Dirofilaria immitis in stray, pet, and working dogs  from Guwahati (Assam) and Aizawl (Mizoram), areas located in two Northeastern States of India. Diagnostic methods applied were microscopy (wet film and Knott's concentration technique), immunological test (Ag ELISA by SNAP 4Dx ELISA kit), and molecular tools (polymerase chain reaction and sequencing), which evidenced 11.38, 18.03, and 13.93% of positive animals, respectively. No significant differences were observed by area (18.23% versus 17.68%) nor by sex (18.1% versus 17.9%), whereas stray dogs proved more infected than other groups (P < 0.05). ELISA test evidenced an overall 22.69% of occult infections, mainly in working dogs (60%), and molecular techniques detected Dirofilaria (Nochtiella) repens in 4 stray dogs from Guwahati. Characterization of D. immitis isolates for ITS-2 region showed close identity with South Asian isolates.The aim of the present study was to determine the prevalence of  Dirofilaria immitis, the heartworm of dog, is one of the most important filaroid nematodes responsible for causing canine dirofilariosis. Heartworm inhabits the right ventricle and pulmonary arteries of dogs and other animals. The heartworm parasite is transmitted by various mosquitoes belonging to the genera Culex, Aedes, and Anopheles. Adult female D. immitis lays microfilariae, which are taken up by suitable mosquito vectors and subsequently develop to the infective 3rd larval stage. Transmission takes place when a potential vector bites dogs or other hosts during a subsequent blood meal. It takes about 6-7 months to become an adult stage. Pathophysiological response to heartworm infection is mainly due to the presence of adult worm. The main clinical symptoms in dirofilariosis include persistent cough, difficult breathing, and poor exercise tolerance followed by ascites, anorexia, and weight loss. The symbiotic relationship with Wolbachia (a rickettsia) along with D. immitis amplifies disease severity [severity . The patseverity . D. immitis, but confirmatory and reliable diagnosis for heartworm disease is dependent on serology and molecular tests. Sometimes, in circulating blood of heartworm infected dogs microfilariae are absent and such condition is termed as “occult infection.” In this case, obviously microscopy and PCR give false negative results. Several commercial ELISA based test kits are available to diagnose heartworm in dogs but these kits are not widely used in India. DNA based techniques provide an alternative approach which is very sensitive and accurate for identification of the filarial parasites [Laboratory diagnosis of dirofilariosis in live animals is always in forefront in terms of demonstration and identification of microfilariae in tested blood sample. Radiography and cardiography aid in the diagnosis ofarasites . D. immitis has also been reported as an emerging zoonosis by several authors [ D. immitis human infections are asymptomatic showing typical coin lesions on chest radiography and are often mistakenly removed as neoplasm [ D. immitis was reported from Mumbai [Heartworm disease due to authors –8. Human authors , 10. Mosneoplasm . In Indim Mumbai . After tm Mumbai . D. immitis in Mizoram, a Northeastern State of India, on the basis of examination of 240 dogs at slaughter [ D. immitis in a hospital population of dogs from Assam [ D. immitis has been carried out in the northeastern part of this country using combination of conventional, serological, and molecular diagnostic techniques.In animals, there have been both epidemiological and clinical case studies of this worm worldwide –20. Prevlaughter . A recen D. immitis in the studied area.The importance of this parasite and the paucity of information about the prevalence of canine heartworm in the Northeastern States of India inspired us to conduct the present plan of work. We studied blood samples from dogs that presented for veterinary attention and from dogs captured by various nongovernmental organizations and maintained in kennels. The prevalence was studied by means of conventional microscopy, an immunological test, and molecular techniques, and their efficacy was compared. Further, we explored some of the molecular characteristics ofThe study was undertaken systematically for a period of one calendar year from August 2011 to July 2012, in dogs from Guwahati and Aizawl. Guwahati, a city of Assam having annual rainfall of 1500–2600 mm, is located at 26°11′0′′ latitude N and 91°44′0′′ longitude E, and Aizawl, the capital city of Mizoram State having annual rainfall of 2400–2962 mm, is located at 23°43′27′′N and 92°43′2′′E. The cities are separated by surface distance of 550 km. The location of epidemiological study undertaken for the present study is shown in the map .In the present investigation, 3 categories of dogs grouped as working dogs of military and paramilitary force, pet dogs, and stray dogs were selected. Pet dogs of different breeds and paramilitary dogs mostly of Labrador and German Shepherd breeds brought to the Teaching Veterinary Clinical Complexes (TVCC) of the College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam, and the College of Veterinary Sciences & A.H., Central Agricultural University, Selesih, Aizawl, Mizoram, during the study period were taken for the study. In case of military dogs, most of the blood samples were directly sent to the Department of Parasitology for routine check-up of heartworm infection. The stray dog population consisted of local nondescript street dogs of either sex captured from different parts of the city for sterilization by local nongovernmental organizations like Peoples for Animal (PFA) and Just Be Friendly (JBF).Three categories of dogs like working (103), pet (266), and stray (413) totaling 782 numbers were examined. Dogs under the study were of both sexes and a total of 488 dogs from different localities of Guwahati and 294 dogs from Aizawl formed the entire base of study during the programme.2EDTA) vacuum tubes and stored at 4°C until further use.Approximately 5 mL of blood was drawn from the cephalic vein collected in disodium salt of ethylene diamine tetraacetic acid  [ D. immitis circulating antigen but positive at KCT were processed for this technique. Fresh adult D. immitis and positive blood samples collected from Aizawl slaughter house were used to standardize techniques and used as positive control for the tests. In addition, blood samples were taken from dogs clinically infected and found positive for D. immitis being confirmed by KCT and SNAP tests. Briefly, the molecular technique for the present study was performed as follows.The prevalence study forue (KCT) . Subsequue (KCT) . The immIsolation of genomic DNA from blood and adult parasite was carried out using the DNeasy Blood and Tissue Kit  as per the protocols provided by the manufacturer. The final templates were kept at −20°C.μL Taq polymerase buffer (10x), 01 μL dNTP (10 mM), 0.5 μL MgCl2 (50 mM), 0.75 μL of each forward and reverse primer (60 pM), 1 U Taq polymerase, and 3.0 μL template DNA (60 ng/μL concentration), by making the final volume up to 25.0 μL with NFW. The cycling condition used for amplifying the targeted product consisted of an initial denaturing step at 94°C for 2 min and 32 cycles of denaturing (30 s at 94°C), annealing (30 s at 60°C for 5.8S-ITS-2-28S-based primers and 30 s at 58°C for ITS-2-based primers), and extension (30 s at 72°C); a final extension (7 min at 72°C or 12 min at 72°C for cloning); and a soak at 4°C in a Techne-5000 thermal cycler (Bibby Scientific). The confirmation of the amplified products was made by gel electrophoresis of the PCR product in 1.5% agarose gel stained with ethidium bromide and visualized under gel doc .PCR technique targeted to amplify the ITS-2 region of filarial worms' rDNA was applied according to previously described protocols , 27. Bri D. immitis target, both on representative positive blood samples and on adult worms, was assessed by amplicon purification followed by cloning and sequencing.The specificity of the PCR amplification for the correspondingα E. coli cell was used for transformation of the plasmid using Transform Aid Bacterial Transformation Kit . Subsequently, clones were confirmed by clones' confirmation PCR.Cloning of the PCR amplicon(s) for the genomic region as described above has been performed using pDrive cloning vector . DH5 D. immitis of dogs by Clustal W method using DNASTAR software and phylogenic analysis was done. Sequences were compared in silico with sequences of D. immitis (ITS-2) rDNA available in the gene bank for each gene examined using the nucleotide-nucleotide “Basic Local Alignment Search Tool.” Sequences were submitted to NCBI to obtain accession numbers.The recombinant clones were sent to the Department of Biochemistry, University of Delhi, South Campus, for automated sequencing. The sequences obtained were aligned and compared with other published sequences of D. immitis detected in 88 animals the 302 bp expected band ( D. immitis DNA (band of 542 bp) in 88 dogs and recognized D. repens DNA (band of 484 bp) in further 4 animals  blood samples analysed proved microfilaraemic under wet film methods, whereas infection rates found by means of the other techniques were higher. As expected, the highest number of positive animals was detected by the ELISA test, which evidenced an overall prevalence of 18.03% , withoutted band  whereas  animals . The pre Dirofilaria immitis was comparedwith additional twelve sequences from the NCBI GenBank by Clustal W of DNASTAR. Sequences from isolates of India (EU087699), Taiwan (AF217800), China , Iran , Brazil , and Turkey (HM126607) were included. All the sequences fell under the same group; however, the sequences from Southeast Asia were more closely related. The phylogenetic tree constructed based on this finding is depicted ( D. immitis (accession numbers JX481279 and JX866681) and for D. repens (accession number JX524743) were obtained from GenBank.Phylogenetic analysis of two Guwahati isolates ofdepicted . Pairwisdepicted . Accessi D. immitis infection in dogs from the Northeastern States of India. The number of dogs proven positive for D. immitis in one or more diagnostic tests was variable according to the test applied. Recording of 6.26% blood samples microfilaraemic under wet film does not give the actual epidemiological situation of the studied areas owing to the low sensitivity of the method and to the failure in microfilarial species differentiation [ D. immitis infection in pet dogs and 25.90% in stray dogs. A similar type of varying from 4.76% to 29.54% D. immitis infection was recorded in Assam in pet and street dogs, respectively [The present study provides the first comprehensive assessment ofntiation . Currentntiation . The preectively . The rec D. immitis in male dogs, but we could not draw a conclusion on the differences of prevalence amongst male and female dogs. Like most record on heartworm prevalence [ D. immitis in male dogs.Our present study also revealed a higher prevalence ofevalence , 30–32 aevalence , 34, ourIn the present investigation, many dogs which were found positive using SNAP 4Dx kit revealed occult infections. The same samples when subjected to PCR studies revealed lesser percent prevalence. Occult infections (amicrofilaraemic infections) could arise due to several causes like low parasite burdens, prepatent infection by young adults, infection of dog by only male worm, geriatric female worm, and immune response from the host against microfilariae or under microfilaricidal therapy. The high percentage of occult infection is not uncommon and was previously reported by several authors from different parts of the world , 36. Hig D. immitis detected by PCR was 13.93% (109/782), lower than that evidenced by ELISA test, probably due to occult infections or to possible failure in DNA extraction. Hence, the main benefit of PCR in epidemiological surveys on dirofilariosis is 100% confirmatory species identification when mixed infections coexist [ D. repens supports the reports of human infection in Northeastern States of India [ D. immitis to the human host, often succeeds in its development to adult worm.The overall prevalence of coexist . Detectiof India  and is a D. immitis isolates of Guwahati showed a close identity with certain South Asian isolates of D. immitis. Pairwise homology analysis revealed 98.6–98.9% identity with a few sequences available at NCBI GenBank. Previously, 92.6% homology of D. immitis of Mizoram isolate with D. immitis of Taiwan isolate (AF217800) was documented [Phylogenetic analysis ofcumented . In the  D. immitis in the northeastern region of India and reestablishes the area as heartworm endemic. KCT along with antigen ELISA detection test confirmed their sensitivity, whereas molecular techniques confirmed their value in identification of canine filarial worms. The presence of both Dirofilaria species should alert physicians to the risk of human infections.This study confirms the predominance of


Scotland is currently implementing a trauma network with four Major Trauma Centres (MTCs). Transforming successful teaching hospitals in to MTCs is likely to generate various beliefs amongst staff. The Theoretical Domains Framework (TDF) is a tool to elicit and analyse beliefs . This stSemi-structured interviews were conducted with 10 participants from a single hospital prior to MTC designation including clinicians, nursing and management staff. A topic guide was designed using the TDF. Interview transcripts were analysed following a framework analysis approach and coded according to TDF domains. Themes were analysed for relevance according to prevalence, expressed importance, discordance and underlying evidence base.e.g. My optimism/pessimism is conditional upon availability of necessary resources), resource demands  becoming a MTC), current capability , knowledge/skill development , trauma teams and a structured trauma pathway (e.g. Someone should lead/coordinate care of trauma patients), and performance improvement processes  supportive and geared towards performance improvement).1728 utterances were coded into 98 themes, of which 57 were classified as relevant barriers/facilitators. Themes addressed 6 key areas: Beliefs towards becoming a MTC (This study identified a range of barriers and facilitators likely to influence the transition of this hospital into a MTC. Findings highlight a need for clear systems, processes, communication and teamwork, and delineate the complexity of participants’ motivation and optimism/pessimism. This provides a basis for developing targeted interventions to facilitate the implementation process. This is a replicable method of evidence-based service-improvement, which can be applied elsewhere throughout acute care.


Background. The spread of USA300 methicillin-resistant Staphylococcus aureus (MRSA) across the United States resulted in an epidemic of infections. In Europe, only sporadic cases or small clusters of USA300 infections are described, and its prevalence in England is unknown. We conducted prospective surveillance for USA300 in the east of England.Methods. We undertook a 12-month prospective observational cohort study of all individuals with MRSA isolated from community and hospital samples submitted to a microbiology laboratory. At least 1 MRSA isolate from each individual underwent whole-genome sequencing. USA300 was identified on the basis of sequence analysis, and phylogenetic comparisons were made between these and USA300 genomes from the United States.Results. Between April 2012 and April 2013, we sequenced 2283 MRSA isolates  from 1465 individuals. USA300 was isolated from 24 cases (1.6%). Ten cases (42%) had skin and soft tissue infection, and 2 cases had invasive disease. Phylogenetic analyses identified multiple introductions and household transmission of USA300.Conclusions. Use of a diagnostic laboratory as a sentinel for surveillance has identified repeated introductions of USA300 in eastern England in 2012–2013, with evidence for limited transmission. Our results show how systematic surveillance could provide an early warning of strain emergence and dissemination. Staphylococcus aureus (CA-MRSA) pulsotype USA300 in the United States in 1999 was followed by widespread dissemination and an epidemic of MRSA infection in otherwise healthy people ). Over half of first positive samples were submitted from first-opinion services , rather than from hospital inpatient stays. Eleven cases had MRSA USA300 identified from carriage screening alone: 11 had MRSA USA300 collected from a SSTI wound specimen, with or without associated carriage, and 1 each had invasive infection resulting in bloodstream infection and osteomyelitis, respectively, with MRSA USA300 identified in blood and bone samples, respectively . Mapping the residential address of the 24 cases demonstrated that 23 cases were scattered across eastern England , while the remaining case was a US resident. Dates of collection of the positive samples were distributed throughout the 12-month study period, with a median time of 21 days  between collection of the first and last sequenced isolate (Supplementary Table S1). The finding that MRSA USA300–positive cases were distributed in time and space is suggestive of numerous independent introduction events.We identified 1465 individuals over a 12-month period with at least 1 sample submitted to the microbiology laboratory at CUH that was culture positive for MRSA, and we sequenced 2283 isolates cultured from multisite screens or clinical samples from these cases. CC8 isolates were identified and further evaluated to identify USA300 . The 56 USA300 genomes from this study were combined with the genomes of 348 MRSA USA300 isolates from New York that have been reported previously [The phylogenetic tree revealed 3 groups of closely related isolates containing isolates from >1 individual . Previous studies have reported that USA300 isolates from the United States segregated into 2 clades based on fluoroquinolone resistance genotypes (with or without gyrA 84L/A and/or grlA 80Y/F mutations) [grlA.The oral antibiotics used to treat MRSA SSTI in the United Kingdom and United States in single or combination regimens are clindamycin, doxycycline and trimethoprim-sulfamethoxazole, rifampicin, trimethoprim, and fusidic acid –36. All tations) . When thThis study represents, to our knowledge, the first prospective surveillance study for USA300 in the United Kingdom. It provides clear evidence of how systematic whole-genome sequencing might assist in monitoring the distribution of a potentially high-risk clone. Using whole-genome sequencing, we detected multiple introductions of USA300 into eastern England. In terms of epidemiological characteristics, most cases were young and presented with SSTIs to first-opinion services. This is reminiscent of the early disease epidemiology of USA300 in the United States, where this was largely associated with SSTIs in the community before becoming introduced and established through US hospitals . GenomicUSA300 is known to have spread readily through communities in the United States, but we found limited evidence for such transmission in our study. We identified 1 two-member pair and 1 cluster of genetically closely related USA300. We found epidemiological links between all but 1 individual within these groups. Household transmission within pair A and cluster C is supported by findings of previous US studies, in which a median of 3 SNPs range, 0–772 SNPs), 6 SNPs , and 5 SNPs  were identified per household in New York, Los Angeles, and Chicago, respectively 72 SNPs, .mecC, a mecA homolog associated with livestock [mec IVa, spa t008, agr group 1, PVL positive) from 300 CA-MRSA across England and Wales over a 2-year period (2004–2005) [Our prospective, systematic surveillance study found a prevalence rate of USA300 of 1.6%, which is >3 times higher than the prevalence of MRSA isolates positive for ivestock . Serial ivestock , with 1 ivestock . Like coivestock . The pot04–2005) , yet we copB locus in raising fitness [The reasons underlying the rapid and widespread dissemination of USA300 in the United States remain unknown, despite hypothesized roles for genetic elements such as ACME and, more recently, the  fitness , 32, 33. fitness , 7. Our  fitness , may havS. aureus isolates, which includes spa typing, toxin gene detection and whole-genome sequencing. However, isolate submission is currently voluntary, highly selective, and in practice tends to only be used to test isolates associated with life-threatening infection [The current methods for surveillance of invasive staphylococcal infections in the United Kingdom do not allow for the monitoring of USA300. Antimicrobial susceptibility profiles have been used as a surrogate marker of community lineages on the basis of greater susceptibility overall, compared with that for previous hospital-adapted lineages. This is prone to increasing inaccuracy over time, since lineages may accumulate drug resistance. For example, in the past, ciprofloxacin susceptibility was used as a marker of United Kingdom community lineages , but a snfection . Review We acknowledge a number of limitations in our study. Without undertaking prevalence studies of all residents within a defined geographical area, studies are biased toward the healthcare-seeking population. We tried to reduce this by including all disease and carriage isolates over a yearlong period. Second, there is likely to be United Kingdom–wide geographic variation in USA300 distribution. Third, ethical constraints mean that epidemiological links between patients have been deduced using electronic medical records, which do not capture all epidemiological links. Despite these limitations, our study has identified the covert presence of USA300 in eastern England. More broadly, it shows that systematic whole-genome sequencing within a sentinel center could function as an effective surveillance mechanism to monitor MRSA lineages. If undertaken systematically, whole-genome sequencing–based sentinel surveillance within a coordinated network could be used to provide an early warning of strain emergence and dissemination; such epidemic intelligence would allow appropriate targeting of resources toward interventions to limit further spread.Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.


Preterm birth (PTB) is the world’s leading cause of death in children under 5 years. In 2013, over one million out of six million child deaths were due to complications of PTB. The rate of decline in child death overall has far outpaced the rate of decline attributable to PTB. Three key reasons for this slow progress in reducing PTB mortality are: (a) the underlying etiology and biological mechanisms remain unknown, presenting a challenge to discovering ways to prevent and treat the condition; (ii) while there are several evidence-based interventions that can reduce the risk of PTB and associated infant mortality, the coverage rates of these interventions in low- and middle-income countries remain very low; and (c) the gap between knowledge and action on PTB—the “know-do gap”—has been a major obstacle to progress in scaling up the use of existing evidence-based child health interventions, including those to prevent and treat PTB.In this review, we focus on the know-do gap in PTB as it applies to policymakers. The evidence-based approaches to narrowing this gap have become known as knowledge transfer and exchange (KTE). In our paper, we propose a research agenda for promoting KTE with policymakers, with an ambitious but realistic goal of reducing the global burden of PTB. We hope that our proposed research agenda stimulates further debate and discussion on research priorities to soon bend the curve of PTB mortality.The online version of this article (doi:10.1186/s12978-016-0146-8) contains supplementary material, which is available to authorized users. Preterm birth (PTB)—birth before 37 weeks of gestation—is the world’s leading cause of death in children under 5 years. In 2013, over one million out of six million child deaths were due to complications of PTB [There are three key reasons for this slow progress in reducing PTB mortality. First, while many risk factors for PTB have been identified Table , the undThe evidence-based approaches to narrowing this gap have become known as knowledge transfer and exchange (KTE). Such exchange involves the “synthesis, exchange, and application of knowledge by relevant stakeholders” in ways that accelerate the benefits of global and local innovation in health . KTE worIn PTB, the stakeholders are a diverse group that includes mothers, fathers, and their babies; doctors, nurses and other health care providers; community-based organizations; people researching the causes of PTB and new ways to prevent and treat its effects; and policymakers. These policymakers are at local, national and international levels, including, for example, individuals creating and enacting health policy in health departments, ministries of health, and international health agencies. Policymakers are critical to enacting the ambitious but realistic goal of reducing the global burden of PTB because they influence the way resources are used to leverage the evidence or the “know” – they are key to closing the know-do gap because they have control over the resources that make enacting the evidence possible. In this paper, we review the literature and recommend a KTE research agenda that could help policymakers close these gaps to reduce the burden of PTB.We begin by examining the state of the current evidence on what works best in KTE with policymakers, and on the barriers and facilitators to successful uptake and use of evidence. We then propose a framework for categorizing where the KTE know-do gaps are positioned in the evidence-to-policy cycle. Finally, we end by making recommendations for researchers seeking to close the gaps in order to reduce the burden of PTB.Over the past few decades, KTE has emerged as a framework to optimize the translation of the best research evidence and authentic stakeholder perspectives into better health outcomes . KTE witThis kind of shift is urgently needed to reduce the burden of PTB. Until recently, PTB was a very low priority on the global health agenda. However, the new Sustainable Development Goal (SDG) for Health, SDG 3, calls for “an end” to avertable child and neonatal deaths by 2030 . SDG 3 iEvidence of successful KTE with policymakers would include increased rates of adopting evidence-based interventions . Successful KTE should also mean that local knowledge—that is, evidence that is shared by members of the affected community— is fully integrated in the decision-making and reflected in policy outcomes.A 2014 systematic review of studies examining facilitators and barriers to the use of evidence in health policymaking found 145 separate studies, including 13 systematic reviews . The mosTo our analysis of barriers and facilitators to KTE implementation, we added an analysis of existing approaches to EIP. We discovered multiple interventions that hold promise for efficiently and effectively transferring and exchanging knowledge about solutions to PTB with policymakers. These clustered into two groups, which we label “linear” and “political economy” approaches for tackling the problem.The “linear” EIP approach, also known as “getting research into policy” or GRIP, starts from the viewpoint that there is a gap between the generation of health evidence on PTB and practical policymaking; closing this “evidence to policy” gap is an important tool for health improvement . The linWith a linear approach, certain forms of evidence, particularly systematic reviews and randomized controlled trials (RCTs), are given high priority, since they are the most reliable forms of evidence and are least prone to bias. They are thus positioned at the top of the “hierarchy of evidence” . Here, tThis approach would recognize that PTB policymaking demands different kinds of input from researchers at different stages in the policy process. It also raises the possibility of earlier engagement of policymakers by researchers so that researchers have a better sense of the types of issues of most concern to their audience. Early on, policymakers often want a menu of options, typically with specific information on the pros and cons of each option. Later in the process, they may want technical assistance in drafting and implementing specific policy choices.A second approach, the “political economy” approach, sees both the research process itself and the transfer of research evidence to policy as heavily influenced by competing economic interests, social values, and power dynamics , 15. Theusing multiple forms of evidence, not just systematic reviews ;acknowledging and understanding the political processes, vested interests, and ethical dimensions that shape policymaking;encouraging scientists and experts to support “open reflection and debate” about the evidence; andencouraging policymakers to routinely question their own roles, relationships, and values in relation to their use of research evidence—for example, by reflecting on whether they include, exclude, or perhaps do not “hear” different bodies of evidence.The political economy perspective ultimately calls for a participatory approach to decision making. The very process of generating scientific evidence, and bringing it to bear on policy decisions, should involve “setting up a democratic and deliberative approach, which is inclusive, explicit, transparent, and accountable to the needs of the people these decisions affect” . Key comOur review of the evidence  the collective impact strategy, which has been used to address conditions such as nutritional deficiencies, communicable diseases, obesity, and substance misuse –21, and Context matters. KTE never happens in a social, political, and economic vacuum. For example, the capacity for policymaking will vary according to all aspects of the local environment, including the economic, financial, regulatory, and social environments, which can vary at both local and country levels. For these reasons, effective KTE cannot follow a “one size fits all” approach. A key avenue of research will be to study how the political, economic, cultural and social environments in targeted areas come into play in designing effective KTE interventions and policies to enact them.Effective KTE does not stop once the knowledge has been transferred. Transferring research evidence is a necessary but insufficient condition for achieving sustainable impacts on PTB. Sustained follow-up, continued engagement with policymakers, and tracking the fidelity of the evidence-to-policy process is likely needed to achieve optimal outcomes. Thus, a key avenue of research should be to study the required duration of researcher engagement needed to support “stickiness” and sustainability of knowledge in policy formation, implementation, and short and longer term outcomes.Based on our literature review and analyses described above, we developed a proposed framework to guide consideration of unanswered research questions about the KTE process for policymaking on PTB. This framework, summarized in Fig. Our proposed KTE research framework incorporates both the “linear” and “political economy” approaches to evidence-informed policymaking. Its five components include: (1) understanding the context for policymaking; (2) systematically reviewing the evidence that needs to be transferred in policymakers; (3) using the best-available KTE strategies for transferring that evidence; (4) ongoing, post-transfer engagement; and (5) evaluation of whether evidence shaped policy.The circular design of the figure underscores the importance of feedback loops and integration of the two approaches. We recognize that the transfer and exchange of evidence to and between policymakers is unlikely to follow the neat, step-by-step pattern and the sequence of steps implied by Fig. Using our KTE research framework, we offer an initial “long list” of research questions that could guide refinements in KTE strategies focused on PTB. We also prioritize those likely to be the most important and impactful, and then we propose appropriate methodological approaches to tackle each these questions Table .Table 5RWe conclude that an important early priority is to gather baseline information across research sites being studied on: 1) the current “evidence” sources that policymakers already use, 2) current policy efforts at tackling PTB that are already in place, 3) existing perceptions and misperceptions of the root causes of PTB held by policymakers, consumers, and providers, 4) prior successes with decreasing PTB, and 5) the readiness of policymakers to review evidence of effective practices. Qualitative methods, including in-depth interviews and focus groups, are best suited to capture these baseline data, as well as content analyses of existing policies and protocols. Case study designs will further provide opportunities to understand the complex context of political, cultural and economic conditions in a holistic way, while integrating what is already known through previous research and using between-site comparisons. This “landscaping” should also track preliminary specific areas of concern and interest to policymakers prior to attempting any interventions.Once the context and strategies of KTE are understood, researchers should then focus on evaluation of the effectiveness of a KTE strategy. To understand how a KTE strategy has been previously applied, retrospective designs may work well. The advantage of this type of study is that it provides an opportunity to explore in-depth the implementation of a strategy of interest. Randomized controlled trials, the “gold standard” in research, may be difficult to conduct at a policy level. Randomization can be helpful if there are enough settings in which the policy strategy is being considered to warrant this design. When a policy strategy is being rolled out on a broad level—across a country for example—KTE strategies could be applied differentially across local policy decision-making units  and the processes and outcomes studied and compared. The advantage of well-designed randomized studies is that they have more power to causally associate a KTE strategy with a policy change, as opposed to changes that resulted from other factors present in the social, political and economic context.Conducting an RCT of a policy intervention at the national level is typically very costly. RCTs would also require intensive policymaker and citizen engagement . RCTs are probably most practical when sufficient resources are available. For example, with adequate funding, an RCT would be a valuable design to test a “one-stop shop” of information on PTB, a rapid response team to provide rapid provision of evidence on PTB when policymakers request it, and twinning policymakers with local technical assistance providers. Such a trial could help to ascertain the “dosage” of policy maker engagement to test the question of KTE sustainability.In this article, we have identified and characterized a set of important research questions related to KTE with policymakers that could potentially be used to reduce the global burden of PTB. We have also proposed some study ideas and strategies that hold promise for the field. Discovering the right strategies, or bundles of strategies, to optimize KTE for this group of stakeholders is crucial for the success of the PTBI. We hope that our proposed research agenda stimulates further debate and discussion on research priorities to soon bend the curve of PTB mortality.


An earlier attempt to adapt the REALM (Rapid Estimate of Adult Literacy in Medicine) word recognition test to Dutch was not entirely successful due to ceiling effects. In contrast to REALM, the Short Assessment of Health Literacy (SAHL) assesses both word recognition and comprehension in the health domain. The aim of this study was to design, test and validate a SAHL for Dutch patients (SAHL-D).We pretested 95 health-related terms (n = 127) and selected 33 best performing items for validation in a quantitative survey (n = 329). For each item, a correct recognition (1 point) and comprehension (1 point) contributed to the total score . Internal consistency was assessed using Cronbach’s alpha. Construct validity was examined by analyzing association patterns of SAHL-D with educational level, objective and subjective health literacy, prose literacy, and vocabulary. Receiver operating characteristic (ROC) curves, with prose literacy as the reference standard, determined optimal cut-off scores.Cronbach’s alpha was 0.77 for recognition, 0.79 for comprehension, and 0.86 for the total score. Scores significantly differed substantially by educational level. Association patterns mostly confirmed a priori expectations in direction and strength, thereby supporting the construct validity of the SAHL-D. The optimal cut-off scores for differentiating between adequate and low literacy lie between 52.5 and 55.5. A shorter SAHL-D version presenting 22 terms offers a comparable prediction performance.The results provide positive evidence for the reliability and validity of the SAHL-D. The SAHL-D can be applied to analyze the role of health literacy in health and healthcare, and for the development and evaluation of targeted interventions.The online version of this article (doi:10.1186/1471-2458-14-990) contains supplementary material, which is available to authorized users. In our current information society, individuals are increasingly required to participate in complex decision-making processes. For example, managing health and finances involves obtaining and processing complex information, and making decisions in interaction with domain experts such as physicians and financial planners. To succeed in these tasks, individuals need to be ‘literate’ in various ways. Rapid and reliable assessments of these literacy levels are needed, not only to help professional communicators, but also to study the effects of literacy deficiencies and evaluate literacy-focused interventions. This paper presents a new health literacy assessment for Dutch patients.In its general sense, literacy refers to the ability to read and write. At the basic level, this ability is associated with reading fluency and word recognition as measured by standard reading tests. At an advanced level, this ability is associated with vocabulary, i.e. knowledge of word meanings. Both word recognition and vocabulary are essential for reading comprehension . A broadIn addition to these general literacy concepts, there is a growing interest in domain-specific literacies, which has provided concepts such as financial literacy , media lIn the field of HL, broad conceptual definitions go hand in hand with specific operational definitions , 7. In akidney had to be associated with either urine or fever. In order to receive one point for an item, both the pronunciation and the association had to be correct. The SAHLSA produced a more balanced score distribution, was reliable and unidimensional, and correlated well (Pearson 0.65) with the Test of Functional Health Literacy in Adults (TOFHFLA). Lee et al. also presented an 18-item version of the SAHLSA [Valid and reliable measurement of HL is essential to investigate the impact of low HL on population health and healthcare use, to analyze the differential effectiveness of health interventions by HL level, and to develop, evaluate and implement effective evidence-based interventions targeting people with low HL. Clinical applications of HL assessment intend to enable clinicians to effectively adapt their communication strategies to patients with low HL. Brief and easy-to-use HL measures have been developed in English, including the Rapid Estimate of Adult Literacy in Medicine (REALM) . Fransene SAHLSA .Since Dutch resembles Spanish in its relatively transparent orthography, adding a semantic component to a pronunciation task is assumed to produce a more powerful Dutch HL measure than the REALM-D. The aim of this study was to design and test a SAHL for Dutch patients (SAHL-D), as well as to validate it against various other literacy measures, including a prose comprehension test.http://www.thesauruszorgenwelzijn.nl[The authors HPM and MF selected 95 candidate SAHL-D terms from a Dutch thesaurus of health terms lzijn.nl, of whicPotential participants for the pretest were approached by undergraduate students (Language and communication) in the waiting room of the outpatient clinic of Internal Medicine at a large university hospital. Inclusion criteria were aged ≥ 18 years and able to communicate in Dutch. Those willing to participate signed an informed consent form, filled in a questionnaire and participated in a personal interview with one of the students.The questionnaire assessed general vocabulary skills based on a written multiple choice vocabulary test used in the 8th grade of Dutch pre-vocational secondary education . Each itIn the personal interview, the SAHL-D was administered by handing the participant the 95 cards, one by one. Word recognition was assessed by asking the participant to read the word out loud. The instructions for students contained information on correct phonetic pronunciation and the correct stress of each syllable in each word. Word comprehension was assessed by asking participants to choose the correct word associated with the ‘target’ word, or to use the ‘Do not know’ option; participants were encouraged not to guess the answer.In the pretest we analyzed item scores and distributions of proportions correct to select the items with the best discriminative ability. Reliability of the set of 95 items was analyzed by Cronbach’s alpha. Analyses of variance (ANOVA) were used to assess relations between educational level and scores. The feasibility was assessed by noting the administration time for a subset of participants. Finally, we examined whether word features (such as opaque orthography and corpus frequency) were related to recognition and comprehension of each word.We selected a subset of the pretest item pool by rejecting items that were scored correctly for recognition or comprehension by at least 95% of the participants. This left 33 items that mainly refer to medical specialties, tests and treatments on the one hand, and diseases and symptoms on the other  in health care; and how often they had contact with a professional care provider in the past year. Following the International Standard Classification of Education (ISCED), educational level was categorized as low ; intermediate ; and high . For the resulting 48-item test, alpha was 0.87.Prose literacy: In this study, we sought to validate our literacy measure by comparing it to a general test of higher-order reading skills, especially the contextual reconstruction of meaning in prose contexts (as opposed to word knowledge). Prose literacy was assessed by a subset of items from a reading comprehension test widely used for 9th graders in Dutch pre-university secondary education  [on total  items [1Health Literacy Survey-Europe Q16: A short version of the Health Literacy Survey-Europe [y-Europe  was usedy-Europe .The following variables were assessed in the online questionnaire:The HLS-EU-Q16 consists of 16 items scored on a 4-point scale (very difficult to very easy). For each item the option ‘Do not know’ was also provided .Newest Vital Sign (NVS): The NVS is a 6-question tool to assess an individual’s ability to find and interpret information  on an ice cream nutrition label [on label . Earlieron label  translatIn a telephonic interview, NVS-D and SAHL-D were administered. These tests were sent as pdf files by email, not beforehand but upon starting the interview. As soon as the mail arrived, the participant started working on the NVS-D, followed by SAHL-D.SAHL-D: SAHL-D started with a title page and provided a single word per page, with the candidate meanings underneath it. The participant proceeded page by page. The item order was kept on, except in rare cases when words were skipped accidentally (by pressing the arrow button more than once). In those cases, the interviewer steered the participant back to the omitted word after the current item has been completed. At any time of the test, the participant saw only a single target word on the screen. Upon opening a new page, participants were given 5 seconds to pronounce the word, after which a multiple choice option was to be chosen immediately. This procedure practically rules out the possibility of using dictionaries. The participants worked alone (possible consultations with others would have been overheard). Administration of the SAHL-D took (on average) 6.39 min.During the interview, we sent one file with the ice cream label and another one with the questions; respondents were asked to open both files on their screen. The interviewer read the questions out loud while the respondents read the questions and looked at the label on their screen.In the validation study we assessed the proportions of correct answers and score distributions of the SAHL-D. Feasibility was assessed by calculating percentage refusals and acceptance and the time to complete the SAHL-D. Reliability was tested with Cronbach’s alpha.To explore the possibility of a shorter SAHL-D, we created an item subset by first discarding recognition items with rest-item correlations of ≤ 0.10 in the 33-item reliability analysis and/or a proportion correct of ≥ 0.95. This left 22 recognition items. We included the shorter 22-item set (SAHL-D22) in the analyses to illustrate the potential for a briefer SAHL-D.Construct validity was examined by analyzing association patterns of the SAHL-D, NVS-D, HLS-EU-Q16, educational level, prose literacy and vocabulary scores in relation to predefined expectations about the size and pattern of the associations.Regarding known-groups validity, we expected the SAHL-D to be able to distinguish between low, intermediate and high levels of education based on significant differences in the mean scores.Because of partly overlapping constructs, we expected a strong correlation between general vocabulary, prose literacy, NVS-D and the SAHL-D.We expected a significant (but not sizeable) correlation between the SAHL-D (objective measure) and the HLS-EU-Q16 (subjective measure).Regarding associations with socio-demographic variables, earlier literacy research , 23 led The following hypotheses were formulated:ANOVA pairwise comparisons with Bonferroni correction were used for multiple testing to test differences in the SAHL-D scores by educational level, age, gender, and profession . The association between the SAHL-D with general vocabulary, prose literacy, NVS-D, and HLS-EU-Q16 was tested with Pearson’s correlations and stepwise linear regression analyses to correct for background variables.We used receiver operating characteristic (ROC) curves with adequate prose literacy as the reference standard to determine optimal cut-off scores for identifying objective HL.Of the 127 patients participating in the pretest, 51% was male, 20% had a low and 34% had an intermediate educational level; the age range was 20–85 years with a mean of 50.4 (SD 14.4) years.recognition task proved to be relatively easy, with a mean proportion correct of 0.93. Of the 95 words, 5 were correctly pronounced by all participants and another 53 items were correct for ≥ 95% of the participants. Cronbach’s alpha for the recognition test was 0.94. The comprehension test was of similar difficulty (mean proportion correct 0.90). Of the 95 items, 4 were correctly scored by all participants and another 40 items were correct for ≥ 95% of the participants. Cronbach’s alpha for the comprehension test was 0.93.On average, the 95-word test took 9 min. The r). Correlations between SAHL-D recognition and comprehension with general vocabulary were similar, i.e. 0.66 and 0.57, respectively. The total correct score for the candidate items varied with educational level, although the effect size was modest .The correlation between recognition performance and comprehension performance was 0.83 . There was a significant difference in gender between participants and non-participants: 41% of the participants was male compared with 50% of the non-participants (p < 0.01).2 = 0.183) was more robust than for the pretest item set.Table Cronbach’s alpha’s for SAHL-D recognition, comprehension and total were 0.77, 0.79 and 0.86, respectively; for SAHL-D22, these alpha’s were .74, .73 and .83 respectively. Table Table We determined the potential of the SAHL-D and SAHL-D22 to correctly identify individuals with adequate and inadequate HL. Inadequate literacy was defined as a prose literacy correct score of 6 or lower. This threshold was chosen to be well below the mean correct score for the lowest educational level (8.3); under this definition, 18% of the participants is inadequately literate.The area under the ROC curve was 0.80 (CI 0.73-0.88) for SAHL-D. In the various uses of SAHIL, we may choose different cutoffs, i.e. the SAHL-D score below which the test taker is considered to be inadequately health literate. High cut-offs help to correctly identify low literacy (as not many of the low-literacy participants reach the threshold), but are not useful in identifying adequate literacy levels as many literate participants do not reach the threshold either. Reversely, low cut-off points better identify adequately literate individuals, but fare badly in detecting low literacy, as a considerable number of low-literacy participant outscore the threshold. Optimal cutoffs are to be found in the middle of the curve. For example, a cut-off score of 52.5 would correctly classify 66% of the test takers with inadequate HL as such and 86% of the test takers with adequate HL. For a cut-off value of 54.5 these values are 74% and 76% respectively; a cut-off of 55.5 gives values of 80% and 69%. While a high detection rate for low literacy seems preferable, higher cutoffs also imply larger numbers of false positives (i.e. people incorrectly ‘diagnosed’ with low literacy). The final cutoff choice depends on the use of the test, and the priorities in a given setting, especially the estimated costs of false-positive and false-negative results.Like other objective HL measures, the SAHL-D remains close to the basic literacy concept. The REALM  and MediA strength of this study is that the SAHL-D was based on a careful selection and pretest of health-related words that are frequently applied in The Netherlands. Considerable effort was required to find items that were sufficiently demanding for the test, given that Dutch has a fairly transparent orthography; this may explain why the earlier REALM-D test was less successful. Furthermore, adding a comprehension component to the test yielded more discriminative power, at least in the more demanding format used in the main study.A limitation of the present study is that, in the validation study, the sample was restricted to persons able to write and speak Dutch and having access to internet. This probably means that on average, our research sample is somewhat more literate than the general population. Therefore, we recommend that the SAHL-D be implemented in various clinical contexts and different populations to further investigate its reliability and validity. Another limitation is that there is no objective  literacy test available in Dutch. We therefore used an item sample taken from prose literacy tests used in Dutch higher secondary education. Since cut-off points were not available for these items, we defined adequate and inadequate prose literacy with reference to the mean proportion for the lowest educational group.The SAHL-D represents a new HL assessment tool in Dutch, consisting of a recognition and comprehension test for 33 (or 22) health-related words. The results of the first validation study provide positive evidence for the reliability and validity of the SAHL-D.As hypothesized, we found a strong correlation between SAHL-D with general vocabulary, prose literacy and the NVS-D; substantial correlations were found between all literacy measures, ranging from 0.53-0.61. We expected a significant (but not sizeable) correlation between the SAHL-D and the HLS-EU-Q16, since HL is subjectively measured in the HLS-EU and the SAHL-D is an objective measure; in fact a lower correlation was found between the SAHL-D and the HLS-EU-Q16, that was not significant after correction for educational level and other background variables. As expected we found a significant correlation between the SAHL-D and educational level and age; the correlation with education being stronger than that with age. All these results support the construct validity of the SAHL-D. After adjustment for educational level, age was no longer significant in the regression model, indicating that differences in age could be explained by differences in educational level.Although we did not expect gender differences in SAHL-D scores, our regression analyses found that women scored higher than men, also after correcting for age and educational level. As our general vocabulary and prose literacy scores show no gender differences, this difference seems to be specific to the health domain. Discussion of related evidence can be found in Peerson & Saunders .In conclusion, our results indicate that the SAHL-D is a valid Dutch-language measure of functional HL that can be applied in research on the role of objective HL in health and healthcare use, the differential effectiveness of (preventive) health intervention by HL, and the development of targeted interventions in healthcare. Implementation of the SAHL-D in various contexts in public health and health care is necessary to further investigate its reliability and validity.Words included in the SAHL-D.(PDF 80 KB)Additional file 1:Sample items of the SAHL-D comprehension test.(PDF 83 KB)Additional file 2:


Motor vehicle accident (MVA) victims may suffer both acute and post-traumatic stress disorders (PTSD). With PTSD affecting social, interpersonal and occupational functioning, clinicians as well as the National Institute of Health are very interested in identifying the most effective psychological treatment to reduce PTSD. From research findings, eye movement desensitization and reprocessing (EMDR) therapy is considered as one of the effective treatment of PTSD. In this paper, we present the results of a meta-analysis of fMRI studies on PTSD after MVA through activation likelihood estimation. We found that PTSD following MVA is characterized by neural modifications in the anterior cingulate cortex (ACC), a cerebral structure involved in fear-conditioning mechanisms. Basing on previous findings in both humans and animals, which demonstrate that desensitization techniques and extinction protocols act on the limbic system, the effectiveness of EMDR and of cognitive behavioral therapies (CBT) may be related to the fact that during these therapies the ACC is stimulated by desensitization. Traumatic events  are an important cause of psychological distress and psychiatric disorders. Harvey and Bryant  reports PTSD is a relatively common psychiatric disorder occurring as a consequence of a major traumatic event. It is clinically characterized by the following symptoms: involuntarily re-experiencing phenomena ; avoidance of talking about or being reminded of the traumatic event, negative alterations in thoughts and mood, emotional numbing and hyperarousal symptoms  . fMRI studies mainly adopted trauma script-driven imagery paradigms, during which participants were required to remember olfactory, auditory, somato-sensory and visual sensations that were associated with their traumatic event  and non-emotional pictures were presented in the delay phase, PTSD-MVA patients showed higher activation in the emotion-processing regions, including the amygdala, precuneus and fusiform gyrus, but lower activation in the inferior frontal cortex, insula and left supramarginal gyrus than the control group , with an increased ALFF in the left medial prefrontal cortex and the right ACC  in the left medial prefrontal cortex, in the orbitofrontal cortex and in the middle frontal gyrus as well as in the right STG. These areas are also involved in different degree of changes also in PTSD caused by other traumas, however, the limbic system and in particular of the ACC are more involved in PTSD due to MVA with respect to other traumas. However, more fMRI studies should be performed to better understand neural mechanisms underlying different type of traumas and above all to state that an area is involved only in one type of trauma with respect to another one it should be performed a meta-analysis in which neural correlates coming from different studies on different traumas are analyzed with the aim to individuate cluster of activations.While several studies have assessed the behavioral effect of EMDR therapy (as reported above), less attention has been given to neural correlates of recovery after EMDR. One of the most important findings comes from neurophysiological studies, using electroencephalography (EEG). It has recently been found that EMDR facilitates the processing of traumatic memories in eye movement desensitization by improving interhemispheric coherence, measured by EEG  OR PTSD PET. Searches of this database were limited to articles written in English. This produced 615 papers. Our a-priori inclusion criteria for papers were: (1) Inclusion of whole-brain analysis performed using magnetic resonance imaging (MRI) or positron emission tomography (PET); this excluded papers that reported only results from ROI analysis. (2) Providing of coordinates of activation foci, either in Montreal Neurological Institute (MNI) or Talairach reference space. (3) All participants in the studies were diagnosed with PTSD after MVA. (4) PTSD was diagnosed according to the DSM-IV diagnostic criteria, including symptoms persisting for 6 months or more. (5) Other psychiatric disorders or other trauma-related PTSD were excluded. (6) Only group studies were included. Two authors (L.P. and M.B.) independently identified studies matching with the criteria reported above. Only published studies were included in our meta-analysis.Using these criteria, we selected only 6 papers reporting 9 individual experiments on PTSD-MVA patients . Meta-analysis was carried out on selected studies using the “activation likelihood estimation” (ALE) analysis. Studies are summarized in Table Activation likelihood estimation (ALE) analyzes the probability that a voxel will contain at least one of the activation foci; it is calculated at each voxel, and results in a thresholded ALE map. ALE assesses the overlap between foci by modeling the probability distributions centered at the coordinates of each one , used to assess the functional connectivity of specific brain regions  at the 0.05 level of significance . Moreover, a minimum cluster size of 200 mm3 was chosen. A cluster analysis was performed on the thresholded map. The ALE results were registered on an MNI-normalized template (brainmap.org) using Mricro.The ALE meta-analysis was performed using GingerALE 2.1.1 (brainmap.org) with MNI coordinates . According to Eickhoff et al.’s  modifiedx = 8; y = 46; z = 12)  Figure . In deta) Figure .The main aim of the present study is to provide neurobiological evidence on which to base a biological-founded treatment model for PTSD after MVA. Neuroimaging evidence reviewed in the introduction section appears to suggest that by modulating the neural circuits altered in PTSD after MVA, EMDR may represent a “treatment of choice” for PTSD-MVA. Thus, we performed a comprehensive meta-analysis of neuroimaging studies of PTSD-MVA to assess (a) brain areas showing consistent modifications in PTSD after MVA; and (b) their functional connectivity.Global results from the present meta-analysis demonstrate that PTSD-MVA is correlated with consistent neural modifications mainly located at the level of the right ACC, whose activity is in turn strongly correlated with activity in the frontal, parietal and limbic lobes. Also based on previous neuroimaging evidence of neural underpinnings of EMDR  is exposure to the feared object or context  and EMDR. This result emerged from 70 studies involving a total of 4,761 participants, demonstrating more effective TFCBT and EMDR than waitlist/usual care (Bisson et al., EMDR on the other hand has repeatedly been found to rely on the ACC (Yamasaki et al., Anyway, EMDR cannot be considered suitable just for MVA-PTSD. Actually, EMDR has been successfully used, to treat PTSD after different traumatic events (Lansing et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Providing psychological first aid (PFA) is generally considered to be an important element in preliminary care of disaster victims. Using the best available scientific basis for courses and educational materials, the Belgian Red Cross-Flanders wants to ensure that its volunteers are trained in the best way possible.To identify effective PFA practices, by systematically reviewing the evidence in existing guidelines, systematic reviews and individual studies.Systematic literature searches in five bibliographic databases  were conducted from inception to July 2013.Five practice guidelines were included which were found to vary in the development process (AGREE II score 20–53%) and evidence base used. None of them provides solid evidence concerning the effectiveness of PFA practices. Additionally, two systematic reviews of PFA were found, both noting a lack of studies on PFA. A complementary search for individual studies, using a more sensitive search strategy, identified 11 237 references of which 102 were included for further full-text examination, none of which ultimately provides solid evidence concerning the effectiveness of PFA practices.The scientific literature on psychological first aid available to date, does not provide any evidence about the effectiveness of PFA interventions. Currently it is impossible to make evidence-based guidelines about which practices in psychosocial support are most effective to help disaster and trauma victims. In the first few moments and hours after a disaster, survivors may have medical, material, social, and emotional needs. After the traditional steps to guarantee physical safety, it became common practice to also offer immediate psychosocial support Training people in PFA improves their confidence in applying it In this paper, a search for evidence was performed to develop an evidence-based guideline on PFA, aimed at improving our own trainings. We started by critically evaluating the evidence base included in existing guidelines and systematic reviews on PFA. We then performed an extensive literature search, applying a more sensitive approach compared to previous searches, to identify studies on the effectiveness of PFA.Considering the broad and unclear terminology used within the PFA domain, we will first set out some key definitions relevant to this paper, in order to be transparent in our own terminology.Psychological first aid: We conceive PFA as an intervention approach aimed at helping people deal with the experience and the consequences of a disaster or adversity. Our practice is strongly based on the five principles of Hobfoll Immediate aftermath of a disaster: a couple of hours until 7 days after the eventMental health professionals: people with a psychological/social degreeLaypeople: People without any previous training in the field of psychological or social supportA primary search (until July 2013) was performed to identify PFA guidelines (using the G-I-N database and MEDLINE) and to identify systematic reviews with a focus on PFA (using The Cochrane Library and MEDLINE) . GuideliThe search for individual studies focused on the following question: “In people affected by a disaster or trauma do certain PFA interventions, promote safety, connectedness, self and collective efficacy, calm and hope?”. Search strategies were developed by the BRC Centre for Evidence-Based Practice in cooperation with the BRC Psychosocial Intervention Service. Electronic searches of literature were conducted in the following databases: The Cochrane Library, MEDLINE (PubMed interface), PsycINFO, PILOTS , from the date of inception until July 2013. Highly sensitive search strategies have been used in order to be as complete as possible in the search for scientific literature. Search terms such as ‘PFA’ and its synonyms were extended with terms referring to the five essential elements individually  as described by Hobfoll et al. Articles were included when containing the following characteristics: (1) population: victims of a disaster or traumatic event; not diagnosed or referred by a health professional; (2) intervention: community-based interventions; by laypeople, first responders, health care professionals, the victim himself; taking place the first few hours/days of a disaster, single interventions; feasible for laypersons (3) measuring mental health parameters  or physiological parameters (blood pressure or heart rate). Studies must have a controlled experimental or observational study design in order to be included. Studies on individual treatment or one-to-one sessions, therapeutic interventions after a diagnosis by a health care professional; medical interventions, long term interventions and psychological debriefing were excluded.The specific data extracted were (1) methodological development and content for guidelines and systematic reviews; (2) details on the study type, population, intervention and outcomes for individual studies. The Appraisal of Guidelines for Research and Evaluation II (AGREE II) instrument A total of five published practice guidelines were identified by the search strategy Next to the 5 guidelines, the search identified two systematic reviews: one developed by Fox et al. (from 1990 to September 2010) supported by the American Red Cross and another by Bisson & Lewis (from inception of the database to 2009) commissioned by the World Health Organization Analysis of the search strategies, included in the guidelines and systematic reviews, revealed rather specific searches yielding less than thousand references for screening. To further explore the presence or absence of studies on PFA, we built a more sensitive search strategy and conducted our own systematic literature search.The highly sensitive search identified 11 237 titles, with 10 097 remaining after deduplication. A total of 104 records were defined as potentially eligible based on title and abstract. At the base of evidence-based guideline development lies the use of systematic literature reviews to assess existing research regarding the effectiveness of interventions. The aim of the present article was to more sensitively investigate the evidence supporting PFA guidelines and materials. A detailed analysis of 5 available guidelines unravels that they refer to different references or sources as a base for their recommendations The lack of research evaluating non-therapeutic PFA interventions, might find its origin on different levels. First of all, scientific evidence in the field of prehospital care is scarce in general, and the available studies often use flawed methods leading to evidence of a very low quality Evidence-based approaches for psychosocial support after adversities are increasingly being demanded The limitations of this systematic literature search are important to recognize. Even though a very sensitive search strategy was adopted, there is no absolute guarantee of not having missed relevant articles as much of the literature is not well-indexed in the bibliographic databases. Secondly, the systematic search for papers was restricted to five databases, most relevant to our topic . Thirdly, rigorous selection criteria were used to ensure that only highly relevant evidence was retrieved, focusing on non-therapeutic PFA interventions within the first seven days following a disaster which might explain why no studies could be included in the systematic review. The lack of evidence for PFA interventions obviously does not prove evidence of absence of a useful effect of PFA, but it does reveal the need for future studies on the effectiveness of early PFA interventions to support the relatively new concept of PFA.Although PFA is considered to be an important approach for disaster-affected populations, there is a complete lack of high-quality experimental and observational studies on the effectiveness of PFA in the immediate aftermath of a disaster. Consequently, research is needed to determine the most effective, efficient, and acceptable interventions before evidence-based PFA guidelines on how to train laypeople and professionals can be developed.S1 ChecklistPRISMA Checklist.(DOC)Click here for additional data file.S1 AppendixSearch strategies.(DOC)Click here for additional data file.S2 AppendixExcluded studies after full text evaluation.(DOC)Click here for additional data file.


A range of evidence informs healthcare decision-making, from formal research findings to ‘soft intelligence’ or local data, as well as practical experience or tacit knowledge. However, cultural and organisational factors often prevent the translation of such evidence into practice. Using a multi-level framework, this project will analyse how interactions between the evidence available and processes at the micro  and meso  levels influence decisions to introduce or diffuse innovations in acute and primary care within the National Health Service in the UK.This study will use a mixed methods design, combining qualitative and quantitative methods, and involves four interdependent work streams: (1) rapid evidence synthesis of relevant literature with stakeholder feedback; (2) in-depth case studies of ‘real-world’ decision-making in acute and primary care; (3) a national survey and discrete choice experiment; and (4) development of guidance for decision-makers and evaluators to support the use of evidence in decision-making.This study will enhance the understanding of decision-makers’ use of diverse forms of evidence. The findings will provide insights into how and why some evidence does inform decisions to introduce healthcare innovations, and why barriers persist in other cases. It will also quantify decision-makers’ preferences, including the ‘tipping point’ of evidence needed to shift stakeholders’ views. Practical guidance will be shared with healthcare decision-makers and evaluators on uses of evidence to enable the introduction and diffusion of innovation. A range of evidence informs healthcare decision-making, from formal research findings  to ‘softA preliminary, scoping review of the health services research literature on evidence use in decision-making concerning innovation suggests a need for new research in three areas. First, at the micro  level, there is a need to determine the combinations of evidence, including practical or local evidence , used bySecond, at the meso  level, there is a need to examine how evidence informs ‘real-world’ decision-making processes through in-depth case studies , taking The inclusion of both micro and meso level processes reflects the theorised interaction between levels in quality improvement processes , i.e. orThird, studies have shown that stakeholders prefer different types of evidence  , 9, but To identify, using a literature review and stakeholder feedback, the factors that influence the use of evidence in decision-making on the introduction and diffusion of innovations in health care;To assess the use of evidence in informing decision-making on the introduction and diffusion of innovation using ‘real-world’ case studies in acute and primary care;To establish decision-makers’ preferences for evidence  to inform the introduction and diffusion of innovations;To develop guidance for decision-makers and evaluators to support the evaluation and application of evidence to enable innovation.To address the gaps identified in the current literature, this study will answer the following research question: what is the role of evidence in decision-making on the introduction and diffusion of service innovations in acute and primary care? In order to address this research question, the following objectives for the study have been defined:This multidisciplinary study uniquely brings together different methodological and disciplinary perspectives  to study the role of evidence in decision-making with the aim of meeting these objectives. The following work streams, which are interdependent and will inform one another, are planned in relation to each objective. Methods for data collection and analysis are described by work stream.The rapid review, with stakeholder feedback, has two purposes: (a) to map the types of information used to inform decision-making in different contexts and (b) to identify factors at the micro and meso levels that influence how this information informs decision-making on innovation. We will then obtain stakeholder feedback on the compiled evidence in relation to these purposes using focus groups to identify any gaps or themes that need to be developed further.The literature review will identify examples of evidence use in decisions to adopt innovation from studies conducted on the NHS and health systems internationally. To identify papers, relevant social science and biomedical databases will be searched, including PubMed, ISI Web of Knowledge, and Business Source Complete, using search terms in the title or abstract. Search terms, that may be combined, will include the following: ‘decision-making’, ‘service innovation’, ‘evidence use’, ‘innovation adoption or diffusion’, ‘professional roles’, and ‘organisational factors or processes’. A hand search of selected management and health policy journals and books will be undertaken; bibliographies of recent and highly relevant papers will also be consulted. Studies of decision-making at the national health system level and in public health will be excluded. The review will be based predominantly on primary qualitative studies and systematic and other types of review. To map types of information used in decision-making, we will include quantitative studies that provide descriptive statistics on evidence use, as appropriate.Through thematic analysis of the compiled literature , we willThe views of four groups of stakeholders will be obtained through the focus groups: (1) acute care providers, (2) primary care providers, (3) service commissioners, and (4) patient representatives. Four focus groups, with 8–10 participants in each group, will be structured using discussion points derived from the rapid literature review. Telephone interviews will be used for participants not able to attend a face-to-face focus group. National participants will be identified using different channels. Through the National Institute of Health Research Collaborations for Leadership in Applied Health Research and Care (CLAHRC) North Thames, which has 54 partners including hospitals, local authorities, and commissioners, we have links across the 13 CLAHRCs nationally which will be used to reach participants.In-depth case studies will be conducted on the use of evidence in ‘real world’ decision-making concerning the introduction or diffusion of three service innovations in acute and primary care. Case studies were chosen because they allow complex phenomena to be studied in-depth, allowing both the case  and the context  to be taken into account, as well as interactions between the two . This apAs shown in Table At the micro level: how both stroke clinicians and senior managers within individual provider organisations’ use, and negotiate understandings of, research evidence relative to other information, e.g. financial impact and local need, when considering reconfiguration.At the meso level: how individual ‘champions’ and collective decision-making groups influence how evidence is used and consensus reached among providers and commissioners across health systems considering reconfiguration.Evidence produced by members of the research team has shown that centralising stroke services to create a smaller number of high-volume units in London has improved patient outcomes , 19. ThiAt the micro level: how general practitioners within primary care practices with historically lower and higher referral rates for suspected cancer have responded to new guidance, including profession-specific barriers and facilitators.At the meso level: how involvement and interaction between different organisations, including clinical networks, commissioners, third sector, and service providers, have influenced responses to the referral guidance and its use to inform service planning.In 2015, NICE updated its clinical guidelines  for recoWe will study innovations in outpatient services for treating chronic eye disease where demographic change is placing increasing pressure on hospitals. New service configurations are being developed by a number of providers within the NHS. The study will be conducted with a large specialist provider of ophthalmic services in England, which delivers 470,000 outpatient appointments per year through an organisational network (including partnerships with other providers) with multiple clinics across a large metropolitan area and the surrounding region. The Trust has piloted ‘virtual review’ clinics for stable glaucoma patients to improve resource use and enable provision closer to patients’ homes, although barriers to implementation were encountered .At the micro level: types of evidence required by different professional groups, including clinical and managerial staff, to support roll-out of new clinics.At the meso level: processes across the organisational network used to acquire, share, and interpret evidence to inform decision-making. This will include analysis of how counter evidence and other factors, e.g. perceived resource implications and local need, are negotiated and inform decision-making.A prospective study will analyse how evidence informs decision-making by the Trust and other providers within the network to move from the pilot phase to a wider implementation of virtual clinics across other sites, including responses to evidence for change by different sites and professional groups. This case will focus on the following:Case study data will be collected through semi-structured interviews, non-participant observation, and documentary analysis Table . InterviThe analysis of qualitative data from the case studies will combine inductive and deductive approaches , as themUtilising the literature review, stakeholder feedback, and case study data, a survey of providers and commissioners will assess how preferences to introduce or diffuse innovations are influenced by characteristics of the evidence for change relative to other contextual factors (objective 3). The first part of the questionnaire will elicit preferences for different types and quality of evidence. The second part will be a DCE that will compare different decision-making groups’ preferences for the strength of evidence to examine tipping points in preferring one option over another.We will develop a survey questionnaire for distribution to stakeholders . The questionnaire will have two parts. The first will contain questions about preferences for different types of evidence and quality of the evidence when judging whether or not to implement a new innovation. Types of evidence will include impact on health ; behaviour; knowledge; use of services; budget; and incremental cost-effectiveness. Quality of evidence will capture internal and external validities  and contextual factors (e.g. credibility of the person or organisation providing the evidence). We will ask respondents whether or not different types and quality of evidence are important and to rank them in order of importance. The different types and quality of evidence will be drawn from findings of the rapid review, focus groups, and case studies.The second part will be a DCE to capture preferences of stakeholders for the strength of evidence needed  to implement new innovations and how types of evidence are traded against one another.We will identify types of evidence as described in the first part of the questionnaire. In the context of a DCE, these are referred to as attributes.We will assign levels to these attributes  based on real-world examples of innovations, derived from objectives 1 and 2 and systematic literature reviews.We will design the DCE questionnaire using a pairwise choice framework. We will compile a set of pairwise scenarios that describe the feasible combinations of levels and attributes of new innovations. The number of pairwise choices will be reduced to a practical number for participants to answer.The DCE will follow international best-practice guidelines  and be dThe questionnaire will be piloted then administered to three groups of respondents: (1) acute care providers, (2) local commissioners, and (3) primary care providers (GPs and practice staff). Both managerial and clinical representatives will be sampled in the questionnaire.Responses will be collected by online survey tools and hard-copy questionnaires delivered at face-to-face meetings. We will sample questionnaire respondents nationally and will use similar sampling methods to objective 1. We will also recruit respondents via newsletters distributed by Royal Colleges, professional organisations, and at professional conferences and meetings.Sample size calculations for DCEs are not straightforward, depending on question format, complexity of choice tasks, desired precision of the results, degree of heterogeneity in the target population, availability of respondents, and need for subgroup analyses. A sample size of 300 is commonly recommended , which wTo analyse the data from the first part of the questionnaire, frequency tables will be created to describe whether or not different types and quality of evidence are important. We will cross-tabulate the responses by type of respondent and explore differences using chi-square tests. We will investigate differences in the ranking of different types and quality of evidence using rank-sum tests and Kendall’s coefficient of concordance.The DCE data will be analysed using conditional logit regression analysis. The results will indicate which attribute (type of evidence) is most important to respondents and how this compares with the other attributes. We will explore the trade-offs the participants are willing to make between attributes, quantified using the marginal rates of substitution, which summarise how respondents trade-off values of the different attributes, e.g. what increase in total spending decision-makers are willing to trade-off for a 1 % reduction in mortality. We will model interaction effects, allowing us to investigate how preferences for one attribute vary dependent on another . We will also determine the predicted probability that a set of new innovations derived from different combinations of attributes would be selected based on the regression results. This will estimate the probability that innovations with particular values for the types of evidence will be selected and allows them to be ranked in terms of their order of preference.Combining findings from the literature review, focus groups, case studies, and DCE, factors to take into account when seeking, using, and producing evidence will be distilled into guidance for decision-makers and evaluators to inform decisions to introduce or adopt innovations. This may build on Lavis et al.’s five questions to inform strategies to improve the use of evidence by decision-makers  . The guiOur overall approach is to use mixed or ‘merged’ methods, which involves the integration of quantitative and qualitative methods . This wiThis study will enhance understanding of decision-makers’ use of diverse forms of evidence and the importance of contextual factors in shaping the ways in which evidence is used to inform decision-making on innovation. The findings will enhance understanding of how and why some evidence does inform decisions to introduce health care innovations and why barriers persist in other cases. It will also quantify decision-makers’ preferences, including the tipping point of evidence needed to shift stakeholders’ views. Guidance will be shared with key groups of decision-makers at different levels of the NHS, as well as evaluators, to inform how evidence is produced and used to satisfy a range of stakeholders when making decisions on adopting innovations. The study’s findings will improve understanding of how evidence can inform practice, including barriers and facilitators to its use. The study will produce generalisable knowledge on how to optimize the use of evidence to inform decisions on innovation adoption which, in turn, may help to accelerate and maximise the implementation of practices that improve patient outcomes.The evidence needed to reach the tipping point for different groups will be determined using a DCE with providers and commissioners within both acute and primary care settings. A strength of this study is the use of quantitative and qualitative data to determine decision-makers’ preferences. The DCE will seek to measure the influence of diverse evidence and contextual factors, including professional interests and relationships, on decision-making. Thus, a practical challenge is translating insights from the qualitative data into appropriate options that can be used for eliciting participants’ preferences. For instance, if the qualitative data suggests that decision-making is a distributed process that unfolds over time, this might suggest the need to adapt the model of the lone, contemplative decision-maker typically used in a DCE .The study’s findings will provide new insights into the ways in which evidence informs both the adoption of new innovations and the diffusion of existing innovations to new contexts, taking into account diverse types of evidence , different types of stakeholder, and contextual factors involved in decision-making. By combining qualitative and quantitative methods, the study will generate new knowledge regarding the types and quality of evidence that decision-makers prefer and the tipping point of evidence necessary to shift the perspectives of different groups of decision-makers. This includes groups or contexts that may typically be less receptive to change, e.g. medical professionals , 35, andThis study was considered by the Chair of the UCL Research Ethics Committee on 29 February 2016 and is exempt from the requirement to obtain ethical approval.


Tolerant to Chilling and Freezing 1 (TCF1), interacts with histones H3 and H4 and associates with chromatin containing a target gene, BLUE-COPPER-BINDING PROTEIN (BCB), encoding a glycosylphosphatidylinositol-anchored protein that regulates lignin biosynthesis. Loss of TCF1 function leads to reduced BCB transcription through affecting H3K4me2 and H3K27me3 levels within the BCB gene, resulting in reduced lignin content and enhanced freezing tolerance. Furthermore, plants with knocked-down BCB expression (amiRNA-BCB) under cold acclimation had reduced lignin accumulation and increased freezing tolerance. The pal1pal2 double mutant (lignin content reduced by 30% compared with WT) also showed the freezing tolerant phenotype, and TCF1 and BCB act upstream of PALs to regulate lignin content. In addition, TCF1 acts independently of the CBF (C-repeat binding factor) pathway. Our findings delineate a novel molecular pathway linking the TCF1-mediated cold-specific transcriptional program to lignin biosynthesis, thus achieving cell wall remodeling with increased freezing tolerance.Cell water permeability and cell wall properties are critical to survival of plant cells during freezing, however the underlying molecular mechanisms remain elusive. Here, we report that a specifically cold-induced nuclear protein,  TCF1 on lignin content during cold acclimation in Arabidopsis. TCF1 is specifically induced by cold and is required for chromatin based gene regulation of cold responsive genes such as BCB (a GAP) that regulates lignin genes. Further evidence shows that reduction in lignin dramatically increases plant freezing tolerance, while lignin maintenance required for cold acclimation is regulated by TCF-mediated signaling. Thus, our study has revealed, for the first time, lignin remodeling as a key function of cold acclimation and freezing tolerance. The findings provide the first direct molecular evidence that freezing tolerance is directly related to cell wall properties during cold acclimation and extra/intercellular freezing upon and freezing/thawing process.Cold acclimation is a well-known adaptive process through which plants can dramatically increase their tolerance to freezing temperature. Modifications of cell wall have been recognized as a key characteristic during plant acclimation to low temperature. However, the molecular mechanism responsible for such cellular adaptation still remains a mystery. Here, we report an unexpected regulatory role of  Arabidopsis, expression profiling of cold-treated plants revealed that up to 20% of genes in the genome are regulated by cold. Characterization of a group of cold-regulated (COR) genes which are highly induced by cold stress using forward and reverse genetics has led to identification of a key CBF  signaling pathway. CBF transcription factors  can activate expression of the COR genes by binding to cis-elements in their promoters and induce cold acclimation and freezing tolerance  and HTR9 [histone H3 (AT5g10400)] coding regions were amplified and cloned in-frame into pGADT7 between the NdeI and XhoI sites to generate prey constructs. pACT2-HTB1 (histone H2B) and pACT2-HFO1 (histone H4) were from Dr. R. A. Bressan at Purdue University. For analysis of specific histone H4 interaction with TCF1, TCF1 coding region was cloned in-frame between the EcoRI and BamHI sites of pGADT7, another two histone H4 variants (At5g59690 and At1g07820) with high expression in tcf1-1 were introduced into pGBKT7 at EcoRI and SalI sites. Plasmid DNA of bait and prey constructs was transformed into the S. cerevisiae strain Y190. Individual transformants were streaked on plates containing a synthetic, minimal (SD) medium lacking tryptophan and leucine and grown for 24 h. Yeast cells were transferred onto a filter paper, and β-galactosidase  filter assays were performed [The erformed .Nuclei were isolated as described by . Twenty Escherichia coli as fusions with GST. The Ran clone was provided by Dr. Murray Stewart . Assays of guanine nucleotide exchange activity were performed by using [3H]GDP to load 30 pmol GST-Ran and subsequent incubation with 0.5 nM recombinant RCC1 or TCF1 for 3 min. The exchange activity was calculated as ln (Ct/C0), where C0 and Ct are radioactive counts at the start and end of the reaction, respectively. The GEF assays were repeated four times. Analysis of TCF1-histone interaction was performed as previously described [GEF activity assay was performed as described in . Brieflyescribed . Competiescribed .BCB and ACT2 genes, using primers shown in Chromatin was isolated and the chromatin immunoprecipitation assay was carried out ,69 by usCell wall fractions were isolated as described by Tokunaga et al . BrieflyS1 Fig(A) Arabidopsis eFP Browser online software (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi) shows the TCF1 gene expression was induced by cold stress. (B) Clustal W2 analysis of identity between RCC1 and TCF1 protein . Arrows in different colors with letter ABCD stands marked seven RCC1 repeat domains. (C) Expasy online software (http://www.expasy.org/scanprosite) predicted that Homo sapiens RCC1 contains 7 RCC1 repeats. Using the same program, we found that the TCF1 protein contains six predicted RCC1 repeats that are located as follows: 83–135, 178–252, 253–329, 330–381, 382–435, and 436–486.(TIF)Click here for additional data file.S2 Figprp20 mutant was transformed with the indicated vector. Individual colonies of transformants were streaked on SD plates lacking Ura with galactose as carbon source and put in 23°C /37°C for 24 h simultaneously. prp20+RCC1, prp20 transformed with human RCC1 gene. prp20+Vector, prp20 transformed with empty vector pMB150. prp20+TCF1, prp20 transformed with TCF1 gene.Yeast (TIF)Click here for additional data file.S3 Fig(A) Freezing analysis of 3-week-old tcf1-1 and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod without cold acclimation. Quantification of survival rate was taken at 7th days after treatments. (B) RT-PCR analysis of TCF1 expression in two TCF1-RNAi lines (TCF1-RNAi-2 and TCF1-RNAi-6) at 4°C for 7 day. (C) Freezing analysis of 3-week-old TCF1-RNAi lines and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod without cold acclimation. Quantification of survival rate was taken at 7th days after treatments. (D) Tolerance of 3-week-old TCF1-RNAi lines (TCF1-RNAi-2 and TCF1-RNAi-6) and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod with cold acclimation for 7 days. The pictures were taken 7 days after treatments. (E) Quantification of survival rate of the treated plants in (C), . (F) Leakage of electrolytes in TCF1-RNAi lines and WT plants treated at indicated temperatures below freezing. Error bars are standard deviation (n = 8), .(TIF)Click here for additional data file.S4 Fig(A) The three week-old non-acclimated plants were treated at -8°C for 2 h followed by a seven day recovery. These include tcf1-1, WT, a representative line of tcf1-1 transformed with an empty vector pEZR(K)LC (tcf1-1Vector-2), and three independent homozygous lines of tcf1-1 transformed with TCF1 gene  without cold acclimation. (B) Semi-quantitative RT-PCR for TCF1 expression in the wild type and transgenic plants. The three-week-old plants were treated with or without a seven-day cold acclimation at 4°C, respectively. Lane 1: WT, lane 2: tcf1-1, lane 3: tcf1-1Vector-2, lane 4: tcf1-1TCF1-3, lane 5: tcf1-1TCF1-12, lane 6: tcf1-1TCF1-13. (C) Quantification of survival rate of cold acclimated (black) and non-acclimated (grey) plants after freezing treatment at -8°C for 2 h and a seven day period of recovery. 1: WT, 2: tcf1-1, 3: tcf1-1Vector-2, 4: tcf1-1TCF1-3, 5: tcf1-1TCF1-12, 6: tcf1-1TCF1-13. Error bars are standard deviation (n = 80–100). Three biological experiments were performed and the data are expressed as mean ±S.E. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis. (D) Leakage of electrolytes in WT, tcf1-1 and TCF1 complementary lines  treated at indicated temperatures below freezing. 3-week-old plants were cold-acclimated at 4°C for 7 day (Cold) or without treatment. Error bars are standard deviation (n = 10), .(TIF)Click here for additional data file.S5 Fig(A) The three week-old tcf1-1 and wild type plants were treated with cold at 4°C and rosettes were harvested at the indicated time points for RNA extraction and semi-quantitative RT-PCR. Rosettes of the plants treated with 100 μM ABA, 400 mM mannitol or 300 mM NaCl for 3 h were also collected for gene expression analysis. Transcript levels were assayed for CBF genes and loading control ACTIN2. (B) Semi-quantitative RT-PCR for CBF2 expression in a T-DNA insertion line. Three-week-old SALK_025203 and Col-0 seedlings grown on soil were subjected to low temperature (4°C) for 3 h, and the shoots were collected for the expression analysis. ACT2 gene was used as a loading control. The result showed that the mutant is a null mutation for the CBF2 gene, and was named as cbf2.(TIF)Click here for additional data file.S6 Figtcf1-1 grown on MS medium plates were treated with cold stress for 7 day, and the levels of four genes’ transcripts were analyzed. The genes showed altered transcript levels in tcf1-1 in the microarray analysis were selected. ACT2 gene was used as a loading control. CA: Cold acclimation, NA: None cold acclimation.Three-week-old WT and (TIF)Click here for additional data file.S7 Fig(A) Levels of H3K4me2 and H3K27me3 by ChIP-PCR analysis with wild-type (WT), tcf1-1, tcf1-1TCF1-3 and tcf1-1TCF1-12 plants normalized to ACT2 or AGAMOUS in BCBc1 fragment with and without a 7-day cold treatment. (B) to (E) Levels of H3K9me2, H3K9ac, H4Ac , H3K14ac and H3K36me3 by ChIP-PCR analysis normalized to indicated control in BCBc1 fragment in wild type and tcf1-1 plants with a seven-day cold acclimation.(TIF)Click here for additional data file.S8 FigBCB transcripts in wild-type (Black) and amiRNA-BCB (Grey) plants. The plants were treated with cold at 4°C for 7 days. Three biological experiments were performed and the data are expressed as mean ±S.E. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis.The levels of (TIF)Click here for additional data file.S9 Fig(A) Freezing treatment of three-week-old amiRNA-BCB transgenic plants (#4–9 and #9–2) and wild-type (WT) plants at the indicated temperature without cold acclimation. The pictures were taken 7-days after treatments. (B) Quantification of survival of the plants in (A). Error bars are standard deviation (n = 80–100).(TIF)Click here for additional data file.S10 Fig(A) Three week-old pal1pal2 and wild type (WT) plants without a 7-day cold treatment were used for freezing treatments at indicated time. The pictures were taken 7 days after treatments. (B) Quantification of survival of the wild type (Black) and pal1pal2 plants (Grey) in (A). Error bars represent standard deviation (n = 80–100).(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.