bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three‐dimensional organization of local excitatory and inhibitory inputs to neurons in laminae III–IV of the spinal dorsal horn
Authors: Kato Go , Kosugi Masafumi , Mizuno Masaharu , Strassman Andrew M. 
Published: August 2013

Key points
Axons of sensory neurons that detect painful and non‐painful stimulation of body tissues project centrally to the dorsal horn of the spinal cord, where they are partially segregated in the superficial and deep laminae, respectively.
Interneuronal connections between superficial and deep laminae could potentially modulate sensory transmission and contribute to alterations that occur under conditions of pain hypersensitivity.
This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of local interneuronal synaptic connections to laminae III–IV neurons, combined with intracellular staining for morphological analysis, in an in vitro‘slice’ preparation of the rat lumbar spinal cord.
Synaptic input from superficial laminae (I–II) was received by laminae III–IV neurons with long dorsal dendrites, supporting the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and, more generally, that local connectivity is governed by rules that are specific to the laminar position and morphology of the postsynaptic neuron.
Abstract  Laser scanning photostimulation was used to map the distribution of the synaptic input zones (sites that give local synaptic inputs) for dorsal horn laminae III–IV neurons, in parasagittal and transverse slices of the rat lumbar spinal cord, and examine how these inputs differed for neurons of different morphologies. All neurons received local excitatory and inhibitory synaptic inputs from within laminae III–IV, while a subset of neurons also received excitatory input from the superficial laminae, especially lamina IIi, as well as the II/III border region. Two anatomical properties were found to be predictive of the dorsoventral position of a neuron's input zone relative to its soma: (1) both excitatory and inhibitory input zones were more dorsal for neurons with longer dorsal dendrites, and (2) excitatory, but not inhibitory, input zones were more dorsal (relative to the soma) for more ventral neurons, with the transition between the dorsal input zones of laminae III–IV neurons and the ventral input zones of lamina II neurons occurring at the II/III border. The observed morphophysiological correlations support the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and that, more generally, local connectivity within the dorsal horn is governed by rules relating the position of a neuron's soma and dendrites to the position of the local presynaptic neurons from which it receives inputs, which are specific to the axis and direction (dorsal vs. ventral), whether the input is excitatory or inhibitory, and the laminar position of the postsynaptic neuron.
Dynamics of Defective Hepatitis C Virus Clones in Reinfected Liver Grafts in Liver Transplant Recipients: Ultradeep Sequencing Analysis
Authors: Ohtsuru Shigeru , Ueda Yoshihide , Marusawa Hiroyuki , Inuzuka Tadashi , Nishijima Norihiro , Nasu Akihiro , Shimizu Kazuharu , Koike Kaoru , Uemoto Shinji , Chiba Tsutomu 
Published: August 2013

ABSTRACT
Hepatitis C virus (HCV) reinfects liver allografts in transplant recipients by replicating immediately after transplantation, causing a rapid increase in blood serum HCV RNA levels. We evaluated dynamic changes in the viral genetic complexity after HCV reinfection of the graft liver; we also identified the characteristics of replicating HCV clones using a massively parallel ultradeep sequencing technique to determine the full-genome HCV sequences in the liver and serum specimens of five transplant recipients with genotype 1b HCV infection before and after liver transplantation. The recipients showed extremely high genetic heterogeneity before transplantation, and the HCV population makeup was not significantly different between the liver and blood serum specimens of the individuals. Viral quasispecies complexity in serum was significantly lower after liver transplantation than before it, suggesting that certain HCV clones selectively proliferated after transplantation. Defective HCV clones lacking the structural region of the HCV genome did not increase in number, and full-genome HCV clones selectively increased in number immediately after liver transplantation. A re-increase in the same defective clone existing before transplantation was detected 22 months after transplantation in one patient. Ultradeep sequencing technology revealed that the genetic heterogeneity of HCV was reduced after liver transplantation. Dynamic changes in defective HCV clones after liver transplantation indicate that these clones have important roles in the HCV life cycle.
Splitting of a Prevalent Mycobacterium bovis Spoligotype by Variable-Number Tandem-Repeat Typing Reveals High Heterogeneity in an Evolving Clonal Group
Authors: Rodriguez-Campos Sabrina , Navarro Yurena , Romero Beatriz , de Juan Lucía , Bezos Javier , Mateos Ana , Golby Paul , Smith Noel H. , Hewinson Glyn R. , Domínguez Lucas , García-de-Viedma Darío , Aranaz Alicia 
Published: August 2013

ABSTRACT

Mycobacterium bovis
populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115
M. bovis
isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by
M. bovis
SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.
The Aptima HPV Assay Fulfills the Cross-Sectional Clinical and Reproducibility Criteria of International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening
Authors: Heideman D. A. M. , Hesselink A. T. , van Kemenade F. J. , Iftner T. , Berkhof J. , Topal F. , Agard D. , Meijer C. J. L. M. , Snijders P. J. F. 
Published: August 2013

ABSTRACT

The Aptima HPV assay (Hologic Gen-Probe, San Diego, CA) is an FDA-approved assay for detecting human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types. This study evaluated the clinical performance of the Aptima HPV assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+), relative to the high-risk HPV GP5+/GP6+ PCR, in a cross-sectional clinical equivalence analysis using the noninferiority score test with cervical samples from population-based screening, i.e., 69 cervical scraping samples from women with CIN2+ and 843 from women without evidence of CIN2+. In addition, intralaboratory reproducibility over time and interlaboratory agreement of the Aptima HPV assay results were assessed with another set of 548 cervical samples. The Aptima HPV assay showed a clinical sensitivity for CIN2+ of 94.2% (95% confidence interval [CI], 85.5 to 97.8%) and a clinical specificity for CIN2+ of 94.5% (95% CI, 92.8 to 95.9%); by comparison, these figures were 97.1% (95% CI, 89.1 to 99.3%) (67/69 samples) and 93.6% (95% CI, 91.7 to 95.0%) (785/839 samples), respectively, for GP5+/GP6+ PCR. The clinical sensitivity and specificity of the Aptima HPV assay were noninferior to those of GP5+/GP6+ PCR (
P
= 0.039 and 0.00016, respectively). In addition, high reproducibility of the Aptima HPV assay, as reflected by the intralaboratory reproducibility over time of 96.0% (95% CI, 94.4 to 97.3%) (526/548 samples; kappa = 0.89) and interlaboratory agreement of 96.7% (95% CI, 95.4 to 98.1%) (531/548 samples; kappa = 0.91), was found. Altogether, these data show that the Aptima HPV assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements for cervical screening. Longitudinal data are needed to ensure that the long-term negative predictive value of this mRNA assay is similar to those of validated HPV DNA tests.
Novel Approach for Differentiating Shigella Species and Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Authors: Khot Prasanna D. , Fisher Mark A. 
Published: August 2013

ABSTRACTShigellaspecies are so closely related toEscherichia colithat routine matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) cannot reliably differentiate them. Biochemical and serological methods are typically used to distinguish these species; however, “inactive” isolates ofE. coliare biochemically very similar toShigellaspecies and thus pose a greater diagnostic challenge. We used ClinProTools (Bruker Daltonics) software to discover MALDI-TOF MS biomarker peaks and to generate classification models based on the genetic algorithm to differentiate betweenShigellaspecies andE. coli.Sixty-sixShigellaspp. and 72E. coliisolates were used to generate and test classification models, and the optimal models contained 15 biomarker peaks for genus-level classification and 12 peaks for species-level classification. We were able to identify 90% ofE. coliandShigellaclinical isolates correctly to the species level. Only 3% of tested isolates were misidentified. This novel MALDI-TOF MS approach allows laboratories to streamline the identification ofE. coliandShigellaspecies.
Comparison of Nucleic Acid Amplification Assays with BD Affirm VPIII for Diagnosis of Vaginitis in Symptomatic Women
Authors: Cartwright Charles P. , Lembke Bryndon D. , Ramachandran Kalpana , Body Barbara A. , Nye Melinda B. , Rivers Charles A. , Schwebke Jane R. 
Published: August 2013

ABSTRACT

A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII
Gardnerella
vaginalis
test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting
Candida albicans
and
Candida glabrata
(CAN-PCR) was compared with the Affirm VPIII
Candida
test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (
n
= 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima
Trichomonas vaginalis
assay; ATV) for
T. vaginalis
were compared with the Affirm VPIII
Trichomonas vaginalis
test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.
Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase
Authors: Lamp Benjamin , Riedel Christiane , Wentz Eveline , Tortorici Maria-Alejandra , Rümenapf Till 
Published: August 2013

ABSTRACT

Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus
Pestivirus
within the
Flaviviridae
family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu
1781
and Met
1782
, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In
trans
-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu
1781
results in inactivation. A second intramolecular cleavage was mapped to the Leu
1748
/Lys
1749
peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of
Flaviviridae
.
TLR3- and MyD88-Dependent Signaling Differentially Influences the Development of West Nile Virus-Specific B Cell Responses in Mice following Immunization with RepliVAX WN, a Single-Cycle Flavivirus Vaccine Candidate
Authors: Xia Jingya , Winkelmann Evandro R. , Gorder Summer R. , Mason Peter W. , Milligan Gregg N. 
Published: August 2013

ABSTRACT
Recognition of conserved pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) results in the activation of innate signaling pathways that drive the innate immune response and ultimately shape the adaptive immune response. RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine candidate derived from West Nile virus (WNV), is intrinsically adjuvanted with multiple PAMPs and induces a vigorous anti-WNV humoral response. However, the innate mechanisms that link pattern recognition and development of vigorous antigen-specific B cell responses are not completely understood. Moreover, the roles of individual PRR signaling pathways in shaping the B cell response to this live attenuated SCFV vaccine have not been established. We examined and compared the role of TLR3- and MyD88-dependent signaling in the development of anti-WNV-specific antibody-secreting cell responses and memory B cell responses induced by RepliVAX WN. We found that MyD88 deficiency significantly diminished B cell responses by impairing B cell activation, development of germinal centers (GC), and the generation of long-lived plasma cells (LLPCs) and memory B cells (MBCs). In contrast, TLR3 deficiency had more effect on maintenance of GCs and development of LLPCs, whereas differentiation of MBCs was unaffected. Our data suggest that both TLR3- and MyD88-dependent signaling are involved in the intrinsic adjuvanting of RepliVAX WN and differentially contribute to the development of vigorous WNV-specific antibody and B cell memory responses following immunization with this novel SCFV vaccine.
Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein
Authors: Niskanen Einari A. , Kalliolinna Olli , Ihalainen Teemu O. , Häkkinen Milla , Vihinen-Ranta Maija 
Published: August 2013

ABSTRACT
The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.
Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication
Authors: Orav Marit , Henno Liisi , Isok-Paas Helen , Geimanen Jelizaveta , Ustav Mart , Ustav Ene 
Published: August 2013

ABSTRACT
We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.
Inhibition of CUL4A Neddylation Causes a Reversible Block to SAMHD1-Mediated Restriction of HIV-1
Authors: Hofmann Henning , Norton Thomas D. , Schultz Megan L. , Polsky Sylvie B. , Sunseri Nicole , Landau Nathaniel R. 
Published: August 2013

ABSTRACT
The deoxynucleoside triphosphohydrolase SAMHD1 restricts retroviral replication in myeloid cells. Human immunodeficiency virus type 2 (HIV-2) and a simian immunodeficiency virus from rhesus macaques (SIVmac) encode Vpx, a virion-packaged accessory protein that counteracts SAMHD1 by inducing its degradation. SAMHD1 is thought to work by depleting the pool of intracellular deoxynucleoside triphosphates but has also been reported to have exonuclease activity that could allow it to degrade the viral genomic RNA or viral reverse-transcribed DNA. To induce the degradation of SAMHD1, Vpx co-opts the cullin4a-based E3 ubiquitin ligase, CRL4. E3 ubiquitin ligases are regulated by the covalent attachment of the ubiquitin-like protein Nedd8 to the cullin subunit. Neddylation can be prevented by MLN4924, a drug that inhibits the nedd8-activating enzyme. We report that MLN4924 inhibits the neddylation of CRL4, blocking Vpx-induced degradation of SAMHD1 and maintaining the restriction. Removal of the drug several hours postinfection released the block. Similarly, Vpx-containing virus-like particles and deoxynucleosides added to the cells more than 24 h postinfection released the SAMHD1-mediated block. Taken together, these findings support deoxynucleoside triphosphate pool depletion as the primary mechanism of SAMHD1 restriction and argue against a nucleolytic mechanism, which would not be reversible.
EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases
Authors: Jha Hem Chandra , Lu Jie , Saha Abhik , Cai Qiliang , Banerjee Shuvomoy , Prasad Mahadesh A. J. , Robertson Erle S. 
Published: August 2013

ABSTRACT
Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis.
Redox and Light Control the Heme-Sensing Activity of AppA
Authors: Yin Liang , Dragnea Vladimira , Feldman George , Hammad Loubna A. , Karty Jonathan A. , Dann Charles E. , Bauer Carl E. 
Published: August 2013

ABSTRACTThe DNA binding activity of the photosystem-specific repressor PpsR is known to be repressed by the antirepressor AppA. AppA contains a blue-light-absorbing BLUF domain and a heme-binding SCHIC domain that controls the interaction of AppA with PpsR in response to light and heme availability. In this study, we have solved the structure of the SCHIC domain and identified the histidine residue that is critical for heme binding. We also demonstrate that dark-adapted AppA binds heme better than light-excited AppA does and that heme bound to the SCHIC domain significantly reduces the length of the BLUF photocycle. We further show that heme binding to the SCHIC domain is affected by the redox state of a disulfide bridge located in the Cys-rich carboxyl-terminal region. These results demonstrate that light, redox, and heme are integrated inputs that control AppA’s ability to disrupt the DNA binding activity of PpsR.IMPORTANCEPhotosynthetic bacteria must coordinate synthesis of the tetrapyrroles cobalamin, heme, and bacteriochlorophyll, as overproduction of the latter two is toxic to cells. A key regulator controlling tetrapyrrole biosynthesis is PpsR, and the activity of PpsR is controlled by the heme-binding and light-regulated antirepressor AppA. We show that AppA binds heme only under dark conditions and that heme binding significantly affects the length of the AppA photocycle. Since AppA interacts with PpsR only in the dark, bound heme thus stimulates the antirepressor activity of PpsR. This causes the redirection of tetrapyrrole biosynthesis away from heme into the bacteriochlorophyll branch.