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1.  Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis 
Journal of Bacteriology  2014;196(6):1143-1154.
In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.
doi:10.1128/JB.00739-13
PMCID: PMC3957716  PMID: 24363347
2.  Left Ventricular Aneurysm Repair with Use of a Bovine Pericardial Patch 
Texas Heart Institute Journal  2014;41(4):407-410.
Left ventricular aneurysm, which can impair systolic function, has a reported incidence of 10% to 35% in patients after myocardial infarction. In a 58-year-old woman who had a history of myocardial infarction, we excised a large left ventricular aneurysm and restored left ventricular geometry with use of a bovine pericardial patch. The aneurysm's characteristics and the patient's preoperative left ventricular ejection fraction of 0.25 had indicated surgical intervention. The patient had an uneventful postoperative course, and her left ventricular ejection fraction was 0.50 to 0.55 on the 4th postoperative day. This case illustrates the value of surgical treatment for patients who have a debilitating left ventricular aneurysm.
doi:10.14503/THIJ-13-3726
PMCID: PMC4120504  PMID: 25120394
Cardiac surgical procedures; heart aneurysm/prevention & control/surgery; heart ventricles/surgery; myocardial infarction/complications; treatment outcome; ventricular dysfunction, left/surgery
3.  Pentavalent Single-Domain Antibodies Reduce Campylobacter jejuni Motility and Colonization in Chickens 
PLoS ONE  2013;8(12):e83928.
Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.
doi:10.1371/journal.pone.0083928
PMCID: PMC3877120  PMID: 24391847
4.  Whole genome sequencing and comparative genomic analyses of two Vibrio cholerae O139 Bengal-specific Podoviruses to other N4-like phages reveal extensive genetic diversity 
Virology Journal  2013;10:165.
Background
Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group.
Methods
Host-range analysis and plaque morphology screening were done for both ϕJA1 and ϕVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed.
Results
The host specificity of ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ϕJA1 and ϕVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family.
Conclusions
The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.
doi:10.1186/1743-422X-10-165
PMCID: PMC3670811  PMID: 23714204
Vibrio cholerae O139; N4-like virus; Genome comparison; Terminal repeats; Intein; Phylogenetic relationship
5.  Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2 
PLoS ONE  2012;7(11):e48228.
In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.
doi:10.1371/journal.pone.0048228
PMCID: PMC3486847  PMID: 23133618
6.  Development of a high throughput assay for indirectly measuring phage growth using the OmniLogTM system 
Bacteriophage  2012;2(3):159-167.
The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. Here we describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. Here we show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, we show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.
doi:10.4161/bact.21440
PMCID: PMC3530525  PMID: 23275867
Bacillus anthracis; OmniLogTM; bacteriophage; in vitro lytic assay; phage
7.  Genome and Proteome of Campylobacter jejuni Bacteriophage NCTC 12673▿† 
Applied and Environmental Microbiology  2011;77(23):8265-8271.
Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.
doi:10.1128/AEM.05562-11
PMCID: PMC3233066  PMID: 21965409
8.  Alternative Medicine Techniques Have Non-Linear Effects on Radiation Response and Can Alter the Expression of Radiation Induced Bystander Effects 
Dose-Response  2012;11(1):82-98.
Many so-called “alternative medicine” techniques such as Reiki and acupuncture produce very good outcomes for intractable pain and other chronic illnesses but the efficacy is often dismissed as being psychosomatic. However a plausible mechanism does exist i.e. that the treatments alter the electromagnetic fields in living organisms and thereby prevent or reduce activity of neurons which lead to the pain. Low doses of ionising radiation have similar effects on electromagnetic fields and are known to induce signaling cascades in tissues due to ion gradients. To test this hypothesis cell cultures were exposed to Reiki – like and to acupuncture – like treatments, both performed by qualified practitioners. The cells were exposed either before or after the treatment to x-rays and were monitored for production of direct damage or bystander signals. The data suggest that the alternative techniques altered the response of cells to direct irradiation and altered bystander signal mechanisms. We conclude that alternative medicine techniques involving electromagnetic perturbations may modify the response of cells to ionizing radiation. In addition to the obvious implications for mechanistic studies of low dose effects, this could provide a novel target to exploit in radiation protection and in optimizing therapeutic gain during radiotherapy.
doi:10.2203/dose-response.11-048.Mothersill
PMCID: PMC3578456  PMID: 23550268
radiation-induced bystander effects; complementary and alternative medicine; Reiki; acupuncture; non-linear dose response; bioenergy
9.  Orally Administered P22 Phage Tailspike Protein Reduces Salmonella Colonization in Chickens: Prospects of a Novel Therapy against Bacterial Infections 
PLoS ONE  2010;5(11):e13904.
One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.
doi:10.1371/journal.pone.0013904
PMCID: PMC2989905  PMID: 21124920
10.  DNA Damage-Induced G1 Arrest in Hematopoietic Cells Is Overridden following Phosphatidylinositol 3-Kinase-Dependent Activation of Cyclin-Dependent Kinase 2 
Molecular and Cellular Biology  2001;21(18):6113-6121.
Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G1 checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that γ-irradiated murine myeloid 32D cells arrest in G1 with active cyclin D–cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21Cip1 and p27Kip1, yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G1 arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.
doi:10.1128/MCB.21.18.6113-6121.2001
PMCID: PMC87328  PMID: 11509654
11.  Effects of Miconazole and Dodecylimidazole on Sterol Biosynthesis in Ustilago maydis 
Miconazole at minimal fungitoxic concentrations inhibits ergosterol biosynthesis in sporidia of Ustilago maydis by interference with sterol C14 demethylation. The action is analogous to that of the fungicides triarimol and fenarimol. The fungicide 1-dodecylimidazole at low concentrations (0.1 to 0.25 μg/ml) inhibits sterol C14 demethylation; however, at higher concentrations (1.0 μg/ml or greater) it also inhibits 2,3-oxidosqualene cyclization and subsequent transmethylation. It is postulated that this diversity of effects of 1-dodecylimidazole results from binding of the inhibitor to sterol carrier protein(s).
PMCID: PMC352718  PMID: 464593

Results 1-11 (11)