Outcomes of fracture-related infections - do organism, depth of involvement, and temporality count?

Purpose: To determine mortality and outcomes of patients diagnosed with fracture-related infections (FRIs).Methods: FRI patients treated at a trauma centre between 2001 and 2020 were analysed. The primary outcome was 1-year mortality; mortality associations with FRI organism, depth of involvement, and temporality were investigated with multivariable survival analysis. Healthcare-associated and serological outcomes were reported as secondary outcomes. Results: 311 FRIs with mean age of 67.0 and median Charlson comorbidity index of 0 were analysed. Methicillin-sensitive Staphylococcus aureus (MSSA) (29.9%) was the most frequently implicated organism. The majority of FRIs were deep infections (62.7%). FRIs were diagnosed at a median of 40 (IQR 15-200) days post index surgery. The mean follow-up was 5.9 years. One-year mortality amounted to 17.7%. MSSA FRIs were associated with better survival (adj HR 0.34, 95%CI 0.15-0.76, p = 0.008). There was no difference in survivorship between deep or superficial FRI (adj HR 0.86, 95%CI 0.62-1.19, p = 0.353) or in relation to onset time (adj HR 1.0, 95%CI 0.99-1.00, p = 0.943). Implant removal or debridement alone was performed in 61.7% and 17% respectively. Antibiotics was prescribed for 53 (IQR 23-110) days, and patients were hospitalised for 39 (IQR 19-78) days. CRP and ESR normalised in 70.3% (median 46 days) and 53.8% (median 86 days) patients respectively. Conclusion: Fracture-related infections are associated with significant mortality and morbidity regardless of depth and temporality. Non-MSSA FRIs are associated with inferior survival.

Hip Fracture as a Predictive Marker for the Risk of Dementia: A Population-Based Cohort Study.

OBJECTIVES: This study aimed to investigate the association between hip fracture and the risk of dementia. DESIGN: A retrospective real-world propensity score-matched cohort study was conducted using the real-world hip fracture cohort (RHFC). SETTING AND PARTICIPANTS: Electronic health record data from the Clinical Data Analysis and Reporting System (CDARS) in Hong Kong were used. A total of 52,848 patients aged ≥65 years and with at least an event of fall from 2006 to 2015 were included in the RHFC. METHODS: The incidence of fall, hip fracture, and dementia was determined using their International Classification of Diseases, Ninth Revision (ICD-9) codes. Competing risk regression models were used to estimate hazard ratios (HRs) and 95% CIs. RESULTS: Hip fracture was associated with an increased risk of dementia (HR 1.09, 95% CI 1.04-1.15, P < .001). The subgroup analysis showed that association was significant in women but not in men. CONCLUSIONS AND IMPLICATIONS: Hip fracture was associated with the increased risk of dementia among older adults. Further studies investigating the potential roles of hip fracture in the development of dementia could benefit the management of both conditions in older adults.

Low Bone Mineral Density With Risk of Dementia: A Prospective Cohort Study.

OBJECTIVES: Osteoporosis and dementia often coexist, but the association between the 2 diseases remains unclear. This study aimed to investigate the relationship between bone mineral density (BMD) and the risk of incident dementia. DESIGN: Prospective cohort study, the Hong Kong Osteoporosis Study (HKOS). SETTING AND PARTICIPANTS: Data were from the HKOS and the Clinical Data Analysis and Reporting System (CDARS) in Hong Kong. A total of 5803 participants aged ≥40 years and free of dementia were included in the HKOS. METHODS: The baseline BMD at the lumbar spine, femoral neck, trochanter, and total hip were measured using dual-energy x-ray absorptiometry (DXA). The incidence of dementia was identified using their International Classification of Diseases, Ninth Revision, codes. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% CIs. RESULTS: The median follow-up time of the HKOS was 16.8 years. Higher BMD T scores at the lumbar spine, trochanter, and total hip were significantly associated with the reduced risk of dementia with the respective HR of 0.85 (95% CI 0.76-0.95; P = .004), 0.78 (95% CI 0.68-0.90; P < .001), and 0.82 (95% CI 0.72-0.93; P = .003). The subgroup analyses showed that associations were significant in women but not in men, whereas the associations were unaltered after adjusting for serum estradiol. CONCLUSIONS AND IMPLICATIONS: Low BMD was associated with an increased risk of dementia, particularly in women. Future studies evaluating the clinical usefulness of BMD on dementia prediction and management are warranted.

Immediate Risk for Cardiovascular Events in Hip Fracture Patients: A Population-Based Cohort Study.

BACKGROUND: Emerging evidence showed that bone metabolism and cardiovascular disease (CVD) are closely related. We previously observed a potential immediate risk of cardiovascular mortality after hip fracture. However, whether there is an immediate risk of cardiovascular events after hip fracture is unclear. The aim of this study was to evaluate the risk for major adverse cardiovascular events (MACEs) between patients having experienced falls with and without hip fracture. METHODS: This retrospective population-based cohort study used data from a centralized electronic health record database managed by Hong Kong Hospital Authority. Patients having experienced falls with and without hip fracture were matched by propensity score (PS) at a 1:1 ratio. Adjusted associations between hip fracture and risk of MACEs were evaluated using competing risk regression after accounting for competing risk of death. RESULTS: Competing risk regression showed that hip fracture was associated with increased 1-year risk of MACEs (hazard ratio [HR], 1.27; 95% confidence interval [CI], 1.21-1.33; p < .001), with a 1-year cumulative incidence difference of 2.40% (1.94%-2.87%). The HR was the highest in the first 90-days after hip fracture (HR of 1.32), and such an estimate was continuously reduced in 180 days, 270 days, and 1 year after hip fracture. CONCLUSIONS: Hip fracture was associated with increased immediate risk of MACEs. This study suggested that a prompt evaluation of MACE among older adults aged 65 years and older who are diagnosed with hip fracture irrespectively of cardiovascular risk factors may be important, as early management may reduce subsequent risk of MACE.

Quality of Surgery and Patient Selection Are Keys to Success in Hip Stabilization Surgery in Cerebral Palsy.

BACKGROUND: Cerebral palsy patients are at risk of hip instability, to which various soft tissue and bony surgeries are performed should conservative management fail. We aim to identify factors associated with treatment failure to guide surgical management. METHODS: Cerebral palsy patients treated at 2 university-affiliated tertiary pediatric orthopaedic referral centers with hip stabilization surgery performed for subluxation in 1998 to 2015 with minimum of 5 years follow-up were reviewed. Failure was defined as reoperation to the same hip because of recurrent subluxation. Age, sex, Gross Motor Function Classification System level, tone abnormality, operation type, Reimer's migration index (RMI), and acetabular index (AI) were assessed. Cut-off values were identified through Youden index on receiver operating characteristic curve. RESULTS: Eighty-nine hips from 55 patients with mean follow-up of 12.4 years were analyzed. Revision surgery was performed in 14 hips. Postoperative hip subluxation (P<0.001) and acetabular dysplasia (P=0.001) were predictive of failure, with postoperative RMI conferring an adjusted hazard ratio of 1.13 (95% confidence interval: 1.08-1.19, P<0.001) on multivariable survival analysis. Achieving a postoperative RMI of <27.5% predicts success with 92.9% sensitivity and 72% specificity with area under curve of 0.916 (P<0.001), while postoperative AI of <23.1 degrees predicts success with 92.3% sensitivity and 62.2% specificity with area under curve of 0.796 (P=0.001). In subgroup analysis of soft-tissue-only procedures, RMI >44% preoperative and >32% postoperative were associated with reoperation. In femur-only osteotomies, preoperative RMI >48% and postoperative RMI >28% were associated with failure. In pelvic and combined osteotomies, postoperative RMI >32% and AI >30 degrees were associated with failure. Other factors analyzed were not associated with reoperation. CONCLUSIONS: Patient selection and quality of surgery in terms of residual postoperative hip subluxation and acetabular dysplasia are associated with need for remedial surgery. Soft-tissue-only procedures should aim to correct RMI to <32%. Bony surgery should be considered when preoperative RMI >44%, and pelvic osteotomies if RMI >48%. Pelvic osteotomies should target postoperative RMI <32% and AI <30 degrees. LEVEL OF EVIDENCE: Level II-prognostic study.

[Hospital paramedic. An interprofessional blended learning concept to qualify paramedics and medical personnel for deployment in intensive care units and emergency departments during the COVID-19 pandemic]. / Kliniksanitäter. Ein interprofessionelles Blended-Learning-Konzept zur Weiterqualifikation von Rettungsdienst- und medizinischem Personal zum Einsatz auf Intensivstationen und in Notaufnahmen während der COVID-19-Pandemie.

BACKGROUND: The COVID-19 pandemic necessitated a time-critical expansion of medical staff in intensive care units (ICU) and emergency rooms (ER). OBJECTIVE: This article describes the development, performance and first results of an interprofessional blended learning concept called hospital paramedics, qualifying paramedics and additional medical personnel to support ICUs and ERs. MATERIAL AND METHODS: The Protestant Hospital of the Bethel Foundation (EvKB), University Hospital OWL, University of Bielefeld in cooperation with the Study Institute Westfalen-Lippe, developed a 2-stage blended learning concept (stage 1 e­learning with online tutorials, stage 2 practical deployment) comprising 3 modules: ICU, ER and in-hospital emergency medicine. At the beginning, the participants were asked about their sociodemographic data (age, gender, type of medical qualifications) and subjective feeling of confidence. At the end, a final discussion with the participant, the practice instructor and the supervising physician took place and an evaluation of the deployment by the head of the practice and the hospital paramedic was carried out using questionnaires. RESULTS: Within 6 weeks 58 (63%) of the 92 participants completed the online course and 17 (29%) additionally completed their traineeship. In the ICU they assisted with preparing catheter systems, medication and nursing, performed Manchester triage and initial care in the ER. After completion hospital paramedics were significantly more confident when working in a hospital, catheterization and tracheostoma care (p < 0.05). Of the supervisors 94% deemed the deployment as useful and 100% of the participants were prepared to be available at short notice in their areas as compensation for the COVID-19-pandemic in the event of a staff shortage. Through the provision of additional intensive care ventilators and monitoring units in the period from March to the beginning of May 2020 and the personnel management that was carried out, the EvKB was in a position to increase the number of previously provided ventilator beds by potentially >40 ventilation places. CONCLUSION: Blended learning concepts, such as hospital paramedics, can quickly qualify medical personnel for use in system-relevant settings, relieve nursing staff and thus create an expansion of intensive care capacities. Existing or pending pandemic and contingency plans should be complemented by such blended learning training so that they are immediately available in case of a second pandemic wave, future pandemics or other crisis situations.

Effect of elevated temperature and UV radiation on molecular structure of linoleic acid by ATR-IR and two-dimensional correlation spectroscopy.

The effect of elevated temperature (44 °C) and ultraviolet (UV) radiation on molecular structure of linoleic acid (LA) was studied by Attenuated Total Reflection Infrared (ATR-IR) spectroscopy. To obtain more detailed information on molecular mechanism of these changes we applied moving-window analysis and two-dimensional correlation spectroscopy (2DCOS). Analysis of the time-dependent ATR-IR spectra of LA before and after UV irradiation revealed the structural changes in molecules of LA. The extent of these changes was significantly higher after an application of UV radiation. During 24 h experiment temperature was constant, therefore the spectral changes result from relatively slow processes (and requiring more energy), e.g. cis/trans isomerization, disruption of the C=C double bonds and partial breaking of hydrogen bonds in the cyclic dimers. As a side effect of these structural changes one can observe variations in the orientation of the chains. It is of note that the methyl and methylene groups reveal slightly different behaviour.

Anti-Tremor Action of Subtype Selective Positive Allosteric Modulators of GABAA Receptors in a Rat Model of Essential Tremors.

Essential tremor (ET) is among the most prevalent neurological disorders and the most common cause of abnormal tremors. It is characterized by postural and action tremors ranging from 4 to 12 Hz. The treatments of choice for ET are propranolol and primidone, but their use is associated with adverse effects like hypotension, depression, and cognitive impairments. Benzodiazepines, which nonselectively enhances the effect of GABA at the GABAA α1/2/3/5 receptors, have been shown to be effective in treating ET. Their use, however, is limited due to sedation, ataxia, tolerance development and memory impairment. Sedation and ataxia are attributed to the activity at the α1 subunit while cognitive impairment is ascribed to the action on the α5 subunit of the GABAA receptors. It can be hypothesized that subtype selective GABAA receptor modulators only acting via the α2, and α3 subunits may have an improved side effect profile while retaining the beneficial effects. Here, we have evaluated the effect of subtype selective GABAA α2/3/5 receptor modulators on harmaline-induced tremors in rats. The tremors were automatically quantified in tremor boxes. We show that the GABAA α2/3 subtype selective modulator NS16085 significantly and dose-dependently inhibits harmaline-induced tremors in rats, indicating that potentiation of α2- and α3-containing GABAA receptors is sufficient to ameliorate harmaline-induced tremors. These results provide the first support for a therapeutic role of a subtype selective GABAA α2/3 modulator in the treatment of ET.

Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML.

The key myeloid transcription factor (TF), CEBPA, is frequently mutated in acute myeloid leukemia (AML), but the direct molecular effects of this leukemic driver mutation remain elusive. To investigate CEBPA mutant AML, we performed microscale, in vivo chromatin immunoprecipitation sequencing and identified a set of aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. Comparing gene expression changes in human CEBPA mutant AML and the corresponding Cebpa Lp30 mouse model, we identified Nt5e, encoding CD73, as a cross-species AML gene with an upstream leukemic enhancer physically and functionally linked to the gene. Increased expression of CD73, mediated by the CEBPA-p30 isoform, sustained leukemic growth via the CD73/A2AR axis. Notably, targeting of this pathway enhanced survival of AML-transplanted mice. Our data thus indicate a first-in-class link between a cancer driver mutation in a TF and a druggable, direct transcriptional target.

ATR-IR study of skin components: Lipids, proteins and water. Part II: Near infrared radiation effect.

Near infrared (NIR) radiation has been widely used in medicine and biomedical engineering. In spite of numerous studies the molecular mechanism of NIR radiation on biological systems has not been established as yet. The objective of this work was examination of the effect of NIR irradiation on the skin components. Modifications of lipid organization after NIR exposure vs. temperature (from 20 to 90 °C) have been investigated using Attenuated Total Reflectance Infrared (ATR-IR) spectroscopy. This work is a continuation of our previous studies on the temperature effect on skin components [1]. After NIR exposure a temperature shift of the phase transition from the orthorhombic to hexagonal packing (≈40 °C) has been observed. In contrast, the second phase transition temperature (≈70 °C) is almost invariable. The phase transitions in lipids were correlated with modifications of the structure of water and proteins. To our knowledge, for the first time the temperatures of the phase transitions after NIR exposure were investigated.

Change in psychotropic drug use in Norwegian nursing homes between 2004 and 2011.

ABSTRACTBackground:We aimed to assess whether there were any changes in the use of psychotropic drugs in Norwegian nursing homes between 2004 and 2011. Also, we investigated whether the predictors of use of specific psychotropic drug groups have changed. METHODS: We conducted a secondary analysis of two cohort studies of two Norwegian nursing home samples (2004/05 and 2010/11). Multivariate models were applied. RESULTS: We found a significant decrease in the prescription of antipsychotic drugs between 2004 and 2011 (0.63 OR, 95%CI = 0.49-0.82, p < 0.001) even after adjusting for relevant demographic and clinical variables. There are only minor changes for the other psychotropic drugs. We found that (1) the use of specific psychotropic drug groups as well as the number of psychotropic drugs used was associated with more affective symptoms and (2) the use of specific psychotropic drug groups as well as the number of psychotropic drugs used was associated with lower scores on the Physical Self-Maintenance scale. CONCLUSION: This is the first study to show a robust decrease in antipsychotic drug use in nursing home patients with dementia unrelated to possible changes in case mix. The change might be explained by treatment recommendations against its use except in the most severe conditions of aggression or psychosis. Our findings indicate that it takes several years to implement scientific knowledge in clinical practice in nursing homes.

ATR-IR study of skin components: Lipids, proteins and water. Part I: Temperature effect.

In this work we report the studies of the effect of temperature on skin components, such as lipids, proteins and water. Modifications of lipids structure induced by increasing temperature (from 20 to 90°C) have been studied using ATR-IR (Attenuated Total Reflectance Infrared) spectroscopy, which is a powerful tool for characterization of the molecular structure and properties of tissues, such as skin. Due to the small depth of penetration (0.6-5.6µm), ATR-IR spectroscopy probes only the outermost layer of the skin, i.e. the stratum corneum (SC). The assignment of main spectral features of skin components allows for the determination of phase transitions from the temperature dependencies of band intensities [e.g. νas(CH2) and νs(CH2)]. The phase transitions were determined by using two methods: the first one was based on the first derivative of the Boltzmann function and the second one employed tangent lines of sigmoidal, aforementioned dependencies. The phase transitions in lipids were correlated with modifications of the structure of water and proteins.

Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs.

Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq.

BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre-selection for nucleosome-containing chromatin or pre-amplification of precipitated DNA, making them prone to introduce experimental biases.

C/EBPα is required for long-term self-renewal and lineage priming of hematopoietic stem cells and for the maintenance of epigenetic configurations in multipotent progenitors.

Transcription factors are key regulators of hematopoietic stem cells (HSCs) and act through their ability to bind DNA and impact on gene transcription. Their functions are interpreted in the complex landscape of chromatin, but current knowledge on how this is achieved is very limited. C/EBPα is an important transcriptional regulator of hematopoiesis, but its potential functions in HSCs have remained elusive. Here we report that C/EBPα serves to protect adult HSCs from apoptosis and to maintain their quiescent state. Consequently, deletion of Cebpa is associated with loss of self-renewal and HSC exhaustion. By combining gene expression analysis with genome-wide assessment of C/EBPα binding and epigenetic configurations, we show that C/EBPα acts to modulate the epigenetic states of genes belonging to molecular pathways important for HSC function. Moreover, our data suggest that C/EBPα acts as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options.

UPF2 is a critical regulator of liver development, function and regeneration.

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a post-transcriptional RNA surveillance process that facilitates the recognition and destruction of mRNAs bearing premature terminations codons (PTCs). Such PTC-containing (PTC+) mRNAs may arise from different processes, including erroneous processing and expression of pseudogenes, but also from more regulated events such as alternative splicing coupled NMD (AS-NMD). Thus, the NMD pathway serves both as a silencer of genomic noise and a regulator of gene expression. Given the early embryonic lethality in NMD deficient mice, uncovering the full regulatory potential of the NMD pathway in mammals will require the functional assessment of NMD in different tissues. METHODOLOGY/PRINCIPAL FINDINGS: Here we use mouse genetics to address the role of UPF2, a core NMD component, in the development, function and regeneration of the liver. We find that loss of NMD during fetal liver development is incompatible with postnatal life due to failure of terminal differentiation. Moreover, deletion of Upf2 in the adult liver results in hepatosteatosis and disruption of liver homeostasis. Finally, NMD was found to be absolutely required for liver regeneration. CONCLUSION/SIGNIFICANCE: Collectively, our data demonstrate the critical role of the NMD pathway in liver development, function and regeneration and highlights the importance of NMD for mammalian biology.

A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle development.

Nk-2 proteins are essential developmental regulators from flies to humans. In Drosophila, the family member tinman is the major regulator of cell fate within the dorsal mesoderm, including heart, visceral, and dorsal somatic muscle. To decipher Tinman's direct regulatory role, we performed a time course of ChIP-on-chip experiments, revealing a more prominent role in somatic muscle specification than previously anticipated. Through the combination of transgenic enhancer-reporter assays, colocalization studies, and phenotypic analyses, we uncovered two additional factors within this myogenic network: by activating eyes absent, Tinman's regulatory network extends beyond developmental stages and tissues where it is expressed; by regulating stat92E expression, Tinman modulates the transcriptional readout of JAK/STAT signaling. We show that this pathway is essential for somatic muscle development in Drosophila and for myotome morphogenesis in zebrafish. Taken together, these data uncover a conserved requirement for JAK/STAT signaling and an important component of the transcriptional network driving myogenesis.

Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network.

Smooth muscle plays a prominent role in many fundamental processes and diseases, yet our understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. We present a transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity, as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program.

A temporal map of transcription factor activity: mef2 directly regulates target genes at all stages of muscle development.

Dissecting components of key transcriptional networks is essential for understanding complex developmental processes and phenotypes. Genetic studies have highlighted the role of members of the Mef2 family of transcription factors as essential regulators in myogenesis from flies to man. To understand how these transcription factors control diverse processes in muscle development, we have combined chromatin immunoprecipitation analysis with gene expression profiling to obtain a temporal map of Mef2 activity during Drosophila embryonic development. This global approach revealed three temporal patterns of Mef2 enhancer binding, providing a glimpse of dynamic enhancer use within the context of a developing embryo. Our results provide mechanistic insight into the regulation of Mef2's activity at the level of DNA binding and suggest cooperativity with the bHLH protein Twist. The number and diversity of new direct target genes indicates a much broader role for Mef2, at all stages of myogenesis, than previously anticipated.