Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity

BACKGROUND: Two component lantibiotics, such as the plasmid-encoded lacticin 3147 produced by Lactococcus lactis DPC3147 and staphylococcin C55 produced by Staphylococcus aureus C55, represent an emerging subgroup of bacteriocins. These two bacteriocins are particularly closely related, exhibiting 86% (LtnA1 and C55α) and 55% (LtnA2 and C55β) identity in their component peptides. The aim of this study was to investigate, for the first time for any two component bacteriocins, the significance of the relatedness between these two systems. RESULTS: So close is this relatedness that the hybrid peptide pairs LtnA1:C55β and C55α:LtnA2 were found to have activities in the single nanomolar range, comparing well with the native pairings. To determine whether this flexibility extended to the associated post-translational modification/processing machinery, the staphylococcin C55 structural genes were directly substituted for their lacticin 3147 counterparts in the ltn operon on the large conjugative lactococcal plasmid pMRC01. It was established that the lacticin LtnA1 post-translational and processing machinery could produce functionally active C55α, but not C55β. In order to investigate in closer detail the significance of the differences between LtnA1 and C55α, three residues in LtnA1 were replaced with the equivalent residues in C55α. Surprisingly, one such mutant LtnA1-Leu21Ala was not produced. This may be significant given the positioning of this residue in a putative lipid II binding loop. CONCLUSION: It is apparent, despite sharing striking similarities in terms of structure and activity, that these two complex bacteriocins display some highly dedicated features particular to either system

Medical communication and technology: a video-based process study of the use of decision aids in primary care

Background: much of the research on decision-making in health care has focused on consultation outcomes. Less is known about the process by which clinicians and patients come to a treatment decision. This study aimed to quantitatively describe the behaviour shown by doctors and patients during primary care consultations when three types of decision aids were used to promote treatment decision-making in a randomised controlled trial.Methods: a video-based study set in an efficacy trial which compared the use of paper-based guidelines (control) with two forms of computer-based decision aids (implicit and explicit versions of DARTS II). Treatment decision concerned warfarin anti-coagulation to reduce the risk of stroke in older patients with atrial fibrillation. Twenty nine consultations were video-recorded. A ten-minute 'slice' of the consultation was sampled for detailed content analysis using existing interaction analysis protocols for verbal behaviour and ethological techniques for non-verbal behaviour.Results: median consultation times (quartiles) differed significantly depending on the technology used. Paper-based guidelines took 21 (19–26) minutes to work through compared to 31 (16–41) minutes for the implicit tool; and 44 (39–55) minutes for the explicit tool. In the ten minutes immediately preceding the decision point, GPs dominated the conversation, accounting for 64% (58–66%) of all utterances and this trend was similar across all three arms of the trial. Information-giving was the most frequent activity for both GPs and patients, although GPs did this at twice the rate compared to patients and at higher rates in consultations involving computerised decision aids. GPs' language was highly technically focused and just 7% of their conversation was socio-emotional in content; this was half the socio-emotional content shown by patients (15%). However, frequent head nodding and a close mirroring in the direction of eye-gaze suggested that both parties were active participants in the conversationConclusion: irrespective of the arm of the trial, both patients' and GPs' behaviour showed that they were reciprocally engaged in these consultations. However, even in consultations aimed at promoting shared decision-making, GPs' were verbally dominant, and they worked primarily as information providers for patients. In addition, computer-based decision aids significantly prolonged the consultations, particularly the later phases. These data suggest that decision aids may not lead to more 'sharing' in treatment decision-making and that, in their current form, they may take too long to negotiate for use in routine primary car

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Site directed mutagenesis of the structural backbone of the lantibiotic peptide LtnA1

<p><b>Copyright information:</b></p><p>Taken from "Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity"</p><p>http://www.biomedcentral.com/1471-2180/7/24</p><p>BMC Microbiology 2007;7():24-24.</p><p>Published online 2 Apr 2007</p><p>PMCID:PMC1858699.</p><p></p> Mutants N15K, A17N and L21A are indicated by arrows. Well diffusion assays indicating the activity (AU/ml) of concentrated purified supernatants of the mutants (Varian C18 column) and mass spectrometry profiles accompany each mutant generated. The scale on the y-axis of the mass-spec is L21A: 2000–3700 mass/charge, A17N: 2700–3400 mass/charge, N15K: 2000–3600 mass/charge. The Mutant L21A has no activity (0 AU/ml), the + indicates that this mutant was combined with LtnA1 and LtnA2, and a complementation effect was evident with LtnA1. The mutant A27S could not be generated but represents the fourth amino acid difference between staphylococcin C55α and LtnA1. The shaded broken circle represents the putative lipid II binding region of LtnA1

Genetic organisation of the gene clusters of the closely related lantibiotics lacticin 3147 (A) and staphylococcin C55 (B)

<p><b>Copyright information:</b></p><p>Taken from "Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity"</p><p>http://www.biomedcentral.com/1471-2180/7/24</p><p>BMC Microbiology 2007;7():24-24.</p><p>Published online 2 Apr 2007</p><p>PMCID:PMC1858699.</p><p></p> C depicts a comparison of the amino acid sequence of the propeptide of one of the structural components of lacticin 3147 (LtnA1) and staphylococcin C55 (C55α). D depicts a comparison of the amino acid sequence of the propeptide of one of the structural components of lacticin 3147 (LtnA2) and staphylococcin C55 (C55β). Homologous amino acids are indicated in bold, differences are indicated in red. E and F indicates evolutionary divergence (measured in the number of substitution events) between the unmodified lantibiotic propeptides (E) LtnA1 and C55α and (F) LtnA2 and C55β. (G) Visualization of homology modeling of LtnA1 (gold) and C55α (purple)

(A and B) Illustration of the requirement for synergistic activity of both structural components of the lantibiotics lacticin 3147 (LtnA1 and LtnA2) and staphylococcin C55 (C55α and C55β) for full activity of the lantibiotic

<p><b>Copyright information:</b></p><p>Taken from "Relatedness between the two-component lantibiotics lacticin 3147 and staphylococcin C55 based on structure, genetics and biological activity"</p><p>http://www.biomedcentral.com/1471-2180/7/24</p><p>BMC Microbiology 2007;7():24-24.</p><p>Published online 2 Apr 2007</p><p>PMCID:PMC1858699.</p><p></p> C and D illustrate cross complementation of the structural peptides of lacticin 3147 with those of staphylococcin C55. Isobologram graphs show nanomolar concentrations of purified peptides of lacticin 3147 with those of staphylococcin C55. Each point on the graph represents 50% growth inhibition of the indicator HP. Isobolograms of (E) represent purified lacticin peptides LtnA1 and LtnA2 titrated against each other, (F) represent purified staphylococcin C55 peptides C55α and C55β titrated against each other, (G) represent purified lacticin LtnA1 peptide titrated against staphylococcin C55β and (H) represent purified staphylococcin C55α titrated against lacticin LtnA2 peptide

Prebiotic supplementation in frail older people affects specific gut microbiota taxa but not global diversity

Background: There are complex interactions between aging, frailty, diet, and the gut microbiota; modulation of the gut microbiota by diet could lead to healthier aging. The purpose of this study was to test the effect of diets differing in sugar, fat, and fiber content upon the gut microbiota of mice humanized with microbiota from healthy or frail older people. We also performed a 6-month dietary fiber supplementation in three human cohorts representing three distinct life-stages. Methods: Mice were colonized with human microbiota and then underwent an 8-week dietary intervention with either a high-fiber/low-fat diet typical of elderly community dwellers or a low-fiber/high-fat diet typical of long-stay residential care subjects. A cross-over design was used where the diets were switched after 4 weeks to the other diet type to identify responsive taxa and innate immunity changes. In the human intervention, the subjects supplemented their normal diet with a mix of five prebiotics (wheat dextrin, resistant starch, polydextrose, soluble corn fiber, and galactooligo-saccharide) at 10 g/day combined total, for healthy subjects and 20 g/day for frail subjects, or placebo (10 g/day maltodextrin) for 26 weeks. The gut microbiota was profiled and immune responses were assayed by T cell markers in mice, and serum cytokines in humans. Results: Humanized mice maintained gut microbiota types reflecting the respective healthy or frail human donor. Changes in abundance of specific taxa occurred with the diet switch. In mice with the community type microbiota, the observed differences reflected compositions previously associated with higher frailty. The dominance of Prevotella present initially in community inoculated mice was replaced by Bacteroides, Alistipes, and Oscillibacter. Frail type microbiota showed a differential effect on innate immune markers in both conventional and germ-free mice, but a moderate number of taxonomic changes occurring upon diet switch with an increase in abundance of Parabacteroides, Blautia, Clostridium cluster IV, and Phascolarctobacterium. In the human intervention, prebiotic supplementation did not drive any global changes in alpha- or beta-diversity, but the abundance of certain bacterial taxa, particularly Ruminococcaceae (Clostridium cluster IV), Parabacteroides, Phascolarctobacterium, increased, and levels of the chemokine CXCL11 were significantly lower in the frail elderly group, but increased during the wash-out period. Conclusions: Switching to a nutritionally poorer diet has a profound effect on the microbiota in mouse models, with changes in the gut microbiota from healthy donors reflecting previously observed differences between elderly frail and non-frail individuals. However, the frailty-associated gut microbiota did not reciprocally switch to a younger healthy-subject like state, and supplementation with prebiotics was associated with fewer detected effects in humans than diet adjustment in animal model