bluuebunny/crossref_metadata_2025_split huggingface dataset:
Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses
Authors: Kommedal Øyvind , Wilhelmsen Marianne Thulin , Skrede Steinar , Meisal Roger , Jakovljev Aleksandra , Gaustad Peter , Hermansen Nils Olav , Vik-Mo Einar , Solheim Ole , Ambur Ole Herman , Sæbø Øystein , Høstmælingen Christina Teisner , Helland Christian 
Published: March 2014

ABSTRACT

Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture- and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications.
Aggregatibacter aphrophilus
,
Fusobacterium nucleatum
, and
Streptococcus intermedius
or combinations of them were found in all spontaneous polymicrobial abscesses.
F. nucleatum
was systematically detected in samples with anaerobic flora. The increased detection rate for
Actinomyces
spp. and facultative Gram-negative rods further revealed several species associations. We suggest that
A. aphrophilus
,
F. nucleatum
, and
S. intermedius
are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition,
F. nucleatum
seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop.
In Vitro
Antimicrobial Susceptibility of Aerococcus urinae
Authors: Humphries Romney M. , Hindler Janet A. 
Published: March 2014

ABSTRACT

Aerococcus urinae
may cause urinary tract infections, bacteremia, and endocarditis. No standardized susceptibility test methods or interpretive criteria have been proposed for this organism. This study reports the MIC results for 128
A. urinae
isolates tested by broth microdilution. The isolates had low MICs to amoxicillin, cefotaxime, ceftriaxone, doxycycline, linezolid, meropenem, penicillin, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. However, 55% of the isolates had MICs to clindamycin of >0.25 μg/ml, 44% had MICs to erythromycin of >0.25 μg/ml, and 16% had MICs to levofloxacin of >2 μg/ml.
Ureaplasma parvum Prosthetic Joint Infection Detected by PCR
Authors: Farrell John J. , Larson Joshua A. , Akeson Jeffrey W. , Lowery Kristin S. , Rounds Megan A. , Sampath Rangarajan , Bonomo Robert A. , Patel Robin 
Published: March 2014

ABSTRACT

We describe the first reported case of
Ureaplasma parvum
prosthetic joint infection (PJI) detected by PCR.
Ureaplasma
species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material).
U. parvum
is a relatively new species name for certain serovars of
Ureaplasma urealyticum
, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected
U. parvum
in all samples.
U. parvum
-specific PCR testing was performed on all synovial fluid samples to confirm the
U. parvum
detection.
Rapid Detection of New Delhi Metallo-β-Lactamase Gene and Variants Coding for Carbapenemases with Different Activities by Use of a PCR-Based
In Vitro
Protein Expression Method
Authors: Huang Li , Hu Xiumei , Zhou Man , Yang Yinmei , Qiao Jinjuan , Wang Dianbing , Yu Junping , Cui Zongqiang , Zhang Zhiping , Zhang Xian-En , Wei Hongping 
Published: March 2014

ABSTRACT

New Delhi metallo-β-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on β-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based
in vitro
protein expression (PCR-P) method was used to detect
bla
NDM-1
and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with
in vitro
cell-free expression to convert the
bla
NDM-1
amplicons into NDM for carbapenemase assay. The method could screen for
bla
NDM-1
within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent
bla
NDM-1
variants, 2 functional variants,
bla
NDM-4
and
bla
NDM-5
, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing
bla
NDM-1
. This novel method provides the first integrated approach for rapidly detecting the full-length
bla
NDM-1
and revealing its functional variants in clinical isolates.
NEMO Is Essential for Kaposi's Sarcoma-Associated Herpesvirus-Encoded vFLIP K13-Induced Gene Expression and Protection against Death Receptor-Induced Cell Death, and Its N-Terminal 251 Residues Are Sufficient for This Process
Authors: Tolani Bhairavi , Matta Hittu , Gopalakrishnan Ramakrishnan , Punj Vasu , Chaudhary Preet M. 
Published: March 2014

ABSTRACT

Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 was originally believed to protect virally infected cells against death receptor-induced apoptosis by interfering with caspase 8/FLICE activation. Subsequent studies revealed that K13 also activates the NF-κB pathway by binding to the NEMO/inhibitor of NF-κB (IκB) kinase gamma (IKKγ) subunit of an IKK complex and uses this pathway to modulate the expression of genes involved in cellular survival, proliferation, and the inflammatory response. However, it is not clear if K13 can also induce gene expression independently of NEMO/IKKγ. The minimum region of NEMO that is sufficient for supporting K13-induced NF-κB has not been delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis remains to be determined. In this study, we used microarray analysis on K13-expressing wild-type and
NEMO
-deficient cells to demonstrate that NEMO is required for modulation of K13-induced genes. Reconstitution of
NEMO
-null cells revealed that the N-terminal 251 amino acid residues of NEMO are sufficient for supporting K13-induced NF-κB but fail to support tumor necrosis factor alpha (TNF-α)-induced NF-κB. K13 failed to protect
NEMO
-null cells against TNF-α-induced cell death but protected those reconstituted with the NEMO mutant truncated to include only the N-terminal 251 amino acid residues [the NEMO(1-251) mutant]. Taken collectively, our results demonstrate that NEMO is required for modulation of K13-induced genes and the N-terminal 251 amino acids of NEMO are sufficient for supporting K13-induced NF-κB. Finally, the ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB.

IMPORTANCE
Kaposi's sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptor-induced apoptosis and to activate the NF-κB pathway by binding to adaptor protein NEMO/IKKγ. However, whether K13 can also induce gene expression independently of NEMO and the minimum region of NEMO that is sufficient for supporting K13-induced NF-κB remain to be delineated. Furthermore, the contribution of NEMO and NF-κB to the protective effect of K13 against death receptor-induced apoptosis is not clear. We demonstrate that NEMO is required for modulation of K13-induced genes and its N-terminal 251 amino acids are sufficient for supporting K13-induced NF-κB. The ability of K13 to protect against TNF-α-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-κB. Our results suggest that K13-based gene therapy approaches may have utility for the treatment of patients with NEMO mutations and immunodeficiency.
A Complex Comprising Phosphatidylinositol 4-Kinase IIIβ, ACBD3, and Aichi Virus Proteins Enhances Phosphatidylinositol 4-Phosphate Synthesis and Is Critical for Formation of the Viral Replication Complex
Authors: Ishikawa-Sasaki Kumiko , Sasaki Jun , Taniguchi Koki 
Published: March 2014

ABSTRACT

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754–766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the
in vitro
kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated.

IMPORTANCE
The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus
Kobuvirus
, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication and individual viral proteins enhance the level of cellular PI4P, and suggested that the viral protein/ACBD3/PI4KB complex plays an important role in forming a functional replication complex. Thus, the present study provides a new example of modulation of cellular lipid metabolism by viruses to support the replication of their genomes.
Lysophosphatidylcholine Reversibly Arrests Pore Expansion during Syncytium Formation Mediated by Diverse Viral Fusogens
Authors: Ciechonska Marta , Duncan Roy 
Published: March 2014

ABSTRACT
Using lysophosphatidylcholine, a curvature-inducing lysolipid, we have isolated a reversible, “stalled pore” phenotype during syncytium formation induced by the p14 fusion-associated small transmembrane (FAST) protein and influenza virus hemagglutinin (HA) fusogens. This is the first evidence that lateral propagation of stable fusion pores leading to syncytiogenesis mediated by diverse viral fusogens is inhibited by promotion of positive membrane curvature in the outer leaflets of the lipid bilayer surrounding intercellular fusion pores.
An Allometric Relationship between the Genome Length and Virion Volume of Viruses
Authors: Cui Jie , Schlub Timothy E. , Holmes Edward C. 
Published: March 2014

ABSTRACTVirions vary in size by at least 4 orders of magnitude, yet the evolutionary forces responsible for this enormous diversity are unknown. We document a significant allometric relationship, with an exponent of approximately 1.5, between the genome length and virion volume of viruses and find that this relationship is not due to geometric constraints. Notably, this allometric relationship holds regardless of genomic nucleic acid, genome structure, or type of virion architecture and therefore represents a powerful scaling law. In contrast, no such relationship is observed at the scale of individual genes. Similarly, after adjusting for genome length, no association is observed between virion volume and the number of proteins, ruling out protein number as the explanation for the relationship between genome and virion sizes. Such a fundamental allometric relationship not only sheds light on the constraints to virus evolution, in that increases in virion size but not necessarily structure are associated with concomitant increases in genome size, but also implies that virion sizes in nature can be broadly predicted from genome sequence data alone.IMPORTANCEViruses vary dramatically in both genome and virion sizes, but the factors responsible for this diversity are uncertain. Through a comparative and quantitative investigation of these two fundamental biological parameters across diverse viral taxa, we show that genome length and virion volume conform to a simple allometric scaling law. Notably, this allometric relationship holds regardless of the type of virus, including those with both RNA and DNA genomes, and encompasses viruses that exhibit more than 3 logs of genome size variation. Accordingly, this study helps to reveal the basic rules of virus design.
NTPDASE4
Gene Products Cooperate with the Adenovirus E4orf4 Protein through PP2A-Dependent and -Independent Mechanisms and Contribute to Induction of Cell Death
Authors: Avital-Shacham Meirav , Sharf Rakefet , Kleinberger Tamar 
Published: March 2014

ABSTRACT

The adenovirus E4orf4 protein induces nonclassical apoptosis in mammalian cells through at least two complementing pathways regulated by the interactions of E4orf4 with protein phosphatase 2A (PP2A) and Src kinases. In
Saccharomyces cerevisiae
cells, which do not express Src, E4orf4 induces PP2A-dependent toxicity. The yeast Golgi apyrase Ynd1 was found to contribute to E4orf4-mediated toxicity and to interact with the PP2A-B55α regulatory subunit. In addition, a mammalian Ynd1 orthologue, the
NTPDASE4
gene product Golgi UDPase, was shown to physically interact with E4orf4. Here we report that knockdown of
NTPDASE4
suppressed E4orf4-induced cell death. Conversely, overexpression of the
NTPDASE4
gene products Golgi UDPase and LALP70 enhanced E4orf4-induced cell killing. We found that similarly to results obtained in yeast, the apyrase activity of mammalian UDPase was not required for its contribution to E4orf4-induced toxicity. The interaction between E4orf4 and UDPase had two consequences: a PP2A-dependent one, resulting in increased UDPase levels, and a PP2A-independent outcome that led to dissociation of large UDPase-containing protein complexes. The present report extends our findings in yeast to E4orf4-mediated death of mammalian cells, and combined with previous results, it suggests that the E4orf4-NTPDase4 pathway, partly in association with PP2A, may provide an alternative mechanism for the E4orf4-Src pathway to contribute to the cytoplasmic death function of E4orf4.

IMPORTANCE
The adenovirus E4orf4 protein contributes to regulation of the progression of virus infection from the early to the late phase, and when expressed alone, it induces a unique caspase-independent programmed cell death which is more efficient in cancer cells than in normal cells. The interactions of E4orf4 with cellular proteins that mediate its functions, such as PP2A and Src kinases, are highly conserved in evolution. The results presented here reveal that the
NTPDASE4
gene product Golgi UDPase, first discovered to contribute to E4orf4 toxicity in
Saccharomyces cerevisiae
, associates with E4orf4 and plays a role in induction of cell death in mammalian cells. Details of the functional interaction between E4orf4, PP2A, and the UDPase are described. Identification of the evolutionarily conserved mechanisms underlying E4orf4 activity will increase our understanding of the interactions between the virus and the host cell and will contribute to our grasp of the unique mode of E4orf4-induced cell death.
The Rad6/18 Ubiquitin Complex Interacts with the Epstein-Barr Virus Deubiquitinating Enzyme, BPLF1, and Contributes to Virus Infectivity
Authors: Kumar Ravindra , Whitehurst Christopher B. , Pagano Joseph S. 
Published: March 2014

ABSTRACT

Epstein-Barr virus (EBV) encodes BPLF1, a lytic cycle protein with deubiquitinating activity that is contained in its N-terminal domain and conserved across the
Herpesviridae
. EBV replication is associated with cellular DNA replication and repair factors, and initiation of EBV lytic replication induces a DNA damage response, which can be regulated at least in part by BPLF1. The cellular DNA repair pathway, translesion synthesis (TLS), is disrupted by BPLF1, which deubiquitinates the DNA processivity factor, PCNA, and inhibits the recruitment of the TLS polymerase, polymerase eta (Pol eta), after damage to DNA by UV irradiation. Here we showed that the E3 ubiquitin ligase, which activates TLS repair by monoubiquitination of PCNA, is also affected by BPLF1 deubiquitinating activity. First, BPLF1 interacts directly with Rad18, and overexpression of BPLF1 results in increased levels of the Rad18 protein, suggesting that it stabilizes Rad18. Next, expression of functionally active BPLF1 caused relocalization of Rad18 into nuclear foci, which is consistent with sites of cellular DNA replication that occur during S phase. Also, levels of Rad18 remain constant during lytic reactivation of wild-type virus, but reactivation of BPLF1 knockout virus resulted in decreased levels of Rad18. Finally, the contribution of Rad18 levels to infectious virus production was examined with small interfering RNA (siRNA) targeting Rad18. Results demonstrated that reducing levels of Rad18 decreased production of infectious virus, and infectious titers of BPLF1 knockout virus were partially restored by overexpression of Rad18. Thus, BPLF1 interacts with and maintains Rad18 at high levels during lytic replication, which assists in production of infectious virus.

IMPORTANCE
Characterization of EBV BPLF1's deubiquitinating activity and identification of its targets and subsequent functional effects remain little studied. All members of the
Herpesviridae
contain BPLF1 homologs with conserved enzymatic activity, and findings discovered with EBV BPLF1 are likely applicable to other members of the family. Discovery of new targets of BPLF1 will point to cellular pathways and viral processes regulated by the enzymatic activity of the EBV-encoded deubiquitinating enzyme. Here we determined the importance of the cellular ubiquitin ligase Rad18 in these processes and how it is affected by BPLF1. Our findings demonstrate that EBV can co-opt Rad18 as a novel accessory factor in the production of infectious virus.
The Rhinovirus Subviral A-Particle Exposes 3′-Terminal Sequences of Its Genomic RNA
Authors: Harutyunyan Shushan , Kowalski Heinrich , Blaas Dieter 
Published: March 2014

ABSTRACT

Enteroviruses, which represent a large genus within the family
Picornaviridae
, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (+)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that
in vitro
acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ∼700 bases. However, even after hours of incubation at pH 5.2, 5′-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment
in vivo
. This finding suggests that the exposed 3′-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded.

IMPORTANCE
For host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification
in vitro
results in RNA exit starting from the 3′-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.