bluuebunny/crossref_metadata_2025 huggingface dataset:
Adjustment of polyamine contents in Escherichia coli
Authors: Kashiwagi K , Igarashi K
Published: November 2016

Adjustment of polyamine contents in Escherichia coli was studied with strains of Escherichia coli producing normal (DR112) and excessive amounts of ornithine decarboxylase [DR112(pODC)] or S-adenosylmethionine decarboxylase [DR112(pSAMDC)]. Although DR112(pODC) produced approximately 70 times more ornithine decarboxylase than DR112 did, the amounts of polyamines in the cells of both strains did not change significantly. The amounts of polyamines in DR112(pODC) were adjusted by excretion of excessive amounts of putrescine to the medium. When ornithine was deficient in cells, polyamine contents in DR112(pODC) were much higher than those in DR112, although polyamine contents were low in both strains. This indicates that large amounts of ornithine decarboxylase increased the utilization of ornithine for putrescine synthesis. During ornithine deficiency, strain DR112 produced 3.4 times more ornithine decarboxylase. Strain DR112(pSAMDC) produced seven times more S-adenosylmethionine decarboxylase than DR112 did. In DR112(pSAMDC) an increase (40%) in spermidine content, a decrease (35%) in putrescine content, and no significant excretion of putrescine and spermidine were observed. The amount of ornithine decarboxylase in DR112(pSAMDC) was approximately 30% less than that in DR112. In addition, S-adenosylmethionine decarboxylase activity was strongly inhibited by spermidine. A possible regulatory mechanism to maintain polyamine contents in Escherichia coli is discussed based on the results.
Identification of two proteins encoded by com, a competence control locus of Streptococcus pneumoniae
Authors: Chandler M S , Morrison D A
Published: November 2016

The com locus, which controls competence for genetic transformation in Streptococcus pneumoniae, was analyzed by construction of a series of subclones, insertion mutations, and deletions of the cloned DNA in Escherichia coli. In vitro transcription-translation of these com plasmids revealed two neighboring genes, comA and comB, encoding proteins of 77,000 and 49,000 daltons, respectively. Their map positions and orientations were determined. Insertions in either gene eliminated the corresponding protein and had no effect on the other. In addition, a 15,000-dalton com protein was tentatively identified, although the exact location of this gene remains to be determined. Features of the DNA adjacent to the com locus are also described.
DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH
Authors: Byrne C R , Monroe R S , Ward K A , Kredich N M
Published: November 2016

Nucleotide sequences of the cysK regions of Salmonella typhimurium and Escherichia coli have been determined. A total of 3,812 and 2,595 nucleotides were sequenced from S. typhimurium and E. coli, respectively. Open reading frames of 323 codons were found in both species and were identified as those of cysK by comparison of deduced amino acid sequences with amino- and carboxyl-terminal amino acid analyses of the S. typhimurium cysK gene product O-acetylserine (thiol)-lyase A. The two cysK DNA sequences were 85% identical, and the deduced amino acid sequences were 96% identical. The major transcription initiation sites for cysK were found to be virtually identical in the two organisms, by using primer extension and S1 nuclease protection techniques. The -35 region corresponding to the major transcription start site was TTCCCC in S. typhimurium and TTCCGC in E. coli. The deviation of these sequences from the consensus sequence TTGACA may reflect the fact that cysK is subject to positive control and requires the cysB regulatory protein for expression. Sequences downstream of cysK were found to include ptsH and a portion of ptsI, thus establishing the exact relationship of cysK with these two genes. A 290-codon open reading frame, which may represent the cysZ gene, was identified upstream of cysK.
Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants
Authors: Eddy C K , Smith O H , Noel K D
Published: November 2016

A library of Zymomonas mobilis genomic DNA was constructed in the broad-host-range cosmid pLAFR1. The library was mobilized into a variety of Escherichia coli and Pseudomonas putida trp mutants by using the helper plasmid pRK2013. Five Z. mobilis trp genes were identified by the ability to complement the trp mutants. The trpF, trpB, and trpA genes were on one cosmid, while the trpD and trpC genes were on two separate cosmids. The organization of the Z. mobilis trp genes seems to be similar to the organization found in Rhizobium spp., Acinetobacter calcoaceticus, and Pseudomonas acidovorans. The trpF, trpB, and trpA genes appeared to be linked, but they were not closely associated with trpD or trpC genes.
Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators
Authors: Wittman V , Wong H C
Published: November 2016

The synthesis of the inducible enzyme penicillinase of Bacillus licheniformis is negatively controlled by a repressor (D.A. Dubnau and M.R. Pollock, J. Gen. Microbiol. 41:7-21, 1965; D. J. Sherratt and J. F. Collins, J. Gen. Microbiol. 76:217-230,1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168:1128-1132, 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (penIc) from strain 749/C that constitutively produces penicillinase. The penIc and penI+ (wild-type) genes were expressed in Escherichia coli. Complementation analysis indicated that the repressor is the only trans-acting protein required to regulate the expression of the penI and penP genes. We purified the wild-type repressor protein, used it in gel retardation and DNase I protection experiments, and identified three operators positioned in the region between the penP and penI coding sequences. The spatial arrangement of the operators and the hierarchy in repressor binding seen in the protection experiments indicate that (i) the penI gene product represses the expression of the penP gene by physically blocking the RNA polymerase-binding site and (ii) the penI gene is autoregulated.
Absence of a characteristic cell wall lipopolysaccharide in the phototrophic bacterium Chloroflexus aurantiacus
Authors: Meissner J , Krauss J H , Jürgens U J , Weckesser J
Published: November 2016

Two strains of the gliding phototrophic bacterium Chloroflexus aurantiacus were investigated for the presence of lipopolysaccharide (LPS). With both strains, all fractions of hot phenol-water extracts and the extracted cell residues from whole cells or cell homogenates were found to be free from characteristic LPS constituents, such as 3-hydroxy fatty acids, 2-keto-3-deoxyoctonate, heptoses, or O-chain sugars. Phenolchloroform-petroleum ether extracts were also free from precipitable LPS. A lipid A fraction could not be obtained, and there was no hint for glucosamine as a possible lipid A backbone amino sugar. Absence of LPS was confirmed by sodium deoxycholate gel electrophoresis.
Expression of the divergent tricarboxylate transport operon (tctI) of Salmonella typhimurium
Authors: Widenhorn K A , Somers J M , Kay W W
Published: November 2016

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.
Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT
Authors: Flynn J L , Ohman D E
Published: November 2016

Pseudomonas aeruginosa can convert to a mucoid colony morphology by a genetic mechanism called alginate conversion; this results in the production of copious amounts of the exopolysaccharide alginate. The mucoid phenotype of P. aeruginosa is commonly associated with its ability to cause chronic pulmonary tract infections in patients with cystic fibrosis. In this study we isolated the cis-acting locus involved in alginate conversion, called algS, from both mucoid and nonmucoid isogenic strains. We then examined the role of algS in the control of algT, a trans-active gene required for alginate production in P. aeruginosa. We used a new cosmid cloning vector, called pEMR2, that permitted both the cloning of large DNA fragments and their subsequent gene replacement in P. aeruginosa. To verify the predicted properties of this vector, we isolated and tested a pEMR2 hisI+ clone. Using cloned algS-containing DNA and a method for gene replacement, we constructed isogenic strains of P. aeruginosa that had Tn501 adjacent to algS on the chromosome. Two pEMR2 clone banks containing genomic fragments from isogenic algS(On) (exhibiting the alginate production phenotype) and algS(Off) (exhibiting the non-alginate production phenotype) strains were constructed, and Tn501 served as an adjacent marker to select for clones containing the respective algS allele. The pEMR2 algS(On) and pEMR2 algS(Off) clones were shown to contain the indicated algS allele by gene replacement with the chromosome of strains that carried the opposite allele. To test whether algS controls the expression of the adjacent algT gene, we constructed a pLAFR1 algS(Off)T clone and showed it to be unable to complement an algT::Tn501 mutation in trans. In contrast, a pLAFR1 algS(On)T clone did complement algT::Tn501 in trans. Thus, algS appears to control the activation of algT expression, bringing about alginate conversion.
Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3
Authors: Toba M , Masaki H , Ohta T
Published: November 2016

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, ColE8-J was shown to be situated between ColE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids.
Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium
Authors: Bower S G , Hove-Jensen B , Switzer R L
Published: November 2016

The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to two rat isozyme sequences. Strains containing plasmid-borne prsA have been used to overproduce and purify the enzyme. The promoter for the S. typhimurium prsA gene was identified by deletion analysis and by similarity to the promoter for the E. coli prsA gene. The location of the prsA promoter results in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional significance.
Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants
Authors: Rollo E E , Oliver D B
Published: November 2016

SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant. An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants.
Mechanism of biosynthesis of 2-oxo-3-methylvalerate in Chlorobium vibrioforme
Authors: Nesbakken T , Kolsaker P , Ormerod J
Published: November 2016

The biosynthesis of 2-oxo-3-methylvalerate in Chlorobium vibrioforme was investigated by 13C nuclear magnetic resonance spectroscopy of the oxoacid formed from 13C-labeled acetate by washed suspensions. The threonine pathway could be excluded, and the results are in accord with a mechanism for the formation of 2-oxobutyrate from acetyl coenzyme A and pyruvate via citramalate.
Thiol-sensitive promoters of Escherichia coli
Authors: Javor G T , Stringer C D , Ryu J
Published: November 2016

Mu dX(lac) insertion mutants of Escherichia coli CSH50 in which the expression of the lacZ gene was sensitive to the presence of exogenous 1-thioglycerol or dithiothreitol were isolated. Both stimulatory and inhibitory mutants were found. The existence of several thiol-sensitive promoters suggests that exogenous thiols may provoke global stress responses in E. coli.
Construction of an Escherichia coli K-12 ada deletion by gene replacement in a recD strain reveals a second methyltransferase that repairs alkylated DNA
Authors: Shevell D E , Abou-Zamzam A M , Demple B , Walker G C
Published: November 2016

We constructed an ada deletion by gene replacement in a recD1014 strain of Escherichia coli. Characterization of a delta ada-25 recD+ strain revealed the presence of a second DNA methyltransferase activity in E. coli K-12 which transfers a methyl group from methylated DNA to a protein with a molecular weight of 18,000 to 20,000.
Construction of a fol mutant strain of Escherichia coli for use in dihydrofolate reductase mutagenesis experiments
Authors: Ahrweiler P M , Frieden C
Published: November 2016

A strain of Escherichia coli with the fol gene deleted and a kan gene inserted in its place was created for use in cloning and isolation of mutant dihydrofolate reductase. Southern blot analysis and dihydrofolate reductase enzyme assays confirmed the delta fol::kan genotype. A thyA mutation accompanied the fol deletion and is required for survival of a dihydrofolate reductase-deficient strain.
Detection of an Escherichia coli Sequence Type 167 Strain with Two Tandem Copies of
bla
NDM-1
in the Chromosome
Authors: Shen Ping , Yi Maoli , Fu Ying , Ruan Zhi , Du Xiaoxing , Yu Yunsong , Xie Xinyou
Published: November 2016

New Delhi metallo-β-lactamase-1 (NDM-1)-producing
Enterobacteriaceae
has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the
bla
NDM-1
gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant
Escherichia coli
strain Y5, originating from a urine sample and containing the
bla
NDM-1
gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of
bla
NDM-1
, the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with
Escherichia coli
6409 (GenBank accession no.
CP010371
), which lacks the same
bla
NDM-1
gene-surrounding structure as Y5. The
bla
NDM-1
gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (IS
CR1
) element (
sul1-ARR-3-cat-bla
NDM-1
-
bleo
-IS
CR1
), which appears intact in the plasmid from
Proteus mirabilis
(GenBank accession no.
KP662515
). The genomic context indicates that the IS
CR1
element mediated the
bla
NDM-1
transposition from a single source plasmid to the chromosome. Our study is the first report of an
Enterobacteriaceae
strain harboring a chromosomally integrated
bla
NDM-1
, which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor
bla
NDM-1
.
Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR
Authors: Kim Young-gon , Yun Seung Gyu , Kim Min Young , Park Kwisung , Cho Chi Hyun , Yoon Soo Young , Nam Myung Hyun , Lee Chang Kyu , Cho Yun-Jung , Lim Chae Seung
Published: November 2016

Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (
P
= 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (
P
= 0.1574). Adenovirus was detected more frequently in saliva samples (
P
< 0.0001), whereas influenza virus type A and human rhinovirus were detected more frequently in NPS specimens (
P
= 0.0001 and
P
= 0.0289, respectively). The possibility of false-positive adenovirus detection from saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies.
Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses
Authors: Crosby Catherine M. , Matchett William E. , Anguiano-Zarate Stephanie S. , Parks Christopher A. , Weaver Eric A. , Pease Larry R. , Webby Richard J. , Barry Michael A.
Published: November 2016

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen
in vitro
. SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as “needle-free” mucosal vaccines.

IMPORTANCE
Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors.
Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes
Authors: Sánchez-Aparicio M. T. , Ayllón J. , Leo-Macias A. , Wolff T. , García-Sastre A.
Published: November 2016

The retinoic acid-inducible gene 1 (RIG-I) signaling pathway is essential for the recognition of viruses and the initiation of host interferon (IFN)-mediated antiviral responses. Once activated, RIG-I interacts with polyubiquitin chains generated by TRIM25 and binds mitochondrial antiviral signaling protein (MAVS), leading to the production of type I IFN. We now show specific interactions among these key partners in the RLR pathway through the use of bimolecular fluorescence complementation (BiFC) and super-resolution microscopy. Dimers of RIG-I, TRIM25, and MAVS localize into different compartments. Upon activation, we show that TRIM25 is redistributed into cytoplasmic dots associated with stress granules, while RIG-I associates with TRIM25/stress granules and with mitochondrial MAVS. In addition, MAVS competes with TRIM25 for RIG-I binding, and this suggests that upon TRIM25-mediated activation of RIG-I, RIG-I moves away from TRIM25 to interact with MAVS at the mitochondria. For the first time, the distribution of these three proteins was analyzed at the same time in virus-infected cells. We also investigated how specific viral proteins modify some of the protein complexes in the pathway. The protease NS3/4A from hepatitis C virus redistributes the complexes RIG-I/MAVS and MAVS/MAVS but not RIG-I/TRIM25. In contrast, the influenza A virus NS1 protein interacts with RIG-I and TRIM25 in specific areas in the cell cytoplasm and inhibits the formation of TRIM25 homocomplexes but not the formation of RIG-I/TRIM25 heterocomplexes, preventing the formation of RIG-I/MAVS complexes. Thus, we have localized spatially in the cell different complexes formed between RIG-I, TRIM25, and MAVS, in the presence or absence of two viral IFN antagonistic proteins.

IMPORTANCE
The first line of defense against viral infections is the innate immune response. Viruses are recognized by pathogen recognition receptors, such as the RIG-I like receptor family, that activate a signaling cascade that induces IFN production. In the present study, we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal, leading to an efficient antiviral state. In addition, this work presents a new means by how, when, and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response.
Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function
Authors: Marvin Shauna A. , Russier Marion , Huerta C. Theodore , Russell Charles J. , Schultz-Cherry Stacey
Published: November 2016

Whether influenza virus replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian, and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks. The first block is during viral entry, where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion, resulting in lysosomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG-opsonized bioparticles and Fc receptor CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and plays an important role in pathogenesis during infection
in vivo
.

IMPORTANCE
Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet-to-be-identified mechanism(s) to overcome a postnuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.
Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses
Authors: Wang Xiaoquan , Ilyushina Natalia A. , Lugovtsev Vladimir Y. , Bovin Nicolai V. , Couzens Laura K. , Gao Jin , Donnelly Raymond P. , Eichelberger Maryna C. , Wan Hongquan
Published: November 2016

Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the United States in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses.

IMPORTANCE
Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the United States since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the U.S. swine population in the mid-1990s, but they are different from both these ancestral viruses and current circulating human seasonal H3N2 strains in terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In this study, we identified amino acids in antigenic site B of the surface glycoprotein hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 variants which pose a threat to public health.
Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly
Authors: Alam Syed Benazir , Rochon D'Ann
Published: November 2016

Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of
Cucumber necrosis virus
(CNV), a nonenveloped positive-sense single-stranded RNA [(+)ssRNA] virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on
Chenopodium quinoa
. In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant
Nicotiana benthamiana
Hsc70-2 with virus particles
in vitro
leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus.

IMPORTANCE
Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host HSP70 homologs play multiple roles in the CNV infection cycle. We therefore examined the potential role of these cellular components in the CNV disassembly process. We show that the HSP70 family member Hsc70-2 is physically associated with CNV virions and that HSP70 antibody reduces the ability of CNV to establish infection. Statistically significantly fewer lesions are produced when virions having undetectable HSc70-2 are used as an inoculum. Finally incubation of Hsc70-2 with CNV particles results in conformational changes in particles. Taken together, our data point to an important role of the host factor Hsc70-2 in CNV disassembly.
The Short Form of the Zinc Finger Antiviral Protein Inhibits Influenza A Virus Protein Expression and Is Antagonized by the Virus-Encoded NS1
Authors: Tang Qiannan , Wang Xinlu , Gao Guangxia
Published: November 2016

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses. There are two ZAP isoforms arising from alternative splicing, which differ only at the C termini. It was recently reported that the long isoform (ZAPL) promotes proteasomal degradation of influenza A virus (IAV) proteins PA and PB2 through the C-terminal poly(ADP-ribose) polymerase (PARP) domain, which is missing in the short form (ZAPS), and that this antiviral activity is antagonized by the viral protein PB1. Here, we report that ZAP inhibits IAV protein expression in a PARP domain-independent manner. Overexpression of ZAPS inhibited the expression of PA, PB2, and neuraminidase (NA), and downregulation of the endogenous ZAPS enhanced their expression. We show that ZAPS inhibited PB2 protein expression by reducing the encoding viral mRNA levels and repressing its translation. However, downregulation of ZAPS only modestly enhanced the early stage of viral replication. We provide evidence showing that the antiviral activity of ZAPS is antagonized by the viral protein NS1. A recombinant IAV carrying an NS1 mutant that lost the ZAPS-antagonizing activity replicated better in ZAPS-deficient cells. We further provide evidence suggesting that NS1 antagonizes ZAPS by inhibiting its binding to target mRNA. These results uncover a distinct mechanism underlying the interactions between ZAP and IAV.

IMPORTANCE
ZAP is a host antiviral factor that has been extensively reported to inhibit the replication of certain viruses by repressing the translation and promoting the degradation of the viral mRNAs. There are two ZAP isoforms, ZAPL and ZAPS. ZAPL was recently reported to promote IAV protein degradation through the PARP domain. Whether ZAPS, which lacks the PARP domain, inhibits IAV and the underlying mechanisms remained to be determined. Here, we show that ZAPS posttranscriptionally inhibits IAV protein expression. This antiviral activity of ZAP is antagonized by the viral protein NS1. The fact that ZAP uses two distinct mechanisms to inhibit IAV infection and that the virus evolved different antagonists suggests an important role of ZAP in the host effort to control IAV infection and the importance of the threat of ZAP to the virus. The results reported here help us to comprehensively understand the interactions between ZAP and IAV.
Clinically Approved Ion Channel Inhibitors Close Gates for Hepatitis C Virus and Open Doors for Drug Repurposing in Infectious Viral Diseases
Authors: Pietschmann Thomas
Published: November 2016

Chronic hepatitis C virus (HCV) infection causes severe liver disease and affects ca. 146 million individuals. Novel directly acting antivirals targeting HCV have revolutionized treatment. However, high costs limit access to therapy. Recently, several related drugs used in humans to treat allergies or as neuroleptics emerged as potent HCV cell entry inhibitors. Insights into their antiviral modes of action may increase opportunities for drug repurposing in hepatitis C and possibly other important human viral infections.
Camp-based family treatment of childhood obesity: randomised controlled trial
Authors: Benestad Beate , Lekhal Samira , Småstuen Milada Cvancarova , Hertel Jens Kristoffer , Halsteinli Vidar , Ødegård Rønnaug Astri , Hjelmesæth Jøran
Published: November 2016

Objective

To compare the effectiveness of a 2-year camp-based family treatment programme and an outpatient programme on obesity in two generations.

Design

Pragmatic randomised controlled trial.

Setting

Rehabilitation clinic, tertiary care hospital and primary care.

Patients

Families with at least one child (7–12 years) and one parent with obesity.

Interventions

Summer camp for 2 weeks and 4 repetition weekends or lifestyle school including 4 days family education.Behavioural techniques motivating participants to healthier lifestyle.

Main outcome measures

Children: 2-year changes in body mass index (BMI) SD score (SDS). Parents: 2-year change in BMI. Main analyses: linear mixed models.

Results

Ninety children (50% girls) were included. Baseline mean (SD) age was 9.7 (1.2) years, BMI 28.7 (3.9) kg/m2and BMI SDS 3.46 (0.75). The summer-camp children had a lower adjusted estimated mean (95% CI) increase in BMI (−0.8 (−3.5 to −0.2) kg/m2), but the BMI SDS reductions did not differ significantly (−0.11 (−0.49 to 0.05)). The 2-year baseline adjusted BMI and BMI SDS did not differ significantly between summer-camp and lifestyle-school completers, BMI 29.8 (29.1 to 30.6) vs 30.7 (29.8 to 31.6) kg/m2and BMI SDS 2.96 (2.85 to 3.08) vs 3.11 (2.97 to 3.24), respectively. The summer-camp parents had a small reduction in BMI (−0.9 (−1.8 to −0.03) vs −0.8 (−2.1 to 0.4) in the lifestyle-school group), but the within-group changes did not differ significantly (0.3 (−1.7 to 2.2)).

Conclusions

A 2-year family camp-based obesity treatment programme had no significant effect on BMI SDS in children with severe obesity compared with an outpatient family-based treatment programme.

Trial registration number

NCT01110096.
Post-traumatic stress, anxiety and depression following miscarriage or ectopic pregnancy: a prospective cohort study
Authors: Farren Jessica , Jalmbrant Maria , Ameye Lieveke , Joash Karen , Mitchell-Jones Nicola , Tapp Sophie , Timmerman Dirk , Bourne Tom
Published: November 2016

Objectives

This is a pilot study to investigate the type and severity of emotional distress in women after early pregnancy loss (EPL), compared with a control group with ongoing pregnancies. The secondary aim was to assess whether miscarriage or ectopic pregnancy impacted differently on the type and severity of psychological morbidity.

Design

This was a prospective survey study. Consecutive women were recruited between January 2012 and July 2013. We emailed women a link to a survey 1, 3 and 9 months after a diagnosis of EPL, and 1 month after the diagnosis of a viable ongoing pregnancy.

Setting

The Early Pregnancy Assessment Unit (EPAU) of a central London teaching hospital.

Participants

We recruited 186 women. 128 had a diagnosis of EPL, and 58 of ongoing pregnancies. 11 withdrew consent, and 11 provided an illegible or invalid email address.

Main outcome measures

Post-traumatic stress disorder (PTSD) was measured using the Post-traumatic Diagnostic Scale (PDS), and anxiety and depression using the Hospital Anxiety and Depression Scale (HADS).

Results

Response rates were 69/114 at 1 month and 44/68 at 3 months in the EPL group, and 20/50 in controls. Psychological morbidity was higher in the EPL group with 28% meeting the criteria for probable PTSD, 32% for anxiety and 16% for depression at 1 month and 38%, 20% and 5%, respectively, at 3 months. In the control group, no women met criteria for PTSD and 10% met criteria for anxiety and depression. There was little difference in type or severity of distress following ectopic pregnancy or miscarriage.

Conclusions

We have shown a large number of women having experienced a miscarriage or ectopic pregnancy fulfil the diagnostic criteria for probable PTSD. Many suffer from moderate-to-severe anxiety, and a lesser number depression. Psychological morbidity, and in particular PTSD symptoms, persists at least 3 months following pregnancy loss.
Alcohol consumption over time and mortality in the Swedish Women’s Lifestyle and Health cohort
Authors: Licaj Idlir , Sandin Sven , Skeie Guri , Adami Hans-Olov , Roswall Nina , Weiderpass Elisabete
Published: November 2016

Background

Alcohol consumption is steadily increasing in high-income countries but the harm and possible net benefits of light-to-moderate drinking remain controversial. We prospectively investigated the association between time-varying alcohol consumption and overall and cause-specific mortality among middle-aged women.

Methods

Among 48 249 women at baseline (33 404 at follow-up) in the prospective Swedish Women's Lifestyle and Health cohort, age 30–49 years at baseline, we used repeated information on alcohol consumption and combined this method with multiple imputation in order to maximise the number of participants and deaths included in the analyses. Multivariable Cox regression models were used to calculate HRs for overall and cause-specific mortality.

Results

During >900 000 person/years, a total of 2100 deaths were recorded through Swedish registries. The median alcohol consumption increased from 2.3 g/day in 1991/1992 (baseline) to 4.7 g/day in 2004 (follow-up). Compared with light drinkers (0.1–1.5 g/day), a null association was observed for all categories of alcohol consumption with the exception of never drinkers. The HR comparing never with light drinkers was 1.46 (95% CI 1.22 to 1.74). There was a statistically significant negative trend between increasing alcohol consumption and cardiovascular and ischaemic heart diseases mortality. The results were similar when women with prevalent conditions were excluded.

Conclusions

In conclusion, in a cohort of young women, light alcohol consumption was protective for cardiovascular and ischaemic heart disease mortality but not for cancer and overall mortality.
Surgical ablation of atrial fibrillation: a protocol for a systematic review and meta-analysis of randomised controlled trials
Authors: McClure Graham R , Belley-Cote Emilie P , Singal Rohit K , Jaffer Iqbal H , Dvirnik Nazari , An Kevin R , Fortin Gabriel , Spence Jessica , Whitlock Richard P
Published: November 2016

Introduction

Atrial fibrillation (AF) affects 10% of patients undergoing cardiac surgery and is an independent risk factor for all-cause mortality, ischaemic stroke and heart failure. Surgical AF ablation has been shown to significantly improve maintenance of sinus rhythm, however, small to medium size trials conducted to date lack the power required to assess patient-important outcomes such as mortality, stroke, heart failure and health-related quality of life. Moreover, a recent randomised trial (RCT) suggested harm by surgical AF ablation with an almost threefold increase in the requirement for permanent pacemaker postablation. We aim to perform a systematic review and meta-analysis to evaluate efficacy and safety of surgical AF ablation compared to no surgical ablation.

Methods and analysis

We will search Cochrane CENTRAL, MEDLINE and EMBASE for RCTs evaluating the use of surgical AF ablation, including any lesion set, versus no surgical AF ablation in adults with AF undergoing any type of cardiac surgery. Outcomes of interest include mortality, embolic events, quality of life, rehospitalisation, freedom from AF and adverse events, including need for pacemaker and worsening heart failure. Independently and in duplicate, reviewers will screen references, assess eligibility of potentially relevant studies using predefined eligibility criteria and collect data using prepiloted forms. We will pool data using a random effects model and present results as relative risk with 95% CIs for dichotomous outcomes and as mean difference with 95% CI for continuous outcomes. We will assess risk of bias using the Cochrane Collaboration tool, and quality of evidence with the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach.

Ethics and dissemination

Our results will help guide clinical practice by providing the most comprehensive analysis of risks and benefits associated with the procedure. Our results will be disseminated through publication in peer-reviewed journals and conference presentations.

Trial registration number

CRD42015025988.
Cohort profile: Pacific Islands Families (PIF) growth study, Auckland, New Zealand
Authors: Rush E , Oliver M , Plank L D , Taylor S , Iusitini L , Jalili-Moghaddam S , Savila F , Paterson J , Tautolo E
Published: November 2016

Purpose

This article profiles a birth cohort of Pacific children participating in an observational prospective study and describes the study protocol used at ages 14–15 years to investigate how food and activity patterns, metabolic risk and family and built environment are related to rates of physical growth of Pacific children.

Participants

From 2000 to 2015, the Pacific Islands Families Study has followed, from birth, the growth and development of over 1000 Pacific children born in Auckland, New Zealand. In 2014, 931 (66%) of the original cohort had field measures of body composition, blood pressure and glycated haemoglobin. A nested subsample (n=204) was drawn by randomly selecting 10 males and 10 females from each decile of body weight. These participants had measurement of body composition by dual-energy X-ray absorptiometry, food frequency, 6 min walk test and accelerometer-determined physical activity and sedentary behaviours, and blood biomarkers for metabolic disease such as diabetes. Built environment variables were generated from individual addresses.

Findings to date

Compared to the Centres for Disease Control and Prevention (CDC) reference population with mean SD scores (SDS) of 0, this cohort of 931 14-year-olds was taller, weighed more and had a higher body mass index (BMI) (mean SDS height >0.6, weight >1.6 and BMI >1.4). 7 of 10 youth were overweight or obese. The nested-sampling frame achieved an even distribution by body weight.

Future plans

Cross-sectional relationships between body size, fatness and growth rate, food patterns, activity patterns, pubertal development, risks for diabetes and hypertension and the family and wider environment will be examined. In addition, analyses will investigate relationships with data collected earlier in the life course and measures of the cohort in the future. Understanding past and present influences on child growth and health will inform timely interventions to optimise future health and reduce inequalities for Pacific people.
Complementary role of cardiac CT in the assessment of aortic valve replacement dysfunction
Authors: Moss Alastair J , Dweck Marc R , Dreisbach John G , Williams Michelle C , Mak Sze Mun , Cartlidge Timothy , Nicol Edward D , Morgan-Hughes Gareth J
Published: November 2016

Aortic valve replacement is the second most common cardiothoracic procedure in the UK. With an ageing population, there are an increasing number of patients with prosthetic valves that require follow-up. Imaging of prosthetic valves is challenging with conventional echocardiographic techniques making early detection of valve dysfunction or complications difficult. CT has recently emerged as a complementary approach offering excellent spatial resolution and the ability to identify a range of aortic valve replacement complications including structural valve dysfunction, thrombus development, pannus formation and prosthetic valve infective endocarditis. This review discusses each and how CT might be incorporated into a multimodal cardiovascular imaging pathway for the assessment of aortic valve replacements and in guiding clinical management.