bluuebunny/crossref_metadata_2025 huggingface dataset:
Bayesian inference of elastic properties with resonant ultrasound spectroscopy
Authors: Bales Ben , Petzold Linda , Goodlet Brent R. , Lenthe William C. , Pollock Tresa M.
Published: January 2018

Bayesian modeling and Hamiltonian Monte Carlo (HMC) are utilized to formulate a robust algorithm capable of simultaneously estimating anisotropic elastic properties and crystallographic orientation of a specimen from a list of measured resonance frequencies collected via Resonance Ultrasound Spectroscopy (RUS). Unlike typical optimization procedures which yield point estimates of the unknown parameters, computing a Bayesian posterior yields probability distributions for the unknown parameters, and HMC is an efficient way to compute this posterior. The algorithms described are demonstrated on RUS data collected from two parallelepiped specimens of structural metal alloys. First, the elastic constants for a specimen of fine-grain polycrystalline Ti-6Al-4 V with random crystallographic texture and isotropic elastic symmetry are estimated. Second, the elastic constants and crystallographic orientation for a single crystal Ni-based superalloy CMSX-4 specimen are accurately determined, using only measurements of the specimen geometry, mass, and resonance frequencies. The unique contributions of this paper are as follows: the application of HMC for sampling the Bayesian posterior of a probabilistic RUS model, and the procedure for simultaneous estimation of elastic constants and lattice-specimen misorientation. Compared to previous approaches these algorithms demonstrate superior convergence behavior, particularly when the initial parameterization is unknown, and enable substantially simplified experimental procedures.
Enhanced Efflux Pump Activity in Old Candida glabrata Cells
Authors: Bhattacharya Somanon , Fries Bettina C.
Published: January 2018

We investigated the effect of replicative aging on antifungal resistance in
Candida glabrata
. Our studies demonstrate significantly increased transcription of ABC transporters and efflux pump activity in old versus young
C. glabrata
cells of a fluconazole-sensitive and -resistant strain. In addition, higher tolerance to killing by micafungin and amphotericin B was noted and is associated with higher transcription of glucan synthase gene
FKS1
and lower ergosterol content in older cells.
IncX3 Epidemic Plasmid Carrying
bla
NDM-5
in Escherichia coli from Swine in Multiple Geographic Areas in China
Authors: Ho Pak-Leung , Wang Ya , Liu Melissa Chun-Jiao , Lai Eileen Ling-Yi , Law Pierra Ying-Tung , Cao Huiluo , Chow Kin-Hung
Published: January 2018

Six imported pigs originating from Guangdong, Henan, and Hunan provinces in China during October 2015 to February 2017 were cultured and found to be positive for meropenem-resistant
Escherichia coli
. The samples yielded 9
E. coli
isolates of diverse sequence types carrying
bla
NDM-5
on IncX3 (8 isolates from 5 farms) or IncFII (1 isolate from 1 farm) plasmids. The
mcr-1
gene was coharbored by 4 isolates. The IncX3 plasmids (∼46 kb) carrying
bla
NDM-5
were identical or nearly identical to each other.
In Vitro
and
In Vivo
Evaluation of the Antifungal Activity of APX001A/APX001 against Candida auris
Authors: Hager Christopher L. , Larkin Emily L. , Long Lisa , Zohra Abidi Fatima , Shaw Karen J. , Ghannoum Mahmoud A.
Published: January 2018

Candida auris
is an emerging multidrug-resistant yeast that has been responsible for invasive infections associated with high morbidity and mortality.
C. auris
strains often demonstrate high fluconazole and amphotericin B MIC values, and some strains are resistant to all three major antifungal classes. We evaluated the susceptibility of 16
C. auris
clinical strains, isolated from a wide geographical area, to 10 antifungal agents, including APX001A, a novel agent that inhibits the fungal protein Gwt1 (glycosylphosphatidylinositol-anchored wall transfer protein 1). APX001A demonstrated significantly lower MIC
50
and MIC
90
values (0.004 and 0.031 μg/ml, respectively) than all other agents tested. The efficacy of the prodrug APX001 was evaluated in an immunocompromised murine model of disseminated
C. auris
infection. Significant efficacy (80 to 100% survival) was observed in all three APX001 treatment groups versus 50% survival for the anidulafungin treatment group. In addition, APX001 showed a significant log reduction in CFU counts in kidney, lung, and brain tissue (1.03 to 1.83) versus the vehicle control. Anidulafungin also showed a significant log reduction in CFU in the kidneys and lungs (1.5 and 1.62, respectively) but did not impact brain CFU. These data support further clinical evaluation of this new antifungal agent.
Detection of
mcr-1
Gene among Escherichia coli Isolates from Farmed Fish and Characterization of
mcr-1
-Bearing IncP Plasmids
Authors: Lv Luchao , Cao Yuping , Yu Peixin , Huang Raorao , Wang Jing , Wen Qiaoling , Zhi Chanping , Zhang Qianhui , Liu Jian-Hua
Published: January 2018

The presence of the
mcr-1
gene in
Escherichia coli
isolated from retail freshwater fish was investigated. Seven (3.65%) clonally unrelated original
E. coli
isolates from grass carp were positive for
mcr-1
. The
mcr-1
genes were encoded by either chromosomes (
n
= 2) or conjugative plasmids (2 IncI2, 2 IncP, and 1 IncX4). The IncP plasmids were similar to other
mcr-1
-harboring IncP plasmids from China, though the insertion sites varied. Our report warrants further surveillance of resistance genes in aquaculture.
Transposition of Tn
1213
Encoding the PER-1 Extended-Spectrum β-Lactamase
Authors: Mancini Stefano , Poirel Laurent , Kieffer Nicolas , Nordmann Patrice
Published: January 2018

PER-1 is an extended-spectrum β-lactamase that is encoded by a gene located in composite transposon Tn
1213
made by two distinct insertion sequences, namely, IS
Pa12
and IS
Pa13. In vitro
mobilization performed in
Escherichia coli
shows that Tn
1213
is functional and is able to mobilize the
bla
PER-1
gene, although at a very low frequency (ca. 1 × 10
−9
).
How Much Do rRNA Gene Surveys Underestimate Extant Bacterial Diversity?
Authors: Rodriguez-R Luis M. , Castro Juan C. , Kyrpides Nikos C. , Cole James R. , Tiedje James M. , Konstantinidis Konstantinos T.
Published: January 2018

The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by ∼12%, on average, compared to the ANI-based approach (∼14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera
Pseudomonas
,
Burkholderia
,
Escherichia
,
Campylobacter
, and
Citrobacter
. These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary.

IMPORTANCE
Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA gene sequences at some threshold of nucleotide identity, typically 97% or 98.5%. Here, we explore how well this strategy reflects the more accurate whole-genome-based proxies and determine the frequency with which the high conservation of 16S rRNA sequences masks substantial species-level diversity.
Sapovirus in Wastewater Treatment Plants in Tunisia: Prevalence, Removal, and Genetic Characterization
Authors: Varela Miguel F. , Ouardani Imen , Kato Tsuyoshi , Kadoya Syunsuke , Aouni Mahjoub , Sano Daisuke , Romalde Jesús L.
Published: January 2018

Sapovirus(SaV), from theCaliciviridaefamily, is a genus of enteric viruses that cause acute gastroenteritis. SaV is shed at high concentrations with feces into wastewater, which is usually discharged into aquatic environments or reused for irrigation without efficient treatments. This study analyzed the incidence of human SaV in four wastewater treatment plants from Tunisia during a period of 13 months (December 2009 to December 2010). Detection and quantification were carried out using reverse transcription-quantitative PCR (RT-qPCR) methods, obtaining a prevalence of 39.9% (87/218). Sixty-one positive samples were detected in untreated water and 26 positive samples in processed water. The Dekhila plant presented the highest contamination levels, with a 63.0% prevalence. A dominance of genotype I.2 was observed on 15 of the 24 positive samples that were genetically characterized. By a Bayesian estimation algorithm, the SaV density in wastewater was estimated using left-censored data sets. The mean value of log SaV concentration in untreated wastewater ranged between 2.7 and 4.5 logs. A virus removal efficiency of 0.2 log was calculated for the Dekhila plant as the log ratio posterior distributions between untreated and treated wastewater. Multiple quantitative values obtained in this study must be available in quantitative microbial risk assessment in Tunisia as parameter values reflecting local conditions.

IMPORTANCEHuman sapovirus (SaV) is becoming more prevalent worldwide and organisms in this genus are recognized as emerging pathogens associated with human gastroenteritis. The present study describes novel findings on the prevalence, seasonality, and genotype distribution of SaV in Tunisia and Northern Africa. In addition, a statistical approximation using Bayesian estimation of the posterior predictive distribution (“left-censored” data) was employed to solve methodological problems related with the limit of quantification of the quantitative PCR (qPCR). This approach would be helpful for the future development of quantitative microbial risk assessment procedures for wastewater.
The eIF2α Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking
Authors: Bahnan Wael , Boucher Justin C. , Gayle Petoria , Shrestha Niraj , Rosen Mark , Aktas Bertal , Adkins Becky , Ager Arba , Khan Wasif N. , Schesser Kurt
Published: January 2018

The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for
Listeria monocytogenes
, there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that
Hri
−/−
mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of
L. monocytogenes
infection, there was much greater pathogen proliferation in the liver of
Hri
−/−
mice than in the liver of
Hri
+/+
mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in
Hri
+/+
mice in the first few hours of infection whereas the increase in IL-6 levels in
Hri
−/−
mice was notably delayed. Consistent with these
in vivo
findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected
Hri
−/−
macrophages and fibroblasts was significantly higher than the rate seen with infected
Hri
+/+
cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent
L. monocytogenes
in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection.
Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling
Authors: Sahay Bikash , Bashant Kathleen , Nelson Nicole L. J. , Patsey Rebeca L. , Gadila Shiva Kumar , Boohaker Rebecca , Verma Ashutosh , Strle Klemen , Sellati Timothy J.
Published: January 2018

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacteriumBorrelia burgdorferi. C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated withB. burgdorferiproduce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences ofB. burgdorferi-induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.
Evaluation of CpxRA as a Therapeutic Target for Uropathogenic Escherichia coli Infections
Authors: Dbeibo Lana , van Rensburg Julia J. , Smith Sara N. , Fortney Kate R. , Gangaiah Dharanesh , Gao Hongyu , Marzoa Juan , Liu Yunlong , Mobley Harry L. T. , Spinola Stanley M.
Published: January 2018

CpxRA is an envelope stress response system found in all members of the family
Enterobacteriaceae
; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR in
Escherichia coli
by inhibiting CpxA phosphatase activity. As a prelude to testing such compounds
in vivo
, here we constructed
cpxA
(in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) and
cpxR
(system absent) deletion mutants of uropathogenic
E. coli
(UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between the
cpxR
mutant and its parent, but in the
cpxA
mutant, several UPEC virulence determinants were downregulated, including the
fim
and
pap
operons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cells
in vitro
. In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented in
trans
. Unexpectedly, in single-strain challenges, only the
cpxA
mutant was attenuated for virulence in the kidney but not in the bladder or urine. For the
cpxA
mutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.
Evaluation of the Amplidiag CarbaR+VRE Kit for Accurate Detection of Carbapenemase-Producing Bacteria
Authors: Oueslati Saoussen , Girlich Delphine , Dortet Laurent , Naas Thierry
Published: January 2018

As carbapenemase-producing Gram-negative bacilli (CP-GNB) (
Enterobacteriaceae
,
Pseudomonadaceae
, and
Acinetobacter
spp.) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The Amplidiag CarbaR+VRE assay is a multiplex nucleic acid-based
in vitro
diagnostic test intended for the detection of CP-GNB and vancomycin-resistant enterococci (VRE) from cultured colonies. We have evaluated its ability to detect carbapenemase genes in 100 well-characterized GNB and in 200 consecutive enterobacterial isolates with reduced susceptibility to carbapenems that were referred to the French National Reference Center for carbapenem resistance. The assay has been validated on purified DNA but also directly on colonies. The Amplidiag CarbaR+VRE assay could detect all KPC, NDM, VIM, IMP, and OXA-48-like variants tested and all acquired carbapenem-hydrolyzing oxacillinases from
Acinetobacter baumannii
(OXA-23, OXA-24/-40, and OXA-58) as well as the overexpressed chromosomally encoded OXA-51-like β-lactamase associated with an upstream inserted IS
Aba1
. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES-5, and GES-14), GIM-1, AIM-1, SPM-1, DIM-1, OXA-198 in
Pseudomonas aeruginosa
, or OXA-143-like in
A. baumannii
were not detected. Amplidiag CarbaR+VRE's performance values were high (100% sensitivity and 99% specificity) as it could detect the five major carbapenemases—NDM, VIM, IMP, KPC, and OXA-48—as well as OXA-type carbapenemases from
Acinetobacter
spp. that are currently emerging also among
Proteus mirabilis
and other enterobacterial isolates. It can provide a result directly from colonies growing on Mueller-Hinton (MH) agar or on selective screening medium in less than 2 h. Further evaluations are now necessary to determine the performance values directly on rectal swabs.
Arabidopsis RNA Polymerase V Mediates Enhanced Compaction and Silencing of Geminivirus and Transposon Chromatin during Host Recovery from Infection
Authors: Coursey Tami , Regedanz Elizabeth , Bisaro David M.
Published: January 2018

Plants employ RNA-directed DNA methylation (RdDM) and dimethylation of histone 3 lysine 9 (H3K9me2) to silence geminiviruses and transposable elements (TEs). We previously showed that canonical RdDM (Pol IV-RdDM) involving RNA polymerases IV and V (Pol IV and Pol V) is required for
Arabidopsis thaliana
to recover from infection with
Beet curly top virus
lacking a suppressor protein that inhibits methylation (BCTV
L2
−
). Recovery, which is characterized by reduced viral DNA levels and symptom remission, allows normal floral development. Here, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to confirm that >90% of BCTV
L2
−
chromatin is highly compacted during recovery, and a micrococcal nuclease-chromatin immunoprecipitation assay showed that this is largely due to increased nucleosome occupancy. Physical compaction correlated with augmented cytosine and H3K9 methylation and with reduced viral gene expression. We additionally demonstrated that these phenomena are dependent on Pol V and by extension the Pol IV-RdDM pathway. BCTV
L2
−
was also used to evaluate the impact of viral infection on host loci, including repressed retrotransposons
Ta3
and
Athila6A
. Remarkably, an unexpected Pol V-dependent hypersuppression of these TEs was observed, resulting in transcript levels even lower than those detected in uninfected plants. Hypersuppression is likely to be especially important for natural recovery from wild-type geminiviruses, as viral L2 and AL2 proteins cause ectopic TE expression. Thus, Pol IV-RdDM targets both viral and TE chromatin during recovery, simultaneously silencing the majority of viral genomes and maintaining host genome integrity by enforcing tighter control of TEs in future reproductive tissues.

IMPORTANCE
In plants, RdDM pathways use small RNAs to target cytosine and H3K9 methylation, thereby silencing DNA virus genomes and transposable elements (TEs). Further, Pol IV-RdDM involving Pol IV and Pol V is a key aspect of host defense that can lead to recovery from geminivirus infection. Recovery is characterized by reduced viral DNA levels and symptom remission and thus allows normal floral development. Studies described here demonstrate that the Pol V-dependent enhanced viral DNA and histone methylation observed during recovery result in increased chromatin compaction and suppressed gene expression. In addition, we show that TE-associated chromatin is also targeted for hypersuppression during recovery, such that TE transcripts are reduced below the already low levels seen in uninfected plants. Thus, Pol IV-RdDM at once silences the majority of viral genomes and enforces a tight control over TEs which might otherwise jeopardize genome integrity in future reproductive tissue.
A Cyclin-Binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation
Authors: St. Gelais Corine , Kim Sun Hee , Maksimova Victoria V. , Buzovetsky Olga , Knecht Kirsten M. , Shepard Caitlin , Kim Baek , Xiong Yong , Wu Li
Published: January 2018

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity
in vitro
and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.

IMPORTANCE
SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.
TIP60 Complex Inhibits Hepatitis B Virus Transcription
Authors: Nishitsuji Hironori , Ujino Saneyuki , Harada Keisuke , Shimotohno Kunitada
Published: January 2018

Hepatitis B virus (HBV) is a global major health problem, with over one million deaths annually caused by chronic liver damage. Understanding host factors that modulate HBV replication may aid the development of anti-HBV therapies. Our recent genome-wide small interfering RNA screen using recombinant HBV demonstrated that TIP60 inhibited HBV infection. Here, we show that TIP60 complex contributes to anti-HBV defense. The TIP60 complex bound to the HBV promoter and suppressed HBV transcription driven by the precore/core promoter. The silencing of EP400, TRRAP, BAF53a, RUVBL1, and RUVBL2, which form the TIP60 complex, also resulted in increased HBV transcription. These results contribute to our enhanced understanding of the molecular mechanism of HBV transcription associated with the chromatin structure of HBV covalently closed circular DNA (cccDNA). Exploiting these intrinsic cellular defenses might help develop new anti-HBV agents.

IMPORTANCE
Investigating the molecular mechanism of HBV replication is important to understand the persistent nature of HBV infection and to aid the development of new HBV agents, which are currently limited to HBV polymerase inhibitors. Previously, we developed a new reporter HBV. By screening host factors using this recombinant virus, we identified several gene products that regulate HBV infection, including TIP60. Here, we showed that TIP60, a catalytic subunit of the NuA4 complex, inhibited HBV replication. Depletion of TIP60 increased the level of HBV mRNA. Moreover, TIP60 localized in the HBV cccDNA chromatin complex catalyzed the acetylation of histone H4 to recruit Brd4. These results suggest that TIP60, in concert with other cellular factors, plays an important role in the regulation of the HBV chromatin structure by acting as a critical component of the intrinsic antiviral defense, which sheds new light on the regulation of HBV replication.
Glycosyl-Phosphatidylinositol-Anchored Anti-HIV Env Single-Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity
Authors: Misra Anisha , Gleeson Emile , Wang Weiming , Ye Chaobaihui , Zhou Paul , Kimata Jason T.
Published: January 2018

In previous studies, we demonstrated that single-chain variable fragments (scFvs) from anti-human immunodeficiency virus (HIV) Env monoclonal antibodies act as entry inhibitors when tethered to the surface of target cells by a glycosyl-phosphatidylinositol (GPI) anchor. Interestingly, even if a virus escapes inhibition at entry, its replication is ultimately controlled. We hypothesized that in addition to functioning as entry inhibitors, anti-HIV GPI-scFvs may also interact with Env in an infected cell, thereby interfering with the infectivity of newly produced virions. Here, we show that expression of the anti-HIV Env GPI-scFvs in virus-producing cells reduced the release of HIV from cells 5- to 22-fold, and infectivity of the virions that were released was inhibited by 74% to 99%. Additionally, anti-HIV Env GPI-scFv X5 inhibited virion production and infectivity after latency reactivation and blocked transmitter/founder virus production and infectivity in primary CD4+T cells. In contrast, simian immunodeficiency virus (SIV) production and infectivity were not affected by the anti-HIV Env GPI-scFvs. Loss of infectivity of HIV was associated with a reduction in the amount of virion-associated Env gp120. Interestingly, an analysis of Env expression in cell lysates demonstrated that the anti-Env GPI-scFvs interfered with processing of Env gp160 precursors in cells. These data indicate that GPI-scFvs can inhibit Env processing and function, thereby restricting production and infectivity of newly synthesized HIV. Anti-Env GPI-scFvs therefore appear to be unique anti-HIV molecules as they derive their potent inhibitory activity by interfering with both early (receptor binding/entry) and late (Env processing and incorporation into virions) stages of the HIV life cycle.

IMPORTANCEThe restoration of immune function and persistence of CD4+T cells in HIV-1-infected individuals without antiretroviral therapy requires a way to increase resistance of CD4+T cells to infection by both R5- and X4-tropic HIV-1. Previously, we reported that anchoring anti-HIV-1 single-chain variable fragments (scFvs) via glycosyl-phosphatidylinositol (GPI) to the surface of permissive cells conferred a high level of resistance to HIV-1 variants at the level of entry. Here, we report that anti-HIV GPI-scFvs also derive their potent antiviral activity in part by blocking HIV production and Env processing, which consequently inhibits viral infectivity even in primary infection models. Thus, we conclude that GPI-anchored anti-HIV scFvs derive their potent blocking activity of HIV replication by interfering with successive stages of the viral life cycle. They may be effectively used in genetic intervention of HIV-1 infection.
The E3 Ubiquitin Ligase Siah-1 Suppresses Avian Reovirus Infection by Targeting p10 for Degradation
Authors: Chen Xiang , He Zhiyuan , Fu Mengjiao , Wang Yongqiang , Wu Haiyang , Li Xiaoqi , Cao Hong , Zheng Shijun J.
Published: January 2018

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. The ARV p10 protein, a viroporin responsible for the induction of cell syncytium formation and apoptosis, is rapidly degraded in host cells. Our previous report demonstrated that cellular lysosome-associated membrane protein 1 (LAMP-1) interacted with p10 and was involved in its degradation. However, the molecular mechanism underlying LAMP-1-mediated p10 degradation remains elusive. We report here that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) is critical for p10 ubiquitylation. Our data show that Siah-1 ubiquitylated p10 and targeted it for proteasome degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 promotes the proteasomal degradation of p10 via interacting with both p10 and the E3 ligase Siah-1. These data establish a novel host defense mechanism where LAMP-1 serves as a scaffold for both Siah-1 and p10 that allows the E3 ligase targeting p10 for ubiquitylation and degradation to suppress ARV infection.

IMPORTANCEAvian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe. The ARV p10 protein is a virulence factor responsible for the induction of cell syncytium formation and apoptosis and is rapidly degraded in host cells. We previously found that cellular lysosome-associated membrane protein 1 (LAMP-1) interacts with p10 and is involved in its degradation. Here we report that the E3 ubiquitin ligase seven in absentia homolog 1 (Siah-1) ubiquitylated p10 and targeted it for proteasomal degradation. Furthermore, the ubiquitylation of p10 by Siah-1 required the participation of LAMP-1 by forming a multicomponent complex. Thus, LAMP-1 serves as an adaptor to allow Siah-1 to target p10 for degradation, thereby suppressing ARV growth in host cells.
Human Placental Syncytiotrophoblasts Restrict
Toxoplasma gondii
Attachment and Replication and Respond to Infection by Producing Immunomodulatory Chemokines
Authors: Ander Stephanie E. , Rudzki Elizabeth N. , Arora Nitin , Sadovsky Yoel , Coyne Carolyn B. , Boyle Jon P.
Published: January 2018

Toxoplasma gondii
is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human midgestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to
T. gondii
infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict
T. gondii
infection at two distinct stages of the parasite lytic cycle—at the time of attachment and also during intracellular replication. Utilizing comparative transcriptome sequencing (RNA-seq) transcriptional profiling, we also show that human placental trophoblasts from both the second and third trimesters respond uniquely to
T. gondii
infection compared to trophoblast cell lines, typified by the upregulation of several immunity-related genes. One of the most differentially induced genes was the chemokine CCL22, which relies on the secretion of a parasite effector(s) either during or after invasion for its induction. Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of
T. gondii
at early and late stages of human pregnancy and demonstrate the existence of at least two interferon-independent pathways that restrict
T. gondii
access to the fetal compartment.

IMPORTANCE
Toxoplasma gondii
is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of
T. gondii
are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetus-derived cells that comprise the primary placental barrier, restrict
T. gondii
infection at two distinct stages of the parasite life cycle and respond to infection by inducing a unique immunomodulatory transcriptional profile. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict
T. gondii
infection at early and late stages of human pregnancy, identify both permissive and resistant human placental cell types, and identify the placenta-enriched signaling pathways induced in response to infection.
Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments
Authors: Hulseberg Christine E. , Fénéant Lucie , Szymańska Katarzyna M. , White Judith M.
Published: January 2018

Lassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection.

IMPORTANCE
Lassa virus is the most clinically important member of the
Arenaviridae
, a family that includes six additional biosafety level 4 (BSL4) hemorrhagic fever viruses. The lack of specific antiviral therapies for Lassa fever drives an urgent need to identify druggable targets, and interventions that block infection at the entry stage are particularly attractive. Lassa virus is only the second virus known to employ an intracellular receptor, the first being Ebola virus. Here we show that interaction with its intracellular receptor, Lamp1, enhances and upwardly shifts the pH dependence of fusion and consistently permits Lassa virus entry into cells through less acidic endosomes. We propose that in this manner, Lamp1 increases the overall efficiency of Lassa virus infection.
The F-Box Protein Fbp1 Shapes the Immunogenic Potential of
Cryptococcus neoformans
Authors: Masso-Silva Jorge , Espinosa Vanessa , Liu Tong-Bao , Wang Yina , Xue Chaoyang , Rivera Amariliz
Published: January 2018

Cryptococcus neoformans
is the main etiologic agent of cryptococcal meningitis and causes a significant number of deadly infections per year. Although it is well appreciated that host immune responses are crucial for defense against cryptococcosis, our understanding of factors that control the development of effective immunity to this fungus remains incomplete. In previous studies, we identified the F-box protein Fbp1 as a novel determinant of
C. neoformans
virulence. In this study, we found that the hypovirulence of the
fbp1
Δ mutant is linked to the development of a robust host immune response. Infection with the
fbp1
Δ mutant induces a rapid influx of CCR2
+
monocytes and their differentiation into monocyte-derived dendritic cells (mo-DCs). Depletion of CCR2
+
monocytes and their derivative mo-DCs resulted in impaired activation of a protective inflammatory response and the rapid death of mice infected with the
fbp1
Δ mutant. Mice lacking B and T cells also developed fungal meningitis and succumbed to infection with the
fbp1
Δ mutant, demonstrating that adaptive immune responses to the
fbp1
Δ mutant help to maintain the long-term survival of the host. Adaptive immune responses to the
fbp1
Δ mutant were characterized by enhanced differentiation of Th1 and Th17 CD4
+
T cells together with diminished Th2 responses compared to the H99 parental strain. Importantly, we found that the enhanced immunogenicity of
fbp1
Δ mutant yeast cells can be harnessed to confer protection against a subsequent infection with the virulent H99 parental strain. Altogether, our findings suggest that Fbp1 functions as a novel virulence factor that shapes the immunogenicity of
C. neoformans
.

IMPORTANCE
Cryptococcus neoformans
is the most common cause of deadly fungal meningitis, with over 270,000 infections per year. Immune responses are critically required for the prevention of cryptococcosis, and patients with impaired immunity and low CD4
+
T cell numbers are at high risk of developing these deadly infections. Although it is well appreciated that the development of protective immunity is shaped by the interactions of the host immune system with fungal cells, our understanding of fungal products that influence this process remains poor. In this study, we found that the activity of F-box protein 1 (Fbp1) in highly virulent
C. neoformans
clinical strain H99 shapes its immunogenicity and thus affects the development of protective immune responses in the host. The identification of this new mechanism of virulence may facilitate the future development of therapeutic interventions aimed at boosting antifungal host immunity.
O-Mannosylation of Proteins Enables
Histoplasma
Yeast Survival at Mammalian Body Temperatures
Authors: Garfoot Andrew L. , Goughenour Kristie D. , Wüthrich Marcel , Rajaram Murugesan V. S. , Schlesinger Larry S. , Klein Bruce S. , Rappleye Chad A.
Published: January 2018

The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen
Histoplasma capsulatum
, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection.
Histoplasma
yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the β-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils
in vivo
does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress
in vitro
and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of
Histoplasma
yeast cells, which facilitates infection of mammalian hosts.

IMPORTANCE
For dimorphic fungal pathogens, mammalian body temperature can have contrasting roles. Mammalian body temperature induces differentiation of the fungal pathogen
Histoplasma capsulatum
into a pathogenic state characterized by infection of host phagocytes. On the other hand, elevated temperatures represent a significant barrier to infection by many microbes. By functionally characterizing cells lacking O-linked mannosylation enzymes, we show that protein mannosylation confers thermotolerance on
H. capsulatum
, enabling infection of mammalian hosts.
Human Clade 2.3.4.4 A/H5N6 Influenza Virus Lacks Mammalian Adaptation Markers and Does Not Transmit via the Airborne Route between Ferrets
Authors: Herfst Sander , Mok Chris K. P. , van den Brand Judith M. A. , van der Vliet Stefan , Rosu Miruna E. , Spronken Monique I. , Yang Zifeng , de Meulder Dennis , Lexmond Pascal , Bestebroer Theo M. , Peiris J. S. Malik , Fouchier Ron A. M. , Richard Mathilde
Published: January 2018

Avian influenza A viruses are a threat to human health, as they cross the species barrier and infect humans occasionally, often with severe outcome. The antigenic and genetic diversity of A/H5 viruses from the A/goose/Guangdong/1/96 lineage is increasing, due to continued circulation and reassortment in poultry, posing a constant risk for public health and requiring regular risk assessments. Here we performed an in-depth characterization of the properties of the newly emerged zoonotic A/H5N6 virus
in vitro
and in ferrets. The lack of airborne transmission in the ferret model indicates that A/H5N6 virus does not pose a direct public health threat, despite the fact that it can replicate to high titers throughout the respiratory tracts of ferrets and cause more severe disease than other clade 2.3.4.4 viruses.
Recombinant Zika Virus Subunits Are Immunogenic and Efficacious in Mice
Authors: To Albert , Medina Liana O. , Mfuh Kenji O. , Lieberman Michael M. , Wong Teri Ann S. , Namekar Madhuri , Nakano Eileen , Lai Chih-Yun , Kumar Mukesh , Nerurkar Vivek R. , Lehrer Axel T.
Published: January 2018

The recent outbreaks of Zika virus (ZIKV) infection in French Polynesia, the Caribbean, and the Americas have highlighted the severe neuropathological sequelae that such an infection may cause. The development of a safe, effective ZIKV vaccine is critical for several reasons: (i) the difficulty in diagnosing an active infection due to common nonspecific symptoms, (ii) the lack of a specific antiviral therapy, and (iii) the potentially devastating pathological effects ofin uteroinfection. Moreover, a vaccine with an excellent safety profile, such as a nonreplicating, noninfectious vaccine, would be ideal for high-risk people (e.g., pregnant women, immunocompromised patients, and elderly individuals). This report describes the development of a recombinant subunit protein vaccine candidate derived from stably transformed insect cells expressing the ZIKV envelope proteinin vitro, the primary antigen to which effective virus-neutralizing antibodies are engendered by immunized animals for several other flaviviruses; the vaccine candidate elicits effective virus-neutralizing antibodies against ZIKV and provides protection against ZIKV infection in mice.
Microbiota Reconstitution Does Not Cause Bone Loss in Germ-Free Mice
Authors: Quach Darin , Collins Fraser , Parameswaran Narayanan , McCabe Laura , Britton Robert A.
Published: January 2018

The microbiota has been shown to be an important regulator of health and development. With regard to its effect on bone health, a previous study has suggested that gut microbes negatively impact bone density. However, we show here that this is not generalizable to all microbial communities and mouse strain backgrounds. Our results demonstrate that colonization of mice, both outbred and inbred strains, did not have a major impact on bone health. The identification of microbial communities that do not negatively impact bone health may provide a foundation for future investigations that seek to identify microbes that are either beneficial or detrimental to bone metabolism.