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1.  Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System 
PLoS Pathogens  2016;12(2):e1005422.
Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.
Author Summary
Streptococcus pneumoniae is an opportunistic pathogen with high carriage rates in children. Pneumococci express pneumocins that kill competing bacteria. Pneumocin expression is controlled by a pheromone-induced two-component system (BlpR/H) but the triggers for the system are poorly understood. We show that the pheromone-induced two-component system driving competence for genetic transformation, ComD/E, also controls expression of BlpC, the peptide pheromone activating BlpR/H-dependent gene expression. Importantly, we show that the competence pheromone exporter, ComAB, also exports BlpC. Since antibiotics that disrupt protein quality control or DNA replication trigger competence, it follows that the same antibiotics activate pneumocin expression. Our experiments show that this dual-quorum sensing system ensures that pneumocins are expressed at the end of exponential growth when nutrients become limiting. Pneumocin expression might thus be used to liberate nutrients by lysing competing bacteria. Antibiotic-induced pneumocin production might also aid in clearing the niche after antibiotic stress. Any free DNA can then be used for transformation to acquire antibiotic-resistance.
doi:10.1371/journal.ppat.1005422
PMCID: PMC4739728  PMID: 26840404
2.  Analysis of Published Criteria for Clinically Inactive Disease in a Large Juvenile Dermatomyositis Cohort Shows That Skin Disease Is Underestimated 
Almeida, Beverley | Campanilho‐Marques, Raquel | Arnold, Katie | Pilkington, Clarissa A. | Wedderburn, Lucy R. | Nistala, Kiran | Armon, Kate | Briggs, Vanja | Ellis‐Gage, Joe | Roper, Holly | Watts, Joanna | Baildam, Eileen | Hanna, Louise | Lloyd, Olivia | McCann, Liza | Roberts, Ian | McGovern, Ann | Riley, Phil | Al‐Abadi, Eslam | Ryder, Clive | Scott, Janis | Southwood, Taunton | Thomas, Beverley | Amin, Tania | Burton, Deborah | Jackson, Gillian | Van Rooyen, Vanessa | Wood, Mark | Wyatt, Sue | Browne, Michael | Davidson, Joyce | Ferguson, Sue | Gardner‐Medwin, Janet | Martin, Neil | Waxman, Liz | Foster, Helen | Friswell, Mark | Jandial, Sharmila | Qiao, Lisa | Sen, Ethan | Smith, Eve | Stevenson, Vicky | Swift, Alison | Wade, Debbie | Watson, Stuart | Crate, Lindsay | Frost, Anna | Jordan, Mary | Mosley, Ellen | Satyapal, Rangaraj | Stretton, Elizabeth | Venning, Helen | Warrier, Kishore | Almeida, Beverley | Arnold, Katie | Beard, Laura | Brown, Virginia | Campanilho‐Marques, Raquel | Enayat, Elli | Glackin, Yvonne | Halkon, Elizabeth | Hasson, Nathan | Juggins, Audrey | Kassoumeri, Laura | Lunt, Sian | Maillard, Sue | Nistala, Kiran | Pilkington, Clarissa | Simou, Stephanie | Smith, Sally | Varsani, Hemlata | Wedderburn, Lucy | Murray, Kevin | Ioannou, John | Suffield, Linda | Al‐Obaidi, Muthana | Leach, Sam | Lee, Helen | Smith, Helen | Inness, Emma | Kendall, Eunice | Mayers, David | Wilkinson, Nick | Clinch, Jacqui | Pluess‐Hall, Helen
Objective
The Pediatric Rheumatology International Trials Organisation (PRINTO) recently published criteria for classification of patients with juvenile dermatomyositis (DM) as having clinically inactive disease. The criteria require that at least 3 of 4 conditions be met, i.e., creatine kinase level ≤150 units/liter, Childhood Myositis Assessment Scale score ≥48, Manual Muscle Testing in 8 muscles score ≥78, and physician's global assessment of overall disease activity (PGA) ≤0.2. The present study was undertaken to test these criteria in a UK cohort of patients with juvenile DM.
Methods
We assessed 1,114 patient visits for the 4 items in the PRINTO criteria for clinically inactive disease. Each visit was analyzed to determine whether skin disease was present. The Disease Activity Score (DAS) for juvenile DM was determined in 59 patients.
Results
At 307 of the 1,114 visits, clinically inactive disease was achieved based on the 3 muscle criteria (but with a PGA of >0.2); rash was present at 65.8% of these visits and nailfold capillary abnormalities at 35.2%. When PGA ≤0.2 was one of the 3 criteria that were met, the frequency of skin signs was significantly lower (rash in 23.1% and nailfold capillary abnormalities in 8.7%). If PGA was considered an essential criterion for clinically inactive disease (P‐CID), patients with active skin disease were less likely to be categorized as having clinically inactive disease (a median DAS skin score of 0 [of a possible maximum of 9] in visits where the PGA was ≤0.2, versus a median DAS skin score of 4 in patients meeting the 3 muscle criteria [with a PGA of >0.2]; P < 0.001). Use of the P‐CID led to improvements in the positive predictive value and the positive likelihood ratio (85.4% and 11.0, respectively, compared to 72.9% and 5.1 with the current criteria).
Conclusion
There was a high frequency of skin disease among patients with juvenile DM who did not meet the PGA criterion for inactive disease but met the other 3 criteria. Incorporating PGA as an essential criterion for clinically inactive disease helps prevent the misclassification of patients with active skin disease.
doi:10.1002/art.39200
PMCID: PMC4737199  PMID: 25988361
3.  Exploring redundant research into the effect of tranexamic acid on surgical bleeding: further analysis of a systematic review of randomised controlled trials 
BMJ Open  2015;5(8):e009460.
Objectives
We examined whether apparent redundancy in a cumulative meta-analysis of trials is justified by concern about bias, random error or generalisability of the results.
Design
Cumulative meta-analysis, risk of bias assessment, trial sequential analysis, description of study participants over time and a review of rationales for conducting trials.
Data source
126 randomised trials included in a systematic review assessing of tranexamic acid on blood transfusion in surgery.
Results
The cumulative meta-analysis including all trials shows that the pooled estimate first reached statistical significance after the second trial in 1993. When the analysis was limited to the 38 high-quality trials and adjusted to account for potential systematic and random errors, the uncertainty was resolved after the 22nd trial in 2008. When the analysis was restricted to the two high-quality, prospectively registered trials, the cumulative z-curve crossed p=0.05 but not the monitoring boundary, suggesting an early potentially spurious statistically significant result. As precision of the pooled estimate increased, the number of trials initiated increased, although trial activity appeared to move to other surgery types. Most (62%) reports cited at least one systematic review. Of 118 reports examined, concern about generalisability was the reason for initiating the trial in 60%. Other reasons were to address a question other than the effect on bleeding (26%) and to confirm previously observed results (4%). Unawareness of previous research was apparent in 4% trials, while the rationale was unclear in 3%.
Conclusions
Our results indicate that poor quality is a more important cause of redundant research than the failure to review existing evidence. Concerns about generalisability of results is the main motivation for new trials. Contrary to previous claims, our results suggest that systematic reviews showing treatment effects can stimulate an increase in trial activity rather than reduce it.
doi:10.1136/bmjopen-2015-009460
PMCID: PMC4550739  PMID: 26303335
systematic reviews; meta-analysis; tranexamic acid
5.  Lamellipodin Is Important for Cell-to-Cell Spread and Actin-Based Motility in Listeria monocytogenes 
Infection and Immunity  2015;83(9):3740-3748.
Listeria monocytogenes is a foodborne pathogen capable of invading a broad range of cell types and replicating within the host cell cytoplasm. This paper describes the colocalization of host cell lamellipodin (Lpd) with intracellular L. monocytogenes detectable 6 h postinfection of epithelial cells. The association was mediated via interactions between both the peckstrin homology (PH) domain in Lpd and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] on the bacterial surface and by interactions between the C-terminal EVH1 (Ena/VASP [vasodilator-stimulated phosphoprotein] homology domain 1) binding domains of Lpd and the host VASP (vasodilator-stimulated phosphoprotein) recruited to the bacterial cell surface by the listerial ActA protein. Depletion of Lpd by short interfering RNA (siRNA) resulted in reduced plaque size and number, indicating a role for Lpd in cell-to-cell spread. In contrast, overexpression of Lpd resulted in an increase in the number of L. monocytogenes-containing protrusions (listeriopods). Manipulation of the levels of Lpd within the cell also affected the intracellular velocity of L. monocytogenes, with a reduction in Lpd corresponding to an increase in intracellular velocity. These data, together with the observation that Lpd accumulated at the interface between the bacteria and the developing actin tail at the initiation of actin-based movement, indicate a possible role for Lpd in the actin-based movement and the cell-to-cell spread of L. monocytogenes.
doi:10.1128/IAI.00193-15
PMCID: PMC4534642  PMID: 26169271
6.  Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation 
Bioresource Technology  2015;187:136-143.
Highlights
•Steam explosion of copier paper reduces xylose and produces inhibitors.•Steam explosion at SF 3.6 and 3.9 increased initial rates of saccharification.•Steam explosion at moderate severities may reduce processing times.•Co-steam explosion of waste paper and wheat straw reduces inhibitor production.
This study evaluated steam (SE) explosion on the saccharification and simultaneous saccharification and fermentation (SSF) of waste copier paper. SE resulted in a colouration, a reduction in fibre thickness and increased water absorption. Changes in chemical composition were evident at severities greater than 4.24 resulting in a loss of xylose and the production of breakdown products known to inhibit fermentation (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol, and at severities 4.53 and 4.83 reduced yields probably due to the effect of breakdown products and fermentation inhibitors. However, at moderate severities of 3.6 and 3.9 there was an increase in initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose.
doi:10.1016/j.biortech.2015.03.089
PMCID: PMC4504980  PMID: 25846183
Steam explosion; Enzyme saccharification; Fermentation; SSF; Cellulosic biomass
7.  Video Evidence That London Infants Can Resettle Themselves Back to Sleep After Waking in the Night, as well as Sleep for Long Periods, by 3 Months of Age 
This article has supplementary material on the Web site: www.jdbp.org.
ABSTRACT:
Objective:
Most infants become settled at night by 3 months of age, whereas infants not settled by 5 months are likely to have long-term sleep-waking problems. We assessed whether normal infant development in the first 3 months involves increasing sleep-period length or the ability to resettle autonomously after waking in the night.
Methods:
One hundred one infants were assessed at 5 weeks and 3 months of age using nighttime infrared video recordings and parental questionnaires.
Results:
The clearest development was in sleep length; 45% of infants slept continuously for ≥5 hours at night at 3 months compared with 10% at 5 weeks. In addition, around a quarter of infants woke and resettled themselves back to sleep in the night at each age. Autonomous resettling at 5 weeks predicted prolonged sleeping at 3 months suggesting it may be a developmental precursor. Infants reported by parents to sleep for a period of 5 hours or more included infants who resettled themselves and those with long sleeps. Three-month olds fed solely breast milk were as likely to self-resettle or have long sleep bouts as infants fed formula or mixed breast and formula milk.
Conclusions:
Infants are capable of resettling themselves back to sleep in the first 3 months of age; both autonomous resettling and prolonged sleeping are involved in “sleeping through the night” at an early age. Findings indicate the need for physiological studies of how arousal, waking, and resettling develop into sustained sleeping and of how environmental factors support these endogenous and behavioral processes.
doi:10.1097/DBP.0000000000000166
PMCID: PMC4459553  PMID: 26035139
infant sleeping; infant crying; settling; parenting
8.  Comparison of routes for achieving parenteral access with a focus on the management of patients with Ebola virus disease 
Background
Dehydration is an important cause of death in patients with Ebola virus disease (EVD). Parenteral fluids are often required in patients with fluid requirements in excess of their oral intake. The peripheral intravenous route is the most commonly used method of parenteral access, but inserting and maintaining an intravenous line can be challenging in the context of EVD. Therefore it is important to consider the advantages and disadvantages of different routes for achieving parenteral access (e.g. intravenous, intraosseous, subcutaneous and intraperitoneal).
Objectives
To compare the reliability, ease of use and speed of insertion of different parenteral access methods.
Search methods
We ran the search on 17 November 2014. We searched the Cochrane Injuries Group's Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations, Ovid MEDLINE(R) Daily, Ovid MEDLINE(R) and Ovid OLDMEDLINE(R), Embase Classic + Embase (OvidSP), CINAHL (EBSCOhost), clinicaltrials.gov and screened reference lists.
Selection criteria
Randomised controlled trials comparing different parenteral routes for the infusion of fluids or medication.
Data collection and analysis
Two review authors examined the titles and abstracts of records obtained by searching the electronic databases to determine eligibility. Two review authors extracted data from the included trials and assessed the risk of bias. Outcome measures of interest were success of insertion; time required for insertion; number of insertion attempts; number of dislodgements; time period with functional access; local site reactions; clinicians' perception of ease of administration; needlestick injury to healthcare workers; patients' discomfort; and mortality. For trials involving the administration of fluids we also collected data on the volume of fluid infused, changes in serum electrolytes and markers of renal function. We rated the quality of the evidence as 'high', 'moderate', 'low' or 'very low' according to the GRADE approach for the following outcomes: success of insertion, time required for insertion, number of dislodgements, volume of fluid infused and needlestick injuries.
Main results
We included 17 trials involving 885 participants. Parenteral access was used to infuse fluids in 11 trials and medications in six trials. None of the trials involved patients with EVD. Intravenous and intraosseous access was compared in four trials; intravenous and subcutaneous access in 11; peripheral intravenous and intraperitoneal access in one; saphenous vein cutdown and intraosseous access in one; and intraperitoneal with subcutaneous access in one. All of the trials assessing the intravenous method involved peripheral intravenous access.
We judged few trials to be at low risk of bias for any of the assessed domains.
Compared to the intraosseous group, patients in the intravenous group were more likely to experience an insertion failure (risk ratio (RR) 3.89, 95% confidence interval (CI) 2.39 to 6.33; n = 242; GRADE rating: low). We did not pool data for time to insertion but estimates from the trials suggest that inserting intravenous access takes longer (GRADE rating: moderate). Clinicians judged the intravenous route to be easier to insert (RR 0.15, 95% CI 0.04 to 0.61; n = 182). A larger volume of fluids was infused via the intravenous route (GRADE rating: moderate). There was no evidence of a difference between the two routes for any other outcomes, including adverse events.
Compared to the subcutaneous group, patients in the intravenous group were more likely to experience an insertion failure (RR 14.79, 95% CI 2.87 to 76.08; n = 238; GRADE rating: moderate) and dislodgement of the device (RR 3.78, 95% CI 1.16 to 12.34; n = 67; GRADE rating: low). Clinicians also judged the intravenous route as being more difficult to insert and patients were more likely to be agitated in the intravenous group. Patients in the intravenous group were more likely to develop a local infection and phlebitis, but were less likely to develop erythema, oedema or swelling than those in the subcutaneous group. A larger volume of fluids was infused into patients via the intravenous route. There was no evidence of a difference between the two routes for any other outcome.
There were insufficient data to reliably determine if the risk of insertion failure differed between the saphenous vein cutdown (SVC) and intraosseous method (RR 4.00, 95% CI 0.51 to 31.13; GRADE rating: low). Insertion using SVC took longer than the intraosseous method (MD 219.60 seconds, 95% CI 135.44 to 303.76; GRADE rating: moderate). There were no data and therefore there was no evidence of a difference between the two routes for any other outcome.
There were insufficient data to reliably determine the relative effects of intraperitoneal or central intravenous access relative to any other parenteral access method.
Authors' conclusions
There are several different ways of achieving parenteral access in patients who are unable meet their fluid requirements with oral intake alone. The quality of the evidence, as assessed using the GRADE criteria, is somewhat limited because of the lack of adequately powered trials at low risk of bias. However, we believe that there is sufficient evidence to draw the following conclusions: if peripheral intravenous access can be achieved easily, this allows infusion of larger volumes of fluid than other routes; but if this is not possible, the intraosseous and subcutaneous routes are viable alternatives. The subcutaneous route may be suitable for patients who are not severely dehydrated but in whom ongoing fluid losses cannot be met by oral intake.
A film to accompany this review can be viewed here (http://youtu.be/ArVPzkf93ng).
PLAIN LANGUAGE SUMMARY
Comparison of the different ways of giving fluids to patients who cannot drink enough, such as patients with Ebola virus disease
Background
Many patients with Ebola virus disease (EVD) die because they are dehydrated. Patients with EVD often experience severe vomiting and diarrhoea, which causes them to lose fluids that are difficult to replace by drinking alone. It is possible to give fluids in ways that do not involve the digestive tract; this is known as parenteral access. This includes infusing fluids into a vein (intravenously), into bone marrow (intraosseously), into fatty tissue under the skin (subcutaneously) or into the abdominal space (intraperitoneally). Giving fluids intravenously is the usual method, but can be problematic in patients with EVD because starting intravenous fluids can be difficult in very dehydrated patients, and infection control practices may make maintaining the infusion challenging. It is therefore useful if those caring for patients with EVD know the advantages and disadvantages of the other ways to give fluids, so that they can decide which is the most suitable for their patients.
Searches for trials
We carried out searches for trials comparing different parenteral access methods on 17 November 2014.
Trial characteristics
We found 17 trials involving 885 participants. None involved patients with EVD. Fifteen trials involved patients who required parenteral access for the infusion of fluids or medicines and two trials assessed different methods under simulated conditions, such as on a training manikin. Many trials were of poor quality.
Key results
When the results of these trials were gathered together, they suggested that both the intraosseous and subcutaneous routes may be easier and quicker to insert into patients than the intravenous route, but more fluid can be given intravenously than by either the intraosseous or subcutaneous method. There has not been enough research into the intraperitoneal method to know how it compares to the other methods.
Conclusions
Healthcare workers caring for patients with EVD should be aware of the alternative ways of giving fluids. The trials we found were not of very high quality, therefore we need to be cautious when drawing conclusions based on their results. However, together they suggest if intravenous access can be achieved easily, then this should be used as it allows the infusion of larger volumes of fluid. However, if intravenous access is not possible, intraosseous and subcutaneous routes are alternatives that can be inserted quickly. Many of the trials conducted so far are of poor quality and none involved patients with EVD, therefore more trials should be carried out.
A film to accompany this review can be viewed here.
doi:10.1002/14651858.CD011386.pub2
PMCID: PMC4455225  PMID: 25914907
9.  Chronic Trichuris muris Infection in C57BL/6 Mice Causes Significant Changes in Host Microbiota and Metabolome: Effects Reversed by Pathogen Clearance 
PLoS ONE  2015;10(5):e0125945.
Trichuris species are a globally important and prevalent group of intestinal helminth parasites, in which Trichuris muris (mouse whipworm) is an ideal model for this disease. This paper describes the first ever highly controlled and comprehensive investigation into the effects of T. muris infection on the faecal microbiota of mice and the effects on the microbiota following successful clearance of the infection. Communities were profiled using DGGE, 454 pyrosequencing, and metabolomics. Changes in microbial composition occurred between 14 and 28 days post infection, resulting in significant changes in α and β- diversity. This impact was dominated by a reduction in the diversity and abundance of Bacteroidetes, specifically Prevotella and Parabacteroides. Metabolomic analysis of stool samples of infected mice at day 41 showed significant differences to uninfected controls with a significant increase in the levels of a number of essential amino acids and a reduction in breakdown of dietary plant derived carbohydrates. The significant reduction in weight gain by infected mice probably reflects these metabolic changes and the incomplete digestion of dietary polysaccharides. Following clearance of infection the intestinal microbiota underwent additional changes gradually transitioning by day 91 towards a microbiota of an uninfected animal. These data indicate that the changes in microbiota as a consequence of infection were transitory requiring the presence of the pathogen for maintenance. Interestingly this was not observed for all of the key immune cell populations associated with chronic T. muris infection. This reflects the highly regulated chronic response and potential lasting immunological consequences of dysbiosis in the microbiota. Thus infection of T. muris causes a significant and substantial impact on intestinal microbiota and digestive function of mice with affects in long term immune regulation.
doi:10.1371/journal.pone.0125945
PMCID: PMC4418675  PMID: 25938477
10.  Phenotypic Heterogeneity in Expression of the K1 Polysaccharide Capsule of Uropathogenic Escherichia coli and Downregulation of the Capsule Genes during Growth in Urine 
Infection and Immunity  2015;83(7):2605-2613.
Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTI). The K1 capsule on the surface of UPEC strains is a key virulence factor, and its expression may be important in the onset and progression of UTI. In order to understand capsule expression in more detail, we analyzed its expression in the UPEC strain UTI89 during growth in rich medium (LB medium) and urine and during infection of a bladder epithelial cell line. Comparison of capsule gene transcription using a chromosomal gfp reporter fusion showed a significant reduction in transcription during growth in urine compared to that during growth in LB medium. When examined at the single-cell level, following growth in both media, capsule gene expression appears to be heterogeneous, with two distinct green fluorescent protein (GFP)-expressing populations. Using anti-K1 antibody, we showed that this heterogeneity in gene expression results in two populations of encapsulated and unencapsulated cells. We demonstrated that the capsule hinders attachment to and invasion of epithelial cells and that the unencapsulated cells within the population preferentially adhere to and invade bladder epithelial cells. We found that once internalized, UTI89 starts to produce capsule to aid in its intracellular survival and spread. We propose that this observed phenotypic diversity in capsule expression is a fitness strategy used by the bacterium to deal with the constantly changing environment of the urinary tract.
doi:10.1128/IAI.00188-15
PMCID: PMC4468546  PMID: 25870229
11.  Endovascular strategy or open repair for ruptured abdominal aortic aneurysm: one-year outcomes from the IMPROVE randomized trial 
Braithwaite, Bruce | Cheshire, Nicholas J. | Greenhalgh, Roger M. | Grieve, Richard | Hassan, Tajek B. | Hinchliffe, Robert | Howell, Simon | Moore, Fionna | Nicholson, Anthony A. | Soong, Chee V. | Thompson, Matt M. | Thompson, Simon G. | Ulug, Pinar | Heatley, Francine | Anjum, Aisha | Kalinowska, Gosia | Sweeting, Michael J. | Thompson, Simon G. | Gomes, Manuel | Grieve, Richard | Powell, Janet T. | Ashleigh, Ray | Gomes, Manuel | Greenhalgh, Roger M. | Grieve, Richard | Hinchliffe, Robert | Sweeting, Michael | Thompson, Matt M. | Thompson, Simon G. | Ulug, Pinar | Roberts, Ian | Bell, Peter R. F. | Cheetham, Anne | Stephany, Jenny | Warlow, Charles | Lamont, Peter | Moss, Jonathan | Tijssen, Jan | Braithwaite, Bruce | Nicholson, Anthony A. | Thompson, Matthew | Ashleigh, Ray | Thompson, Luke | Cheshire, Nicholas J. | Boyle, Jonathan R. | Serracino-Inglott, Ferdinand | Thompson, Matt M. | Hinchliffe, Robert J. | Bell, Rachel | Wilson, Noel | Bown, Matt | Dennis, Martin | Davis, Meryl | Ashleigh, Ray | Howell, Simon | Wyatt, Michael G. | Valenti, Domenico | Bachoo, Paul | Walker, Paul | MacSweeney, Shane | Davies, Jonathan N. | Rittoo, Dynesh | Parvin, Simon D. | Yusuf, Waquar | Nice, Colin | Chetter, Ian | Howard, Adam | Chong, Patrick | Bhat, Raj | McLain, David | Gordon, Andrew | Lane, Ian | Hobbs, Simon | Pillay, Woolagasen | Rowlands, Timothy | El-Tahir, Amin | Asquith, John | Cavanagh, Steve | Dubois, Luc | Forbes, Thomas L. | Ashworth, Emily | Baker, Sara | Barakat, Hashem | Brady, Claire | Brown, Joanne | Bufton, Christine | Chance, Tina | Chrisopoulou, Angela | Cockell, Marie | Croucher, Andrea | Dabee, Leela | Dewhirst, Nikki | Evans, Jo | Gibson, Andy | Gorst, Siobhan | Gough, Moira | Graves, Lynne | Griffin, Michelle | Hatfield, Josie | Hogg, Florence | Howard, Susannah | Hughes, Cían | Metcalfe, David | Lapworth, Michelle | Massey, Ian | Novick, Teresa | Owen, Gareth | Parr, Noala | Pintar, David | Spencer, Sarah | Thomson, Claire | Thunder, Orla | Wallace, Tom | Ward, Sue | Wealleans, Vera | Wilson, Lesley | Woods, Janet | Zheng, Ting
European Heart Journal  2015;36(31):2061-2069.
Aims
To report the longer term outcomes following either a strategy of endovascular repair first or open repair of ruptured abdominal aortic aneurysm, which are necessary for both patient and clinical decision-making.
Methods and results
This pragmatic multicentre (29 UK and 1 Canada) trial randomized 613 patients with a clinical diagnosis of ruptured aneurysm; 316 to an endovascular first strategy (if aortic morphology is suitable, open repair if not) and 297 to open repair. The principal 1-year outcome was mortality; secondary outcomes were re-interventions, hospital discharge, health-related quality-of-life (QoL) (EQ-5D), costs, Quality-Adjusted-Life-Years (QALYs), and cost-effectiveness [incremental net benefit (INB)]. At 1 year, all-cause mortality was 41.1% for the endovascular strategy group and 45.1% for the open repair group, odds ratio 0.85 [95% confidence interval (CI) 0.62, 1.17], P = 0.325, with similar re-intervention rates in each group. The endovascular strategy group and open repair groups had average total hospital stays of 17 and 26 days, respectively, P < 0.001. Patients surviving rupture had higher average EQ-5D utility scores in the endovascular strategy vs. open repair groups, mean differences 0.087 (95% CI 0.017, 0.158), 0.068 (95% CI −0.004, 0.140) at 3 and 12 months, respectively. There were indications that QALYs were higher and costs lower for the endovascular first strategy, combining to give an INB of £3877 (95% CI £253, £7408) or €4356 (95% CI €284, €8323).
Conclusion
An endovascular first strategy for management of ruptured aneurysms does not offer a survival benefit over 1 year but offers patients faster discharge with better QoL and is cost-effective.
Clinical trial registration
ISRCTN 48334791.
doi:10.1093/eurheartj/ehv125
PMCID: PMC4553715  PMID: 25855369
Aneurysm; Aorta; Rupture; Surgery; Stent grafts; Cost-effectiveness
12.  Investigating flavour characteristics of British ale yeasts: techniques, resources and opportunities for innovation 
Yeast (Chichester, England)  2014;32(1):281-287.
Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour-related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd.
doi:10.1002/yea.3052
PMCID: PMC4383656  PMID: 25361168
13.  Identification of furfural resistant strains of Saccharomyces cerevisiae and Saccharomyces paradoxus from a collection of environmental and industrial isolates 
Background
Fermentation of bioethanol using lignocellulosic biomass as a raw material provides a sustainable alternative to current biofuel production methods by utilising waste food streams as raw material. Before lignocellulose can be fermented, it requires physical, chemical and enzymatic treatment in order to release monosaccharides, a process that causes the chemical transformation of glucose and xylose into the cyclic aldehydes furfural and hydroxyfurfural. These furan compounds are potent inhibitors of Saccharomyces fermentation, and consequently furfural tolerant strains of Saccharomyces are required for lignocellulosic fermentation.
Results
This study investigated yeast tolerance to furfural and hydroxyfurfural using a collection of 71 environmental and industrial isolates of the baker’s yeast Saccharomyces cerevisiae and its closest relative Saccharomyces paradoxus. The Saccharomyces strains were initially screened for growth on media containing 100 mM glucose and 1.5 mg ml−1 furfural. Five strains were identified that showed a significant tolerance to growth in the presence of furfural, and these were then screened for growth and ethanol production in the presence of increasing amounts (0.1 to 4 mg ml−1) of furfural.
Conclusions
Of the five furfural tolerant strains, S. cerevisiae National Collection of Yeast Cultures (NCYC) 3451 displayed the greatest furfural resistance and was able to grow in the presence of up to 3.0 mg ml−1 furfural. Furthermore, ethanol production in this strain did not appear to be inhibited by furfural, with the highest ethanol yield observed at 3.0 mg ml−1 furfural. Although furfural resistance was not found to be a trait specific to any one particular lineage or population, three of the strains were isolated from environments where they might be continually exposed to low levels of furfural through the ongoing natural degradation of lignocelluloses, and would therefore develop elevated levels of resistance to these furan compounds. Thus, these strains represent good candidates for future studies of genetic variation relevant to understanding and manipulating furfural resistance and in the development of tolerant ethanologenic yeast strains for use in bioethanol production from lignocellulose processing.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0217-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13068-015-0217-z
PMCID: PMC4389715  PMID: 25861389
Saccharomyces cerevisiae; Saccharomyces paradoxus; Furfural; Furan; Ethanol; Lignocellulose
14.  The science of clinical practice: disease diagnosis or patient prognosis? Evidence about “what is likely to happen” should shape clinical practice 
BMC Medicine  2015;13:20.
Background
Diagnosis is the traditional basis for decision-making in clinical practice. Evidence is often lacking about future benefits and harms of these decisions for patients diagnosed with and without disease. We propose that a model of clinical practice focused on patient prognosis and predicting the likelihood of future outcomes may be more useful.
Discussion
Disease diagnosis can provide crucial information for clinical decisions that influence outcome in serious acute illness. However, the central role of diagnosis in clinical practice is challenged by evidence that it does not always benefit patients and that factors other than disease are important in determining patient outcome. The concept of disease as a dichotomous ‘yes’ or ‘no’ is challenged by the frequent use of diagnostic indicators with continuous distributions, such as blood sugar, which are better understood as contributing information about the probability of a patient’s future outcome. Moreover, many illnesses, such as chronic fatigue, cannot usefully be labelled from a disease-diagnosis perspective. In such cases, a prognostic model provides an alternative framework for clinical practice that extends beyond disease and diagnosis and incorporates a wide range of information to predict future patient outcomes and to guide decisions to improve them. Such information embraces non-disease factors and genetic and other biomarkers which influence outcome.
Summary
Patient prognosis can provide the framework for modern clinical practice to integrate information from the expanding biological, social, and clinical database for more effective and efficient care.
doi:10.1186/s12916-014-0265-4
PMCID: PMC4311412  PMID: 25637245
Clinical decision-making; Contested diagnoses; Diagnosis; Evidence-based medicine; Information; Outcomes of care; Overdiagnosis; Prognosis; Stratified medicine
15.  Methodology for enabling high-throughput simultaneous saccharification and fermentation screening of yeast using solid biomass as a substrate 
Background
High-throughput (HTP) screening is becoming an increasingly useful tool for collating biological data which would otherwise require the employment of excessive resources. Second generation biofuel production is one such process. HTP screening allows the investigation of large sample sets to be undertaken with increased speed and cost effectiveness. This paper outlines a methodology that will enable solid lignocellulosic substrates to be hydrolyzed and fermented at a 96-well plate scale, facilitating HTP screening of ethanol production, whilst maintaining repeatability similar to that achieved at a larger scale.
Results
The results showed that utilizing sheets of biomass of consistent density (handbills), for paper, and slurries of pretreated biomass that could be pipetted allowed standardized and accurate transfers to 96-well plates to be achieved (±3.1 and 1.7%, respectively). Processing these substrates by simultaneous saccharification and fermentation (SSF) at various volumes showed no significant difference on final ethanol yields, either at standard shake flask (200 mL), universal bottle (10 mL) or 96-well plate (1 mL) scales. Substrate concentrations of up to 10% (w/v) were trialed successfully for SSFs at 1 mL volume. The methodology was successfully tested by showing the effects of steam explosion pretreatment on both oilseed rape and wheat straws.
Conclusions
This methodology could be used to replace large shake flask reactions with comparatively fast 96-well plate SSF assays allowing for HTP experimentation. Additionally this method is compatible with a number of standardized assay techniques such as simple colorimetric, High-performance liquid chromatography (HPLC) and Nuclear magnetic resonance (NMR) spectroscopy. Furthermore this research has practical uses in the biorefining of biomass substrates for second generation biofuels and novel biobased chemicals by allowing HTP SSF screening, which should allow selected samples to be scaled up or studied in more detail.
doi:10.1186/s13068-014-0181-z
PMCID: PMC4314751  PMID: 25648300
High-throughput screening; Enzyme saccharification; Simultaneous saccharification and fermentation; Cellulosic biomass; Cellulosic ethanol; Biorefining; Biomass
16.  Mechanism of action of tranexamic acid in bleeding trauma patients: an exploratory analysis of data from the CRASH-2 trial 
Critical Care  2014;18(6):685.
Introduction
To investigate the mechanism of action of tranexamic acid (TXA) in bleeding trauma patients, we examined the timing of its effect on mortality. We hypothesised that if TXA reduces mortality by decreasing blood loss, its effect should be greatest on the day of the injury when bleeding is most profuse. However, if TXA reduces mortality via an anti-inflammatory mechanism its effect should be greater over the subsequent days.
Methods
Exploratory analysis, including per-protocol analyses, of data from the CRASH-2 trial, a randomised placebo controlled trial of the effect of TXA on mortality in 20,211 trauma patients with, or at risk of, significant bleeding. We examined hazard ratios (HR) and 95% confidence intervals for all-cause mortality, deaths due to bleeding and non-bleeding deaths, according to the day since injury. The CRASH-2 trial is registered as ISRCTN86750102 and ClinicalTrials.gov NCT00375258.
Results
The effect of TXA on mortality is greatest for deaths occurring on the day of the injury (HR all-cause mortality = 0.83, 0.73 to 0.93). This survival benefit is only evident in patients in whom treatment is initiated within 3 hours of their injury (HR ≤3 hours = 0.78, 0.68 to 0.90; HR >3 hours = 1.02, 0.76 to 1.36). Initiation of TXA treatment within 3 hours of injury reduced the hazard of death due to bleeding on the day of the injury by 28% (HR = 0.72, 0.60 to 0.86). TXA treatment initiated beyond 3 hours of injury appeared to increase the hazard of death due to bleeding, although the estimates were imprecise.
Conclusions
Early administration of tranexamic acid appears to reduce mortality primarily by preventing exsanguination on the day of the injury.
Electronic supplementary material
The online version of this article (doi:10.1186/s13054-014-0685-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13054-014-0685-8
PMCID: PMC4277654  PMID: 25498484
17.  Next-generation sequencing to dissect hereditary nephrotic syndrome in mice identifies a hypomorphic mutation in Lamb2 and models Pierson’s syndrome 
The Journal of Pathology  2014;233(1):18-26.
The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson’s syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd
doi:10.1002/path.4308
PMCID: PMC4241031  PMID: 24293254
proteinuria; nephrotic syndrome; congenital nephrosis; kidney glomerulus; glomerular basement membrane; next-generation sequencing; mutagenesis; animal model
18.  HALT-IT - tranexamic acid for the treatment of gastrointestinal bleeding: study protocol for a randomised controlled trial 
Trials  2014;15:450.
Background
Gastrointestinal bleeding is a common emergency that causes substantial mortality worldwide. Acute upper and lower gastrointestinal bleeding accounts for about 75,000 hospital admissions each year in the UK and causes the death of about 10% of these patients.
Tranexamic acid has been shown to reduce the need for blood transfusion in surgical patients and to reduce mortality in bleeding trauma patients, with no apparent increase in thromboembolic events.
A systematic review of clinical trials of upper gastrointestinal bleeding shows a reduction in the risk of death with tranexamic acid but the quality of the trials was poor and the estimates are imprecise. The trials were also too small to assess the effect of tranexamic acid on thromboembolic events.
Methods
HALT-IT is a pragmatic, randomised, double-blind, placebo-controlled trial which will determine the effect of tranexamic acid on mortality, morbidity (re-bleeding, non-fatal vascular events), blood transfusion, surgical intervention, and health status in patients with acute gastrointestinal bleeding. Eight thousand adult patients who fulfil the eligibility criteria will be randomised to receive tranexamic acid or placebo.
Adults with significant acute upper or lower gastrointestinal bleeding can be included if the responsible doctor is substantially uncertain as to whether or not to use tranexamic acid in that particular patient.
Trial treatment consists of a loading dose of tranexamic acid (1 g by intravenous injection) or placebo (sodium chloride 0.9%) given as soon as possible after randomisation, followed by an intravenous infusion of 3 g tranexamic acid or placebo (sodium chloride 0.9%) over 24 hours.
The main analyses will compare those allocated tranexamic acid with those allocated placebo, on an intention-to-treat basis. Results will be presented as effect estimates with a measure of precision (95% confidence intervals). Subgroup analyses for the primary outcome will be based on time to treatment, source of bleeding (upper versus lower), suspected variceal bleeding and severity of bleeding. A study with 8,000 patients will have over 90% power to detect a 25% reduction in mortality from 10% to 7.5%.
Trial registration
Current Controlled Trials ISRCTN11225767 (registration date: 3 July 2012); Clinicaltrials.gov NCT01658124 (registration date: 26 July 2012).
Electronic supplementary material
The online version of this article (doi:10.1186/1745-6215-15-450) contains supplementary material, which is available to authorized users.
doi:10.1186/1745-6215-15-450
PMCID: PMC4253634  PMID: 25409738
Gastrointestinal bleeding; Tranexamic acid; Clinical trials
19.  Applying results from clinical trials: tranexamic acid in trauma patients 
This paper considers how results from clinical trials should be applied in the care of patients, using the results of the Clinical Randomisation of an Antifibrinolytic in Significant Haemorrhage (CRASH-2) trial of tranexamic acid in bleeding trauma patients as a case study. We explain why an understanding of the mechanisms of action of the trial treatment, and insight into the factors that might be relevant to this mechanism, is critical in order to properly apply (generalise) trial results and why it is not necessary that the trial population is representative of the population in which the medicine will be used. We explain why cause (mechanism)-specific mortality is more generalizable than all-cause mortality and why the risk ratio is the generalizable measure of the effect of the treatment. Overall, we argue that a biological insight into how the treatment works is more relevant when applying research results to patient care than the application of statistical reasoning.
doi:10.1186/s40560-014-0056-1
PMCID: PMC4336134  PMID: 25705414
Evidence-based medicine; Generalizability; Clinical trials; Tranexamic acid; Trauma
20.  Interventions for preventing injuries caused by impaired alertness in individuals with jet lag and shift work disorder 
This is the protocol for a review and there is no abstract. The objectives are as follows:
To assess the effects of interventions for preventing injuries caused by impaired alertness in persons with jet lag or shift work disorder.
doi:10.1002/14651858.CD007741
PMCID: PMC4176681  PMID: 25267894
21.  Caffeine for the prevention of injuries and errors in shift workers 
Background
Sleepiness leads to a deterioration in performance and attention, and is associated with an increased risk of injury. Jet lag and shift work disorder are circadian rhythm sleep disorders which result in sleepiness and can elevate injury risk. They create a need for individuals to operate at times which are different to those dictated by their circadian rhythms. Consequently there is also a need for interventions to help ensure that these persons can do so safely. Caffeine has a potential role in promoting alertness during times of desired wakefulness in persons with jet lag or shift work disorder, however its effects on injury and error are unclear.
Objectives
To assess the effects of caffeine for preventing injuries caused by impaired alertness in persons with jet lag or shift work disorder.
Search methods
We searched the Cochrane Injuries Group Specialised Register, CENTRAL (The Cochrane Library), MEDLINE, EMBASE, PsycINFO, CINAHL, TRANSPORT (to July 2008); and PubMed databases (to April 2010). We also searched the Internet and checked reference lists of relevant papers.
Selection criteria
Randomised controlled trials investigating the effects of caffeine on injury, error or cognitive performance in people with jet lag or shift work disorder.
Data collection and analysis
Two authors independently screened search results and assessed full texts for inclusion. Data were extracted and risk of bias was assessed. Estimates of treatment effect (odds ratio and standardised mean difference (SMD)) and 95% confidence intervals (CI) were calculated and pooled using the fixed-effect model.
Main results
Thirteen trials were included. None measured an injury outcome. Two trials measured error, and the remaining trials used neuropsychological tests to assess cognitive performance. The trials assessing the impact on errors found that caffeine significantly reduced the number of errors compared to placebo. The pooled effect estimates on performance by cognitive domain suggest that, when compared to placebo, caffeine improved concept formation and reasoning (SMD −0.41; 95% CI −1.04 to 0.23), memory (SMD −1.08; 95% CI −2.07 to −0.09), orientation and attention (SMD −0.55; 95% CI −0.83 to −0.27) and perception (SMD −0.77; 95% CI −1.73 to 0.20); although there was no beneficial effect on verbal functioning and language skills (SMD 0.18; 95% CI −0.50 to 0.87). One trial comparing the effects of caffeine with a nap found that there were significantly less errors made in the caffeine group. Other trials comparing caffeine with other active interventions (for example nap, bright light, modafinil) found no significant differences. There is a high risk of bias for the adequacy of allocation concealment and presence of selective outcome reporting amongst the trials.
Authors’ conclusions
Caffeine may be an effective intervention for improving performance in shift workers however, there are no trials from which we can assess its effect on injuries. The results largely originate from studies involving young participants under simulated conditions, and the extent to which the findings are generalisable to older workers and real world shift work is unclear. Based on the current evidence, there is no reason for healthy individuals who already use caffeine within recommended levels to improve their alertness to stop doing so. The assessment of the relative effects of caffeine to other potential countermeasures should be a focus of future research.
doi:10.1002/14651858.CD008508
PMCID: PMC4160007  PMID: 20464765
Accidents, Occupational [*prevention & control]; Caffeine [*therapeutic use]; Central Nervous System Stimulants [*therapeutic use]; Cognition Disorders [*prevention & control]; Neuropsychological Tests; Psychomotor Performance [drug effects; physiology]; Randomized Controlled Trials as Topic; Sleep Disorders, Circadian Rhythm [drug therapy]; Work Schedule Tolerance [*physiology]; Humans
22.  Haemostatic drugs for traumatic brain injury 
Background
Traumatic brain injury (TBI) is a leading cause of death and disability. Intracranial bleeding is a common complication of TBI, and intracranial bleeding can develop or worsen after hospital admission. Haemostatic drugs may reduce the occurrence or size of intracranial bleeds and consequently lower the morbidity and mortality associated with TBI.
Objectives
To assess the effects of haemostatic drugs on mortality, disability and thrombotic complications in patients with traumatic brain injury.
Search methods
We searched the electronic databases: Cochrane Injuries Group Specialised Register (3 February 2009), CENTRAL (The Cochrane Library 2009, Issue 1), MEDLINE (1950 to Week 3 2009), PubMed (searched 3 February 2009 (last 180 days)), EMBASE (1980 to Week 4 2009), CINAHL (1982 to January 2009), ISI Web of Science: Science Citation Index Expanded (SCI-EXPANDED) (1970 to January 2009), ISI Web of Science: Conference Proceedings Citation Index - Science (CPCI-S) (1990 to January 2009).
Selection criteria
We included published and unpublished randomised controlled trials comparing haemostatic drugs (antifibrinolytics: aprotinin, tranexamic acid (TXA), aminocaproic acid or recombined activated factor VIIa (rFVIIa)) with placebo, no treatment, or other treatment in patients with acute traumatic brain injury.
Data collection and analysis
Two review authors independently examined all electronic records, and extracted the data. We judged that there was clinical heterogeneity between trials so we did not attempt to pool the results of the included trials. The results are reported separately.
Main results
We included two trials. One was a post-hoc analysis of 30 TBI patients from a randomised controlled trial of rFVIIa in blunt trauma patients. The risk ratio for mortality at 30 days was 0.64 (95% CI 0.25 to 1.63) for rFVIIa compared to placebo. This result should be considered with caution as the subgroup analysis was not pre-specified for the trial. The other trial evaluated the effect of rFVIIa in 97 TBI patients with evidence of intracerebral bleeding in a computed tomography (CT) scan. The corresponding risk ratio for mortality at the last follow up was 1.08 (95% CI 0.44 to 2.68). The quality of the reporting of both trials was poor so it was difficult to assess the risk of bias.
Authors’ conclusions
There is no reliable evidence from randomised controlled trials to support the effectiveness of haemostatic drugs in reducing mortality or disability in patients with TBI. New randomised controlled trials assessing the effects of haemostatic drugs in TBI patients should be conducted. These trials should be large enough to detect clinically plausible treatment effects.
doi:10.1002/14651858.CD007877.pub2
PMCID: PMC4066582  PMID: 20091656
23.  Red Blood Cell Transfusion and Mortality in Trauma Patients: Risk-Stratified Analysis of an Observational Study 
PLoS Medicine  2014;11(6):e1001664.
Using a large multicentre cohort, Pablo Perel and colleagues evaluate the association of red blood cell transfusion with mortality according to the predicted risk of death for trauma patients.
Please see later in the article for the Editors' Summary
Background
Haemorrhage is a common cause of death in trauma patients. Although transfusions are extensively used in the care of bleeding trauma patients, there is uncertainty about the balance of risks and benefits and how this balance depends on the baseline risk of death. Our objective was to evaluate the association of red blood cell (RBC) transfusion with mortality according to the predicted risk of death.
Methods and Findings
A secondary analysis of the CRASH-2 trial (which originally evaluated the effect of tranexamic acid on mortality in trauma patients) was conducted. The trial included 20,127 trauma patients with significant bleeding from 274 hospitals in 40 countries. We evaluated the association of RBC transfusion with mortality in four strata of predicted risk of death: <6%, 6%–20%, 21%–50%, and >50%. For this analysis the exposure considered was RBC transfusion, and the main outcome was death from all causes at 28 days. A total of 10,227 patients (50.8%) received at least one transfusion. We found strong evidence that the association of transfusion with all-cause mortality varied according to the predicted risk of death (p-value for interaction <0.0001). Transfusion was associated with an increase in all-cause mortality among patients with <6% and 6%–20% predicted risk of death (odds ratio [OR] 5.40, 95% CI 4.08–7.13, p<0.0001, and OR 2.31, 95% CI 1.96–2.73, p<0.0001, respectively), but with a decrease in all-cause mortality in patients with >50% predicted risk of death (OR 0.59, 95% CI 0.47–0.74, p<0.0001). Transfusion was associated with an increase in fatal and non-fatal vascular events (OR 2.58, 95% CI 2.05–3.24, p<0.0001). The risk associated with RBC transfusion was significantly increased for all the predicted risk of death categories, but the relative increase was higher for those with the lowest (<6%) predicted risk of death (p-value for interaction <0.0001). As this was an observational study, the results could have been affected by different types of confounding. In addition, we could not consider haemoglobin in our analysis. In sensitivity analyses, excluding patients who died early; conducting propensity score analysis adjusting by use of platelets, fresh frozen plasma, and cryoprecipitate; and adjusting for country produced results that were similar.
Conclusions
The association of transfusion with all-cause mortality appears to vary according to the predicted risk of death. Transfusion may reduce mortality in patients at high risk of death but increase mortality in those at low risk. The effect of transfusion in low-risk patients should be further tested in a randomised trial.
Trial registration
www.ClinicalTrials.gov NCT01746953
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Trauma—a serious injury to the body caused by violence or an accident—is a major global health problem. Every year, injuries caused by traffic collisions, falls, blows, and other traumatic events kill more than 5 million people (9% of annual global deaths). Indeed, for people between the ages of 5 and 44 years, injuries are among the top three causes of death in many countries. Trauma sometimes kills people through physical damage to the brain and other internal organs, but hemorrhage (serious uncontrolled bleeding) is responsible for 30%–40% of trauma-related deaths. Consequently, early trauma care focuses on minimizing hemorrhage (for example, by using compression to stop bleeding) and on restoring blood circulation after blood loss (health-care professionals refer to this as resuscitation). Red blood cell (RBC) transfusion is often used for the management of patients with trauma who are bleeding; other resuscitation products include isotonic saline and solutions of human blood proteins.
Why Was This Study Done?
Although RBC transfusion can save the lives of patients with trauma who are bleeding, there is considerable uncertainty regarding the balance of risks and benefits associated with this procedure. RBC transfusion, which is an expensive intervention, is associated with several potential adverse effects, including allergic reactions and infections. Moreover, blood supplies are limited, and the risks from transfusion are high in low- and middle-income countries, where most trauma-related deaths occur. In this study, which is a secondary analysis of data from a trial (CRASH-2) that evaluated the effect of tranexamic acid (which stops excessive bleeding) in patients with trauma, the researchers test the hypothesis that RBC transfusion may have a beneficial effect among patients at high risk of death following trauma but a harmful effect among those at low risk of death.
What Did the Researchers Do and Find?
The CRASH-2 trail included 20,127 patients with trauma and major bleeding treated in 274 hospitals in 40 countries. In their risk-stratified analysis, the researchers investigated the effect of RBC transfusion on CRASH-2 participants with a predicted risk of death (estimated using a validated model that included clinical variables such as heart rate and blood pressure) on admission to hospital of less than 6%, 6%–20%, 21%–50%, or more than 50%. That is, the researchers compared death rates among patients in each stratum of predicted risk of death who received a RBC transfusion with death rates among patients who did not receive a transfusion. Half the patients received at least one transfusion. Transfusion was associated with an increase in all-cause mortality at 28 days after trauma among patients with a predicted risk of death of less than 6% or of 6%–20%, but with a decrease in all-cause mortality among patients with a predicted risk of death of more than 50%. In absolute figures, compared to no transfusion, RBC transfusion was associated with 5.1 more deaths per 100 patients in the patient group with the lowest predicted risk of death but with 11.9 fewer deaths per 100 patients in the group with the highest predicted risk of death.
What Do These Findings Mean?
These findings show that RBC transfusion is associated with an increase in all-cause deaths among patients with trauma and major bleeding with a low predicted risk of death, but with a reduction in all-cause deaths among patients with a high predicted risk of death. In other words, these findings suggest that the effect of RBC transfusion on all-cause mortality may vary according to whether a patient with trauma has a high or low predicted risk of death. However, because the participants in the CRASH-2 trial were not randomly assigned to receive a RBC transfusion, it is not possible to conclude that receiving a RBC transfusion actually increased the death rate among patients with a low predicted risk of death. It might be that the patients with this level of predicted risk of death who received a transfusion shared other unknown characteristics (confounders) that were actually responsible for their increased death rate. Thus, to provide better guidance for clinicians caring for patients with trauma and hemorrhage, the hypothesis that RBC transfusion could be harmful among patients with trauma with a low predicted risk of death should be prospectively evaluated in a randomised controlled trial.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001664.
This study is further discussed in a PLOS Medicine Perspective by Druin Burch
The World Health Organization provides information on injuries and on violence and injury prevention (in several languages)
The US Centers for Disease Control and Prevention has information on injury and violence prevention and control
The National Trauma Institute, a US-based non-profit organization, provides information about hemorrhage after trauma and personal stories about surviving trauma
The UK National Health Service Choices website provides information about blood transfusion, including a personal story about transfusion after a serious road accident
The US National Heart, Lung, and Blood Institute also provides detailed information about blood transfusions
MedlinePlus provides links to further resources on injuries, bleeding, and blood transfusion (in English and Spanish)
More information in available about CRASH-2 (in several languages)
doi:10.1371/journal.pmed.1001664
PMCID: PMC4060995  PMID: 24937305
24.  Ribosomal DNA Sequence Heterogeneity Reflects Intraspecies Phylogenies and Predicts Genome Structure in Two Contrasting Yeast Species 
Systematic Biology  2014;63(4):543-554.
The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of closely related organisms, and discuss how it could be extended to future studies of multilocus rDNA systems. [concerted evolution; genome hydridisation; phylogenetic analysis; ribosomal DNA; whole genome sequencing; yeast]
doi:10.1093/sysbio/syu019
PMCID: PMC4055870  PMID: 24682414
25.  Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines 
PLoS ONE  2014;9(2):e88144.
Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections.
doi:10.1371/journal.pone.0088144
PMCID: PMC3912152  PMID: 24498433

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