bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of the stability and pharmacokinetics in dogs of modified ciclosporin capsules stored at −20°C and room temperature
Authors: Bachtel Jeremy C. , Pendergraft Jennifer S. , Rosychuk Rodney A. W. , Gustafson Daniel L. , Hansen Ryan J. , Lunghofer Paul J. 
Published: July 2015

BackgroundPlacement of ciclosporin (Atopica®; Novartis Animal Health, Greensboro, NC, USA) capsules in a freezer prior to administration may reduce the incidence of vomiting in dogs. However, its impact on ciclosporin stability and pharmacokinetics is unknown.Hypothesis/ObjectivesThe purpose of this study was to evaluate the stability of Atopica® capsules and pharmacokinetics of ciclosporin in dogs after storage at −20°C in comparison with storage of capsules at 15–25°C. We hypothesized that there would be no difference in stability or pharmacokinetic parameters between freezer‐stored and room‐temperature Atopica® capsules.AnimalsEight healthy research beagle dogs received one 5.0 mg/kg oral dose each of freezer‐stored and room‐temperature Atopica® capsules with a 1 week washout period between.MethodsCiclosporin concentrations of all available Atopica® capsule strengths were assessed for stability after −20°C storage at five time points over 30 days and at room temperature (15–25°C). A blinded, randomized cross‐over study was also performed to compare blood concentrations of ciclosporin after capsule storage for 28 days at −20 versus 15–25°C. Blood samples were obtained over a 24 h period after administration. Capsule and whole‐blood ciclosporin concentrations were assessed via high‐performance liquid chromatography–tandem mass spectrometry.ResultsThere was no significant difference in stability between freezer‐stored and room‐temperature Atopica® capsules at any time point. In the cross‐over study, there were no significant differences in pharmacokinetic parameters assessed.Conclusions and clinical importancePlacing Atopica® capsules in a −20°C freezer for 28 days does not affect stability or absorption in the dog.
Effect of topical latanoprost 0.005% on intraocular pressure and pupil diameter in normal and glaucomatous cats
Authors: McDonald Jessica E. , Kiland Julie A. , Kaufman Paul L. , Bentley Ellison , Ellinwood N. Matthew , McLellan Gillian J. 
Published: July 2015

AbstractObjectiveTo determine the effects of latanoprost on intraocular pressure (IOP) and pupil diameter (PD) in cats with inherited primary congenital glaucoma (PCG) and normal cats.Animals studied and proceduresIOPandPDwere measured in both eyes (OU) of 12 adult cats (six normal, sixPCG), three times per week for 3 weeks prior to, for 3 weeks during, and for 2 weeks following twice‐daily treatment with 0.005% latanoprost to the right eye (OD) and vehicle to the left (control) eye (OS).IOPandPDwere measured hourly, for 8 h, 1 day prior to, and on the first and last days of treatment. Aqueous humor flow rate (AHF) was determined at baseline and at the end of the treatment phase in six normal cats.ResultsMeanIOPwas significantly lower in treated vs. control eyes ofPCGcats, for up to 8 h following a single latanoprost treatment, and a maximalIOPreduction of 63% occurred in treated eyes at 3 h. Latanoprost acutely loweredIOPin cats withPCG, but this effect appeared to diminish over 3 weeks of treatment.AHFwas modestly increased in the treated eyes of normal cats after 3 weeks of latanoprost treatment, althoughIOPwas not significantly affected. Latanoprost caused miosis, with rebound mydriasis at 24 h posttreatment, in the treated eyes of all cats.ConclusionsFurther research is needed to determine the suitability and efficacy of latanoprost treatment for long‐termIOP‐lowering in cats withPCGor other forms of glaucoma.
Calibration of GafChromic EBT3 for absorbed dose measurements in 5 MeV proton beam and 60Co γ‐rays
Authors: Vadrucci M. , Esposito G. , Ronsivalle C. , Cherubini R. , Marracino F. , Montereali R. M. , Picardi L. , Piccinini M. , Pimpinella M. , Vincenti M. A. , De Angelis C. 
Published: July 2015

Purpose:To study EBT3 GafChromic film in low‐energy protons, and for comparison purposes, in a reference 60Co beam in order to use it as a calibrated dosimetry system in the proton irradiation facility under construction within the framework of the Oncological Therapy with Protons (TOP)‐Intensity Modulated Proton Linear Accelerator for RadioTherapy (IMPLART) Project at ENEA‐Frascati, Italy.Methods:EBT3 film samples were irradiated at the Istituto Nazionale di Fisica Nucleare—Laboratori Nazionali di Legnaro, Italy, with a 5 MeV proton beam generated by a 7 MV Van de Graaff CN accelerator. The nominal dose rates used were 2.1 Gy/min and 40 Gy/min. The delivered dose was determined by measuring the particle fluence and the energy spectrum in air with silicon surface barrier detector monitors. A preliminary study of the EBT3 film beam quality dependence in low‐energy protons was conducted by passively degrading the beam energy. EBT3 films were also irradiated at ENEA‐National Institute of Ionizing Radiation Metrology with gamma radiation produced by a 60Co source characterized by an absorbed dose to water rate of 0.26 Gy/min as measured by a calibrated Farmer type ionization chamber. EBT3 film calibration curves were determined by means of a set of 40 film pieces irradiated to various doses ranging from 0.5 Gy to 30 Gy absorbed dose to water. An EPSON Expression 11000XL color scanner in transmission mode was used for film analysis. Scanner response stability, intrafilm uniformity, and interfilm reproducibility were verified. Optical absorption spectra measurements were performed on unirradiated and irradiated EBT3 films to choose the most sensitive color channel to the dose range used.Results:EBT3 GafChromic films show an under response up to about 33% for low‐energy protons with respect to 60Co gamma radiation, which is consistent with the linear energy transfer dependence already observed with higher energy protons, and a negligible dose‐rate dependence in the 2–40 Gy/min range. Short‐ and long‐term scanner stabilities were 0.5% and 1.5%, respectively; film uniformity and reproducibility were better than 0.5%.Conclusions:The main purpose of this study was to implement EBT3 dosimetry in the proton low‐energy radiobiology line of the TOP‐IMPLART accelerator, having a maximum energy of 7 MeV. Low‐energy proton and 60Co calibrated sources were used to investigate the behavior of film response vs to be written in italicum dose. The calibration in 5 MeV protons is currently used for dose assessment in the radiobiological experiments at the TOP‐IMPLART accelerator carried out at that energy value.
Epidemiology, Clinical Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections
Authors: Crump John A. , Sjölund-Karlsson Maria , Gordon Melita A. , Parry Christopher M. 
Published: July 2015

SUMMARYSalmonella entericainfections are common causes of bloodstream infection in low-resource areas, where they may be difficult to distinguish from other febrile illnesses and may be associated with a high case fatality ratio. Microbiologic culture of blood or bone marrow remains the mainstay of laboratory diagnosis. Antimicrobial resistance has emerged inSalmonella enterica, initially to the traditional first-line drugs chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole. Decreased fluoroquinolone susceptibility and then fluoroquinolone resistance have developed in association with chromosomal mutations in the quinolone resistance-determining region of genes encoding DNA gyrase and topoisomerase IV and also by plasmid-mediated resistance mechanisms. Resistance to extended-spectrum cephalosporins has occurred more often in nontyphoidal than in typhoidalSalmonellastrains. Azithromycin is effective for the management of uncomplicated typhoid fever and may serve as an alternative oral drug in areas where fluoroquinolone resistance is common. In 2013, CLSI lowered the ciprofloxacin susceptibility breakpoints to account for accumulating clinical, microbiologic, and pharmacokinetic-pharmacodynamic data suggesting that revision was needed for contemporary invasiveSalmonellainfections. Newly established CLSI guidelines for azithromycin andSalmonella entericaserovar Typhi were published in CLSI document M100 in 2015.
Diagnostic Performance of the New Version (v2.0) of GenoType MTBDR
sl
Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study
Authors: Tagliani Elisa , Cabibbe Andrea M. , Miotto Paolo , Borroni Emanuele , Toro Juan Carlos , Mansjö Mikael , Hoffner Sven , Hillemann Doris , Zalutskaya Aksana , Skrahina Alena , Cirillo Daniela M. 
Published: July 2015

ABSTRACT

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDR
sl
assay compared to phenotypic DST and sequencing on a panel of 228
Mycobacterium tuberculosis
isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the e
is
promoter region in the MTBDR
sl
v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDR
sl
v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDR
sl
v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.
Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type
Authors: Smither Sophie J. , Weller Simon A. , Phelps Amanda , Eastaugh Lin , Ngugi Sarah , O'Brien Lyn M. , Steward Jackie , Lonsdale Steve G. , Lever Mark S. 
Published: July 2015

ABSTRACT

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10
8
50% tissue culture infective dose per milliliter [TCID
50
· ml
−1
]) and murine blood (EBOV concentration of 1 × 10
7
TCID
50
· ml
−1
) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.
Expansion of Simian Immunodeficiency Virus (SIV)-Specific CD8 T Cell Lines from SIV-Naive Mauritian Cynomolgus Macaques for Adoptive Transfer
Authors: Mohns Mariel S. , Greene Justin M. , Cain Brian T. , Pham Ngoc H. , Gostick Emma , Price David A. , O'Connor David H. 
Published: July 2015

ABSTRACTCD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). However, the specific qualities and characteristics of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further investigation is needed to determine the precise contributions of these various attributes to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques (MCM). These cells exhibited an effector memory phenotype, produced cytokines in response to cognate antigen, and suppressed viral replicationin vitro. We further cultured cell lines specific for four SIV-derived epitopes, Nef103–111RM9, Gag389–394GW9, Env338–346RF9, and Nef254–262LT9. These cell lines were up to 94.4% pure, as determined by major histocompatibility complex (MHC) tetramer analysis. After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in peripheral blood and lung tissue for at least 24 weeks and trafficked to multiple extralymphoid tissues. However, these cells did not impact the acute-phase SIV load after challenge compared to historic controls. The expansion and autologous transfer of SIV-specific T cells into naive animals provide a unique model for exploring cellular immunity and the control of SIV infection and facilitate a systematic evaluation of therapeutic adoptive transfer strategies for eradication of the latent reservoir.IMPORTANCECD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by SIV challenge may help define the critical elements of an effective T cell response to HIV and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques. This is an important first step toward the development of autologous transfer strategies to explore cellular immunity and potential therapeutic applications in the SIV model.
Similarities between Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Genetic and Phenotypic Protease Quasispecies Diversity
Authors: Martinez Miguel Angel , Nevot Maria , Jordan-Paiz Ana , Franco Sandra 
Published: July 2015

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are two highly variable RNA viruses that cause chronic infections in humans. Although HCV likely preceded the AIDS epidemic by some decades, the global spread of both viruses is a relatively recent event. Nevertheless, HCV global diversity is higher than that of HIV-1. To identify differences in mutant diversity, we compared the HIV-1 protease and HCV NS3 protease quasispecies. Three protease gene quasispecies samples per virus, isolated from a total of six infected patients, were genetically and phenotypically analyzed at high resolution (HIV-1, 308 individual clones; HCV, 299 clones). Single-nucleotide variant frequency did not differ between quasispecies from the two viruses (HIV-1, 2.4 × 10
−3
± 0.4 × 10
−3
; HCV, 2.1 × 10
−3
± 0.5 × 10
−3
) (
P
= 0.1680). The proportion of synonymous substitutions to potential synonymous sites was similar (3.667 ± 0.6667 and 2.183 ± 0.9048, respectively) (
P
= 0.2573), and Shannon's entropy values did not differ between HIV-1 and HCV (0.84 ± 0.02 and 0.83 ± 0.12, respectively) (
P
= 0.9408). Of note, 65% (HIV-1) and 67% (HCV) of the analyzed enzymes displayed detectable protease activity, suggesting that both proteases have a similar mutational robustness. In both viruses, there was a rugged protease enzymatic activity landscape characterized by a sharp peak, representing the master sequence, surrounded by a collection of diverse variants present at lower frequencies. These results indicate that nucleotide quasispecies diversification during chronic infection is not responsible for the higher worldwide genetic diversity observed in HCV.

IMPORTANCE
HCV global diversity is higher than that of HIV-1. We asked whether HCV genetic diversification during infection is responsible for the higher worldwide genetic diversity observed in HCV. To this end, we analyzed and compared the genotype and enzymatic activities of HIV-1 and HCV protease quasispecies existing in infected individuals. Our results indicate that HIV-1 and HCV protease quasispecies have very similar genetic diversity and comparable rugged enzymatic activity landscapes. Therapy for HCV has expanded, with new therapeutic agents such as the direct-acting antivirals (DAAs). DAAs, which target HCV NS3 protease and other virus proteins, have improved cure rates. However, major questions remain to be elucidated regarding the virologic correlates of HCV eradication. The findings shown here may help our understanding of the different therapeutic responses observed during chronic HCV infection.
pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication
Authors: Thomas Marco , Sonntag Eric , Müller Regina , Schmidt Stefanie , Zielke Barbara , Fossen Torgils , Stamminger Thomas 
Published: July 2015

ABSTRACT
The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus.

IMPORTANCE
The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy.
Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k
Authors: Condezo Gabriela N. , Marabini Roberto , Ayora Silvia , Carazo José M. , Alba Raúl , Chillón Miguel , San Martín Carmen 
Published: July 2015

ABSTRACT
Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.

IMPORTANCE
Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.