urothelial carcinoma of the upper urinary tract (UCUT).MethodsThis study analysed 156 cases of primary localised UCUT. Pathological slides were reviewed and clinical findings were collected. An immunohistochemical study was performed and the cytoplasmic and nuclear staining results of UCUT were recorded. Expressions of ANLN were analysed to identify correlations with various clinicopathological parameters, disease-specific survival (DSS) and metastasis-free survival (MeFS).ResultsOverexpression of ANLN in the nucleus had significant positive associations with tumour stage (p=0.017), histological grade (p=0.040), mitotic count (p=0.023), tumour necrosis (p=0.009), invasion patterns (p<0.001) and simultaneous involvement of the renal pelvis and ureter (p=0.032). Overexpression of ANLN in the cytoplasm had a significant negative correlation with patient age (p=0.004), tumour grade (p=0.021) and vascular invasion (p=0.013). Notably, univariable analysis showed that overexpression of ANLN in the nucleus was significantly associated with a poor DSS (p=0.006) and MeFS (p=0.010), and multivariable analysis showed that it was an independent predictor of adverse DSS outcome (p=0.031, relative risk 1.535). Low expression of ANLN in the cytoplasm was strongly associated with a poor DSS (p=0.045) and MeFS (p=0.041) in univariable analysis but not in Cox regression analysis.ConclusionsSubcellular localisation of ANLN is correlated with different tumour phenotypes and probably confers different tumorigenicity. Since high nuclear expression of ANLN is also an independent predictor of poor DSS, it is a useful prognostic marker of UCUT.