bluuebunny/crossref_metadata_2025 huggingface dataset:
To Modulate Survival under Secondary Stress Conditions, Listeria monocytogenes 10403S Employs RsbX To Downregulate σ
B
Activity in the Poststress Recovery Stage or Stationary Phase
Authors: Xia Ye , Xin Yongping , Li Xiaoliang , Fang Weihuan
Published: December 2015

Listeria monocytogenes
is a saprophytic bacterium that thrives in diverse environments and causes listeriosis via ingestion of contaminated food. RsbX, a putative sigma B (σ
B
) regulator, is thought to maintain the ready state in the absence of stress and reset the bacterium to the initial state in the poststress stage in
Bacillus subtilis
. We wondered whether RsbX is functional in
L. monocytogenes
under different stress scenarios. Genetic deletion and complementation of the
rsbX
gene were combined with survival tests and transcriptional and translational analyses of σ
B
expression in response to stresses. We found that deletion of
rsbX
increased survival under secondary stress following recovery of growth after primary stress or following stationary-phase culturing. The Δ
rsbX
mutant had higher expression of σ
B
than its parent strain in the recovery stage following primary sodium stress and in stationary-phase cultures. Apparently, increased σ
B
expression had contributed to improved survival in the absence of RsbX. There were no significant differences in survival rates or σ
B
expression levels in response to primary stresses between the
rsbX
mutant and its parent strain during the exponential phase. Therefore, we provide clear evidence that RsbX is a negative regulator of
L. monocytogenes
σ
B
during the recovery period after a primary stress or in the stationary phase, thus affecting its survival under secondary stress.
Alterations in the Quinolone Resistance-Determining Regions and Fluoroquinolone Resistance in Clinical Isolates and Laboratory-Derived Mutants of Mycoplasma bovis: Not All Genotypes May Be Equal
Authors: Khalil Dima , Becker Claire A. M. , Tardy Florence
Published: December 2015

Mycoplasma bovis
is considered a major contributor to respiratory diseases in young cattle. Resistant
M. bovis
isolates have increasingly been reported worldwide due to extensive use of antimicrobials to treat bovine pneumonia. The frequency of isolates resistant to fluoroquinolones varies considerably from one country to another. The MICs of isolates collected in France have only increased from “very low” to “low.” The present study was conducted to investigate whether alterations in the quinolone resistance-determining regions (QRDRs) could account for this slight modification in susceptibility. No correlation between QRDR alterations and increased MICs was evidenced in clinical isolates. In addition, all clinical isolates were subtyped, and the tendencies of the different sequence types to develop resistance through mutations in QRDRs under selective pressure
in vitro
were examined.
In vitro
, 3 hot spots for mutations in QRDRs (position 83 in GyrA and positions 80 and 84 in ParC) were associated with a high level of resistance when cumulated. We showed that the point mutations in the QRDRs observed
in vitro
were different (in location and selection rapidity) between the different subtypes. Our
in vitro
observations were corroborated by the recent detection of a clinical isolate highly resistant to fluoroquinolones (MIC ≥ 16 μg/ml) and belonging to the subtype which easily accumulates QRDR alterations
in vitro
. The current increased prevalence of this subtype in clinical isolates highlights the urgent need to control fluoroquinolone usage in veterinary medicine.
Phytic Acid and Sodium Chloride Show Marked Synergistic Bactericidal Effects against Nonadapted and Acid-Adapted Escherichia coli O157:H7 Strains
Authors: Kim Nam Hee , Rhee Min Suk
Published: December 2015

The synergistic antimicrobial effects of phytic acid (PA), a natural extract from rice bran, plus sodium chloride againstEscherichia coliO157:H7 were examined. Exposure to NaCl alone at concentrations up to 36% (wt/wt) for 5 min did not reduce bacterial populations. The bactericidal effects of PA alone were much greater than those of other organic acids (acetic, citric, lactic, and malic acids) under the same experimental conditions (P< 0.05). Combining PA and NaCl under conditions that yielded negligible effects when each was used alone led to marked synergistic effects. For example, whereas 0.4% PA or 3 or 4% NaCl alone had little or no effect on cell viability, combining the two completely inactivated both nonadapted and acid-adapted cells, reducing their numbers to unrecoverable levels (>7-log CFU/ml reduction). Flow cytometry confirmed that PA disrupted the cell membrane to a greater extent than did other organic acids, although the cells remained viable. The combination of PA and NaCl induced complete disintegration of the cell membrane. By comparison, none of the other organic acids acted synergistically with NaCl, and neither did NaCl-HCl solutions at the same pH values as the test solutions of PA plus NaCl. These results suggest that PA has great potential as an effective bacterial membrane-permeabilizing agent, and we show that the combination is a promising alternative to conventional chemical disinfectants. These findings provide new insight into the utility of natural compounds as novel antimicrobial agents and increase our understanding of the mechanisms underlying the antibacterial activity of PA.
Genomic Features of Environmental and Clinical Vibrio parahaemolyticus Isolates Lacking Recognized Virulence Factors Are Dissimilar
Authors: Ronholm J. , Petronella N. , Chew Leung C. , Pightling A. W. , Banerjee S. K.
Published: December 2015

Vibrio parahaemolyticus
is a bacterial pathogen that can cause illness after the consumption or handling of contaminated seafood. The primary virulence factors associated with
V. parahaemolyticus
illness are thermostable direct hemolysin (TDH) and Tdh-related hemolysin (TRH). However, clinical strains lacking
tdh
and
trh
have recently been isolated, and these clinical isolates are poorly understood. To help understand the emergence of clinical
tdh
- and
trh
-negative isolates, a genomic approach was used to comprehensively compare 4 clinical
tdh
- and
trh
-negative isolates with 16 environmental
tdh
- and
trh
-negative isolates and 34 clinical isolates positive for
tdh
or
trh
, or both, with the objective of identifying genomic features that are unique to clinical
tdh
- and
trh
-negative isolates. The prevalence of pathogenicity islands (PAIs) common to clinical isolates was thoroughly examined in each of the clinical
tdh
- and
trh
-negative isolates. The
tdh
PAI was not present in any clinical or environmental
tdh
- and
trh
-negative isolates. The
trh
PAI was not present in any environmental isolates; however, in clinical
tdh
- and
trh
-negative isolate 10-4238, the majority of the
trh
PAI including a partial
trh1
gene was present, which resulted in reclassification of this isolate as a
tdh
-negative and
trh
-positive isolate. In the other clinical
tdh
- and
trh
-negative isolates, neither the
trh
gene nor the
trh
PAI was present. We identified 862 genes in clinical
tdh
- and
trh
-negative isolates but not in environmental
tdh
- and
trh
-negative isolates. Many of these genes are highly homologous to genes found in common enteric bacteria and included genes encoding a number of chemotaxis proteins and a novel putative type VI secretion system (T6SS) effector and immunity protein (T6SS1). The availability of genome sequences from clinical
V. parahaemolyticus
tdh
- and
trh
-negative isolates and the comparative analysis may help provide an understanding of how this pathotype is able to survive
in vivo
during clinical illness.
Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans
Authors: Xu Xinping , Zhao Youbao , Kirkman Elyssa , Lin Xiaorong
Published: December 2015

Adaptation to stress by eukaryotic pathogens is often accompanied by a transition in cellular morphology. The human fungal pathogen
Cryptococcus neoformans
is known to switch between the yeast and the filamentous form in response to amoebic predation or during mating. As in the classic dimorphic fungal pathogens, the morphotype is associated with the ability of cryptococci to infect various hosts. Many cryptococcal factors and environmental stimuli, including pheromones (small peptides) and nutrient limitation, are known to induce the yeast-to-hypha transition. We recently discovered that secreted matricellular proteins could also act as intercellular signals to promote the yeast-to-hypha transition. Here we show that the secreted acyl coenzyme A (acyl-CoA)-binding protein Acb1 plays an important role in enhancing this morphotype transition. Acb1 does not possess a signal peptide. Its extracellular secretion and, consequently, its function in filamentation are dependent on an unconventional GRASP (Golgi reassembly stacking protein)-dependent secretion pathway. Surprisingly, intracellular recruitment of Acb1 to the secretory vesicles is independent of Grasp. In addition to Acb1, Grasp possibly controls the secretion of other cargos, because the
grasp
Δ mutant, but not the
acb1
Δ mutant, is defective in capsule production and macrophage phagocytosis. Nonetheless, Acb1 is likely the major or the sole effector of Grasp in terms of filamentation. Furthermore, we found that the key residue of Acb1 for acyl binding, Y80, is critical for the proper subcellular localization and secretion of Acb1 and for cryptococcal morphogenesis.
Metagenomic Approach for Identification of the Pathogens Associated with Diarrhea in Stool Specimens
Authors: Zhou Yanjiao , Wylie Kristine M. , El Feghaly Rana E. , Mihindukulasuriya Kathie A. , Elward Alexis , Haslam David B. , Storch Gregory A. , Weinstock George M.
Published: December 2015

The potential to rapidly capture the entire microbial community structure and/or gene content makes metagenomic sequencing an attractive tool for pathogen identification and the detection of resistance/virulence genes in clinical settings. Here, we assessed the consistency between PCR from a diagnostic laboratory, quantitative PCR (qPCR) from a research laboratory, 16S rRNA gene sequencing, and metagenomic shotgun sequencing (MSS) for
Clostridium difficile
identification in diarrhea stool samples. Twenty-two
C. difficile
-positive diarrhea samples identified by PCR and qPCR and five
C. difficile
-negative diarrhea controls were studied.
C. difficile
was detected in 90.9% of
C. difficile
-positive samples using 16S rRNA gene sequencing, and
C. difficile
was detected in 86.3% of
C. difficile
-positive samples using MSS. CFU inferred from qPCR analysis were positively correlated with the relative abundance of
C. difficile
from 16S rRNA gene sequencing (
r
2
= −0.60) and MSS (
r
2
= −0.55).
C. difficile
was codetected with
Clostridium perfringens
, norovirus, sapovirus, parechovirus, and anellovirus in 3.7% to 27.3% of the samples. A high load of
Candida
spp. was found in a symptomatic control sample in which no causative agents for diarrhea were identified in routine clinical testing. Beta-lactamase and tetracycline resistance genes were the most prevalent (25.9%) antibiotic resistance genes in these samples. In summary, the proof-of-concept study demonstrated that next-generation sequencing (NGS) in pathogen detection is moderately correlated with laboratory testing and is advantageous in detecting pathogens without
a priori
knowledge.
Evaluation of the BYG Carba Test, a New Electrochemical Assay for Rapid Laboratory Detection of Carbapenemase-Producing Enterobacteriaceae
Authors: Bogaerts Pierre , Yunus Sami , Massart Marion , Huang Te-Din , Glupczynski Youri
Published: December 2015

Accurate detection of carbapenemase-producing
Enterobacteriaceae
(CPE) constitutes a major laboratory diagnostic challenge. We evaluated an electrochemical technique (the BYG Carba test) which allows detection of CPE in less than 35 min. The BYG Carba test was first validated in triplicate against 57 collection isolates with previously characterized β-lactam resistance mechanisms (OXA-48,
n
= 12; KPC,
n
= 8; NDM,
n
= 8; VIM,
n
= 8; IMP,
n
= 3; GIM,
n
= 1; GES-6,
n
= 1; no carbapenemase,
n
= 16) and against a panel of 10 isolates obtained from the United Kingdom National External Quality Assessment Service (NEQAS). The test was then evaluated prospectively against 324 isolates referred to the national reference center for suspicion of CPE. The BYG Carba test results were compared with those obtained with the Carba NP test using multiplex PCR sequencing as the gold standard. Of the 57 collection and the 10 NEQAS isolates, all but one GES-6-producing isolate were correctly identified by the Carba BYG test. Among the 324 consecutive
Enterobacteriaceae
isolates tested prospectively, 146 were confirmed as noncarbapenemase producers by PCR while 178 harbored a carbapenemase gene (OXA-48,
n
= 117; KPC,
n
= 25; NDM,
n
= 23; and VIM,
n
= 13). Prospectively, in comparison with PCR results, the BYG Carba test displayed 95% sensitivity and 100% specificity versus 89% and 100%, respectively, for the Carba NP test. The BYG Carba test is a novel, rapid, and efficient assay based on an electro-active polymer biosensing technology discriminating between CPE and non-CPE. The precise electrochemical signal (electrochemical impedance variations) allows the establishment of real-time objective measurement and interpretation criteria which should facilitate the accreditation process of this technology.
Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells
Authors: Weiler Andrea M. , Das Arpita , Akinyosoye Oluwasayo , Cui Sherry , O'Connor Shelby L. , Scheef Elizabeth A. , Reed Jason S. , Panganiban Antonito T. , Sacha Jonah B. , Rakasz Eva G. , Friedrich Thomas C. , Maness Nicholas J.
Published: December 2015

Nef-specific CD8
+
T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive
nef
variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef
165–173
IW9 epitope revealed highly restricted evolution. A common acute escape variant, T
170
I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.
Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication
Authors: Nakamura Shoko , Horie Masayuki , Daidoji Tomo , Honda Tomoyuki , Yasugi Mayo , Kuno Atsushi , Komori Toshihisa , Okuzaki Daisuke , Narimatsu Hisashi , Nakaya Takaaki , Tomonaga Keizo
Published: December 2015

Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from
galnt3
-knockout mice. Interestingly, IAV-infected
galnt3
-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells.

IMPORTANCE
Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.
Antibody Binding Modulates Conformational Exchange in Domain III of Dengue Virus E Protein
Authors: Moraes Adolfo H. , Simonelli Luca , Pedotti Mattia , Almeida Fabio C. L. , Varani Luca , Valente Ana P.
Published: December 2015

Domain III of dengue virus E protein (DIII) participates in the recognition of cell receptors and in structural rearrangements required for membrane fusion and ultimately viral infection; furthermore, it contains epitopes for neutralizing antibodies and has been considered a potential vaccination agent. In this work, we addressed various structural aspects of DIII and their relevance for both the dengue virus infection mechanism and antibody recognition. We provided a dynamic description of DIII at physiological and endosomal pHs and in complex with the neutralizing human antibody DV32.6. We observed conformational exchange in the isolated DIII, in regions important for the packing of E protein dimers on the virus surface. This conformational diversity is likely to facilitate the partial detachment of DIII from the other E protein domains, which is required to achieve fusion to the host cellular membranes and to expose the epitopes of many anti-DIII antibodies. A comparison of DIII of two dengue virus serotypes revealed many common features but also some possibly unexpected differences. Antibody binding to DIII of dengue virus serotype 4 attenuated the conformational exchange in the epitope region but, surprisingly, generated exchange in other parts of DIII through allosteric effects.

IMPORTANCE
Many studies have provided extensive structural information on the E protein and particularly on DIII, also in complex with antibodies. However, there is very scarce information regarding the molecular dynamics of DIII, and almost nothing is available on the dynamic effect of antibody binding, especially at the quantitative level. This work provides one of the very rare descriptions of the effect of antibody binding on antigen dynamics.
Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury
Authors: Sugiyama Michael G. , Gamage Asela , Zyla Roman , Armstrong Susan M. , Advani Suzanne , Advani Andrew , Wang Changsen , Lee Warren L.
Published: December 2015

Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin α
5
β
1
, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.

IMPORTANCE
There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have recently shown that the virus can infect human lung endothelial cells, but the functional consequences of this infection are unknown (S. M. Armstrong, C. Wang, J. Tigdi, X. Si, C. Dumpit, S. Charles, A. Gamage, T. J. Moraes, and W. L. Lee, PLoS One 7:e47323, 2012,
http://dx.doi.org/10.1371/journal.pone.0047323
). Here, we show that this infection causes platelets to adhere to the lung endothelium. Importantly, blocking platelets using two distinct antiplatelet drugs improved survival in a mouse model of severe influenza infection. Thus, platelet inhibition may constitute a novel therapeutic strategy to improve the host response to severe infections with influenza.
Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein
Authors: Wu Yun-li , Peng Xian-e , Zhu Yi-bing , Yan Xiao-li , Chen Wan-nan , Lin Xu
Published: December 2015

Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3β-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis.

IMPORTANCE
Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism underlying HBV-induced pathogenesis of hepatic steatosis still remains to be elucidated. In this study, we found that expression of liver fatty acid binding protein (FABP1) was dramatically increased in the sera of HBV-infected patients and in both sera and liver tissues of HBV-transgenic mice. Forced expression of HBx led to FABP1 upregulation, whereas knockdown of FABP1 remarkably diminished lipid accumulation in both
in vitro
and
in vivo
models. It is possible that HBx promotes hepatic lipid accumulation through upregulating FABP1 in the development of HBV-induced nonalcoholic fatty liver disease. Therefore, inhibition of FABP1 might have therapeutic value in steatosis-associated chronic HBV infection.
ORF33 and ORF38 of Kaposi's Sarcoma-Associated Herpesvirus Interact and Are Required for Optimal Production of Infectious Progeny Viruses
Authors: Wu Jian-jun , Avey Denis , Li Wenwei , Gillen Joseph , Fu Bishi , Miley Wendell , Whitby Denise , Zhu Fanxiu
Published: December 2015

We recently showed that the interaction between Kaposi's sarcoma-associated herpesvirus (KSHV) tegument proteins ORF33 and ORF45 is crucial for progeny virion production, but the exact functions of KSHV ORF33 during lytic replication were unknown (J. Gillen, W. Li, Q. Liang, D. Avey, J. Wu, F. Wu, J. Myoung, and F. Zhu, J Virol
89:
4918–4931, 2015,
http://dx.doi.org/10.1128/JVI.02925-14
). Therefore, here we investigated the relationship between ORF33 and ORF38, whose counterparts in both alpha- and betaherpesviruses interact with each other. Using specific monoclonal antibodies, we found that both proteins are expressed during the late lytic cycle with similar kinetics and that both are present in mature virions as components of the tegument. Furthermore, we confirmed that ORF33 interacts with ORF38. Interestingly, we observed that ORF33 tightly associates with the capsid, whereas ORF38 associates with the envelope. We generated ORF33-null, ORF38-null, and double-null mutants and found that these mutants apparently have identical phenotypes: the mutations caused no apparent effect on viral gene expression but reduced the yield of progeny virion by about 10-fold. The progeny virions also lack certain virion component proteins, including ORF45. During viral lytic replication, the virions associate with cytoplasmic vesicles. We also observed that ORF38 associates with the membranes of vesicles and colocalizes with the Golgi membrane or early endosome membrane. Further analyses of ORF33/ORF38 mutants revealed the reduced production of virion-containing vesicles, suggesting that ORF33 and ORF38 are involved in the transport of newly assembled viral particles into cytoplasmic vesicles, a process important for viral maturation and egress.

IMPORTANCE
Herpesvirus assembly is an essential step in virus propagation that leads to the generation of progeny virions. It is a complicated process that depends on the delicate regulation of interactions among virion proteins. We previously revealed an essential role of ORF45-ORF33 binding for virus assembly. Here, we report that ORF33 and its binding partner, ORF38, are required for infectious virus production due to their important role in the tegumentation process. Moreover, we found that both ORF33 and ORF38 are involved in the transportation of virions through vesicles during maturation and egress. Our results provide new insights into the important roles of ORF33 and ORF38 during viral assembly, a process critical for virus propagation that is intimately linked to KSHV pathobiology.
A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies
Authors: Ding Shilei , Veillette Maxime , Coutu Mathieu , Prévost Jérémie , Scharf Louise , Bjorkman Pamela J. , Ferrari Guido , Robinson James E. , Stürzel Christina , Hahn Beatrice H. , Sauter Daniel , Kirchhoff Frank , Lewis George K. , Pazgier Marzena , Finzi Andrés
Published: December 2015

Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals.
Equine Infectious Anemia Virus Gag Assembly and Export Are Directed by Matrix Protein through
trans
-Golgi Networks and Cellular Vesicles
Authors: Zhang Zeli , Ma Jian , Zhang Xiang , Su Chao , Yao Qiu-Cheng , Wang Xiaojun
Published: December 2015

Gag intracellular assembly and export are very important processes for lentiviruses replication. Previous studies have demonstrated that equine infectious anemia virus (EIAV) matrix (MA) possesses distinct phosphoinositide affinity compared with HIV-1 MA and that phosphoinositide-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release. In this study, we compared the cellular assembly sites of EIAV and HIV-1. We observed that the assembly of EIAV particles occurred on interior cellular membranes, while HIV-1 was targeted to the plasma membrane (PM) for assembly. Then, we determined that W7 and K9 in the EIAV MA N terminus were essential for Gag assembly and release but did not affect the cellular distribution of Gag. The replacement of EIAV MA with HIV-1 MA directed chimeric Gag to the PM but severely impaired Gag release. MA structural analysis indicated that the EIAV and HIV-1 MAs had similar spatial structures but that helix 1 of the EIAV MA was closer to loop 2. Further investigation indicated that EIAV Gag accumulated in the
trans
-Golgi network (TGN) but not the early and late endosomes. The 9 N-terminal amino acids of EIAV MA harbored the signal that directed Gag to the TGN membrane system. Additionally, we demonstrated that EIAV particles were transported to the extracellular space by the cellular vesicle system. This type of EIAV export was not associated with multivesicular bodies or microtubule depolymerization but could be inhibited by the actin-depolymerizing drug cytochalasin D, suggesting that dynamic actin depolymerization may be associated with EIAV production.

IMPORTANCE
In previous studies, EIAV Gag was reported to localize to both the cell interior and the plasma membrane. Here, we demonstrate that EIAV likely uses the TGN as the assembly site in contrast to HIV-1, which is targeted to the PM for assembly. These distinct assembly features are determined by the MA domain. We also identified two sites in the N terminus of EIAV MA that were important for Gag assembly and release. Furthermore, the observation of EIAV transport by cellular vesicles but not by multivesicular bodies sheds light on the mechanisms underlying EIAV cellular replication.
Translation and cross-cultural adaptation of the PARmed-X for Pregnancy into Brazilian Portuguese
Authors: Bgeginski Roberta , Schuch Felipe Barreto , Mottola Michelle F. , Ramos José Geraldo Lopes
Published: December 2015

We describe the translation and cross-cultural adaptation of the PARmed-X for Pregnancy for use in Brazilian Portuguese. The original instrument was developed in English for health screening prior to and guidelines for prenatal exercise. We followed the ten steps according to the Translation and Cultural Adaptation International Society for Pharmacoeconomics and Outcomes Research guidelines. Our template can be used by other health professionals for translation and verification of the original tool into their native language.
The Importance of Atmospheric Deposition for Ocean Productivity
Authors: Jickells Tim , Moore C. Mark
Published: December 2015

Dust is produced primarily in desert regions and transported long distances through the atmosphere to the oceans. Upon deposition of dust, its dissolution can provide an important source of a range of nutrients, particularly iron, to microbes living in open ocean surface waters. The dust supply is greatest nearest to deserts, hence in the Northern Hemisphere. The Southern Ocean region is farthest from these dust sources and shows clear evidence that phytoplankton primary production is limited, at least in part, by the rate of supply of iron. Iron is also essential for nitrogen fixation. In regions of high atmospheric iron supply, such as the tropical North Atlantic, stimulation of nitrogen fixation drives the phytoplankton population toward a state in which phosphorus supply rates limit primary production. Atmospheric deposition is also an important source of nitrogen to the low latitude ocean, where it stimulates primary production. In this review we consider the sources, transport, and deposition of atmospheric dust/iron and nitrogen to the oceans and their impacts on plankton systems. In conclusion, we suggest key areas for future research.
Impacts from Invasive Reptiles and Amphibians
Authors: Kraus Fred
Published: December 2015

Alien herpetofauna have a broad diversity of ecological and evolutionary impacts, involving seven mechanisms. Ecological impacts usually result from trophic disruptions and may be direct or indirect and top-down or bottom-up; they may vary in scale from single species to communities. A single species may impose impacts involving most or all of these categories. Evolutionary impacts most often result from hybridization and introgression but may include diverse changes in native fauna induced by selection. Impact magnitudes observed to date largely range from moderate to major, but massive impacts (including species extinction) are known for a handful of invasive species. Research remains skewed toward a small sample of all invaders, and major research gaps remain in understanding community-level impacts, the risk posed by competition, determinants of predation impact, the relevance of genetic diversity to impacts, and how to predict impacts.
Toward a Conceptual Understanding of β-Diversity in the Deep-Sea Benthos
Authors: McClain Craig R. , Rex Michael A.
Published: December 2015

We review patterns and causes of β-diversity in the deep-sea benthos at different spatial scales and for different body sizes. Changes in species composition occurring with depth are generally gradual, the rate of change being a function of the rate of descent. This gradual change can be interrupted by abrupt environmental shifts, such as oxygen minimum zones, and by major topographic features that alter oceanographic conditions. Changes in species composition with depth can involve both species replacement and species loss, leading to nestedness. Horizontal β-diversity is more moderate than that occurring with depth, except at upper bathyal zones impacted by coastal influences. At very large oceanic scales, both environmental filtering and dispersal limitation influence β-diversity. Although many ecological and evolutionary–historical factors must shape β-diversity in the deep sea, energy availability appears to structure community makeup at all scales examined. We recommend that standardized sampling protocols, statistical methods, and data archiving be used to direct future research.
Cross talk between AT1receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus
Authors: Biancardi Vinicia Campana , Stranahan Alexis M. , Krause Eric G. , de Kloet Annette D. , Stern Javier E.
Published: December 2015

ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (∼65% increase, P < 0.001), an effect that was blunted in the absence of functional TLR4. ANG II increased ROS production, as indicated by dihydroethidium fluorescence, within the PVN of rats and mice ( P < 0.0001 in both cases), effects that were also dependent on the presence of functional TLR4. The microglial inhibitor minocycline attenuated ANG II-mediated ROS production, yet ANG II effects persisted in PVN single-minded 1-AT1aknockout mice, supporting the contribution of a non-neuronal source (likely microglia) to ANG II-driven ROS production in the PVN. Taken together, these results support functional interactions between AT1receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN.
Contribution of the late sodium current to intracellular sodium and calcium overload in rabbit ventricular myocytes treated by anemone toxin
Authors: Kornyeyev Dmytro , El-Bizri Nesrine , Hirakawa Ryoko , Nguyen Steven , Viatchenko-Karpinski Serge , Yao Lina , Rajamani Sridharan , Belardinelli Luiz
Published: December 2015

Pathological enhancement of late Na+current ( INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INacan be a source of intracellular Na+([Na+]i) overload. Late INawas enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na+]iwas determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22–24°C, resting [Na+]iwas 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa8.7-fold. [Na+]imeasured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM ( P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INawith GS-967 (1 μM) prevented Na+iaccumulation. A strong positive correlation was observed between the late INaand the pacing-induced increase of [Na+]i( R2= 0.88) and between the rise in [Na+]iand the increases in cytosolic Ca2+( R2= 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na+]iin quiescent myocytes suggesting that late INawas solely responsible for triggering the ATX-II effect on [Na+]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na+]ioverload associated with the increase in late INacaused by ATX-II. Enhancement of late INacan cause intracellular Na+overload in ventricular myocytes.
Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles
Authors: Diaz-Otero Janice M. , Garver Hannah , Fink Gregory D. , Jackson William F. , Dorrance Anne M.
Published: December 2015

Artery remodeling, described as a change in artery structure, may be responsible for the increased risk of cardiovascular disease with aging. Although the risk for stroke is known to increase with age, relatively young animals have been used in most stroke studies. Therefore, more information is needed on how aging alters the biomechanical properties of cerebral arteries. Posterior cerebral arteries (PCAs) and parenchymal arterioles (PAs) are important in controlling brain perfusion. We hypothesized that aged (22–24 mo old) C57bl/6 mice would have stiffer PCAs and PAs than young (3–5 mo old) mice. The biomechanical properties of the PCAs and PAs were assessed by pressure myography. Data are presented as means ± SE of young vs. old. In the PCA, older mice had increased outer (155.6 ± 3.2 vs. 169.9 ± 3.2 μm) and lumen (116.4 ± 3.6 vs. 137.1 ± 4.7 μm) diameters. Wall stress (375.6 ± 35.4 vs. 504.7 ± 60.0 dyn/cm2) and artery stiffness (β-coefficient: 5.2 ± 0.3 vs. 7.6 ± 0.9) were also increased. However, wall strain (0.8 ± 0.1 vs. 0.6 ± 0.1) was reduced with age. In the PAs from old mice, wall thickness (3.9 ± 0.3 vs. 5.1 ± 0.2 μm) and area (591.1 ± 95.4 vs. 852.8 ± 100 μm2) were increased while stress (758.1 ± 100.0 vs. 587.2 ± 35.1 dyn/cm2) was reduced. Aging also increased mean arterial and pulse pressures. We conclude that age-associated remodeling occurs in large cerebral arteries and arterioles and may increase the risk of cerebrovascular disease.
Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release
Authors: Thompson Jeremy , Hu Ying , Lesnefsky Edward J. , Chen Qun
Published: December 2015

Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I.
Increased TGF-β: a drawback of tracheal occlusion in human and experimental congenital diaphragmatic hernia?
Authors: Vuckovic Aline , Herber-Jonat Susanne , Flemmer Andreas W. , Ruehl Ina M. , Votino Carmela , Segers Valérie , Benachi Alexandra , Martinovic Jelena , Nowakowska Dorota , Dzieniecka Monika , Jani Jacques C.
Published: December 2015

Survivors of severe congenital diaphragmatic hernia (CDH) present significant respiratory morbidity despite lung growth induced by fetal tracheal occlusion (TO). We hypothesized that the underlying mechanisms would involve changes in lung extracellular matrix and dysregulated transforming growth factor (TGF)-β pathway, a key player in lung development and repair. Pulmonary expression of TGF-β signaling components, downstream effectors, and extracellular matrix targets were evaluated in CDH neonates who died between birth and the first few weeks of life after prenatal conservative management or TO, and in rabbit pups that were prenatally randomized for surgical CDH and TO vs. sham operation. Before tissue harvesting, lung tissue mechanics in rabbits was measured using the constant-phase model during the first 30 min of life. Human CDH and control fetal lungs were also collected from midterm onwards. Human and experimental CDH did not affect TGF-β/Smad2/3 expression and activity. In human and rabbit CDH lungs, TO upregulated TGF-β transcripts. Analysis of downstream pathways indicated increased Rho-associated kinases to the detriment of Smad2/3 activation. After TO, subtle accumulation of collagen and α-smooth muscle actin within alveolar walls was detected in rabbit pups and human CDH lungs with short-term mechanical ventilation. Despite TO-induced lung growth, mediocre lung tissue mechanics in the rabbit model was associated with increased transcription of extracellular matrix components. These results suggest that prenatal TO increases TGF-β/Rho kinase pathway, myofibroblast differentiation, and matrix deposition in neonatal rabbit and human CDH lungs. Whether this might influence postnatal development of sustainably ventilated lungs remains to be determined.
Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension
Authors: Bryant Andrew J. , Carrick Ryan P. , McConaha Melinda E. , Jones Brittany R. , Shay Sheila D. , Moore Christy S. , Blackwell Thomas R. , Gladson Santhi , Penner Niki L. , Burman Ankita , Tanjore Harikrishna , Hemnes Anna R. , Karwandyar Ayub K. , Polosukhin Vasiliy V. , Talati Megha A. , Dong Hui-Jia , Gleaves Linda A. , Carrier Erica J. , Gaskill Christa , Scott Edward W. , Majka Susan M. , Fessel Joshua P. , Haase Volker H. , West James D. , Blackwell Timothy S. , Lawson William E.
Published: December 2015

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.