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Articles by Mark Lawrence in JoVE

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Side Effects of Oceanic Iron Fertilization

Association of Actinobacillus Pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

The Edwardsiella Ictaluri O Polysaccharide Biosynthesis Gene Cluster and the Role of O Polysaccharide in Resistance to Normal Catfish Serum and Catfish Neutrophils

Edwardsiella ictaluri, the causative agent of enteric septicaemia of catfish (ESC), expresses long O polysaccharide (OPS) chains on its surface. The authors previously reported the construction of an isogenic Ed. ictaluri OPS mutant strain and demonstrated that this strain is avirulent in channel catfish. This paper reports the cloning of the Ed. ictaluri OPS biosynthesis gene cluster and identification of the mutated gene in the OPS-negative strain. The sequenced region contains eight complete ORFs and one incomplete ORF encoding LPS biosynthesis enzymes. The mutated gene (designated wbiT) was similar to other bacterial galactose-4-epimerases. Glycosyl composition analysis indicated that wild-type Ed. ictaluri OPS contains higher amounts of galactose and N-acetylgalactosamine than the OPS mutant strain, which correlated well with predicted functions of the genes identified in the OPS biosynthesis cluster. The OPS mutant had a relatively small, but significant, decrease in its ability to survive in normal catfish serum compared to wild-type Ed. ictaluri, but it retained the ability to resist killing by catfish neutrophils.

Comparison of Lipid and Non-lipid Markers of Atherosclerosis in Arterial Versus Venous Blood

Early studies have suggested that there may be differences in the concentration of lipoprotein particles and their associated apolipoproteins in arterial and venous blood and that this gradient might explain a proclivity to develop atherosclerotic lesions. The aim of this study was to use current methods of analysis to determine levels of these components, including particle densities and several common inflammatory markers in arterial and venous blood.

Identification of Listeria Innocua by PCR Targeting a Putative Transcriptional Regulator Gene

Listeria innocua is a common, non-pathogenic bacterial species that shares morphological, biochemical and molecular characteristics with the pathogenic species L. monocytogenes. The presence of L. innocua may cause difficulty or confusion in the laboratory identification of L. monocytogenes or other Listeria spp. In this report, through examining the recently published genome sequence of L. innocua strain CLIP 11262 (serovar 6a), we identified a L. innocua-specific gene (lin0464) encoding a putative transcriptional regulator and evaluated its efficacy for species-specific detection by polymerase chain reaction (PCR). The specificity of the oligonucleotide primers (lin0464F and lin0464R) derived from this gene was confirmed with the formation of a 749-bp fragment in PCR from genomic DNA of L. innocua strains only. We expect that this assay will be useful in confirming identification of L. innocua or in studies where rapid detection of L. innocua is necessary.

Alteration of DNA Adenine Methylase (Dam) Activity in Pasteurella Multocida Causes Increased Spontaneous Mutation Frequency and Attenuation in Mice

Pasteurella multocida is one of the primary bacterial pathogens associated with bovine respiratory disease (BRD) complex. Relatively few virulence factors of P. multocida have been characterized, and there is a need for improved vaccines for prevention of BRD. In other Gram-negative species, DNA adenine methylase (Dam) regulates the expression of virulence genes, and appropriate expression of Dam is required for virulence. In this study, the authors cloned and sequenced the P. multocida A1 dam gene and demonstrated that it is able to restore Dam function in an Escherichia coli dam mutant. When P. multocida dam was placed under the control of a constitutively expressed promoter on a plasmid, it caused an increased spontaneous mutation rate in P. multocida. In addition, the plasmid-mediated alteration of Dam production in P. multocida caused it to be highly attenuated in mice. These findings indicate that appropriate expression of Dam is required for virulence of P. multocida, which is believed to be the first report that Dam is required for virulence of a species in the Pasteurellaceae. Therefore, Dam may function as a virulence gene regulator in the Pasteurellaceae, similar to previously reported findings from other Gram-negative species.

Characterization of Virulent and Avirulent Listeria Monocytogenes Strains by PCR Amplification of Putative Transcriptional Regulator and Internalin Genes

Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

Assessment of Anticoagulation Using Activated Clotting Times in Patients Receiving Intravenous Enoxaparin During Percutaneous Coronary Intervention

Enoxaparin is being used more frequently in patients undergoing percutaneous coronary intervention (PCI). In this study, we determined the effect of intravenous enoxaparin on activated clotting time (ACT) measurements in the setting of PCI. In 67 consecutive patients, either 1 mg/kg intravenous enoxaparin alone was given for anticoagulation or 0.75 mg/kg given in patients receiving eptifibatide. ACT was measured before and 5 min following enoxaparin administration. After 1 mg/kg enoxaparin (n = 22), mean ACT increased from 122 +/- 22 to 199 +/- 20 sec. After 0.75 mg/kg enoxaparin and eptifibatide (n = 45), mean ACT increased from 125 +/- 22 to 194 +/- 24 sec. The mean increase in ACT was 77 +/- 26 sec in the 1 mg/kg group and 69 +/- 23 sec in the 0.75 mg/kg group (both P values < 0.0001). Moreover, in a subgroup of 26 patients, there was an excellent correlation (r = 0.86) between ACTs and the ENOX test, a new point-of-care test for assessing enoxaparin anticoagulation. None of the patients had transient abrupt closure, thrombus formation, major bleeding, or required urgent revascularization. Intravenous enoxaparin at clinically relevant doses with and without eptifibatide increases ACT levels at 5 min in patients undergoing PCI. These data suggest the ACT may be useful in the assessment of anticoagulation by enoxaparin.

Effectiveness and Safety of Manual Hemostasis Facilitated by the SyvekPatch with One Hour of Bedrest After Coronary Angiography Using Six-French Catheters

Manual hemostasis facilitated by a SyvekPatch with 1 hour of bedrest after coronary angiography using 6Fr catheters was evaluated in a study of 200 patients. There were no major adverse events and 2% minor adverse events, all of which were managed successfully with additional bedrest of 1 to 2 hours. The findings suggest that the 1-hour bedrest protocol using the SyvekPatch is safe and effective in low-risk patients.

Use of PCR Primers Derived from a Putative Transcriptional Regulator Gene for Species-specific Determination of Listeria Monocytogenes

Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.

Temporary Coronary Guidewire Pacing During Percutaneous Coronary Intervention

Temporary pacing is occasionally required during percutaneous coronary artery interventions. This can be accomplished by the insertion of a temporary transvenous pacemaker wire into the right ventricle, but there is some risk and inconvenience associated with this approach. Temporary pacing using the coronary artery guidewire was described in 1985 but is used infrequently. Using currently available equipment, we evaluated guidewire pacing in 26 patients. Guidewire pacing was successful in all patients, but not with all coronary guidewires at acceptable ventricular capture thresholds. No complications occurred from guidewire pacing. Bench testing of multiple different wires showed several with very high resistances likely unsuitable for clinical use. Temporary guidewire pacing is easily performed and should be considered as an alternative to the separate placement of a temporary transvenous pacemaker.

PCR Detection of a Putative N-acetylmuramidase Gene from Listeria Ivanovii Facilitates Its Rapid Identification

Listeria ivanovii is a Gram-positive bacterial pathogen that is capable of causing abortions and stillbirths in farm animals, particularly sheep and cattle. In terms of morphological, biochemical and molecular characteristics, L. ivanovii resembles other Listeria species such as L. monocytogenes, a pathogen of both man and animals. In this study, through comparative analysis of genomic DNA from the six Listeria species, a L. ivanovii specific clone (liv22-228) containing a 946 bp insert was isolated. This clone contained the 5' ends of two divergently transcribed L. ivanovii genes and an intergenic spacer region, similar in organization to homologous regions from the L. innocua and L. monocytogenes genomes. Regions of low homology in the clone were identified by comparing to the L. innocua and L. monocytogenes genomes, and oligonucleotide primers (liv22-228F and liv22-228R) were designed. These primers amplified a 463 bp band from genomic DNA of L. ivanovii strains only, but not from other Listeria species or common bacteria. Thus, PCR employing L. ivanovii specific primers (liv22-228F and liv22-228R) provides a useful and straightforward method for rapid and precise determination of L. ivanovii.

Specific PCR Identification of Pasteurella Multocida Based on Putative Transcriptional Regulator Genes

Pasteurella multocida is an important animal pathogen that may also infect humans through animal bites and scratches. After comparison of transcriptional regulator gene sequences from the P. multocida genome with other DNA sequences at GenBank, we identified two genes (i.e., Pm0762 and Pm1231) uniquely present in P. multocida. By using oligonucleotide primers (Pm0762F/R and Pm1231F/R) designed from these genes in PCR, it was found that specific DNA products of expected sizes were obtained with genomic DNA from P. multocida only, but not from other bacteria. These results indicated that the putative transcriptional regulator genes Pm0762 and Pm1231 are species-specific, and that the PCR methods targeting these genes provide a useful means of rapidly and precisely identifying P. multocida from other bacteria. Further elucidation of the roles and functions of these putative transcriptional regulator genes (Pm0762 and Pm1231) and their protein products may help provide valuable insight into the molecular mechanism of P. multocida virulence and pathogenicity.

Species-specific PCR Determination of Listeria Seeligeri

Listeria seeligeri is a non-pathogenic bacterium coming under the genus Listeria. As this bacterium resembles other Listeria species such as L. monocytogenes and L. ivanovii that are pathogenic to man and animals, it is important that rapid and precise identification techniques be available for L. seeligeri in cases where such determination is desirable. A specific molecular test on the basis of a uniquely present gene region in L. seeligeri will be of particular value under the circumstances. In this report, after comparative screening of genomic DNA from six Listeria species by dot blot hybridization, we isolated one L. seeligeri-specific clone (lse24-315) that contains an insert of 1538 bp. Using primers (lse24-315F and lse24-315R) derived from this clone, we showed that a specific PCR product of 375 bp was generated from genomic DNA of L. seeligeri strains only, but not of other Listeria species or common bacteria. Therefore, the PCR employing primers lse24-315F and lse24-315R provides a rapid, sensitive and specific method for distinguishing L. seeligeri from other Listeria and common bacteria.

Comparative Assessment of Acid, Alkali and Salt Tolerance in Listeria Monocytogenes Virulent and Avirulent Strains

Listeria monocytogenes is an opportunistic bacterial pathogen of man and animals that has the capacity to survive under extreme environmental conditions. While our knowledge on L. monocytogenes and its ability to sustain within wide pH and temperature ranges and salt concentrations has been largely built on the virulent strains of this species, relatively little is known about avirulent strains in this regard. In this study, we extend our analysis on avirulent L. monocytogenes strains. By subjecting three virulent (EGD, 874 and ATCC 19196) and three avirulent (ATCC 19114, HCC23 and HCC25) strains to various pH and salt concentrations, it was found that L. monocytogenes recovered well after treatment with 100 mM Tris at pH 12.0, and to a lesser extent at pH 3.0. Interestingly, avirulent L. monocytogenes strains showed a somewhat higher tolerance to alkali than virulent strains. This unique feature of avirulent L. monocytogenes strains may potentially be exploited for the development of a rapid technique for differentiation between avirulent and virulent strains. Furthermore, all L. monocytogenes strains tested were resistant to saturated NaCl (about 7 M, or 40% w/v) for a long period of time (20 h and possibly longer). Together, these results highlight that acid, alkali, and/or salt treatments commonly used in food product processing may not be sufficient to eliminate L. monocytogenes, and therefore stringent quality control measures at the beginning and end of the food manufacturing process is essential to ensure that such food products are free of listerial contamination.

Effects of Sub-minimum Inhibitory Concentration Antibiotic Levels and Temperature on Growth Kinetics and Outer Membrane Protein Expression in Mannheimia Haemolytica and Haemophilus Somnus

The objective of this study was to determine the effects of sub-minimum inhibitory concentrations (sub-MICs) of 2 veterinary antibiotic preparations, chlortetracycline (CTC) and chlortetracycline-sulfamethazine (CTC + SMZ), on growth kinetics and outer membrane protein expression in Mannheimia haemolytica and Haemophilus somnus at normal and febrile body temperatures. Sub-minimum inhibitory concentrations of both antibiotics reduced the growth rates of M. haemolytica and H. somnus. Growth of both species was not inhibited when grown at 41 degrees C compared to 37 degrees C. There was no detectable consistent effect of antibiotic or temperature on outer membrane protein expression for either species. Our study indicates that sub-MIC levels of CTC and CTC + SMZ markedly impair growth of clinical M. haemolytica and H. somnus isolates, potentially allowing more effective host clearance during infection.

Isolation and PCR Amplification of a Species-specific Oxidoreductase-coding Gene Region in Listeria Grayi

Listeria grayi is a nonpathogenic Gram-positive bacterium that demonstrates considerable similarities to other members in the genus Listeria, including the foodborne human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. A rapid diagnostic test to identify and diagnose listeriosis would be valuable, especially in cases where the presence of L. grayi may complicate diagnosis. This test would be based on a unique gene present in L. grayi. In this study, after comparative screening of a recombinant L. grayi DNA library by dot blot hybridization, an L. grayi specific clone (lgr20-246) with an insert of 722 bp was isolated. By applying PCR primers derived from a distinct region of the clone not shared by other bacteria, a specific band of 420 bp was amplified from the genomic DNA of L. grayi only and not of other Listeria species or common bacteria. These results suggest that the PCR assay employing primers lgr20-246F and lgr20-246R provides an independent and precise means of distinguishing L. grayi from other Listeria species and common bacteria. Therefore, it would be another useful technique for laboratory differentiation of Listeria bacteria.

Rapid Identification of Streptococcus Pyogenes with PCR Primers from a Putative Transcriptional Regulator Gene

Streptococcus pyogenes (GAS) is a common bacterial pathogen that has emerged as an increasingly important health concern in many parts of the world. Although GAS may appear harmless in healthy individuals, the ability of this bacterium to take advantage of a weakened or compromised host defense system is extraordinary. Following the recent publication of the genome sequences of several S. pyogenes strains, we undertook an investigation of a specialized gene group in GAS that encodes transcriptional regulators. By screening S. pyogenes transcriptional regulator genes from the complete genome of M1 strain SF370 against other DNA sequences at GenBank by BLAST searches, we identified a gene (i.e., Spy1258) that is uniquely present in the bacterium. Application of PCR primers (spy1258F and spy1258R) derived from this gene facilitated amplification of a 407-bp DNA fragment from S. pyogenes only, but not from other species of the genus Streptococcus and common bacteria. Apart from offering an additional target for specific confirmation of GAS, further analysis of the putative transcriptional regulator gene Spy1258 and its related protein product may lead to new insights into the molecular mechanisms of S. pyogenes maintenance and pathogenicity.

PCR Amplification of a Species-specific Putative Transcriptional Regulator Gene Reveals the Identity of Enterococcus Faecalis

Enterococcus faecalis, an opportunistic bacterial pathogen, has become prominent in recent decades due to its ability to develop resistance to various antibiotics. The ability to rapidly identify E. faecalis, followed by prompt treatment, is therefore vital for the control of this pathogen. In this report, upon comparative BLAST search of the E. faecalis genome against other bacterial DNA sequences at GenBank, an E. faecalis-specific gene Ef0027, that codes for a putative phosphosugar binding transcriptional regulator, was identified. Using PCR primers derived from this gene (i.e., Ef0027F/R), a specific DNA fragment of 518 bp was detected in E. faecalis strains only, and it was not found in other Enterococcus species and common bacteria. These results suggest that PCR amplification of the putative transcriptional regulator gene Ef0027 provides a useful means of identifying E. faecalis from other bacteria.

Gene Correction Reduces Cutaneous Inflammation and Granuloma Formation in Murine X-linked Chronic Granulomatous Disease

Our laboratory previously demonstrated that X-linked chronic granulomatous disease (X-CGD) mice develop exaggerated inflammatory responses and form granulomas following intradermal challenge with sterile Aspergillus fumigatus (AF) hyphae. In this study, we examined the efficacy of retroviral-mediated gene transfer (RMGT) into X-CGD bone marrow stem cells in preventing this abnormal inflammatory response. Sterile AF or saline was injected subcutaneously into the ears of wild-type, female X-CGD carrier, X-CGD, or X-CGD mice chimeric for varying numbers of either wild-type or RMGT-corrected neutrophils. Intradermal AF induced marked inflammation at both 3 and 30 d in the X-CGD mice, but not in the carriers or the wild-type mice. Similar to wild-type mice, chimeric X-CGD mice with >20% oxidase-positive neutrophils displayed a minimal and self-limited inflammatory response. Inflammation in chimeric (both wild-type and RMGT-corrected) mice with <15% oxidase-positive neutrophils was also improved compared to X-CGD mice, although still abnormal. This is the first evidence that partial correction of NADPH oxidase activity by gene therapy is likely to be beneficial in reducing or preventing the chronic inflammatory complications of CGD patients if sufficient numbers of RMGT-corrected neutrophils are obtained.

Assessing the Case for Mandatory Folate Fortification: Policy-making in the Face of Scientific Uncertainties

This paper presents the view that policy-makers face scientific uncertainties in assessing the case for mandatory folate fortification as a policy response to epidemiological evidence of the relationship between folate and neural tube defects. Moreover, the resolution of these uncertainties is confounded by the under-resourced state of nutrition information systems in Australia and New Zealand. The uncertainties relate to potential risks and benefits associated with the intervention for the target group and the population in general. These risks and benefits reflect the mismatch between evidence and policy that arises when addressing a presumed genetic abnormality in at-risk individuals with an intervention that is population-wide in its scope. There is an urgent need to conduct ongoing national nutrition surveys and monitor and evaluate policy interventions to strengthen the capacity of nutrition information systems to inform decision-making for this current, and future, public health nutrition policy.

Proteomic Analysis Using an Unfinished Bacterial Genome: the Effects of Subminimum Inhibitory Concentrations of Antibiotics on Mannheimia Haemolytica Virulence Factor Expression

Here we identify, using nonelectrophoretic proteomics, effects of subminimum inhibitory concentrations (subMIC) of two antibiotic preparations, chlortetracycline (CTC), and chlortetracycline-sulfamethazine (CTC + SMZ), on protein expression in the bovine respiratory pathogen Mannheimia haemolytica. The M. haemolytica genome is currently in draft form, and annotation is incomplete. Relying on the principle of gene sequence conservation across species, we used annotated genomes from closely related species to identify, confirm, and functionally annotate 495 M. haemolytica proteins. To conduct quantitative comparative proteomics, we developed a protein quantitation method based on the cross correlation function of the SEQUEST algorithm. When M. haemolytica was cultivated in the presence of 1/4 MIC of CTC and CTC + SMZ, expression of proteins involved in energy production, nucleotide metabolism, translation, and the bacterial stress response (chaperones) were affected. The most notable subMIC effect was a significant decrease in the expression of leukotoxin A, which is an important M. haemolytica virulence factor. Reduction in leukotoxin expression could be one of the molecular mechanisms responsible for the efficacy of these antibiotics against bovine respiratory disease.

Globalization, Food and Health in Pacific Island Countries

Pacific Island countries (PICs) are experiencing an epidemic of obesity and consequent chronic diseases. Despite investment in the development of National Plans of Action for Nutrition (NPANs) and interventions to promote healthy eating and physical activity, nutritional status appears to show little improvement. This paper presents a synthesis of the findings from two research papers that were prepared for a 2003 food safety and quality meeting in Nadi, Fiji. The findings indicate that although lifestyle behaviours might be the immediate cause of dietary imbalances, greater attention should focus on omnipresent influences of globalization as a critical element of the nutrition transition in the Pacific. In particular, those aspects of globalization mediated through the World Trade Organization (WTO) Agreements that are placing pressures on food security and fostering increased dependence on imported food of poor nutritional quality. Rapid, significant and sustainable improvements in public health in PICs require interventions that can tackle these underlying contributors to ill health. There are opportunities to explore the use of food regulatory approaches to influence the composition, availability and accessibility of food products. Within the context of the WTO Agreements the legitimacy of food regulatory approaches will depend upon the case to demonstrate the relationship between the intervention and the protection of food security and public health nutrition. The challenges in realising these opportunities are: 1) to have the capacity to construct a case, 2) meet the technical and financial demands to administer and enforce regulatory approaches, and 3) to take advantage of opportunities available and to be able to fully participate in the international policy-making process.

Challenges in Translating Scientific Evidence into Mandatory Food Fortification Policy: an Antipodean Case Study of the Folate-neural Tube Defect Relationship

To identify challenges in translating scientific evidence of a nutrient and health relationship into mandatory food fortification policy.

Listeria Monocytogenes Serotype 4b Strains Belonging to Lineages I and III Possess Distinct Molecular Features

A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

Effects of Subminimum Inhibitory Concentrations of Antibiotics on the Pasteurella Multocida Proteome

Subminimum inhibitory concentrations (sub-MICs) of antibiotics can be therapeutically effective, but the underlying molecular mechanisms are not well-characterized. We analyzed the Pasteurella multocida proteome response to sub-MICs of amoxicillin, chlortetracycline, and enrofloxacin using isotope-coded affinity tags (ICAT). There were parallel effects on inhibition of growth kinetics and suppression of protein expression by clusters of orthologous groups (COG) categories. Potential compensatory mechanisms enabling antibiotic adaptation were identified, including increased RecA expression caused by enrofloxacin.

PCR Detection of Pathogenic Leptospira Genomospecies Targeting Putative Transcriptional Regulator Genes

The genus Leptospira comprises multiple genomospecies that demonstrate varied pathogenic potential. The availability of rapid and precise diagnostic procedures to differentiate pathogenic from nonpathogenic Leptospira spp. is therefore essential to prevent an otherwise easily treatable malaise from developing into a life-threatening disease. In this report, we conducted an investigation on the diagnostic potential of Leptospira genes encoding putative transcriptional regulators. While PCR primers derived from transcriptional regulator gene la1137 recognized all 24 pathogenic Leptospira strains representing seven species, those from la1937, la3231, la3825, and la4130 detected 19 of the 24 Leptospira strains. However, none of these primers reacted with four nonpathogenic Leptospira species or other common bacteria. The putative transcriptional regulator genes la1137, la1937, la3231, la3825, and la4130 are present in pathogenic Leptospira strains, making them potential targets for diagnostic applications. Further characterization of these genes and their proteins may help elucidate the molecular mechanisms of leptospiral virulence and pathogenicity and pave the way for potential development of novel control strategies against leptospirosis.

Images in Cardiovascular Medicine. A 54-year-old Woman with Chest Pain, Dyspnea, and Inferior Injury on Electrocardiography

Development of Bioluminescent Edwardsiella Ictaluri for Noninvasive Disease Monitoring

Edwardsiella ictaluri is a facultative intracellular bacterium that causes enteric septicemia of catfish (ESC). In this study, we aimed to develop bioluminescent E. ictaluri that can be monitored by noninvasive bioluminescence imaging (BLI). To accomplish this, the luxCDABE operon of Photorhabdus luminescens was cloned downstream of the lacZ promoter in the broad host range plasmid pBBR1MCS4. Edwardsiella ictaluri strain 93-146 transformed with the new plasmid, pAKlux1, was highly bioluminescent. pAKlux1 was stably maintained in E. ictaluri without any apparent effect on growth or native plasmid stability. To assess the usefulness of the bioluminescent strain in disease studies, catfish were infected with 93-146 pAKlux1 by intraperitoneal injection and by bath immersion, and in vivo bacterial dissemination was observed using BLI. This study demonstrated that bioluminescent E. ictaluri can be used for real-time monitoring of ESC in live fish, which should enable observation of pathogen attachment sites and tissue predilections.

Evaluation of the Implementation of the Folate-neural Tube Defect Health Claim and Its Impact on the Availability of Folate-fortified Food in Australia

To evaluate the implementation of the folate-neural tube defect (NTD) health claim and its impact on the availability of folate-fortified food in Australia.

AgBase: a Functional Genomics Resource for Agriculture

Many agricultural species and their pathogens have sequenced genomes and more are in progress. Agricultural species provide food, fiber, xenotransplant tissues, biopharmaceuticals and biomedical models. Moreover, many agricultural microorganisms are human zoonoses. However, systems biology from functional genomics data is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation and agricultural research communities are smaller with limited funding compared to many model organism communities.

Listeria Monocytogenes Subgroups IIIA, IIIB, and IIIC Delineate Genetically Distinct Populations with Varied Pathogenic Potential

Listeria monocytogenes lineage III strains belonging to subgroups IIIA (n = 8), IIIB (n = 5), and IIIC (n = 6) were examined along with other known serotype strains (n = 11) by PCR and Southern hybridization using several recently described species-, virulence-, and serotype-specific primers and probes. The virulence of seven representative lineage III strains was then evaluated in mice via the intraperitoneal route. The results suggest that subgroup IIIA consists of typical rhamnose-positive avirulent serotype 4a and virulent serotype 4c strains, subgroup IIIC consists of atypical rhamnose-negative virulent serotype 4c strains, and subgroup IIIB consists of atypical rhamnose-negative virulent non-serotype 4a and non-serotype 4c strains, some of which may be related to serotype 7. It is possible that subgroup IIIB (including serotype 7) may represent a novel subspecies within L. monocytogenes.

Mandatory Folic Acid Fortification and the Science of 'sociality'

Broad Host Range Fluorescence and Bioluminescence Expression Vectors for Gram-negative Bacteria

Tagging of bacteria with living colors and living light allows increasingly valuable new imaging and detection technologies to be accessible to researchers. In this study, we aimed to create stable broad host range expression vectors for tagging Gram-negative bacteria with fluorescence and bioluminescence. To accomplish this, a mutated form of promoterless green fluorescent protein (gfp) gene, gfpmut3a, from Aequorea victoria and promoterless bacterial luciferase genes, luxCDABE, from Photorhabdus luminescens were inserted into broad host range plasmid pBBR1MCS4. Expression of gfp and luxCDABE genes was driven by lacZ promoter. In addition, dual versions with both gfpmut3a and luxCDABE genes and inducible versions carrying lacI(q) gene were also constructed. These new broad host range vectors containing a stable broad host range origin of replication and mobility genes can be transferred to Gram-negative bacteria by either electroporation or conjugal mating and maintained stably. Availability of these expression vectors should be useful in developing new approaches to study a broad variety of Gram-negative bacteria, particularly for applications investigating host-pathogen interactions in vivo and in vitro.

Mandatory Fortification with Folic Acid - What Would Hippocrates Say?

In October 2006, the Australian and New Zealand Food Regulation Ministerial Council asked for a review of the proposed food standard permitting mandatory fortification of bread with folic acid. This article contributes to the policy debate associated with the standard's review by discussing the potential benefits and risks to the target population and the wider Australian population with emphasis on recent (2006) literature.

Characteristics of Cell-mediated, Anti-listerial Immunity Induced by a Naturally Avirulent Listeria Monocytogenes Serotype 4a Strain HCC23

The characteristics of cell-mediated, anti-listerial immune response initiated by an avirulent Listeria monocytogenes serotype 4a strain HCC23 was assessed. Similar to virulent strain EGD, avirulent strain HCC23 grew readily within macrophage-like J774 cells, but nonhemolytic strain ATCC 15313 did not. Compared with EGD, HCC23 induced a relatively low level of gamma interferon (IFN-gamma) in mice, and ATCC 15313 stimulated no detectable IFN-gamma. The percentages of gated CD4 T cells from mice immunized with EGD and HCC23 showed a notable drop (to 30%) at 21 days post exposure in comparison with that (about 50%) from ATCC 15313-injected or untreated mice; and the percentage of gated NK cells from EGD-immunized group was markedly higher than those from other treatment groups. Mice immunized with HCC23 and EGD developed an equally strong protective immunity against listeriosis that was effective in both short and long terms, but those injected with ATCC 15313 or saline succumbed to listeriosis within 6 days of challenge.

Synthetic Folic Acid Vs. Food Folates

Toward an Improved Laboratory Definition of Listeria Monocytogenes Virulence

Listeria monocytogenes is an opportunistic foodborne pathogen that encompasses a diversity of strains with varied virulence. The ability to rapidly determine the pathogenic potential of L. monocytogenes strains is integral to the control and prevention campaign against listeriosis. Early methods for assessing L. monocytogenes virulence include in vivo bioassays and in vitro cell assays. While in vivo bioassays provide a measurement of all virulence determinants of L. monocytogenes, they are not applied routinely due to their reliance on experimental animals whose costs have become increasingly prohibitive. As a low cost alternative, in vitro cell assays are useful for estimating the virulence of L. monocytogenes strains. However, these assays are often slow, and at times variable. Prior attempts to ascertain L. monocytogenes virulence by targeting virulence-associated proteins and genes have been largely unsuccessful, since many of the assay targets are present in both virulent and avirulent strains. Recent identification of novel virulence-specific genes (particularly internalin gene inlJ) has opened a new avenue for rapid, sensitive, and precise differentiation of virulent L. monocytogenes strains from avirulent strains. The application of DNA sequencing technique also offers an additional tool for assessing L. monocytogenes virulence potential. By providing an update on the laboratory methods that have been reported for the determination of L. monocytogenes pathogenicity, this review discusses future research needs that may help achieve an improved laboratory definition of L. monocytogenes virulence.

A Multiplex PCR for Species- and Virulence-specific Determination of Listeria Monocytogenes

Listeria monocytogenes internalin gene inlJ has been described previously for differentiation of virulent from avirulent strains. However, a recent report indicated that there exist some unusual lineage IIIB strains (e.g., serotype 7 strain R2-142) that possess no inlJ gene but have the capacity to cause mouse mortality via intraperitoneal inoculation. Therefore, a multiplex PCR incorporating inlA, inlC and inlJ gene primers was developed in this study for rapid speciation and virulence determination of L. monocytogenes. Although inlB gene was also assessed for species-specific recognition, it was not included in the multiplex PCR due to the negative reaction observed between the inlB primers and serotypes 4a-e strains. The species identity of the 36 L. monocytogenes strains under investigation was verified through the amplification of an 800 bp fragment with the inlA primers and the virulence of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ primers, respectively. Whereas L. monocytogenes pathogenic strains with capacity to cause mortality (showing relative virulence of 30-100%) in A/J mice via the intraperitoneal route were invariably detected by the inlC and/or inlJ primers, naturally non-pathogenic strains (showing relative virulence of 0%) were negative with these primers. While 8 of the 10 L. ivanovii strains reacted with the inlC primers, they could be effectively excluded as non-L. monocytogenes through their negative reactions with the inlA primers in the multiplex PCR. Thus, the use of the multiplex PCR targeting inlA, inlC and inlJ genes facilitates simultaneous confirmation of L. monocytogenes species identity and virulence.

Urinary Levels of Matrix Metalloproteinase 9 and 2 and Tissue Inhibitor of Matrix Metalloproteinase in Patients with Coronary Artery Disease

To evaluate the feasibility of an assay for urinary levels of matrix metalloproteinases (MMPs) and the potential usefulness of urinary MMPs as a marker of coronary atherosclerosis or acute coronary syndromes (ACS).

Improved Pulsed-field Gel Electrophoresis Procedure for the Analysis of Flavobacterium Columnare Isolates Previously Affected by DNA Degradation

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host.

Development of Bioluminescent Salmonella Strains for Use in Food Safety

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, we developed bioluminescent Salmonella strains that can be used for real-time monitoring of the pathogen's growth on food products. To accomplish this, twelve Salmonella strains from the broiler production continuum were transformed with the broad host range plasmid pAKlux1, and a chicken skin attachment model was developed.

Conceptualising the Policy Practice and Behavioural Research Relationship

Policy is frequently identified in the behavioural nutrition and physical activity research literature as a necessary component of effective research and practice. The purpose of this commentary is to promote a dialogue to contribute towards the further development of conceptual understandings and theories of the relationship between policy practice and behavioural research and how these two activities might work synergistically to improve public health outcomes.

Hypoxemia Secondary to Right-to-left Interatrial Shunt Through a Patent Foramen Ovale in a Patient with an Elevated Right Hemidiaphragm

Though uncommon, right-to-left shunt through a patent foramen ovale with normal right-side pressure and with a normal interatrial pressure gradient has been reported. The speculated pathophysiology is attributed to directional blood flow streaming from the vena cava to the left atrium. Hypoxemia secondary to right-to-left shunt with normal pulmonary artery pressure has been extensively documented after right pneumonectomy. Five prior cases have documented hypoxemia secondary to a right-to-left shunt through a patent foramen ovale in the presence of an elevated right hemidiaphragm. This is the sixth documented case of right-to-left shunt through a patent foramen ovale in the presence of an elevated right hemidiaphragm with a similar presentation in which closure of the patent foramen ovale resulted in resolution of hypoxemia.

Accelerating String Set Matching in FPGA Hardware for Bioinformatics Research

This paper describes techniques for accelerating the performance of the string set matching problem with particular emphasis on applications in computational proteomics. The process of matching peptide sequences against a genome translated in six reading frames is part of a proteogenomic mapping pipeline that is used as a case-study. The Aho-Corasick algorithm is adapted for execution in field programmable gate array (FPGA) devices in a manner that optimizes space and performance. In this approach, the traditional Aho-Corasick finite state machine (FSM) is split into smaller FSMs, operating in parallel, each of which matches up to 20 peptides in the input translated genome. Each of the smaller FSMs is further divided into five simpler FSMs such that each simple FSM operates on a single bit position in the input (five bits are sufficient for representing all amino acids and special symbols in protein sequences).

Effects of Subminimum Inhibitory Concentrations of Antibiotics on the Pasteurella Multocida Proteome: a Systems Approach

To identify key regulators of subminimum inhibitory concentration (sub-MIC) antibiotic response in the Pasteurella multocida proteome, we applied systems approaches. Using 2D-LC-ESI-MS(2), we achieved 53% proteome coverage. To study the differential protein expression in response to sub-MIC antibiotics in the context of protein interaction networks, we inferred P. multocida Pm70 protein interaction network from orthologous proteins. We then overlaid the differential protein expression data onto the P. multocida protein interaction network to study the bacterial response. We identified proteins that could enhance antimicrobial activity. Overall compensatory response to antibiotics was characterized by altered expression of proteins involved in purine metabolism, stress response, and cell envelope permeability.

Quantitative Analysis of Streptococcus Pneumoniae TIGR4 Response to in Vitro Iron Restriction by 2-D LC ESI MS/MS

Understanding the growth of bacterial pathogens in a micronutrient restricted host environment can identify potential virulence proteins that help overcome this nutritional barrier to productive infection. In this study, we investigated the pneumococcal protein expression response to iron limitation using an in vitro model. We identified S. pneumoniae TIGR4 proteins by 2-D LC ESI MS/MS and determined significant changes in protein expression in response to iron restriction using computer-intensive random resampling methods. Differential protein expression was studied in the context of a S. pneumoniae TIGR4 protein interaction network using Pathway Studio. Our analysis showed that pneumococcal iron restriction response was marked by increased expression of known virulence factors like PsaA. It involved changes in the expression of stress response, and phase variation and biofilm formation proteins. The net effect of changes in all these biological processes could increase the virulence of S. pneumoniae TIGR4 during in vivo infection.

A Systematic Policy Approach to Changing the Food System and Physical Activity Environments to Prevent Obesity

As obesity prevention becomes an increasing health priority in many countries, including Australia and New Zealand, the challenge that governments are now facing is how to adopt a systematic policy approach to increase healthy eating and regular physical activity. This article sets out a structure for systematically identifying areas for obesity prevention policy action across the food system and full range of physical activity environments. Areas amenable to policy intervention can be systematically identified by considering policy opportunities for each level of governance (local, state, national, international and organisational) in each sector of the food system (primary production, food processing, distribution, marketing, retail, catering and food service) and each sector that influences physical activity environments (infrastructure and planning, education, employment, transport, sport and recreation). Analysis grids are used to illustrate, in a structured fashion, the broad array of areas amenable to legal and regulatory intervention across all levels of governance and all relevant sectors. In the Australian context, potential regulatory policy intervention areas are widespread throughout the food system, e.g., land-use zoning (primary production within local government), food safety (food processing within state government), food labelling (retail within national government). Policy areas for influencing physical activity are predominantly local and state government responsibilities including, for example, walking and cycling environments (infrastructure and planning sector) and physical activity education in schools (education sector). The analysis structure presented in this article provides a tool to systematically identify policy gaps, barriers and opportunities for obesity prevention, as part of the process of developing and implementing a comprehensive obesity prevention strategy. It also serves to highlight the need for a coordinated approach to policy development and implementation across all levels of government in order to ensure complementary policy action.

Genome Sequence of the Chemolithoautotrophic Bacterium Oligotropha Carboxidovorans OM5T

Oligotropha carboxidovorans OM5(T) (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium with the capability to utilize carbon monoxide, carbon dioxide, and hydrogen. It is also capable of heterotrophic growth under appropriate environmental conditions. Here we report the annotated genome sequence of the circular chromosome of this organism.

Guidewire Pacing Safely and Effectively Treats Bradyarrhythmias Induced by Rheolytic Thrombectomy and Precludes the Need for Transvenous Pacing: the Scott & White Experience

Rheolytic thrombectomy (RT) is useful in certain percutaneous coronary interventions but may be associated with transient bradyarrhythmias. Clinicians have devised numerous strategies to deal with these arrhythmias apart from transvenous right ventricular pacing, some of which are described in other parts of this supplement. We report the Scott & White experience utilizing guidewire pacing to quickly and safely pace the heart in the event of bradyarrhythmia. We found this method to be safe and reliable (96.2% successful) during RT and now use this technique almost exclusively in the cardiac catheterization lab to deal with transient bradyarrhythmias during RT or due to any other cause.We also report an increased incidence of bradyarrhythmia occurring during RT when it is performed in the right coronary artery, with a trend toward an increased incidence during the clinical presentation of ST-elevation myocardial infarction.

Comparative Proteomic Analysis of Listeria Monocytogenes Strains F2365 and EGD

Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37 degrees C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.

Public Health Nutrition. Dietary Guidelines. We Have Traction in Australia

High-throughput Bioluminescence-based Mutant Screening Strategy for Identification of Bacterial Virulence Genes

A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.

The Politics of Developing Reference Standards for Nutrient Intakes: the Case of Australia and New Zealand

Nutrient Reference Values (NRV) are evidence-based benchmarks for assessing the dietary adequacy of individuals and population groups as well as informing public health nutrition policies and programmes. The present paper presents the findings of an analysis of the views of submitters to a draft document associated with the development of the 2006 NRV for Australia and New Zealand. The aim of the study was to explore how these views were reflected in the policy-making process and final policy document.

Proteome Analysis of Edwardsiella Ictaluri

Edwardsiella ictaluri is a facultative intracellular Gram-negative bacterium causing enteric septicemia of catfish (ESC), the most prevalent disease affecting farm-raised channel catfish in the United States. Despite its economic importance, studies addressing high-throughput proteomics were not possible because of lack of comprehensive protein database. Here, we report the first high-throughput proteomics analysis of E. ictaluri using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS. Proteins identified in this study and predicted from the whole E. ictaluri genome were clustered into functional groups using clusters of orthologous groups (COG), and their subcellular locations were predicted. Possible functional relationships among proteins were determined using pathway analysis. The total number of unique E. ictaluri proteins identified using both 2-D LC and 2-DE approaches was 788, of which 15.48% (122) were identified by both methods while 78.43% (618) and 6.09% (48) were unique in 2-D LC and 2-DE, respectively. COG groupings and subcellular localizations were quite similar between our data set and proteins predicted from the whole genome. Twelve pathways were significantly represented in our dataset (p <0.05). Results from this study provided experimental evidence for many proteins that were predicted from the E. ictaluri genome annotation, and they should accelerate future functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.

Functional Neurons and Melanocytes Induced from Immortal Lines of Postnatal Neural Crest-like Stem Cells

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.

Building Global Alliances for Public Health Nutrition Training

The purpose of this paper is to present a review of opportunities and challenges for future progress in building intercountry, regional, and global alliances for public health nutrition training. Drawing on experiences from developing, implementing, and evaluating public health nutrition training in Australasia, Europe, and the Middle East, suggestions are provided for building a network of global training activities. Opportunities are described in areas such as standardization of course competencies and registration schemes, resource sharing, student and trainer exchange programs, and professional development. Challenges are identified and options presented for building global alliances in public health nutrition training into the future.

Examination of Selected National Policies Towards Mandatory Folic Acid Fortification

The purpose of this paper is to present an examination of the contrasting policies towards mandatory folic acid fortification in six countries from different regions of the world. Three questions are addressed: 1) What is the policy of the country? 2) Why was the policy adopted? 3) What lessons have been learned? Policy contrasts among countries were assessed as reflecting different interpretations of the potential risks and benefits associated with folic acid fortification. Although commonalities were identified, it was considered unlikely that there could be a standard policy response for all countries. Instead, a country-by-country policy response based on national circumstances is indicated.

Food Guides. A Compromise Solution

The Transcriptional Response of Pasteurella Multocida to Three Classes of Antibiotics

Pasteurella multocida is a gram-negative bacterial pathogen that has a broad host range. One of the diseases it causes is fowl cholera in poultry. The availability of the genome sequence of avian P. multocida isolate Pm70 enables the application of functional genomics for observing global gene expression in response to a given stimulus. We studied the effects of three classes of antibiotics on the P. multocida transcriptome using custom oligonucleotide microarrays from NimbleGen Systems. Hybridizations were conducted with RNA isolated from three independent cultures of Pm70 grown in the presence or absence of sub-minimum inhibitory concentration (sub-MIC) of antibiotics. Differentially expressed (DE) genes were identified by ANOVA and Dunnett's test. Biological modeling of the differentially expressed genes (DE) was conducted based on Clusters of Orthologous (COG) groups and network analysis in Pathway Studio.

Moving Beyond 'rates, Roads and Rubbish': How Do Local Governments Make Choices About Healthy Public Policy to Prevent Obesity?

While the causes of obesity are well known traditional education and treatment strategies do not appear to be making an impact. One solution as part of a broader complimentary set of strategies may be regulatory intervention at local government level to create environments for healthy nutrition and increased physical activity. Semi structured interviews were conducted with representatives of local government in Australia. Factors most likely to facilitate policy change were those supported by external funding, developed from an evidence base and sensitive to community and market forces. Barriers to change included a perceived or real lack of power to make change and the complexity of the legislative framework. The development of a systematic evidence base to provide clear feedback on the size and scope of the obesity epidemic at a local level, coupled with cost benefit analysis for any potential regulatory intervention, are crucial to developing a regulatory environment which creates the physical and social environment required to prevent obesity.

Do Food Regulatory Systems Protect Public Health?

Gene Model Detection Using Mass Spectrometry

The utility of a genome sequence in biological research depends entirely on the comprehensive description of all of its functional elements. Analysis of genome sequences is still predominantly gene-centric (i.e., identifying gene models/open reading frames). In this article, we describe a proteomics-based method for identifying open reading frames that are missed by computational algorithms. Mass spectrometry-based identification of peptides and proteins from biological samples provide evidence for the expression of the genome sequence at the protein level. This proteogenomic annotation method combines computationally predicted ORFs and the genome sequence with proteomics to identify novel gene models. We also describe our proteogenomic mapping pipeline - a set of computational tools that automate the proteogenomic annotation work flow. This pipeline is available for download at www.agbase.msstate.edu/tools/ .

Pyrosequencing of the Vir Plasmid of Necrotoxigenic Escherichia Coli

Necrotoxigenic Escherichia coli, or NTEC, are defined as E. coli producing the toxin known as cytotoxic necrotizing factor, or CNF. NTEC are responsible for various diseases of humans and animals, including urinary tract infection, septicemia, and diarrhea. A subgroup of NTEC known as NTEC-2 produce a variant of CNF (CNF-2) whose gene is located on a plasmid known as Vir. Because of its involvement in NTEC-2 pathogenesis and its broad distribution among production animals, a Vir plasmid from a bovine NTEC-2 strain was sequenced and analyzed. This plasmid was found to belong to the RepFIB and RepFIIA replicon types, and it totaled 138,682 base pairs in size. Within this plasmid was an approximately 60-kb pathogenicity island, defined by its possession of multiple virulence factors within distinct genetic regions of lower G+C content bounded by inverted repeats. Within this PAI were a variety of putative virulence factors, including F17b fimbrial genes, genes of a novel fimbrial operon, tibAC, hemolysins, and the cnf2 and cdt toxin-encoding genes. The occurrence of this plasmid's virulence- and replication-associated genes was sought among a collection of 96 CNF-2-positive isolates. The most prevalent genes among this collection included repA (RepFIB), cnf2, an ompP homolog, and the tib-AC genes encoding for aggregation and biofilm formation. The Vir plasmid has evolved from an IncFIB ancestral backbone, with the RepFIB locus apparently driving the acquisition of its accessory virulence-associated elements via site-specific recombination. Overall, the completed sequence of a Vir plasmid increases our understanding of NTEC-2 pathogenomics and IncFIB plasmid evolution.

Evidence-informed Process to Identify Policies That Will Promote a Healthy Food Environment in the Pacific Islands

To implement a systematic evidence-informed process to enable Fiji and Tonga to identify the most feasible and targeted policy interventions which would have most impact on diet-related non-communicable diseases.

Lymphocutaneous Nocardiosis: a Case Report and Review of the Literature

Nocardiosis remains a fairly uncommon disease in the United States. Cutaneous nocardiosis is one of many infections that can spread in a sporotrichoid pattern and therefore can be difficult to diagnose without a high index of suspicion. It is mainly caused by Nocardia asteroides and Nocardia brasiliensis, with N brasiliensis isolated in most cases of lymphocutaneous nocardiosis. We present a case of lymphocutaneous nocardiosis in a 65-year-old immunosuppressed man and review the literature.

Identification of Novel Non-coding Small RNAs from Streptococcus Pneumoniae TIGR4 Using High-resolution Genome Tiling Arrays

The identification of non-coding transcripts in human, mouse, and Escherichia coli has revealed their widespread occurrence and functional importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have shown that non-coding transcripts participate in a broad range of cellular functions like gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an obligate human respiratory pathogen responsible for significant worldwide morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as well as identification of novel transcripts at a high-resolution.

Proteomic Analysis of the Fish Pathogen Flavobacterium Columnare

Flavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.

Development of Stable Reporter System Cloning LuxCDABE Genes into Chromosome of Salmonella Enterica Serotypes Using Tn7 Transposon

Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum.

Genome Sequence of the Solvent-producing Bacterium Clostridium Carboxidivorans Strain P7T

Clostridium carboxidivorans strain P7(T) is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO(2) fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate.

If Bystander Effects for Apoptosis Occur in Spleen After Low-Dose Irradiation In Vivo then the Magnitude of the Effect Falls Within the Range of Normal Homeostatic Apoptosis

Abstract To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by 1.4% 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.

Complete Genome and Comparative Analysis of the Chemolithoautotrophic Bacterium Oligotropha Carboxidovorans OM5

Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium capable of utilizing CO (carbon monoxide) and fixing CO2 (carbon dioxide). We previously published the draft genome of this organism and recently submitted the complete genome sequence to GenBank.

If Bystander Effects for Apoptosis Occur in Spleen After Low-dose Irradiation in Vivo then the Magnitude of the Effect Falls Within the Range of Normal Homeostatic Apoptosis

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by five- to sevenfold 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.

Proteome and Membrane Fatty Acid Analyses on Oligotropha Carboxidovorans OM5 Grown Under Chemolithoautotrophic and Heterotrophic Conditions

Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H(2) to derive energy for fixation of CO(2). Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H(2) generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H(2) as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes.

Importance of Skin Abrasion As a Primary Site of Adhesion for Edwardsiella Ictaluri and Impact on Invasion and Systematic Infection in Channel Catfish Ictalurus Punctatus

The route of entry of Edwardsiella ictaluri into its catfish host has been a subject of investigation since the pathogen was first discovered. There is evidence to support entry through the intestinal tract, the nares, and the gills. Here, we evaluated the role of skin abrasion through a series of experimental challenges using bioluminescent E. ictaluri carrying the plasmid pAKLux1. Our results show that E. ictaluri is able to colonize abrasion sites on catfish skin and that catfish with abrasions developed systematic infection faster. We also found that abrasions are associated with significantly increased mortalities following experimental immersion exposure. Finally, a protocol was developed during this study that allowed for immunohistochemical examination of the tissue layers underneath the abrasion sites, confirming the presence of E. ictaluri in subdermal tissues from abrasion sites. This study constitutes the first report on the role of channel catfish skin as a portal of entry for E. ictaluri and further illustrates how versatile this pathogen can be in its mechanisms of entry.

The Proteogenomic Mapping Tool

High-throughput mass spectrometry (MS) proteomics data is increasingly being used to complement traditional structural genome annotation methods. To keep pace with the high speed of experimental data generation and to aid in structural genome annotation, experimentally observed peptides need to be mapped back to their source genome location quickly and exactly. Previously, the tools to do this have been limited to custom scripts designed by individual research groups to analyze their own data, are generally not widely available, and do not scale well with large eukaryotic genomes.

Policy Change to Create Supportive Environments for Physical Activity and Healthy Eating: Which Options Are the Most Realistic for Local Government?

The objective is to identify and test regulatory options for creating supportive environments for physical activity and healthy eating among local governments in Victoria, Australia. A literature review identified nine potential areas for policy intervention at local government level, including the walking environment and food policy. Discussion documents were drafted which summarized the public health evidence and legal framework for change in each area. Levels of support for particular interventions were identified through semi-structured interviews conducted with key informants from local government. We conducted 11 key informant interviews and found support for policy intervention to create environments supportive of physical activity but little support for policy changes to promote healthy eating. Participants reported lack of relevance and competing priorities as reasons for not supporting particular interventions. Promoting healthy eating environments was not considered a priority for local government above food safety. There is a real opportunity for action to prevent obesity at local government level (e.g. mandate the promotion of healthy eating environments). For local government to have a role in the promotion of healthy food environments, regulatory change and suitable funding are required.

Climate Change Adaptation at the Intersection of Food and Health

Nutritious, safe, affordable, and enjoyable food is a fundamental prerequisite for health. As a nation, Australia is currently classified as food secure with the domestic production exceeding domestic consumption of most major food groups. The domestic system is almost self-sufficient in terms of nutritious plant foods, although these foods have seen steady higher price increases relative to other foods, with nutrition equity implications. However, the viability of Australia's food security sits counter to the continued presence of a stable and supportive climate. This article reviews the current state of science concerning the interface between climate change, food systems, and human health to reveal the key issues that must be addressed if Australia is to advance human health and sustainable food systems under a changing climate.

Genome Sequence of Lineage III Listeria Monocytogenes Strain HCC23

More than 98% of reported human listeriosis cases are caused by Listeria monocytogenes serotypes within lineages I and II. Serotypes within lineage III (4a and 4c) are commonly isolated from environmental and food specimens. We report the first complete genome sequence of a lineage III isolate, HCC23, which will be used for comparative analysis.

Proteomic Expression Profiles of Virulent and Avirulent Strains of Listeria Monocytogenes Isolated from Macrophages

Listeria monocytogenes is able to survive and proliferate within macrophages. In the current study, the ability of three L. monocytogenes strains (serovar 1/2a strain EGDe, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1 was analyzed. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGDe and F2365 proliferated within macrophages for at least 7 h. We further analyzed these strains by comparing their protein expression profiles at 0 h, 3 h, and 5 h post-infection using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. Our results indicated that similar metabolic and cell wall associated proteins were expressed by all three strains at 3 h post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5 h post-infection. By comparing the protein expression patterns of these three L. monocytogenes strains during intracellular growth in macrophages, we were able to detect biological differences that may determine the ability of L. monocytogenes to survive in macrophages.

TAAPP: Tiling Array Analysis Pipeline for Prokaryotes

High-density tiling arrays provide closer view of transcription than regular microarrays and can also be used for annotating functional elements in genomes. The identified transcripts usually have a complex overlapping architecture when compared to the existing genome annotation. Therefore, there is a need for customized tiling array data analysis tools. Since most of the initial tiling arrays were conducted in eukaryotes, data analysis methods are well suited for eukaryotic genomes. For using whole-genome tiling arrays to identify previously unknown transcriptional elements like small RNA and antisense RNA in prokaryotes, existing data analysis tools need to be tailored for prokaryotic genome architecture. Furthermore, automation of such custom data analysis workflow is necessary for biologists to apply this powerful platform for knowledge discovery. Here we describe TAAPP, a web-based package that consists of two modules for prokaryotic tiling array data analysis. The transcript generation module works on normalized data to generate transcriptionally active regions (TARs). The feature extraction and annotation module then maps TARs to existing genome annotation. This module further categorizes the transcription profile into potential novel non-coding RNA, antisense RNA, gene expression and operon structures. The implemented workflow is microarray platform independent and is presented as a web-based service. The web interface is freely available for academic use at http://lims.lsbi.mafes.msstate.edu/TAAPP-HTML/.

Multicenter Phase II Trial of Enzastaurin in Patients with Relapsed or Refractory Advanced Cutaneous T-cell Lymphoma

This multicenter, single-arm, open-label non-randomized phase II trial (NCT00744991) was conducted in patients with recurrent/refractory mycosis fungoides (MF), stage IB-IVB, or Sézary syndrome (SS). A Simon two-stage design required 25 patients enrolled in stage 1 with ≥7 confirmed objective responses for expansion into stage 2. Patients were treated with oral enzastaurin (250 mg twice daily) until disease progression or intolerable toxicity. The primary endpoint was investigator-assessed response rate; secondary endpoints were time to objective response, response duration, time-to-progression, patient-reported pruritus, and safety/tolerability. Twenty-five patients were enrolled. A partial response was observed in one patient with MF. Median time-to-progression was 78 and 44 days in MF and SS, respectively. Self-reported pruritus relief and improved composite pruritus-specific symptom scores were documented in six and four patients, respectively. Enzastaurin was well tolerated with mostly grade 1-2 adverse events, mainly diarrhea and fatigue. There were two adverse event-related drug discontinuations with one possibly treatment-related.

Migratory Pathways and Connectivity in Asian Houbara Bustards: Evidence from 15 Years of Satellite Tracking

Information on migratory pathways and connectivity is essential to understanding population dynamics and structure of migrant species. Our manuscript uses a unique dataset, the fruit of 103 individual Asian houbara bustards captured on their breeding grounds in Central Asia over 15 years and equipped with satellite transmitters, to provide a better understanding of migratory pathways and connectivity; such information is critical to the implementation of biologically sound conservation measures in migrant species. At the scale of the distribution range we find substantial migratory connectivity, with a clear separation of migration pathways and wintering areas between western and eastern migrants. Within eastern migrants, we also describe a pattern of segregation on the wintering grounds. But at the local level connectivity is weak: birds breeding within the limits of our study areas were often found several hundreds of kilometres apart during winter. Although houbara wintering in Arabia are known to originate from Central Asia, out of all the birds captured and tracked here not one wintered on the Arabian Peninsula. This is very likely the result of decades of unregulated off-take and severe habitat degradation in this area. At a time when conservation measures are being implemented to safeguard the long-term future of this species, this study provides critical data on the spatial structuring of populations.

Cutaneous Malignancy in Adolescents

There is compelling epidemiologic evidence that cutaneous malignancies, most notably malignant melanoma and cutaneous T-cell lymphoma, are increasing in incidence. The adolescent population is also affected by this rise in incidence, but can represent both a unique diagnostic and therapeutic challenge. Herein we present up-to-date epidemiology, clinical presentation, risk factors for development, and management options for malignant melanoma, basal cell carcinoma, squamous cell carcinoma, and cutaneous T-cell lymphoma as each pertains to the adolescent population. Prevention in this age group is particularly addressed. One unifying theme that emerges is that a high degree of clinical suspicion and vigilance must be maintained to recognize these entities early on in their presentations.

Food and Nutrition Security in the Australia-New Zealand Region: Impact of Climate Change

Megacity Emissions and Lifetimes of Nitrogen Oxides Probed from Space

Megacities are immense sources of air pollutants, with large impacts on air quality and climate. However, emission inventories in many of them still are highly uncertain, particularly in developing countries. Satellite observations allow top-down estimates of emissions to be made for nitrogen oxides (NO(x) = NO + NO(2)), but require poorly quantified a priori information on the NO(x) lifetime. We present a method for the simultaneous determination of megacity NO(x) emissions and lifetimes from satellite measurements by analyzing the downwind patterns of NO(2) separately for different wind conditions. Daytime lifetimes are ~4 hours at low and mid-latitudes, but ~8 hours in wintertime for Moscow. The derived NO(x) emissions are generally in good agreement with existing emission inventories, but are higher by a factor of 3 for the Saudi Arabian capital Riyadh.

Estimating the Impact of Mandatory Folic Acid Fortification on the Folic Acid Intake of Australian Women of Childbearing Age

The primary aim of this study was to estimate the impact of mandatory folic acid (FA) fortification of bread-making flour on the FA intake of Australian women of childbearing age (16-44 years). The secondary objective was to investigate the relationship between estimated FA intake and socio-economic status (SES) and age.

Balancing the Benefits and Risks of Public-private Partnerships to Address the Global Double Burden of Malnutrition

Transnational food, beverage and restaurant companies, and their corporate foundations, may be potential collaborators to help address complex public health nutrition challenges. While UN system guidelines are available for private-sector engagement, non-governmental organizations (NGO) have limited guidelines to navigate diverse opportunities and challenges presented by partnering with these companies through public-private partnerships (PPP) to address the global double burden of malnutrition.

Genetic Differentiation Among Migrant and Resident Populations of the Threatened Asian Houbara Bustard

The Asian houbara bustard Chlamydotis macqueenii is a partial migrant of conservation concern found in deserts of central Asia and the Middle East. In the southern part of the species range, resident populations have been greatly fragmented and reduced by sustained human pressure. In the north, birds migrate from breeding grounds between West Kazakhstan and Mongolia to wintering areas in the Middle East and south central Asia. Extensive satellite tracking has shown substantial partitioning in migration routes and wintering grounds, suggesting a longitudinal barrier to present-day gene flow among migrants. In this context, we explored genetic population structure using 17 microsatellite loci and sampling 108 individuals across the range. We identified limited but significant overall differentiation (F(CT) = 0.045), which was overwhelmingly due to the differentiation of resident Arabian populations, particularly the one from Yemen, relative to the central Asian populations. Population structure within the central Asian group was not detectable with the exception of subtle differentiation of West Kazakh birds on the western flyway, relative to eastern populations. We interpret these patterns as evidence of recent common ancestry in Asia, coupled with a longitudinal barrier to present-day gene flow along the migratory divide, which has yet to translate into genetic divergence. These results provide key parameters for a coherent conservation strategy aimed at preserving genetic diversity and migration routes.

RNA-Seq Based Transcriptional Map of Bovine Respiratory Disease Pathogen "Histophilus Somni 2336"

Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify "novel" genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method.The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (∼30%) were located in genomic region unique to strain 2336 (∼18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations.

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