bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the Salmonella typhimurium pepT gene by cyclic AMP receptor protein (CRP) and FNR acting at a hybrid CRP-FNR site
Authors: Lombardo M J , Lee A A , Knox T M , Miller C G
Published: November 2016

The Salmonella typhimurium pepT gene is induced nearly 30-fold in response to anaerobiosis. Anaerobic expression is dependent on the transcriptional regulator encoded by fnr (previously oxrA). Primer extension analysis and site-directed mutagenesis experiments show that pepT is transcribed from two sigma 70 promoters. One promoter (P1) is FNR dependent and anaerobically induced, while the other (P2) appears to be constitutive. The potABCD operon is divergently transcribed from a promoter near pepT P2. Sequence analysis of pepT promoter mutations which either elevate anaerobic expression or confer constitutive expression revealed that these mutations affect the -10 region of the P1 or P2 promoter, respectively. The pepT200 mutation, which changes the -10 region of the FNR-dependent P1 promoter to the consensus, has the surprising effect of allowing five- to sevenfold anaerobic induction in the absence of FNR. We have shown that the anaerobic induction of pepT-lacZ in a pepT200 fnr strain is dependent on wild-type alleles of both crp and cya. In a pepT200 pepT-lacZ strain, beta-galactosidase activity was elevated aerobically in the presence of exogenous cyclic AMP (cAMP) and was elevated also in succinate minimal medium relative to its level in glucose minimal medium. Primer extension analysis confirmed that P1 is the cAMP receptor protein (CRP)-dependent promoter. Site-directed mutagenesis experiments indicated that a hybrid CRP-FNR binding site positioned at -41 of the P1 promoter is utilized by both FNR and CRP. CRP-cAMP also appeared to repress FNR-dependent transcription of pepT under anaerobic conditions in both the pepT+ and pepT200 backgrounds. Although both CRP and FNR are capable of binding the hybrid site and activating transcription of pepT, CRP requires the consensus -10 sequence for efficient activation.
Genomic organization of the acidophilic chemolithoautotrophic bacterium Thiobacillus ferrooxidans ATCC 21834
Authors: Irazabal N , Marín I , Amils R
Published: November 2016

The genomic organization of the acidophilic chemolithoautotrophic bacterium Thiobacillus ferrooxidans ATCC 21834 has been studied by pulsed-field gel electrophoresis (PFGE). Analysis of its intact DNA, as well as the restriction patterns obtained with several endonucleases, allowed the characterization of one circular chromosome of 2.9 Mb and one plasmid of 8.6 kb. The first complete and highly resolved physical map (86 restriction sites) of the chromosome of an acidophilic obligate chemolithoautotrophic bacterium has been constructed by using endonucleases PmeI, SwaI, XbaI, and SpeI. The rRNA and str operons have been located on the chromosomal physical map.
Transcriptional activation of the bkd operon of Pseudomonas putida by BkdR
Authors: Madhusudhan K T , Hester K L , Friend V , Sokatch J R
Published: November 2016

Reinvestigation of the transcriptional start site of the bkd operon of Pseudomonas putida revealed that the transcriptional start site was located 86 nucleotides upstream of the translational start. There was a sigma 70 binding site 10 bp upstream of the transcriptional start site. The dissociation constants for BkdR, the transcriptional activator of the bkd operon, were 3.1 x 10(-7) M in the absence of L-valine and 8.9 x 10(-8) M in the presence of L-valine. Binding of BkdR to substrate DNA in the absence of L-valine imposed a bend angle of 92 degrees in the DNA. In the presence of L-valine, the angle was 76 degrees. BkdR did not bind to either of the two fragments of substrate DNA resulting from digestion with AgeI. Because AgeI attacks between three potential BkdR binding sites, this suggests that binding of BkdR is cooperative. P. putida JS110 and JS112, mutant strains which do not express any of the components of branched-chain keto acid dehydrogenase, were found to contain missense mutations in bkdR resulting in R40Q and T22I changes in the putative helix-turn-helix of BkdR. Addition of glucose to the medium repressed expression of lacZ from a chromosomal bkdR-lacZ fusion, suggesting that catabolite repression of the bkd operon was the result of reduced expression of bkdR. These data are used to present a model for the role of BkdR in transcriptional control of the bkd operon.
Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis
Authors: Richter G , Fischer M , Krieger C , Eberhardt S , Lüttgen H , Gerstenschläger I , Bacher A
Published: November 2016

The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.
Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway
Authors: Hyland S A , Eveland S S , Anderson M S
Published: November 2016

The lpxC (envA) gene of Escherichia coli encodes UDP-3-O-acyl-GlcNAc deacetylase, the second and committed step of lipopolysaccharide biosynthesis. Although present in all gram-negative bacteria examined, the deacetylase from E. coli is the only example of this enzyme that has been expressed and purified. In order to examine other variants of this protein, we cloned the Pseudomonas aeruginosa deacetylase structural gene from a lambda library as a 5.1-kb EcoRI fragment. The LpxC reading frame encodes an inferred protein of 33,435 Da that is highly homologous to the E. coli protein and that possesses a nearly identical hydropathy profile. In order to verify function, we subcloned the P. aeruginosa lpxC gene into the T7-based expression vector pET11a. Upon induction at 30 degrees C, this construct yielded active protein to approximately 18% of the soluble fraction. We devised a novel, rapid, and reproducible assay for the deacetylase which facilitated purification of the enzyme in three steps. The purified recombinant protein was found to be highly sensitive to EDTA yet was reactivated by the addition of excess heavy metal, as was the case for crude extracts of P. aeruginosa. In contrast, deacetylase activity in crude extracts of E. coli was insensitive to EDTA, and the extracts of the envA1 mutant were sensitive in a time-dependent manner. The lpxC gene has no significant homology with amidase signature sequences. Therefore, we assign this protein to the metalloamidase family as a member with a novel structure.
A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds
Authors: Pérez-Llarena F J , Liras P , Rodríguez-García A , Martín J F
Published: November 2016

A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid.
Association of the histone-like protein HBsu with the nucleoid of Bacillus subtilis
Authors: Köhler P , Marahiel M A
Published: November 2016

To investigate the physiological role of the essential histone-like protein of Bacillus subtilis (HBsu) in the nucleoid structure, a fusion to the green fluorescent protein (GFP) of Aequorea victoria was constructed. This purified fusion protein, HBsuGFP, showed a threefold-reduced affinity to DNA compared to unmodified HBsu; however, in gel mobility shift experiments HBsuGFP DNA-binding was greatly enhanced in the presence of low HBsu concentrations. Additional production of HBsu also had a positive effect on the retarded growth of a B. subtilis strain, PK9C8, which expresses only hbs-gfp (encoding HBsuGFP). HBsu seemed to influence not only growth but also nucleoid structure, as monitored by DNA staining and fluorescence microscopy. Without HBsu production, strain PK9C8 showed a relaxed nucleoid structure associated with HBsuGFP. However, a highly compact nucleoid structure that coincides with the fluorescence of the fusion protein was visualized when HBsu synthesis was induced. This provides the first evidence for in vivo association of HBsu in DNA packaging and its consequence on cell growth.
Characterization of the tol-pal and cyd region of Escherichia coli K-12: transcript analysis and identification of two new proteins encoded by the cyd operon
Authors: Muller M M , Webster R E
Published: November 2016

Sequence analysis showed that the cyd operon is immediately upstream of the tol-pal region. Northern (RNA) blot analysis demonstrated that the transcript for the cyd operon terminates just before the promoter for transcription of the tol genes. The cyd transcript contains cydA cydB followed by two open reading frames: orfC, encoding a 37-residue peptide, and orfD, encoding a 97-residue peptide. Both OrfC and OrfD are synthesized in minicells.
Growth rate-related regulation of the ilvGMEDA operon of Escherichia coli K-12 is a consequence of the polar frameshift mutation in the ilvG gene of this strain
Authors: Parekh B S , Hatfield G W
Published: November 2016

In Escherichia coli K-12 the intracellular levels of threonine deaminase and transaminase B, products of ilvA and ilvE, respectively, in the ilvGMEDA operon, increase with increasing growth rates (S. Pedersen, P. L. Bloch, S. Reeh, and F. C. Neidhardt, Cell 14:179-190, 1978). However, the transcriptional activities of the upstream ilvpG and the internal ilvpE promoters do not increase. Therefore, the growth rate-related expression of this operon is not regulated at the level of transcription initiation. Unlike other wild-type E. coli strains, E. coli K-12 contains a polar frameshift mutation in the ilvG gene (R. P. Lawther, D. H. Calhoun, C. W. Adams, C. A. Hauser, J. Gray, and G. W. Hatfield, Proc. Natl. Acad. Sci. USA 78:922-925, 1981). In an E. coli K-12 (IlvG+) derivative strain, where the reading frame of the ilvG gene is restored, no growth rate-related expression of the ilvGMEDA operon is observed. Thus, the growth rate-related expression of the ilvGMEDA operon in E. coli K-12 is the fortuitous consequence of the polar frameshift mutation in the ilvG gene of this strain.
The Streptomyces ATP-binding component MsiK assists in cellobiose and maltose transport
Authors: Schlösser A , Kampers T , Schrempf H
Published: November 2016

Streptomyces reticuli harbors an msiK gene which encodes a protein with an amino acid identify of 90% to a corresponding protein previously identified in Streptomyces lividans. Immunological studies revealed that S. lividans and S. reticuli synthesize their highest levels of MsiK during growth with cellobiose, but not with glucose. Moreover, moderate amounts of MsiK are produced by both species in the course of growth with maltose, melibiose, and xylose and by S. lividans in the presence of xylobiose and raffinose. In contrast, a recently identified cellobiose-binding protein and its distantly related homolog were only found if S. reticuli or S. lividans, respectively, was cultivated with cellobiose. Uptake of cellobiose and maltose was tested and ascertained for S. reticuli and S. lividans, but not for an msiK S. lividans mutant. However, transformants of this mutant carrying the S. reticuli or S. lividans msiK gene on a multicopy plasmid had regained the ability to transport both sugars. The data show that MsiK assists two ABC transport systems.
Structure and regulation of expression of the Bacillus subtilis valyl-tRNA synthetase gene
Authors: Luo D , Leautey J , Grunberg-Manago M , Putzer H
Published: November 2016

We have sequenced the valyl-tRNA synthetase gene (valS) of Bacillus subtilis and found an open reading frame coding for a protein of 880 amino acids with a molar mass of 101,749. The predicted amino acid sequence shares strong similarity with the valyl-tRNA synthetases from Bacillus stearothermophilus, Lactobacillus casei, and Escherichia coli. Extracts of B. subtilis strains overexpressing the valS gene on a plasmid have increased valyl-tRNA aminoacylation activity. Northern analysis shows that valS is cotranscribed with the folC gene (encoding folyl-polyglutamate synthetase) lying downstream. The 300-bp 5' noncoding region of the gene contains the characteristic regulatory elements, T box, "specifier codon" (GUC), and rho-independant transcription terminator of a gene family in gram-positive bacteria that encodes many aminoacyl-tRNA synthetases and some amino acid biosynthetic enzymes and that is regulated by tRNA-mediated antitermination. We have shown that valS expression is induced by valine limitation and that the specificity of induction can be switched to threonine by changing the GUC (Val) specifier triplet to ACC (Thr). Overexpression of valS from a recombinant plasmid leads to autorepression of a valS-lacZ transcriptional fusion. Like induction by valine starvation, autoregulation of valS depends on the presence of the GUC specifier codon. Disruption of the valS gene was not lethal, suggesting the existence of a second gene, as is the case for both the thrS and the tyrS genes.
Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly
Authors: Maguire B A , Wild D G
Published: November 2016

The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33. Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33. Neither strain showed major defects in ribosome assembly. However, when the mutations were transferred to other strains of E. coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated. In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced. The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.
Active efflux of bile salts by Escherichia coli
Authors: Thanassi D G , Cheng L W , Nikaido H
Published: November 2016

Enteric bacteria such as Escherichia coli must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. The outer membrane barrier of gram-negative bacteria plays an important role in this resistance, but ultimately it can only retard the influx of bile salts. We therefore examined whether E. coli possessed an energy-dependent efflux mechanism for these compounds. Intact cells of E. coli K-12 appeared to pump out chenodeoxycholate, since its intracellular accumulation increased more than twofold upon deenergization of the cytoplasmic membrane by a proton conductor. Growth inhibition by bile salts and accumulation levels of chenodeoxycholate increased when mutations inactivating the acrAB and emrAB gene clusters were introduced. The AcrAB system especially appeared to play a significant role in bile acid efflux. However, another efflux system(s) also plays an important role, since the accumulation level of chenodeoxycholate increased strongly upon deenergization of acrA emrB double mutant cells. Everted membrane vesicles accumulated taurocholate in an energy-dependent manner, apparently consuming delta pH without affecting delta psi. The efflux thus appears to be catalyzed by a proton antiporter. Accumulation by the everted membrane vesicles was not decreased by mutations in acr and emrB genes and presumably reflects activity of the unknown system seen in intact cells. It followed saturation kinetics with Vmax and Km values in the neighborhood of 0.3 nmol min(-1) mg of protein(-1) and 50 microM, respectively.
Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA
Authors: Leibovitz E , Ohayon H , Gounon P , Béguin P
Published: November 2016

This article reports the characterization of the Clostridium thermocellum SdbA protein thought to anchor the cellulosome to the bacterial cell surface. The NH2-terminal region of SdbA consists of a cohesin domain which specifically binds the dockerin domain of the cellulosomal scaffolding protein CipA. The COOH-terminal region consists of a triplicated segment, termed SLH repeats, which is present in the sequence of many bacterial cell surface polypeptides. The binding parameters of the interaction between the dockerin domain of CipA and the cohesin domain of SdbA were studied by using, as a probe, the chimeric polypeptide CelC-DSCipA, which carries the dockerin domain of CipA fused to endoglucanase CelC. In the presence of Ca2+, CelC-DSCipA bound to SdbA with an affinity constant of 1.26 x 10(7) M(-1). Binding of CelC-DSCipA to SdbA as a function of Ca2+ concentration was sigmoidal, corresponding to a Hill coefficient of 2 and an affinity constant for Ca2+ of 4 x 10(6) M(-2). This suggested the presence of two cooperatively bound Ca2+ ions in the cohesin-dockerin complex. Immunoblotting of C. thermocellum subcellular fractions and electron microscopy of immunocytochemically labeled cells indicated that SdbA is located on the cell surface and is a component of the cellulosome. Together, the data confirm that SdbA could mediate anchoring of the cellulosome to the surface of C. thermocellum cells by interacting with the dockerin domain of CipA.
Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes
Authors: Jessen-Marshall A E , Parker N J , Brooker R J
Published: November 2016

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.
Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis
Authors: Stroeher U H , Parasivam G , Dredge B K , Manning P A
Published: November 2016

The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.
Cloning of the dapB gene, encoding dihydrodipicolinate reductase, from Mycobacterium tuberculosis
Authors: Pavelka M S , Weisbrod T R , Jacobs W R
Published: November 2016

Diaminopimelate (DAP) is used by bacteria for the synthesis of lysine. In many species of bacteria, including mycobacteria, DAP is also used for peptidoglycan biosynthesis. In this report we describe the cloning of the dapB gene encoding dihydrodipicolinate reductase (DHPR), which catalyzes a key branch point reaction in the bacterial DAP biosynthetic pathway, from Mycobacterium tuberculosis. Analyses of the DapB proteins from different bacterial species suggest that two different classes of DHPR enzymes may exist in bacteria.
Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis
Authors: Ropp P A , Nicholas R A
Published: November 2016

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.
Regulation of gene expression by repressor localization: biochemical evidence that membrane and DNA binding by the PutA protein are mutually exclusive
Authors: Muro-Pastor A M , Ostrovsky P , Maloy S
Published: November 2016

The PutA protein from Salmonella typhimurium is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate, a reaction that is coupled to the transfer of electrons to the electron transport chain in the cytoplasmic membrane. The PutA protein is also a transcriptional repressor that regulates the expression of the put operon in response to the availability of proline. Despite extensive genetic and biochemical studies of the PutA protein, it was not known if the PutA protein carries out both of these two opposing functions while membrane associated or if instead it carries them out in different cellular compartments. To distinguish between these alternatives, we directly assayed the binding of purified PutA protein to DNA and membranes in vitro. The results indicate that wild-type PutA does not simultaneously associate with DNA and membranes. In addition, PutA superrepressor mutants that exhibit increased repression of the put genes show a direct correlation between decreased membrane binding and increased DNA binding. These results support a model in which the PutA protein shuttles between the membrane (where it acts as an enzyme but lacks access to DNA-binding sites) and the cytoplasm (where it binds DNA and acts as a transcriptional repressor), depending on the availability of proline.
Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis
Authors: Prüller Sandra , Turni Conny , Blackall Patrick J. , Beyerbach Martin , Klein Günter , Kreienbrock Lothar , Strutzberg-Minder Katrin , Kaspar Heike , Meemken Diana , Kehrenberg Corinna
Published: November 2016

Currently, there is no agreed method available for broth microdilution susceptibility testing ofHaemophilus parasuis, one of the most important bacterial pathogens in pig production. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. Growth determinations using one type strain and three field isolates revealed no visible growth ofH. parasuisin media which have proven to be suitable for susceptibility testing of fastidious organisms. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. The reproducibility of MICs obtained in this medium was evaluated and statistically analyzed, considering a model with two different variables (precondition of five identical MICs and MIC mode accepting a deviation of ±1 dilution step, respectively). No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). Nearly all MICs of quality control strains were in the acceptable range. Subsequently, 47H. parasuisisolates representing 13 serovars were tested with the newly developed medium and TMB. Statistical analysis of all isolates and 15 antimicrobial agents and antimicrobial combinations showed no significant difference between MICs obtained in supplemented CAMHB and TMB. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 24 h.
The Added Cost of Rapid Diagnostic Testing and Active Antimicrobial Stewardship: Is It Worth It?
Authors: McElvania TeKippe Erin
Published: November 2016

Rapid diagnostic testing reduces the turnaround time for pathogen identification in the clinical microbiology laboratory, but the impact on patient care and hospital costs is a matter of speculation. Patel et al. (J. Clin. Microbiol. 55:60–67, 2017,
https://doi.org/10.1128/JCM.01452-16
) investigate the impact of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in conjunction with active antimicrobial stewardship to determine if implementation is indeed worth the added costs.
Crystal Structure of the Marburg Virus VP35 Oligomerization Domain
Authors: Bruhn Jessica F. , Kirchdoerfer Robert N. , Urata Sarah M. , Li Sheng , Tickle Ian J. , Bricogne Gérard , Saphire Erica Ollmann
Published: November 2016

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera
Marburgvirus
and
Ebolavirus
, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.

IMPORTANCE
Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.
PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection
Authors: Marshall Karen E. , Hughson Andrew , Vascellari Sarah , Priola Suzette A. , Sakudo Akikazu , Onodera Takashi , Baron Gerald S.
Published: November 2016

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrP
C
) influences PrP
C
misfolding into the disease-associated isoform, PrP
res
, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrP
C
away from rafts. Previous studies showed that nonraft transmembrane PrP
C
variants resist conversion to PrP
res
when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrP
C
and GPI-anchored PrP
res
in distinct membrane environments. Thus, it remained unclear whether transmembrane PrP
C
might convert to PrP
res
if seeded by an exogenous source of PrP
res
not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrP
C
. To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrP
C
supported persistent PrP
res
propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrP
C
is required for persistent PrP
res
propagation, implicating raft microdomains as a location for conversion.

IMPORTANCE
Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrP
C
) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrP
C
or transmembrane-anchored PrP
C
, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrP
res
. We show that only GPI-anchored PrP
C
was able to convert to PrP
res
and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrP
C
to rafts are crucial to the ability of PrP
C
to propagate as a prion.
Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase
ICP0
Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia
Authors: Pan Dongli , Pesola Jean M. , Li Gang , McCarron Seamus , Coen Donald M.
Published: November 2016

Herpes simplex virus 1 (HSV-1) latency entails the repression of productive (“lytic”) gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to
ICP0
mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of
ICP0
mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of
ICP0
and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting
ICP0
mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency.

IMPORTANCE
Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since miR-H2's discovery as a viral microRNA bearing complete sequence complementarity to the mRNA for the important viral gene activator ICP0, inhibition of ICP0 expression by miR-H2 has been a major hypothesis to help explain the repression of lytic gene expression during latency. However, this hypothesis remained untested in latently infected animals. Using a miR-H2-deficient mutant virus, we found no evidence that miR-H2 represses the expression of
ICP0
or other lytic genes in cells or mice infected with HSV-1. Although miR-H2 can repress
ICP0
expression in transfection assays, such repression is weak. The results suggest that other mechanisms for miR-H2 activity and for the repression of lytic gene expression during latency deserve investigation.
Intracranial venous thrombosis in a man taking clomiphene citrate
Authors: Zahid M , Arshad A , Zafar A , Al-Mohannadi D
Published: November 2016

Clomiphene citrate is widely used by the female population for the management of a wide array of medical conditions especially those related to ovulation, but its use in male population remains controversial. Efficacy of clomiphene is not well established in male hypogonadism and infertility. Its side effects and complications are also poorly studied or reported. We present a case of a man with infertility issues for which he started receiving clomiphene citrate 3 weeks before presenting with progressive symptoms of headache, vomiting, transient blurred vision and diplopia, which eventually turned out to be the symptoms of intracranial venous thrombosis. After excluding the common causes of intracranial venous thrombosis and hypercoagulable state, clomiphene citrate seems the most likely culprit.
Monitoring small animal patients in the intensive care unit
Authors: Humm Karen , Kellett‐Gregory Lindsay
Published: November 2016

All patients in an intensive care unit (ICU) have instability in at least one of the major body systems (cardiovascular, neurological and respiratory systems) or the potential for this to develop. If these systems are not functioning appropriately, death can result. The aim of intensive care is therefore to prevent this by monitoring for, recognising and treating any instability. This article focuses on when intensive care is required and how to monitor a patient in the ICU effectively.
Everyday veterinary ethics in 2016
Authors: McCulloch Steven
Published: November 2016

THE Everyday Ethics series in In Practice gives readers an opportunity to consider and contribute to discussion of some of the ethical dilemmas that can arise in veterinary practice. Each month, a case scenario is presented, followed by discussion of some of the issues involved. In this final contribution for 2016, Steven McCulloch, who coordinates the series, summarises contributions made during the year, and responses to online polls.
Nursing critically ill patients in the intensive care unit
Authors: Haskey Elle
Published: November 2016

Critically ill patients present a challenge to the whole veterinary team because they require invasive diagnostic tests, advanced procedures and intensive nursing care. It is important to start with an assessment of the patient to prioritise concerns and develop a care plan tailored for the individual. The nursing process involves the implementation and delivery of this plan, followed by evaluation of the nursing/treatments. This is a cyclical process that is repeated over and over again as the patient's key parameters are reassessed and a further plan is made. The aim of this article is to explore some key aspects of critical care nursing, including infection control and hand hygiene, the management of tubes and lines, and dealing with recumbent patients.
Impact of the 2008 global financial crisis on the health of Canadians: repeated cross-sectional analysis of the Canadian Community Health Survey, 2007–2013
Authors: Nour Sabrina , Labonté Ronald , Bancej Christina
Published: November 2016

Background

Despite a clear impact on the Canadian economy, little is known about the subsequent health impacts of the 2008 global financial crisis (GFC) in this country. This study fills this gap in knowledge by conducting a repeated cross-sectional analysis of the Canadian Community Health Survey (CCHS).

Methods

Data from 7 cycles (2007–2013) of the CCHS were combined to form a large data set representative of the Canadian working-age population (15–64 years) residing in 1 of 10 provinces. A logistic regression model was used to determine whether exposure to various periods of the GFC resulted in increased odds of reporting poor mental health. Exposure was categorised into 4 periods based on political and economic indicators, as follows: precrisis period (baseline), initial crisis period, stimulus period and austerity period. Other outcomes investigated included: anxiety disorders (AD), mood disorders (MD), poor physical health and health-related behaviours (heavy alcohol drinking (HAD) and decreased fruit/vegetable consumption (FVC)).

Results

A significant increased odds of reporting poor mental health was observed during the austerity period compared with the precrisis period (OR=1.26 (1.16 to 1.32)); findings remain significant when adjusted for sex, marital status and education. Exposure to the austerity period was also significantly associated with increased odds of reporting AD, MD, HAD and decreased odds of FVC. No significant associations were observed for the poor self-perceived physical health variable.

Conclusions

Statistically significant associations were observed between several negative health outcomes and the austerity period when compared with the precrisis period. Austerity has been linked to worsening health in other studies and represents an example of how the policy response can have greater detrimental impact on health than the financial crisis itself.
Why do team-sport athletes drink fluid in excess when exercising in cool conditions?
Authors: Bargh Melissa J. , King Roderick F.G.J. , Gray Michael P. , Jones Ben
Published: November 2016

This study assessed the potential physiological and perceptual drivers of fluid intake and thirst sensation during intermittent exercise. Ten male rugby players (17 ± 1 years, stature: 179.1 ± 4.2 cm, body mass (BM): 81.9 ± 8.1 kg) participated in six 6-min small-sided games, interspersed with 2 min rest, where fluid intake was ad libitum during rest periods. Pre- and postmeasurements of BM, subjective ratings (thirst, thermal comfort, thermal sensation, mouth dryness), plasma osmolality (POsm), serum sodium concentration (S[Na+]), haematocrit and haemoglobin (to calculate plasma volume change; PV) were taken. Fluid intake was measured during rest periods. BM change was –0.17 ± 0.59% and fluid intake was 0.88 ± 0.38 L. Pre- to post-POsm decreased (–3.1 ± 2.3 mOsm·kg−1; p = 0.002) and S[Na+] remained similar (–0.3 ± 0.7 mmol·L−1, p = 0.193). ΔPV was 5.84 ± 3.65%. Fluid intake displayed a relationship with pre-POsm (r = –0.640, p = 0.046), prethermal comfort (r = 0.651; p = –0.041), ΔS[Na+] (r = 0.816, p = 0.004), and ΔPV (r = 0.740; p = 0.014). ΔThirst sensation displayed a relationship with premouth dryness (r = 0.861, p = 0.006) and Δmouth dryness (r = 0.878, p = 0.004). Yet a weak positive relationship between Δthirst sensation and fluid intake was observed (r = 0.085, p = 0.841). These data observed in an ambient temperature of 13.6 ± 0.9 °C, suggest team-sport athletes drink in excess of fluid homeostasis requirements and thirst sensation in cool conditions; however, this was not influenced by thermal discomfort.
Comparison of the antiglycation activity of leaves of eight traditionally used wild blueberry species (Vaccinium L.) from northern Canada and Europe with their phytochemistry
Authors: Ferrier Jonathan , Saleem Ammar , Djeffal Sabrina , Schlarb James , Haddad Pierre S. , Balick Michael J. , Cuerrier Alain , Arnason John T.
Published: November 2016

Blueberry leaves (Vaccinium spp. L.) are used as a natural health product and traditional medicine for health maintenance in type-2 diabetes. In this study, seven blueberry species available to northern communities in North America and one used by the Lukomir Highlanders in Europe were assessed for phytochemical content and antidiabetic activity using an in vitro advanced glycation end product (antiglycation) assay. All Vaccinium spp. were found to have potent antiglycation activity that was greater than that of the outgroup Gaylussacia sp. Inhibitory concentrations of leaf extracts from Vaccinium spp. varied from 4.1 to 79.0 μg/mL. The activity was correlated to phenolic content, and eight phenolic metabolites were found to provide species markers. These species have potential for complementary dietary strategies for type-2 diabetics in northern communities.
Aspirin and (or) omega-3 polyunsaturated fatty acids protect against corticohippocampal neurodegeneration and downregulate lipoxin A4production and formyl peptide receptor-like 1 expression in pentylenetetrazole-kindled rats
Authors: Abd-Elghafour Basma A. , El-Sayed Norhan M. , Ahmed Amal A.M. , Zaitone Sawsan A. , Moustafa Yasser M.
Published: November 2016

There is evidence for a relationship between inflammation and seizures because epilepsy can be caused by or result in inflammation. This study aimed to investigate the effect of aspirin and (or) omega-3 polyunsaturated fatty acids (PUFAs) on seizure activity and neurodegeneration in pentylenetetrazole (PTZ)-kindled rats focusing on their effect on corticohippocampal production of lipoxin A4(LXA4) and expression of formyl peptide receptor-like 1 (FPRL1) receptors. Male rats were injected with PTZ (35 mg/kg, i.p.) 3 times per week for a total of 15 doses. Rats were treated daily with aspirin (20 mg/kg, i.p.), omega-3 PUFAs (85 mg/kg, p.o.), or a combination of them for 35 days. Both LXA4level and expression of FPRL1 receptor in the cortices and hippocampi of rats’ brains were greater in PTZ-kindled rats compared to a saline control group. Cotreatment with aspirin and (or) omega-3 PUFAs reduced convulsive behaviour; reduced levels of LXA4, interleukin-1β, and nuclear factor-κB; and showed a lower percentage of corticohippocampal degenerative cells compared to PTZ-kindled rats. The combination of the 2 therapeutic agents did not provide significant improvement in comparison with the monotherapies. These findings suggest the use of aspirin or omega-3 PUFAs may delay the development of seizures and provide neuroprotection in a clinical setting.
Multi-objective dynamic aperture optimization for storage rings
Authors: Li Yongjun , Yang Lingyun
Published: November 2016

We report an efficient dynamic aperture (DA) optimization approach using multi-objective genetic algorithm (MOGA), which is driven by nonlinear driving terms computation. It was found that having small low order driving terms is a necessary but insufficient condition of having a decent DA. Then direct DA tracking simulation is implemented among the last generation candidates to select the best solutions. The approach was demonstrated successfully in optimizing NSLS-II storage ring DA.