bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrical activity at the sympathetic neuroeffector junction in the guinea‐pig vas deferens.
Authors: Brock J A , Cunnane T C
Published: December 2015

1. The relationship between the nerve terminal action potential and transmitter release from sympathetic postganglionic nerve terminals has been studied in vitro by focal extracellular recording. 2. In the absence of stimulation, 'spontaneous excitatory junction currents' (SEJCs) were recorded with amplitudes up to 500 microV, durations of 50‐80 ms and frequencies of occurrence of 0.3‐0.05 Hz; SEJCs of unusually long time course were also observed. The SEJCs were not recorded in tissues pre‐treated with 6‐hydroxydopamine to destroy sympathetic nerves, were unaffected by tetrodotoxin (TTX), the competitive alpha‐adrenoceptor antagonists, prazosin and phentolamine, the irreversible alpha‐adrenoceptor antagonist benextramine but were blocked by alpha,beta‐methylene ATP which desensitizes P2‐purinoceptors. 3. During trains of supramaximal stimuli at 0.1‐4 Hz stimulus locked 'excitatory junction currents' (EJCs) were evoked intermittently from the population of varicosities located under the suction electrode with a probability of occurrence of 0.005‐0.8. Although EJCs occurred intermittently, they were always preceded by an associated, non‐intermittent, nerve impulse (delay less than or equal to 3 ms). 4. The EJCs reflect transmitter release from nerves because they were abolished by TTX, removal of calcium from the bathing medium, exposure to alpha‐beta‐methylene ATP and exhibited frequency‐dependent facilitation. 5. Amplitude distributions of SEJCs and EJCs recorded in the same attachment were similar and skewed towards low‐amplitude events. Individual SEJCs and EJCs could be found which were identical in amplitude and time course. 6. Locally applied TTX blocked impulse propagation and transmitter release in the terminal region; electrotonic invasion of the terminals from the point of block did not activate the transmitter release process. 7. These studies indicate that (1) intermittence of transmitter release is caused by a low probability of release in the invaded varicosity and is not caused by conduction failure in the terminal regions, (2) only a single quantum is normally secreted when the release mechanism of a varicosity is activated by the nerve impulse and (3) active invasion of the terminals is necessary for transmitter release to occur.
NSF looks for new contractor to finish troubled observatory
Authors: Mervis Jeffrey
Published: December 2015

Reboot sought for management of ecology network after delays, cost overruns, and disputes.
Direct observation of collective modes coupled to molecular orbital–driven charge transfer
Authors: Ishikawa Tadahiko , Hayes Stuart A. , Keskin Sercan , Corthey Gastón , Hada Masaki , Pichugin Kostyantyn , Marx Alexander , Hirscht Julian , Shionuma Kenta , Onda Ken , Okimoto Yoichi , Koshihara Shin-ya , Yamamoto Takashi , Cui Hengbo , Nomura Mitsushiro , Oshima Yugo , Abdel-Jawad Majed , Kato Reizo , Miller R. J. Dwayne
Published: December 2015

The making of a molecular movie

Phase transitions familiar from everyday life, such as boiling or melting, are caused by changing the temperature. In the laboratory, however, researchers can also change the phase of a material by shining intense light on it. During such transitions, changes occur in both the electronic and lattice structure of the material. Ishikawa
et al.
used ultrafast optical and electron diffraction probes to monitor both types of change simultaneously during a photo-induced phase transition in a molecular crystal. The resulting molecular movies showed expansion of the intermolecular distance, flattening of the molecules, and tilting of molecular dimers.

Science
, this issue p.
1501
Connecting strongly correlated superfluids by a quantum point contact
Authors: Husmann Dominik , Uchino Shun , Krinner Sebastian , Lebrat Martin , Giamarchi Thierry , Esslinger Tilman , Brantut Jean-Philippe
Published: December 2015

Simulating electronic transport with atoms

Two superconductors connected by a bridge made out of nonsuperconducting material form a so-called Josephson junction (see the Perspective by Belzig). Valtolina
et al.
replaced the superconductors with two reservoirs of a superfluid Fermi gas and connected them by a weak link to allow atoms to move from one side to the other. Then they made one reservoir more populated than the other and studied the ensuing dynamics as a function of interaction strength between the atoms. In a related experiment, Husmann
et al.
kept the interaction strength at its maximum, but varied the temperature and the properties of the link. As temperature increased, the superfluid disappeared and thermal transport took over.

Science
, this issue p.
1498
, p.
1505
; see also p.
1470
Rice perception of symbiotic arbuscular mycorrhizal fungi requires the karrikin receptor complex
Authors: Gutjahr Caroline , Gobbato Enrico , Choi Jeongmin , Riemann Michael , Johnston Matthew G. , Summers William , Carbonnel Samy , Mansfield Catherine , Yang Shu-Yi , Nadal Marina , Acosta Ivan , Takano Makoto , Jiao Wen-Biao , Schneeberger Korbinian , Kelly Krystyna A. , Paszkowski Uta
Published: December 2015

Early stages of a beneficial relationship

Plants benefit from widespread symbiosis with arbuscular mycorrhizal fungi. This symbiosis between plant and fungus aids plants in capturing mineral and micronutrients from the soil. Gutjahr
et al.
have now identified a component of an intracellular receptor, the hydrolase DWARF 14 LIKE, required in rice roots for initiating the symbiosis. A similar receptor detects karrikins in smoke that signal opportunity for fireweed to grow after a forest fire.

Science
, this issue p.
1521
The planet that wasn't there
The Hunt for Vulcan
Thomas Levenson
Random House, 2015. 247 pp.
Authors: Livio Mario
Published: December 2015

The planet Vulcan wasn't just demoted, it was debunked.
Adrift in an ocean of change
Authors: Vogt Meike
Published: December 2015

Rising temperatures and ocean acidifi cation drive changes in phytoplankton communities

[Also see Reports by

McMahon
et al.

and

Rivero-Calle
et al.

]
A more systematic approach to biological risk
Authors: Palmer Megan J. , Fukuyama Francis , Relman David A.
Published: December 2015

Management of emerging risks in life science and technology requires new leadership and a sober assessment of the legacy of Asilomar
Wonder
Wonder
Renwick Gallery of the Smithsonian American Art Museum, Washington, D.C. Through 10 July 2016.
Authors: Smith Orla M.
Published: December 2015

Wonder
is a new, environmentally themed exhibition that heralds the opening of the elegantly refurbished Renwick gallery.
Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies
Authors: Choi Eun Hwa , Zhang Fan , Lu Ying-Jie , Malley Richard
Published: December 2015

The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is
wzy
dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The
in vitro
-released CPS concentrations per 10
7
CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from
Streptococcus pneumoniae
ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from
S. pneumoniae
ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.
Brief History and Characterization of Enhanced Respiratory Syncytial Virus Disease
Authors: Acosta Patricio L. , Caballero Mauricio T. , Polack Fernando P.
Published: December 2015

In 1967, infants and toddlers immunized with a formalin-inactivated vaccine against respiratory syncytial virus (RSV) experienced an enhanced form of RSV disease characterized by high fever, bronchopneumonia, and wheezing when they became infected with wild-type virus in the community. Hospitalizations were frequent, and two immunized toddlers died upon infection with wild-type RSV. The enhanced disease was initially characterized as a “peribronchiolar monocytic infiltration with some excess in eosinophils.” Decades of research defined enhanced RSV disease (ERD) as the result of immunization with antigens not processed in the cytoplasm, resulting in a nonprotective antibody response and CD4+T helper priming in the absence of cytotoxic T lymphocytes. This response to vaccination led to a pathogenic Th2 memory response with eosinophil and immune complex deposition in the lungs after RSV infection. In recent years, the field of RSV experienced significant changes. Numerous vaccine candidates with novel designs and formulations are approaching clinical trials, defying our previous understanding of favorable parameters for ERD. This review provides a succinct analysis of these parameters and explores criteria for assessing the risk of ERD in new vaccine candidates.
Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis
Authors: Leão Célia , Goldstone Robert J. , Bryant Josephine , McLuckie Joyce , Inácio João , Smith David G. E. , Stevenson Karen
Published: December 2015

Typing of
Mycobacterium avium
subspecies
paratuberculosis
strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization of
Mycobacterium avium
subspecies
paratuberculosis
, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133
Mycobacterium avium
subspecies
paratuberculosis
isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs
Mycobacterium avium
subspecies
paratuberculosis
isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit–variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of
Mycobacterium avium
subspecies
paratuberculosis
isolates.
Optimized Use of the MALDI BioTyper System and the FilmArray BCID Panel for Direct Identification of Microbial Pathogens from Positive Blood Cultures
Authors: Fiori B. , D'Inzeo T. , Giaquinto A. , Menchinelli G. , Liotti F. M. , de Maio F. , De Angelis G. , Quaranta G. , Nagel D. , Tumbarello M. , Posteraro B. , Sanguinetti M. , Spanu T.
Published: December 2015

Despite the current reliance on blood cultures (BCs), the diagnosis of bloodstream infections (BSIs) can be sped up using new technologies performed directly on positive BC bottles. Two methods (the MALDI BioTyper system and FilmArray blood culture identification [BCID] panel) are potentially applicable. In this study, we performed a large-scale clinical evaluation (1,585 microorganisms from 1,394 BSI episodes) on the combined use of the MALDI BioTyper and FilmArray BCID panel compared to a reference (culture-based) method. As a result, the causative organisms of 97.7% (1,362/1,394) of the BSIs were correctly identified by our MALDI BioTyper and FilmArray BCID-based algorithm. Specifically, 65 (5.3%) out of 1,223 monomicrobial BCs that provided incorrect or invalid identifications with the MALDI BioTyper were accurately detected by the FilmArray BCID panel; additionally, 153 (89.5%) out of 171 polymicrobial BCs achieved complete identification with the FilmArray BCID panel. Conversely, full use of the MALDI BioTyper would have resulted in the identification of only 1 causative organism in 97/171 (56.7%) of the polymicrobial cultures. By applying our diagnostic algorithm, the median time to identification was shortened (19.5 h versus 41.7 h with the reference method;
P
< 0.001), and the minimized use of the FilmArray BCID panel led to a significant cost savings. Twenty-six out of 31 microorganisms that could not be identified were species/genera not designed to be detected with the FilmArray BCID panel, indicating that subculture was not dispensable for a few of our BSI episodes. In summary, the fast and effective testing of BC bottles is realistically adoptable in the clinical microbiology laboratory workflow, although the usefulness of this testing for the management of BSIs remains to be established.
Clinical and Molecular Characterization of Invasive Heteroresistant Vancomycin-Intermediate Staphylococcus aureus Infections in Korean Hospitals
Authors: Kim Eu Suk , Bae In-Gyu , Cho Jeong Eun , Choi Yun Jung , Kim Il-Hwan , Kang Gi-Su , Sin Hye-yun , Song Kyoung-Ho , Park Chulmin , Lee Dong-Gun , Kim Moonsuk , Park Kyoung Un , Kim Hong Bin
Published: December 2015

Invasive heteroresistant vancomycin-intermediate
Staphylococcus aureus
(h-VISA) isolates were identified and characterized in 10 Korean hospitals from July 2009 to June 2011. The prevalence of h-VISA infections was 3.3% (42/1,289). Most (41/42) were health care-associated infections caused by strains belonging to sequence type 5. Cases of persistent bacteremia were frequent (17/42), and 30-day mortality was high (16/40).
Impact of Variations in Test Method Parameters on
In Vitro
Activity of Surotomycin against Clostridium difficile and Surotomycin Quality Control Limits for Broth Microdilution and Agar Dilution Susceptibility Testing
Authors: Traczewski Maria M. , Deane Jennifer , Sahm Daniel , Brown Steven D. , Chesnel Laurent
Published: December 2015

Test parameter variations were evaluated for their effects on surotomycin MICs. Calcium concentration was the only variable that influenced MICs; therefore, 50 μg/ml (standard for lipopeptide testing) is recommended. Quality control ranges for
Clostridium difficile
(0.12 to 1 μg/ml) and
Eggerthella lenta
(broth, 1 to 4 μg/ml; agar, 1 to 8 μg/ml) were approved by the Clinical and Laboratory Standards Institute based on these data.
Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function
Authors: Zavala Anamaria G. , O'Dowd John M. , Fortunato Elizabeth A.
Published: December 2015

Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM
−
lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM
−
lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM
−
line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM
−
cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production.
In silico
comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints.

IMPORTANCE
Human cytomegalovirus (HCMV) infection of multiple cell lines lacking ataxia telangiectasia mutated (ATM) protein produced wild-type levels of infectious virus. Interactions between virus strains and the microenvironment of individual ATM
−
lines determined the efficiency of virion production. Infection of one ATM
−
cell line, GM05823, produced large titer differentials dependent on the strain used, Towne or AD169. This discrepancy resolved a disagreement in the literature of a requirement for ATM expression and HCMV reproduction. The titer differentials in GM08523 cells were due, in part, to a decreased capacity of AD169 virions to enter the cell and traffic genomes to the nucleus.
In silico
comparison of the Towne, AD169, and related variant strains' genomes was coupled with serial iterations of a virus entry experiment, narrowing 28 candidate proteins responsible for the phenotype down to 4. Reintroduction of UL148 significantly rescued genome trafficking. Differential behavior of virus strains can be exploited to elucidate gene function.
TRIM5α-Mediated Ubiquitin Chain Conjugation Is Required for Inhibition of HIV-1 Reverse Transcription and Capsid Destabilization
Authors: Campbell Edward M. , Weingart Jared , Sette Paola , Opp Silvana , Sastri Jaya , O'Connor Sarah K. , Talley Sarah , Diaz-Griffero Felipe , Hirsch Vanessa , Bouamr Fadila
Published: December 2015

Rhesus macaque TRIM5α (rhTRIM5α) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5α restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5α, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5α fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5α. Similarly, expression of DUb-rhTRIM5α resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5α induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5α-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5α failed to activate NF-κB signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection.

IMPORTANCE
These studies provide direct evidence that ubiquitin conjugation to rhTRIM5α-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action.
Human Cytomegalovirus Promotes Survival of Infected Monocytes via a Distinct Temporal Regulation of Cellular Bcl-2 Family Proteins
Authors: Collins-McMillen Donna , Kim Jung Heon , Nogalski Maciej T. , Stevenson Emily V. , Chan Gary C. , Caskey Joshua R. , Cieply Stephen J. , Yurochko Andrew D.
Published: December 2015

Monocytes play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to target organ systems. To infect monocytes and reprogram them to deliver infectious virus, HCMV must overcome biological obstacles, including the short life span of monocytes and their antiviral proapoptotic response to infection. We have shown that virally induced upregulation of cellular Mcl-1 promotes early survival of HCMV-infected monocytes, allowing cells to overcome an early apoptotic checkpoint at around 48 h postinfection (hpi). Here, we demonstrate an HCMV-dependent shift from Mcl-1 as the primary antiapoptotic player to the related protein, Bcl-2, later during infection. Bcl-2 was upregulated in HCMV-infected monocytes beginning at 48 hpi. Treatment with the Bcl-2 antagonist ABT-199 only reduced the prosurvival effects of HCMV in target monocytes beginning at 48 hpi, suggesting that Mcl-1 controls survival prior to 48 hpi, while Bcl-2 promotes survival after 48 hpi. Although Bcl-2 was upregulated following viral binding/signaling through cellular integrins (compared to Mcl-1, which is upregulated through binding/activation of epidermal growth factor receptor [EGFR]), it functioned similarly to Mcl-1, adopting the early role of Mcl-1 in preventing caspase-3 cleavage/activation. This distinct, HCMV-induced shift from Mcl-1 to Bcl-2 occurs in response to a cellular upregulation of proapoptotic Bax, as small interfering RNA (siRNA)-mediated knockdown of Bax reduced the upregulation of Bcl-2 in infected monocytes and rescued the cells from the apoptotic effects of Bcl-2 inhibition. Our data demonstrate a distinct survival strategy whereby HCMV induces a biphasic regulation of cellular Bcl-2 proteins to promote host cell survival, leading to viral dissemination and the establishment of persistent HCMV infection.

IMPORTANCEHematogenous dissemination of HCMV via infected monocytes is a crucial component of the viral survival strategy and is required for the establishment of persistent infection and for viral spread to additional hosts. Our system of infected primary human blood monocytes provides us with an opportunity to answer specific questions about viral spread and persistence inin vivo-relevant myeloid cells that cannot be addressed with the more traditionally used replication-permissive cells. Our goal in examining the mechanisms whereby HCMV reprograms infected monocytes to promote viral dissemination is to uncover new targets for therapeutic intervention that would disrupt key viral survival and persistence strategies. Because of this important role in maintaining survival of HCMV-infected monocytes, our new data on the role of Bcl-2 regulation during viral infection represents a promising molecular target for mitigating viral spread and persistence.
Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway
Authors: Pattabhi Sowmya , Wilkins Courtney R. , Dong Ran , Knoll Megan L. , Posakony Jeffrey , Kaiser Shari , Mire Chad E. , Wang Myra L. , Ireton Renee C. , Geisbert Thomas W. , Bedard Kristin M. , Iadonato Shawn P. , Loo Yueh-Ming , Gale Michael
Published: December 2015

The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family
Flaviviridae
, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families
Filoviridae
(Ebola virus),
Orthomyxoviridae
(influenza A virus),
Arenaviridae
(Lassa virus), and
Paramyxoviridae
(respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera.

IMPORTANCE
Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses, including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus, and Ebola virus. Our study thus identifies a class of small molecules with a novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant health care burden and/or that are known to cause infections with high case fatality rates.
Pulmonary C Fibers Modulate MMP-12 Production via PAR2 and Are Involved in the Long-Term Airway Inflammation and Airway Hyperresponsiveness Induced by Respiratory Syncytial Virus Infection
Authors: Zang Na , Zhuang Jianguo , Deng Yu , Yang Zhimei , Ye Zhixu , Xie Xiaohong , Ren Luo , Fu Zhou , Luo Zhengxiu , Xu Fadi , Liu Enmei
Published: December 2015

Children with acute respiratory syncytial virus (RSV) infection often develop sequelae of persistent airway inflammation and wheezing. Pulmonary C fibers (PCFs) are involved in the generation of airway inflammation and resistance; however, their role in persistent airway diseases after RSV is unexplored. Here, we elucidated the pathogenesis of PCF activation in RSV-induced persistent airway disorders. PCF-degenerated and intact mice were used in the current study. Airway inflammation and airway resistance were evaluated. MMP408 and FSLLRY-NH2 were the selective antagonists for MMP-12 and PAR2, respectively, to investigate the roles of MMP-12 and PAR2 in PCFs mediating airway diseases. As a result, PCF degeneration significantly reduced the following responses to RSV infection: augmenting of inflammatory cells, especially macrophages, and infiltrating of inflammatory cells in lung tissues; specific airway resistance (sRaw) response to methacholine; and upregulation of MMP-12 and PAR2 expression. Moreover, the inhibition of MMP-12 reduced the total number of cells and macrophages in bronchiolar lavage fluid (BALF), as well infiltrating inflammatory cells, and decreased the sRaw response to methacholine. In addition, PAR2 was upregulated especially at the later stage of RSV infection. Downregulation of PAR2 ameliorated airway inflammation and resistance following RSV infection and suppressed the level of MMP-12. In all, the results suggest that PCF involvement in long-term airway inflammation and airway hyperresponsiveness occurred at least partially via modulating MMP-12, and the activation of PAR2 might be related to PCF-modulated MMP-12 production. Our initial findings indicated that the inhibition of PCF activity would be targeted therapeutically for virus infection-induced long-term airway disorders.

IMPORTANCE
The current study is critical to understanding that PCFs are involved in long-term airway inflammation and airway resistance after RSV infection through mediating MMP-12 production via PAR2, indicating that the inhibition of PCF activity can be targeted therapeutically for virus infection-induced long-term airway disorders.
Human MicroRNA miR-532-5p Exhibits Antiviral Activity against West Nile Virus via Suppression of Host Genes SESTD1 and TAB3 Required for Virus Replication
Authors: Slonchak Andrii , Shannon Rory P. , Pali Gabor , Khromykh Alexander A.
Published: December 2015

West Nile virus (WNV) is a mosquito-transmitted flavivirus that naturally circulates between mosquitos and birds but can also infect humans, causing severe neurological disease. The early host response to WNV infection in vertebrates primarily relies on the type I interferon pathway; however, recent studies suggest that microRNAs (miRNAs) may also play a notable role. In this study, we assessed the role of host miRNAs in response to WNV infection in human cells. We employed small RNA sequencing (RNA-seq) analysis to determine changes in the expression of host miRNAs in HEK293 cells infected with an Australian strain of WNV, Kunjin (WNV
KUN
), and identified a number of host miRNAs differentially expressed in response to infection. Three of these miRNAs were confirmed to be significantly upregulated in infected cells by quantitative reverse transcription (qRT)-PCR and Northern blot analyses, and one of them, miR-532-5p, exhibited a significant antiviral effect against WNV
KUN
infection. We have demonstrated that miR-532-5p targets and downregulates expression of the host genes SESTD1 and TAB3 in human cells. Small interfering RNA (siRNA) depletion studies showed that both SESTD1 and TAB3 were required for efficient WNV
KUN
replication. We also demonstrated upregulation of mir-532-5p expression and a corresponding decrease in the expression of its targets, SESTD1 and TAB3, in the brains of WNV
KUN
-infected mice. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent a host antiviral response aimed at limiting WNV
KUN
infection and highlight the important role of miRNAs in controlling RNA virus infections in mammalian hosts.

IMPORTANCE
West Nile virus (WNV) is a significant viral pathogen that poses a considerable threat to human health across the globe. There is no specific treatment or licensed vaccine available for WNV, and deeper insight into how the virus interacts with the host is required to facilitate their development. In this study, we addressed the role of host microRNAs (miRNAs) in antiviral response to WNV in human cells. We identified miR-532-5p as a novel antiviral miRNA and showed that it is upregulated in response to WNV infection and suppresses the expression of the host genes TAB3 and SESTD1 required for WNV replication. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent an antiviral response aimed at limiting WNV infection and highlight the important role of miRNAs in controlling virus infections in mammalian hosts.
A Single Mutation at PB1 Residue 319 Dramatically Increases the Safety of PR8 Live Attenuated Influenza Vaccine in a Murine Model without Compromising Vaccine Efficacy
Authors: Cox Andrew , Dewhurst Stephen
Published: December 2015

The live attenuated influenza vaccine (LAIV) is preferentially recommended for use in most children yet remains unsafe for the groups most at risk. Here we have improved the safety of a mouse-adapted live attenuated influenza vaccine containing the same attenuating amino acid mutations as in human LAIV by adding an additional mutation at PB1 residue 319. This results in a vaccine with a 20-fold decrease in protective efficacy and a 10,000-fold increase in safety.
Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain
Authors: Dick Robert A. , Barros Marilia , Jin Danni , Lösche Mathias , Vogt Volker M.
Published: December 2015

The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly
in vitro
dramatically reduced binding of Gag to liposomes.
In vivo
, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize.

IMPORTANCE
The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assembly
in vitro
with purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.
Diverse Array of New Viral Sequences Identified in Worldwide Populations of the Asian Citrus Psyllid (Diaphorina citri) Using Viral Metagenomics
Authors: Nouri Shahideh , Salem Nidá , Nigg Jared C. , Falk Bryce W.
Published: December 2015

The Asian citrus psyllid,
Diaphorina citri
, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and
D. citri
represent a major threat to world citrus production. As there is no cure for HLB, insect vector management is considered one strategy to help control the disease, and
D. citri
viruses might be useful. In this study, we used a metagenomic approach to analyze viral sequences associated with the global population of
D. citri
. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we showed that the virus-like sequences of
D. citri
are diverse. We identified novel viral sequences belonging to the picornavirus superfamily, the
Reoviridae
,
Parvoviridae
, and
Bunyaviridae
families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a
Wolbachia
prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents.

IMPORTANCE
Insects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used high-throughput next-generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with
Diaphorina citri
, the Asian citrus psyllid.
D. citri
is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with
D. citri
.
ICP8 Filament Formation Is Essential for Replication Compartment Formation during Herpes Simplex Virus Infection
Authors: Darwish Anthar S. , Grady Lorry M. , Bai Ping , Weller Sandra K.
Published: December 2015

Herpes simplex virus (HSV) dramatically reorganizes the infected-cell nucleus, leading to the formation of prereplicative sites and replication compartments. This process is driven by the essential viral single-stranded DNA (ssDNA) binding protein ICP8, which can form double-helical filaments in the absence of DNA. In this paper, we show that two conserved motifs, FNF (F1142, N1143, and F1144) and FW (F843 and W844), are essential for ICP8 self-interactions, and we propose that the FNF motif docks into the FW region during filament formation. Mammalian expression plasmids bearing mutations in these motifs (FNF and FW) were unable to complement an ICP8-null mutant for growth and replication compartment formation. Furthermore, FNF and FW mutants were able to inhibit wild-type (WT) virus plaque formation and filament formation, whereas a double mutant (FNF-FW) was not. These results suggest that single mutant proteins are incorporated into nonproductive ICP8 filaments, while the double mutant is unable to interact with WT ICP8 and does not interfere with WT growth. Cells transfected with WT ICP8 and the helicase-primase (H/P) complex exhibited punctate nuclear structures that resemble prereplicative sites; however, the FNF and FW mutants failed to do so. Taken together, these results suggest that the FNF and FW motifs are required for ICP8 self-interactions and that these interactions may be important for the formation of prereplicative sites and replication compartments. We propose that filaments or other higher-order structures of ICP8 may provide a scaffold onto which other proteins can be recruited to form prereplicative sites and replication compartments.

IMPORTANCE
For nuclear viruses such as HSV, efficient DNA replication requires the formation of discrete compartments within the infected-cell nucleus in which replication proteins are concentrated and assembled into the HSV replisome. In this paper, we characterize the role of filament formation by the single-stranded DNA binding protein ICP8 in the formation of prereplicative sites and replication compartments. We propose that ICP8 protein filaments generate a protein scaffold for other cellular and viral proteins, resulting in a structure that concentrates both viral DNA and replication proteins. Replication compartments may be similar to other types of cellular membraneless compartments thought to be formed by phase separations caused by low-affinity, multivalent interactions involving proteins and nucleic acids within cells. ICP8 scaffolds could facilitate the formation of replication compartments by mediating interactions with other components of the replication machinery.
Peripheral T Follicular Helper Cells Are the Major HIV Reservoir within Central Memory CD4 T Cells in Peripheral Blood from Chronically HIV-Infected Individuals on Combination Antiretroviral Therapy
Authors: Pallikkuth Suresh , Sharkey Mark , Babic Dunja Z. , Gupta Sachin , Stone Geoffrey W. , Fischl Margaret A. , Stevenson Mario , Pahwa Savita
Published: December 2015

In this study, we examined the peripheral blood (PB) central memory (T
CM
) CD4
+
T cell subsets designated peripheral T follicular helper cells (pTfh cells) and non-pTfh cells to assess HIV permissiveness and persistence. Purified pTfh and non-pTfh cells from healthy HIV-negative donors were tested for HIV permissiveness using green fluorescent protein (GFP)-expressing HIV-1NL4-3/Ba-L, followed by viral reactivation using beads coated with anti-CD3/anti-CD28 monoclonal antibodies. The role of pTfh cells in HIV persistence was analyzed in 12 chronically HIV-1 infected patients before and 48 weeks after initiation of raltegravir-containing combination antiretroviral therapy (cART). Total cellular HIV-1 DNA and episomes containing two copies of the viral long terminal repeat (2LTR circles) were analyzed in using droplet digital PCR in the purified pTfh and non-pTfh cells. Activation-inducible HIV p24 expression was determined by flow cytometry. Results indicate that pTfh cells, in particular PD1
+
pTfh cells, showed greater permissiveness for HIV infection than non-pTfh cells. At week 48 on cART, HIV DNA levels were unchanged from pre-cART levels, although a significant decrease in 2LTR circles was observed in both cell subsets. Inducible HIV p24 expression was higher in pTfh cells than in non-pTfh cells, with the highest frequencies in the PD1
+
CXCR3
−
pTfh cell subset. Frequencies of HLADR
+
CD38
+
activated CD4 T cells correlated with 2LTR circles in pTfh and non-pTfh cells at both time points and with p24
+
cells at entry. In conclusion, among CD4 T
CM
cells in PB of aviremic patients on cART, pTfh cells, in particular the PD1
+
CXCR3
−
subset, constitute a major HIV reservoir that is sustained by ongoing residual immune activation. The inducible HIV p24 assay is useful for monitoring HIV reservoirs in defined CD4 T cell subsets.

IMPORTANCE
Identification of the type and nature of the cellular compartments of circulating HIV reservoirs is important for targeting of HIV cure strategies. In lymph nodes (LN), a subset of CD4 T cells called T follicular helper (Tfh) cells are preferentially infected by HIV. Central memory (T
CM
) CD4 T cells are the major cellular reservoir for HIV in peripheral blood and contain a subset of CD4 T
CM
cells expressing chemokine receptor CXCR5 similar in function to LN Tfh cells termed peripheral Tfh (pTfh) cells. We found that the circulating pTfh cells are highly susceptible to HIV infection and that in HIV-infected patients, HIV persists in these cells following plasma virus suppression with potent cART. These pTfh cells, which constitute a subset of T
CM
CD4 T cells, can be readily monitored in peripheral blood to assess HIV persistence.
Ear and vestibular symptoms in train operators after sudden air pressure changes in trains
Authors: Francois Hugues M A , Vantrappen Luc , Van Rompaey Vincent , Godderis Lode
Published: December 2015

A healthy 31-year-old train operator presented to our occupational health clinic reporting ear aches, headaches, dizziness, unsteadiness and even slight tinnitus. These symptoms first appeared when the patient started operating from a new train cabin. He described a sudden pressure gradient, experienced on some parts of the trajectory, which might have caused these problems. Although the cabins were equipped with a pressure equalising device, this was usually switched off because of the device creating an uncomfortable feeling in the cabin. The literature describes sudden pressure gradients as possible factors for passenger discomfort.
Short biceps femoris fascicles and eccentric knee flexor weakness increase the risk of hamstring injury in elite football (soccer): a prospective cohort study
Authors: Timmins Ryan G , Bourne Matthew N , Shield Anthony J , Williams Morgan D , Lorenzen Christian , Opar David A
Published: December 2015

Background/aim

To investigate the role of eccentric knee flexor strength, between-limb imbalance and biceps femoris long head (BFlh) fascicle length on the risk of future hamstring strain injury (HSI).

Methods

Elite soccer players (n=152) from eight different teams participated. Eccentric knee flexor strength during the Nordic hamstring exercise and BFlh fascicle length were assessed at the beginning of preseason. The occurrences of HSIs following this were recorded by the team medical staff. Relative risk (RR) was determined for univariate data, and logistic regression was employed for multivariate data.

Results

Twenty seven new HSIs were reported. Eccentric knee flexor strength below 337 N (RR=4.4; 95% CI 1.1 to 17.5) and possessing BFlh fascicles shorter than 10.56 cm (RR=4.1; 95% CI 1.9 to 8.7) significantly increased the risk of a HSI. Multivariate logistic regression revealed significant effects when combinations of age, history of HSI, eccentric knee flexor strength and BFlh fascicle length were explored. From these analyses the likelihood of a future HSI in older athletes or those with a HSI history was reduced if high levels of eccentric knee flexor strength and longer BFlh fascicles were present.

Conclusions

The presence of short BFlh fascicles and low levels of eccentric knee flexor strength in elite soccer players increases the risk of future HSI. The greater risk of a future HSI in older players or those with a previous HSI is reduced when they have longer BFlh fascicles and high levels of eccentric strength.