Strategic food vehicles for vitamin D fortification and effects on vitamin D status: A systematic review and meta-analysis of randomised controlled trials.

There has been growing interest in the potential of vitamin D food fortification in Europe as a means of addressing low vitamin D status. The WHO-FAO suggest that choosing a suitable food vehicle and ensuring the combination of the food vehicle and the fortificant will be efficacious and effective are of key importance to a successful food fortification programme. Our key objective was to conduct a systematic review and meta-analysis to investigate the effect of various animal- and plant-based food vehicles fortified with vitamin D (as D3 or D2) on circulating 25-hydroxyvitamin D [25(OH)D] concentrations. A list of prioritised food vehicles was established and we searched PubMed, Embase, Scopus and Web of Science for randomised controlled trials (RCTs) which used these vehicles individually, and which met prespecified criteria. The searches identified 49 papers which described suitable RCTs using vitamin D-fortified bread/savoury biscuits (n = 5), orange juice (n = 5), UV-mushrooms (n = 8), cheese (n = 3), yogurt (n = 5), fluid milk (n = 13), powdered milk (n = 5), eggs (n = 2), edible oils (n = 4), or breakfast cereal (n = 1). No suitable RCTs were identified for rice, maize flour, butter, margarine or dairy spreads, plant-based milk or yogurt alternatives. Random-effects meta-analyses of each food vehicle individually indicated weighted mean differences (WMD) in 25(OH)D in the range ∼9-35 nmol/L (3-15 RCT arms, depending on vehicle), and all statistically significant (P < 0.01-0.0001), with the exception of UV-mushrooms (P = 0.06). Heterogeneity was variable (I2 =33-99%, depending on vehicle), but subgroup analysis based on vitamer and dose reduced it in some instances. Sub-group analysis on the basis of whether the food vehicles were from plant-based or animal-based origin showed no significant difference in WMD (15.2 versus 15.9 nmol/L, respectively; P = 0.48). These results support the use of various animal- and plant-based food vehicles for vitamin D fortification to improve circulating 25(OH)D concentrations in populations. This work was registered with PROSPERO as CRD42023439883.

High-resolution genotyping of Lymphogranuloma Venereum (LGV) strains of Chlamydia trachomatis in London using multi-locus VNTR analysis-ompA genotyping (MLVA-ompA).

BACKGROUND: Lymphogranuloma venereum (LGV) is caused by Chlamydia trachomatis strains with ompA genotypes L1 to L3. An LGV epidemic associated with the L2b genotype has emerged in the past few decades amongst men who have sex with men (MSM). C. trachomatis genotypes can be discriminated by outer membrane protein A gene (ompA) sequencing, however this method has limited resolution. This study employed a high-resolution genotyping method, namely, multi-locus tandem repeat (VNTR) analysis with ompA sequencing (MLVA-ompA), to assess the distribution of LGV MLVA-ompA genotypes amongst individuals attending genitourinary medicine (GUM) clinics in London. METHODS: Clinical specimens were collected from individuals attending eight London-based GUM clinics. Specimens that tested positive for C. trachomatis by commercial nucleic acid amplification test (NAAT) were confirmed as LGV by pmpH real-time PCR. LGV-positive DNA extracts were subsequently genotyped using MLVA-ompA. RESULTS: Two hundred and thirty DNA extracts were confirmed as LGV, and 162 (70%) yielded complete MLVA-ompA genotypes. Six LGV MLVA-ompA genotypes were identified: 1.9.2b-L2, 1.9.3b-L2b, 1.9.2b-L2b, 1.9.2b-L2b/D, 1.4a.2b-L2b, and 5.9.2b-L1. The following LGV ompA genotypes were identified (in descending order of abundance): L2, L2b, L2b/D, and L1. Eight ompA sequences with the hybrid L2b/D profile were detected. The hybrid sequence was identical to the ompA of a recombinant L2b/D strain detected in Portugal in 2017. CONCLUSIONS: The L2 ompA genotype was found to predominate in the London study population. The study detected an unusual hybrid L2b/D ompA profile that was previously reported in Portugal. We recommend further monitoring and surveillance of LGV strains within the UK population.

Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis.

Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck.  Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. Methods We made E. coli/ C. trachomatis shuttle vectors bearing the Himar1 C9  transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the ß-lactamase gene.  Activity of the transposase was monitored by immunoblot and by DNA sequencing. Results We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar1 C9  transposase under tet promoter control and its transposon.  However, we were unable to recover this plasmid in C. trachomatis following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9  transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli.  Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9  transposase under tet promoter control.  Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis.  Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain   C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Diversity in Chlamydial plasmids.

BACKGROUND: Evolutionary studies have been conducted that have investigated the chromosomal variance in the genus of Chlamydia. However, no all-encompassing genus-wide comparison has been performed on the plasmid. Therefore, there is a gap in the current knowledge on Chlamydia plasmid diversity. AIMS: This project is aimed to investigate and establish the nature and extent of diversity across the entire genus of Chlamydia, by comparing the sequences of all currently available plasmid carrying strains. METHODS: The PUBMED database was used to identify plasmid sequences from all available strains that met the set quality criteria for their inclusion in the study. Alignments were performed on the 51 strains that fulfilled the criteria using MEGA X software. Following that Maximum Likelihood estimation was used to construct 11 phylogenetic trees of the whole plasmid sequence, the individual 8 coding sequences, the iteron and a chromosomal gene ompA as a comparator. RESULTS: The genus-wide plasmid phylogeny produced three distinct lineages labelled as alpha, beta and gamma. Nineteen genotypes were found in the initial whole plasmid analysis. Their distribution was allocated as six C. pecorum, two C. pneumoniae, one C. gallinacea, one C. avium, one C. caviae, one C. felis, two C. psittaci, one C. trachomatis, one C. muridarum, and two C. suis. The chromosomal comparative gene ompA supported this distribution, with the same number of primary clades with the same species distribution. However, ompA sequence comparison resulted in fewer genotypes due to a reduced amount of available sequences (33 out of 51). All results were statistically significant. CONCLUSION: The results of this study indicate that the common bacterial ancestor of all the species had a plasmid, which has diverged over time. Moreover, it suggests that there is a strong evolutionary selection towards these species retaining their plasmids due to its high level of conservation across the genus, with the notable exception of C. pneumoniae. Furthermore, the evolutionary analysis showed that the plasmid and the chromosome have co-evolved.

The Nature and Extent of Plasmid Variation in Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen of humans, causing both the sexually transmitted infection, chlamydia, and the most common cause of infectious blindness, trachoma. The majority of sequenced C. trachomatis clinical isolates carry a 7.5-Kb plasmid, and it is becoming increasingly evident that this is a key determinant of pathogenicity. The discovery of the Swedish New Variant and the more recent Finnish variant highlight the importance of understanding the natural extent of variation in the plasmid. In this study we analysed 524 plasmid sequences from publicly available whole-genome sequence data. Single nucleotide polymorphisms (SNP) in each of the eight coding sequences (CDS) were identified and analysed. There were 224 base positions out of a total 7550 bp that carried a SNP, which equates to a SNP rate of 2.97%, nearly three times what was previously calculated. After normalising for CDS size, CDS8 had the highest SNP rate at 3.97% (i.e., number of SNPs per total number of nucleotides), whilst CDS6 had the lowest at 1.94%. CDS5 had the highest total number of SNPs across the 524 sequences analysed (2267 SNPs), whereas CDS6 had the least SNPs with only 85 SNPs. Calculation of the genetic distances identified CDS6 as the least variable gene at the nucleotide level (d = 0.001), and CDS5 as the most variable (d = 0.007); however, at the amino acid level CDS2 was the least variable (d = 0.001), whilst CDS5 remained the most variable (d = 0.013). This study describes the largest in-depth analysis of the C. trachomatis plasmid to date, through the analysis of plasmid sequence data mined from whole genome sequences spanning 50 years and from a worldwide distribution, providing insights into the nature and extent of existing variation within the plasmid as well as guidance for the design of future diagnostic assays. This is crucial at a time when single-target diagnostic assays are failing to detect natural mutants, putting those infected at risk of a serious long-term and life-changing illness.

Growth kinetics of Chlamydia trachomatis in primary human Sertoli cells.

Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections worldwide and has been associated with male infertility. Recently, it was hypothesized that Ct may infect the epithelium of the seminiferous tubule, formed by Sertoli cells, thus leading to impaired spermatogenesis. To date, there is a lack of data on Ct infection of the seminiferous epithelium; therefore, we aimed to characterize, for the first time, an in vitro infection model of primary human Sertoli cells. We compared Ct inclusion size, morphology and growth kinetics with those in McCoy cells and we studied F-actin fibres, Vimentin-based intermediate filaments and α-tubulin microtubules in Sertoli and McCoy cells. Our main finding highlighted the ability of Ct to infect Sertoli cells, although with a unique growth profile and the inability to exit host cells. Furthermore, we observed alterations in the cytoskeletal fibres of infected Sertoli cells. Our results suggest that Ct struggles to generate a productive infection in Sertoli cells, limiting its dissemination in the host. Nevertheless, the adverse effect on the cytoskeleton supports the notion that Ct may compromise the blood-testis barrier, impairing spermatogenesis.

Mediterranean-Style Diet Improves Systolic Blood Pressure and Arterial Stiffness in Older Adults.

We aimed to determine the effect of a Mediterranean-style diet, tailored to meet dietary recommendations for older adults, on blood pressure and arterial stiffness. In 12 months, randomized controlled trial (NU-AGE [New Dietary Strategies Addressing the Specific Needs of Elderly Population for Healthy Aging in Europe]), blood pressure was measured in 1294 healthy participants, aged 65 to 79 years, recruited from 5 European centers, and arterial stiffness in a subset of 225 participants. The intervention group received individually tailored standardized dietary advice and commercially available foods to increase adherence to a Mediterranean diet. The control group continued on their habitual diet and was provided with current national dietary guidance. In the 1142 participants who completed the trial (88.2%), after 1 year the intervention resulted in a significant reduction in systolic blood pressure (-5.5 mm Hg; 95% CI, -10.7 to -0.4; P=0.03), which was evident in males (-9.2 mm Hg, P=0.02) but not females (-3.1 mm Hg, P=0.37). The -1.7 mm Hg (95% CI, -4.3 to 0.9) decrease in diastolic pressure after intervention did not reach statistical significance. In a subset (n=225), augmentation index, a measure of arterial stiffness, was improved following intervention (-12.4; 95% CI, -24.4 to -0.5; P=0.04) with no change in pulse wave velocity. The intervention also resulted in an increase in 24-hour urinary potassium (8.8 mmol/L; 95% CI, 0.7-16.9; P=0.03) and in male participants (52%) a reduction in pulse pressure (-6.1 mm Hg; 95% CI, -12.0 to -0.2; P=0.04) and 24-hour urinary sodium (-27.1 mmol/L; 95% CI, -53.3 to -1.0; P=0.04). In conclusion, a Mediterranean-style diet is effective in improving cardiovascular health with clinically relevant reductions in blood pressure and arterial stiffness. Clinical Trial Registration- URL: http://www.clinicialtrials.gov . Unique identifier: NCT01754012.

Is vitamin D deficiency a public health concern for low middle income countries? A systematic literature review.

PURPOSE: Vitamin D deficiency has been receiving increasing attention as a potential public health concern in low and lower-middle income countries (LMICs), of which there are currently 83. We aimed to conduct a comprehensive systematic literature review (SLR) of available data on vitamin D status and prevalence of vitamin D deficiency in all 83 LMICs. METHODS: We followed the general methodology for SLRs in the area of serum 25-hydroxyvitamin D. Highest priority was placed on identifying relevant population-based studies, followed by cross-sectional studies, and to a lesser extent case-control studies. We adopted the public health convention that a prevalence of vitamin D deficiency (serum 25-hydroxyvitamin D < 25/30 nmol/L) at > 20% in the entire population and/or at-risk population subgroups (infants, children, women of child-bearing age, pregnancy) constitutes a public health issue that may warrant intervention. RESULTS: Our SLR revealed that of the 83 LMICs, 65% (n = 54 countries) had no published studies with vitamin D data suitable for inclusion. Using data from the remaining third, a number of LMICs had evidence of excess burden of vitamin D deficiency in one or more population subgroup(s) using the above convention (Afghanistan, Pakistan, India, Tunisia and Mongolia) as well as possibly other LMICs, albeit with much more limited data. Several LMICs had no evidence of excess burden. CONCLUSION: Vitamin D deficiency is a public health issue in some, but certainly not all, LMICs. There is a clear need for targeting public health strategies for prevention of vitamin D deficiency in those LMICs with excess burden.

The Chlamydia muridarum plasmid revisited : new insights into growth kinetics.

Background: Research in chlamydial genetics is challenging because of its obligate intracellular developmental cycle. In vivo systems exist that allow studies of different aspects of basic biology of chlamydiae, the murine Chlamydia muridarum model is one of great importance and thus an essential research tool. C. muridarum carries a plasmid that has a role in virulence.  Our aim was to compare and contrast the C. muridarum plasmid-free phenotype with that of a chromosomally isogenic plasmid-bearing strain, through the inclusion phase of the developmental cycle. Methods: We measured infectivity for plasmid bearing and plasmid-cured C. muridarum by inclusion forming assays in McCoy cells and in parallel bacterial chromosome replication by quantitative PCR, throughout the developmental cycle. In addition to these studies, we have carefully monitored chlamydial inclusion formation by confocal microscopy and transmission electron microscopy. A new E.coli/chlamydial shuttle vector (pNigg::GFP) was constructed using standard cloning technology and used to transform C. muridarum for further phenotypic studies. Results: We have advanced the definition of the chlamydial phenotype away from the simple static observation of mature inclusions and redefined the C. muridarum plasmid-based phenotype on growth profile and inclusion morphology. Our observations on the growth properties of plasmid-cured C. muridarum challenge the established interpretations, especially with regard to inclusion growth kinetics. Introduction of the shuttle plasmid pNigg::GFP into plasmid-cured C. muridarum restored the wild-type plasmid-bearing phenotype and confirmed that loss of the plasmid was the sole cause for the changes in growth and chromosomal replication. Conclusions: Accurate growth curves and sampling at multiple time points throughout the developmental cycle is necessary to define plasmid phenotypes.  There are subtle but important (previously unnoticed) differences in the overall growth profile of plasmid-bearing and plasmid-free C. muridarum.  We have proven that the differences described are solely due to the plasmid pNigg.

Genetic Transformation of a C. trachomatis Ocular Isolate With the Functional Tryptophan Synthase Operon Confers an Indole-Rescuable Phenotype.

Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Different strains are associated with ocular or urogenital infections, and a proposed mechanism that may explain this tissue tropism is the active tryptophan biosynthesis pathway encoded by the genomic trpRBA operon in urogenital strains. Here we describe genetic complementation studies that are essential to confirm the role of tryptophan synthase in the context of an ocular C. trachomatis genomic background. Ocular strain A2497 was transformed with the (urogenital) pSW2::GFP shuttle vector showing that there is no strain tropism barrier to this plasmid vector; moreover, transformation had no detrimental effect on the growth kinetics of A2497, which is important given the low transformation efficiency of C. trachomatis. A derivative of the pSW2::GFP vector was used to deliver the active tryptophan biosynthesis genes from a urogenital strain of C. trachomatis (Soton D1) to A2497 with the aim of complementing the truncated trpA gene common to most ocular strains. After confirmation of intact TrpA protein expression in the transformed A2497, the resulting transformants were cultivated in tryptophan-depleted medium with and without indole or tryptophan, showing that complementation of the truncated trpA gene by the intact and functional urogenital trpRBA operon was sufficient to bestow an indole rescuable phenotype upon A2497. This study proves that pSW2::GFP derived vectors do not conform to the cross-strain transformation barrier reported for other chlamydia shuttle vectors, suggesting these as a universal vector for transformation of all C. trachomatis strains. This vector promiscuity enabled us to test the indole rescue hypothesis by transforming ocular strain A2497 with the functional urogenital trpRBA operon, which complemented the non-functional tryptophan synthase. These data confirm that the trpRBA operon is necessary and sufficient for chlamydia to survive in tryptophan-limited environments such as the female urogenital tract.

A predictive model of serum 25-hydroxyvitamin D in UK white as well as black and Asian minority ethnic population groups for application in food fortification strategy development towards vitamin D deficiency prevention.

Within Europe, dark-skinned ethnic groups have been shown to be at much increased risk of vitamin D deficiency compared to their white counterparts. Increasing the dietary supply of vitamin D is potentially the only modifiable environmental component that can be used to prevent vitamin D deficiency among dark-skinned ethnic groups living at high latitude. Empirical data to support development of such strategies is largely lacking. This paper presents the development and validation of an integrated model that may be adapted within the UK population to design fortification strategies for vitamin D, for application in both white and black and Asian minority ethnic (BAME) population groups. Using a step-wise approach, models based on available ultraviolet B (UVB) data, hours of sunlight and two key components (the dose-response of serum 25-hydroxyvitamin D [25(OH)D] to UVB in white and BAME persons and the dose-response of 25(OH)D to vitamin D) were used to predict changes population serum 25(OH)D concentrations throughout the year, stratified by ethnicity, 'via increases' in dietary intake arising from food fortification simulations. The integrated model successfully predicted measured average wintertime 25(OH)D concentrations in addition to the prevalence of serum 25(OH)D <30nmol/L in adult white and BAME individuals (18-70y) in the UK-based National Diet and Nutrition Survey both separately (21.7% and 49.3% predicted versus 20.2% and 50.5% measured, for white and BAME, respectively) and when combined at UK population-relevant proportions of 97% white and 7% BAME (23.2% predicted versus 23.1% measured). Thus this integrated model presents a viable approach to estimating changes in the population concentrations of 25(OH)D that may arise from various dietary fortification approaches.

The impact of fatty acid desaturase genotype on fatty acid status and cardiovascular health in adults.

The aim of this review was to determine the impact of the fatty acid desaturase (FADS) genotype on plasma and tissue concentrations of the long-chain (LC) n-3 PUFA, including EPA and DHA, which are associated with the risk of several diet-related chronic diseases, including CVD. In addition to dietary intakes, which are low for many individuals, tissue EPA and DHA are also influenced by the rate of bioconversion from α-linolenic acid (αLNA). Δ-5 and Δ-6 desaturase enzymes, encoded for by FADS1 and FADS2 genes, are key desaturation enzymes involved in the bioconversion of essential fatty acids (αLNA and linoleic acid (LA)) to longer chained PUFA. In general, carriers of FADS minor alleles tend to have higher habitual plasma and tissue levels of LA and αLNA, and lower levels of arachidonic acid, EPA and also to a lesser extent DHA. In conclusion, available research findings suggest that FADS minor alleles are also associated with reduced inflammation and CVD risk, and that dietary total fat and fatty acid intake have the potential to modify relationships between FADS gene variants and circulating fatty acid levels. However to date, neither the size-effects of FADS variants on fatty acid status, nor the functional SNP in FADS1 and 2 have been identified. Such information could contribute to the refinement and targeting of EPA and DHA recommendations, whereby additional LC n-3 PUFA intakes could be recommended for those carrying FADS minor alleles.

Seasonal Changes in Vitamin D-Effective UVB Availability in Europe and Associations with Population Serum 25-Hydroxyvitamin D.

Low vitamin D status is common in Europe. The major source of vitamin D in humans is ultraviolet B (UVB)-induced dermal synthesis of cholecalciferol, whereas food sources are believed to play a lesser role. Our objectives were to assess UVB availability (Jm(-2)) across several European locations ranging from 35° N to 69° N, and compare these UVB data with representative population serum 25-hydroxyvitamin D (25(OH)D) data from Ireland (51-54° N), Iceland (64° N) and Norway (69° N), as exemplars. Vitamin D-effective UVB availability was modelled for nine European countries/regions using a validated UV irradiance model. Standardized serum 25(OH)D data was accessed from the EC-funded ODIN project. The results showed that UVB availability decreased with increasing latitude (from 35° N to 69° N), while all locations exhibited significant seasonal variation in UVB. The UVB data suggested that the duration of vitamin D winters ranged from none (at 35° N) to eight months (at 69° N). The large seasonal fluctuations in serum 25(OH)D in Irish adults was much dampened in Norwegian and Icelandic adults, despite considerably lower UVB availability at these northern latitudes but with much higher vitamin D intakes. In conclusion, increasing the vitamin D intake can ameliorate the impact of low UVB availability on serum 25(OH)D status in Europe.

Chlamydia trachomatis from Australian Aboriginal people with trachoma are polyphyletic composed of multiple distinctive lineages.

Chlamydia trachomatis causes sexually transmitted infections and the blinding disease trachoma. Current data on C. trachomatis phylogeny show that there is only a single trachoma-causing clade, which is distinct from the lineages causing urogenital tract (UGT) and lymphogranuloma venerum diseases. Here we report the whole-genome sequences of ocular C. trachomatis isolates obtained from young children with clinical signs of trachoma in a trachoma endemic region of northern Australia. The isolates form two lineages that fall outside the classical trachoma lineage, instead being placed within UGT clades of the C. trachomatis phylogenetic tree. The Australian trachoma isolates appear to be recombinants with UGT C. trachomatis genome backbones, in which loci that encode immunodominant surface proteins (ompA and pmpEFGH) have been replaced by those characteristic of classical ocular isolates. This suggests that ocular tropism and association with trachoma are functionally associated with some sequence variants of ompA and pmpEFGH.

Anthocyanins do not influence long-chain n-3 fatty acid status: studies in cells, rodents and humans.

Increased tissue status of the long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) is associated with cardiovascular and cognitive benefits. Limited epidemiological and animal data suggest that flavonoids, and specifically anthocyanins, may increase EPA and DHA levels, potentially by increasing their synthesis from the shorter-chain n-3 PUFA, α-linolenic acid. Using complimentary cell, rodent and human studies we investigated the impact of anthocyanins and anthocyanin-rich foods/extracts on plasma and tissue EPA and DHA levels and on the expression of fatty acid desaturase 2 (FADS2), which represents the rate limiting enzymes in EPA and DHA synthesis. In experiment 1, rats were fed a standard diet containing either palm oil or rapeseed oil supplemented with pure anthocyanins for 8 weeks. Retrospective fatty acid analysis was conducted on plasma samples collected from a human randomized controlled trial where participants consumed an elderberry extract for 12 weeks (experiment 2). HepG2 cells were cultured with α-linolenic acid with or without select anthocyanins and their in vivo metabolites for 24 h and 48 h (experiment 3). The fatty acid composition of the cell membranes, plasma and liver tissues were analyzed by gas chromatography. Anthocyanins and anthocyanin-rich food intake had no significant impact on EPA or DHA status or FADS2 gene expression in any model system. These data indicate little impact of dietary anthocyanins on n-3 PUFA distribution and suggest that the increasingly recognized benefits of anthocyanins are unlikely to be the result of a beneficial impact on tissue fatty acid status.

Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes--a homogenous system for sequence-specific detection.

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.

Whole-genome sequences of Chlamydia trachomatis directly from clinical samples without culture.

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

Enterotoxin-producing staphylococci cause intestinal inflammation by a combination of direct epithelial cytopathy and superantigen-mediated T-cell activation.

BACKGROUND: Enterotoxin-producing Staphylococcus aureus may cause severe inflammatory intestinal disease, particularly in infants or immunodeficient or elderly patients. They are also recognized to be associated with sudden infant death syndrome. Little is known, however, about mucosal responses to staphylococci. METHODS: The mucosal lesion in three infants with staphylococcal enterocolitis was assessed by immunohistochemistry and electron microscopy. The organisms underwent extensive molecular analysis. Their toxins were assessed for capacity to induce T-cell activation and host mucosal responses examined by in vitro organ culture. Epithelial responses were studied by coculture with HEp-2 and Caco-2 cells. RESULTS: Intestinal biopsies from the patients showed marked epithelial damage with mucosal inflammation. The three staphylococci, representing two distinct clones, were methicillin-sensitive, producing SEG/I enterotoxins and Rho-inactivating EDIN toxins. Their enterotoxins potently activated T cells, but only whole organisms could induce in vitro enteropathy, characterized by remarkable epithelial desquamation uninhibited by tacrolimus. EDIN-producing staphylococci, but not their supernatants, induced striking cytopathy in HEp-2 epithelial cells but not in Caco-2 cells. Although HEp-2 and Caco-2 cells produced similar IL-8, CCL20, and cathelicidin LL37 responses upon bacterial exposure, only Caco-2 cells expressed mRNA for the ß-defensins HBD2 and HBD3, while HEp-2 cells were unable to do so. CONCLUSIONS: Staphylococci induce enterocolitis by a combination of direct enterocyte cytopathy mediated by EDIN toxins, disrupting the epithelial barrier, and enterotoxin superantigen-induced mucosal T-cell activation. Gut epithelial production of ß-defensins may contribute to host defense against invasive staphylococcal disease.