| 1. |
- Fox, K L, et al.
(författare)
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Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O-acetyl groups
- 2005
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 58:1, s. 207-216
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Tidskriftsartikel (refereegranskat)abstract
- Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5'-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis.
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| 2. |
- Hood, D W, et al.
(författare)
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Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H-influenzae strain Rd (RM118)
- 2001
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:11, s. 957-967
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Tidskriftsartikel (refereegranskat)abstract
- A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha -D-Hepp-(1-->2)-L-alpha -D-Hepp-(1-->3)-[beta -D-Glcp-(1-->4)-]-L-alpha -D-Hepp-(1-->5)-alpha -Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta -D-GalpNAc-(1-->3)-alpha -D-Galp(1-->4)-beta -D-Galp-(1-->4)-beta -D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D H-1-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes Involved in the addition of each glycose residue.
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| 3. |
- Rowbotham, S. E., et al.
(författare)
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Inositol in the MAnaGemENt of abdominal aortic aneurysm (IMAGEN) : Study protocol for a randomised controlled trial
- 2017
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Ingår i: Trials. - : BioMed Central Ltd.. - 1745-6215. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: An abdominal aortic aneurysm (AAA) is a focal dilation of the abdominal aorta and is associated with a risk of fatal rupture. Experimental studies suggest that myo-inositol may exert beneficial effects on AAAs through favourable changes to biological pathways implicated in AAA pathology. The aim of the Inositol in the MAnaGemENt of abdominal aortic aneurysm (IMAGEN) trial is to assess if myo-inositol will reduce AAA growth. Methods/design: IMAGEN is a multi-centre, prospective, parallel-group, randomised, double-blind, placebo-controlled trial. A total of 164 participants with an AAA measuring ≥ 30 mm will be randomised to either 2 g of myo-inositol or identical placebo twice daily for 12 months. The primary outcome measure will be AAA growth estimated by increase in total infrarenal aortic volume measured on computed tomographic scans. Secondary outcome measures will include AAA diameter assessed by computed tomography and ultrasound, AAA peak wall stress and peak wall rupture index, serum lipids, circulating AAA biomarkers, circulating RNAs and health-related quality of life. All analysis will be based on the intention-to-treat principle at the time of randomisation. All patients who meet the eligibility criteria, provide written informed consent and are enrolled in the study will be included in the primary analysis, regardless of adherence to dietary allocation. Discussion: Currently, there is no known medical therapy to limit AAA progression. The IMAGEN trial will be the first randomised trial, to our knowledge, to assess the value of myo-inositol in limiting AAA growth.
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| 4. |
- Schweda, Elke K H, et al.
(författare)
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Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:12, s. 3902-3913
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Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and H-1 NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp- (1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI.
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| 5. |
- Bateman, Alex, et al.
(författare)
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RNAcentral: A vision for an international database of RNA sequences.
- 2011
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Ingår i: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 17:11, s. 1941-6
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Tidskriftsartikel (refereegranskat)abstract
- During the last decade there has been a great increase in the number of noncoding RNA genes identified, including new classes such as microRNAs and piRNAs. There is also a large growth in the amount of experimental characterization of these RNA components. Despite this growth in information, it is still difficult for researchers to access RNA data, because key data resources for noncoding RNAs have not yet been created. The most pressing omission is the lack of a comprehensive RNA sequence database, much like UniProt, which provides a comprehensive set of protein knowledge. In this article we propose the creation of a new open public resource that we term RNAcentral, which will contain a comprehensive collection of RNA sequences and fill an important gap in the provision of biomedical databases. We envision RNA researchers from all over the world joining a federated RNAcentral network, contributing specialized knowledge and databases. RNAcentral would centralize key data that are currently held across a variety of databases, allowing researchers instant access to a single, unified resource. This resource would facilitate the next generation of RNA research and help drive further discoveries, including those that improve food production and human and animal health. We encourage additional RNA database resources and research groups to join this effort. We aim to obtain international network funding to further this endeavor.
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| 6. |
- Bauer, S H J, et al.
(författare)
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A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae : a survey of 24 non-typeable H-influenzae strains
- 2001
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 335:4, s. 251-260
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Tidskriftsartikel (refereegranskat)abstract
- In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by H-1 NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS /5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.
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| 7. |
- Day, Louise T., et al.
(författare)
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"Every Newborn-BIRTH" protocol : observational study validating indicators for coverage and quality of maternal and newborn health care in Bangladesh, Nepal and Tanzania
- 2019
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Ingår i: Journal of Global Health. - : International Global Health Society. - 2047-2978 .- 2047-2986. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: To achieve Sustainable Development Goals and Universal Health Coverage, programmatic data are essential. The Every Newborn Action Plan, agreed by all United Nations member states and >80 development partners, includes an ambitious Measurement Improvement Roadmap. Quality of care at birth is prioritised by both Every Newborn and Ending Preventable Maternal Mortality strategies, hence metrics need to advance from health service contact alone, to content of care. As facility births increase, monitoring using routine facility data in DHIS2 has potential, yet validation research has mainly focussed on maternal recall surveys. The Every Newborn - Birth Indicators Research Tracking in Hospitals (EN-BIRTH) study aims to validate selected newborn and maternal indicators for routine tracking of coverage and quality of facility-based care for use at district, national and global levels.Methods: EN-BIRTH is an observational study including >20000 facility births in three countries (Tanzania, Bangladesh and Nepal) to validate selected indicators. Direct clinical observation will be compared with facility register data and a pre-discharge maternal recall survey for indicators including: uterotonic administration, immediate newborn care, neonatal resuscitation and Kangaroo mother care. Indicators including neonatal infection management and antenatal corticosteroid administration, which cannot be easily observed, will be validated using inpatient records. Trained clinical observers in Labour/Delivery ward, Operation theatre, and Kangaroo mother care ward/areas will collect data using a tablet-based customised data capturing application. Sensitivity will be calculated for numerators of all indicators and specificity for those numerators with adequate information. Other objectives include comparison of denominator options (ie, true target population or surrogates) and quality of care analyses, especially regarding intervention timing. Barriers and enablers to routine recording and data usage will be assessed by data flow assessments, quantitative and qualitative analyses.Conclusions: To our knowledge, this is the first large, multi-country study validating facility-based routine data compared to direct observation for maternal and newborn care, designed to provide evidence to inform selection of a core list of indicators recommended for inclusion in national DHIS2. Availability and use of such data are fundamental to drive progress towards ending the annual 5.5 million preventable stillbirths, maternal and newborn deaths.
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| 8. |
- Day, Louise T, et al.
(författare)
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"Every Newborn-BIRTH" protocol: observational study validating indicators for coverage and quality of maternal and newborn health care in Bangladesh, Nepal and Tanzania.
- 2019
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Ingår i: Journal of global health. - : International Global Health Society. - 2047-2986 .- 2047-2978. ; 9:1
-
Tidskriftsartikel (refereegranskat)abstract
- To achieve Sustainable Development Goals and Universal Health Coverage, programmatic data are essential. The Every Newborn Action Plan, agreed by all United Nations member states and >80 development partners, includes an ambitious Measurement Improvement Roadmap. Quality of care at birth is prioritised by both Every Newborn and Ending Preventable Maternal Mortality strategies, hence metrics need to advance from health service contact alone, to content of care. As facility births increase, monitoring using routine facility data in DHIS2 has potential, yet validation research has mainly focussed on maternal recall surveys. The Every Newborn - Birth Indicators Research Tracking in Hospitals (EN-BIRTH) study aims to validate selected newborn and maternal indicators for routine tracking of coverage and quality of facility-based care for use at district, national and global levels.EN-BIRTH is an observational study including >20 000 facility births in three countries (Tanzania, Bangladesh and Nepal) to validate selected indicators. Direct clinical observation will be compared with facility register data and a pre-discharge maternal recall survey for indicators including: uterotonic administration, immediate newborn care, neonatal resuscitation and Kangaroo mother care. Indicators including neonatal infection management and antenatal corticosteroid administration, which cannot be easily observed, will be validated using inpatient records. Trained clinical observers in Labour/Delivery ward, Operation theatre, and Kangaroo mother care ward/areas will collect data using a tablet-based customised data capturing application. Sensitivity will be calculated for numerators of all indicators and specificity for those numerators with adequate information. Other objectives include comparison of denominator options (ie, true target population or surrogates) and quality of care analyses, especially regarding intervention timing. Barriers and enablers to routine recording and data usage will be assessed by data flow assessments, quantitative and qualitative analyses.To our knowledge, this is the first large, multi-country study validating facility-based routine data compared to direct observation for maternal and newborn care, designed to provide evidence to inform selection of a core list of indicators recommended for inclusion in national DHIS2. Availability and use of such data are fundamental to drive progress towards ending the annual 5.5 million preventable stillbirths, maternal and newborn deaths.
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| 9. |
- Hood, D W, et al.
(författare)
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Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis
- 2004
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 186:21, s. 7429-7439
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Tidskriftsartikel (refereegranskat)abstract
- It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.
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| 10. |
- Hood, Derek W., et al.
(författare)
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Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide
- 2010
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 156, s. 3421-3431
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Tidskriftsartikel (refereegranskat)abstract
- Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either L-glycero-D-manno-heptose (LD-Hep) or D-glycero-D-manno-heptose (DD-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode DD-heptosyl- and LD-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.
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