Morphological and palaeoecological aspects of fossil insects unveiled by UV-A light.

Studying insect fossils, particularly those preserved as compressions in sedimentary matrices, can be difficult due to the taphonomic processes that often result to poor preservation and contrast of structures compared to the embedding matrix. To address this, we propose a user-friendly and simple methodology based on UV-light to study insect fossils and select specimens of interest for more advanced imagery exploration. While UV-light imaging has been previously applied to compressions of arthropod fossils, it typically involved laser light sources. Our approach allows the investigation of fossils using an affordable, compact, and portable UV-light source, along with a simple and replicable low-cost protocol. •The methodology is based on UV-light induced natural fluorescence of sediment and fossil remains.•UV-light is effective on compression fossils to gain natural contrast and enhance observation of body structures like veins or setae on wings.•UV-light is effective to reveal palaeoecological information such as pollen grains preserved on specimens, especially near or on putative pollinator or pollen-eating taxa.

A twig-like insect stuck in the Permian mud indicates early origin of an ecological strategy in Hexapoda evolution.

Full body impressions and resting traces of Hexapoda can be of extreme importance because they bring crucial information on behavior and locomotion of the trace makers, and help to better define trophic relationships with other organisms (predators or preys). However, these ichnofossils are much rarer than trackways, especially for winged insects. Here we describe a new full-body impression of a winged insect from the Middle Permian of Gonfaron (Var, France) whose preservation is exceptional. The elongate body with short prothorax and legs and long wings overlapping the body might suggests a plant mimicry as for some extant stick insects. These innovations are probably in relation with an increasing predation pressure by terrestrial vertebrates, whose trackways are abundant in the same layers. This discovery would possibly support the recent age estimates for the appearance of phasmatodean-like stick insects, nearly 30 million years older than the previous putative records. The new exquisite specimen is fossilized on a slab with weak ripple-marks, suggesting the action of microbial mats favoring the preservation of its delicate structures. Further prospections in sites with this type of preservation could enrich our understanding of early evolutionary history of insects.

A new Symphoromyia in the Middle Eocene Baltic amber (Diptera: Rhagionidae).

Symphoromyia clerci sp. nov. is described from the Eocene Baltic amber. Even if it has nearly all the characters of the extant species of Symphoromyia subgenus Symphoromyia, it differs from these flies in the short scape, as long as the pedicel (at least twice as long in extant taxa). This fossil is twice as large as those that were previously described from the same amber. These taxa need to be revised to verify their generic attribution. Symphoromyia clerci sp. nov. is the second fossil species attributable to this genus on the basis of 'modern' characters. The extant Symphoromyia are frequently hematophagous on mammals, suggesting a similar biology for the Eocene representatives of this genus.

First fossil Eriocottidae discovered in Eocene Baltic amber (Insecta: Lepidoptera).

Eocompsoctena macroptera gen. et sp. nov., the first fossil eriocottid moth, is described from Baltic amber and attributed to the Compsocteninae. Comparisons are provided with the related families Dryadaulidae, Meessiidae, Psychidae, and Tineidae. The new species confirms the antiquity of 'Tineoidea' grade diversification. The putative Gondwanan origin of Eriocottidae is discussed.

Assessing medical care availability for student athletes of a large urban high school district.

BACKGROUND: The need for medical care for student athletes is mounting, as participation in high school athletics is continuing to rise. This study assessed medical care available to high school student athletes in a large, urban school district in California that has not been studied since 2002. By surveying athletic directors and coaches, we expected to find inadequate availability of medical care in the studied district and predicted that care would be more widely available for student athletes at larger high schools. METHODS: We developed and validated a questionnaire assessing practice and game coverage, emergency preparedness, treatment, and injury prevention measures. The survey was administered to athletic directors and coaches at a school district athletic directors' meeting. RESULTS: Forty-three (57%) of 75 distributed surveys were completed. We found that 70% of schools did not staff a healthcare provider for practices, 28% did not staff home games, and 30% did not staff away games, for any sports. We found no significant differences between school sizes with respect to physician referrals after a student was injured, provision of health education, or implementation of emergency action plans. CONCLUSIONS: Although these data do not support our hypothesis of larger schools providing better medical care, it suggests that there are multiple areas of inadequate healthcare regardless of school size. We identified numerous gaps; thus, future work will examine the impact of these gaps.

Meeting children's needs: a mixed-methods approach to a regionalized pediatric surge plan-the Los Angeles County experience.

INTRODUCTION: Children are one of the most vulnerable populations during mass casualty incidents because of their unique physiological, developmental, and psychological attributes. The objective of this project was to enhance Los Angeles County's (LAC) pediatric surge capabilities. The purpose of this study was threefold: (1) determine gaps in pediatric surge capacity and capabilities; (2) double pediatric inpatient capacity; and (3) document a plan to address gaps and meet pediatric inpatient surge. We hypothesized that LAC would be able to meet the identified pediatric surge target by leveraging resources of hospitals within the region. Deliverables included a pediatric surge plan for LAC, pediatric surge training resources, and pediatric supplies for hospitals participating in LAC's Hospital Preparedness Program (HPP). METHODS: After Institutional Review Board approval, the authors used a mixed-methods approach to explore gaps in hospital capacity and capabilities in a large urban county. Hospitals were surveyed via Qualtrics® on 38 questions regarding capacity, staffing, availability of pediatric supplies, and existing pediatric surge plans. Publicly available inpatient bed data were collected from the Office of Statewide Health Planning and Development for the year ending June 2010 and supplemented by hospital survey responses. Population data was used from US Census 2010. This combined dataset was analyzed for capacity, pediatric designations, and capabilities. To supplement this data, three focus groups were conducted between April 2011 and May 2012. Focus group topics included: supplies and training needed for pediatric surge, surge targets, and plan development and functionality. RESULTS: Hospitals varied in pediatric capacity and capability. Forty-six percent of facilities provide inpatient pediatric services. Forty-one hospitals are designated as an Emergency Department Approved for Pediatrics. Identified gaps included: limited pediatric bed capacity, geographic variability, limited pediatric intensive care unit capacity, limited pediatric specialty physician resources, varying availability of pediatric trained staff, less availability of pediatric critical care supplies, and limited ability to accept and receive children. Focus group stakeholders requested advance and just-in-time training and reference guides to supplement the plan. CONCLUSION: LAC was able to create a pediatric surge plan that doubles pediatric acute and pediatric intensive care bed capacity by using participating HPP hospitals. A tiered system was created based on capacity and capability with varying surge targets and guidance on types of patients that could be cared for at each tier. This plan will assist the LAC Emergency Medical Services Agency distribute pediatric patients during a surge event that disproportionately impacts children.

Randomized controlled trial to evaluate the effectiveness of a video game as a child pedestrian educational tool.

BACKGROUND: Injury is the number one cause of death and disability in children in the United States and an increasingly important public health problem globally. While prevention of injuries is an important goal, prevention efforts are currently fragmented, poorly funded, and rarely studied. Among school-aged children, pedestrian crashes are a major mechanism of injury. We hypothesized that we could develop a game-based educational tool that would be effective in teaching elementary school children the principles of pedestrian safety. METHODS: Between November 2011 and June 2013, second- and third-grade children in Los Angeles Unified School District were randomly assigned to play a unique interactive video game (Ace's Adventure) about pedestrian safety or to a traditional didactic session about pedestrian safety. A pretest and posttest were administered to the study participants. Afterward, study participants were observed for appropriate pedestrian behavior on a simulated street set called Street Smarts. All statistical analyses were performed using SAS version 9.2. RESULTS: A total of 348 study participants took the pretest and posttest. There were 180 who were randomized to the didactic and 168 who were randomized to the video game. The didactic group demonstrated a higher mean score increase (1.01, p < 0.0001) as compared with the video game group (0.44, p < 0.0001). However, observation of study participants revealed that participants who played the video game, as compared with the didactic group, more frequently exhibited appropriate behavior during the following: exiting a parked car (p = 0.01), signaling to a car that was backing up (p = 0.01), signaling to a stopped car (p = 0.0002), and crossing the street (p = 0.01). CONCLUSION: Students who played the educational video game about pedestrian safety performed similarly to those who attended a more traditional and labor-intensive didactic learning. Innovative educational methods, such as game playing, could significantly change our approach to injury prevention and have the potential to decrease the burden of injury among children worldwide.

A mixed-methods pilot study of disaster preparedness and resiliency among faith-based organizations.

INTRODUCTION: Faith-based organizations represent a source of stability and are an established presence in a community. They frequently serve their community following disasters. They are not formally included or identified as a disaster resource; thus, there is an opportunity to increase the effectiveness with which faith-based organizations prepare for and respond to disasters. Problem This pilot study aimed to assess perceptions of the level of disaster preparedness and resiliency among faith-based organizations as a first step in understanding how to improve disaster preparedness and resiliency among these organizations and their communities. METHODS: Survey and semi-structured interviews were conducted with six faith-based organizations, one with a leader and one with a staff member. Frequency distributions of survey questions were obtained. Interviews were transcribed and thematic analysis was supported by analytical software, ATLAS. ti. RESULTS: Results of the survey indicated strong social networks among congregation and community members. However, half of the members indicated that they did not socialize often with other races and other neighborhoods. Additionally, trust of other groups of people was generally low. Themes that emerged from qualitative analysis were: (1) perceived disaster preparedness and resiliency; (2) barriers to community preparedness and resiliency; (3) lessons learned from past disasters; (4) social services and networks; and (5) willingness to be prepared. CONCLUSIONS: The results suggest that there is a need for interventions to improve disaster preparedness and resiliency among faith-based organizations.

Evaluation of a child passenger safety class in increasing parental knowledge.

BACKGROUND: Child passenger restraint systems have been found to greatly reduce the risk of injury and death among child passengers. However, nearly half of the children who died in 2009 as a result of motor vehicle crashes were completely unrestrained. Our global hypothesis is that parents and other caregivers failed to restrain children due to a lack of child passenger seat education and practice. In this report, we postulate that a car seat class will improve the basic understanding of child passenger safety. The objective of the study was to evaluate the effectiveness of a car seat class in increasing parental knowledge about child passenger safety. METHODS: Car seat classes were held at a Level 1 pediatric trauma center every other Tuesday for ten months. The curriculum consisted of: child passenger safety laws discussion, a 21-min video on the use of child safety seats followed by a 15-min discussion about the video, 15min of discussing the different types of car seats, and hands-on training on how to properly install and use child restraints. Free car seats were provided to eligible parents. The pre-test was administered at the beginning of class and the post-test at the end of the class. McNemar's test and a paired t-test were used to compare pre- and post-test scores. Test scores were also stratified by language spoken. RESULTS: Forty-four classes were held and a total of 491 parents/caregivers attended the classes. An increase in knowledge was found for all survey questions. Mean knowledge score for the post-test was 3.10 points higher compared to the mean knowledge score from the pre-test. Mean difference in knowledge scores for English-speaking participants were higher than Spanish-speaking participants. CONCLUSION: Lack of knowledge and low risk perception have frequently been cited as barriers for the use of child passenger restraints. Our intervention attempted to eliminate these barriers. We found that this intervention was effective at increasing parental knowledge about child passenger safety. The results of this study may be used to design and implement future interventions in multicultural settings.

Disaster preparedness education in South Los Angeles faith-based organizations: a pilot study in promoting personal and community preparedness and resiliency.

INTRODUCTION: Faith-based organizations (FBOs) often provide crucial services to their communities during and after disasters, but they largely operate outside of the formal structures that manage disaster preparedness and response. Their status within communities and ability to reach broad populations make them potentially powerful groups to lead and promote health and safety initiatives. Thus, there is opportunity to reduce health and knowledge disparities and increase disaster resiliency by providing disaster preparedness education through FBOs. PROBLEM: This pilot study aims to assess and enhance the current level of disaster knowledge and preparedness of congregation members from six FBOs in the Los Angeles County South Service Planning Area (SPA 6) to promote their community's disaster resiliency. METHODS: A structured basic disaster education curriculum was implemented at six FBOs, and participants completed a questionnaire, pre-test and post-test, and satisfaction survey. Frequency distributions of survey questions and mean differences of pre- and post-test scores were obtained. RESULTS: Results of the questionnaire indicated that more than half of the participants had previous experience with disaster preparedness and had taken some steps toward stockpiling supplies but showed gaps in making concrete evacuation plans and meeting places. The mean disaster knowledge test score increased from 5.15 for the pre-test to 8.04 for the post-test (p < 0.0001). The participants reported high satisfaction with the seminar. CONCLUSIONS: This pilot study demonstrated that there is interest, as well as need, within the faith-based community to increase personal and community disaster preparedness. The implementation of a structured disaster education seminar for congregation members showed significant immediate improvement in disaster knowledge of the participants.

Cell phones and young drivers: a systematic review regarding the association between psychological factors and prevention.

OBJECTIVE: Cell phone use among young drivers has become increasingly common in recent years. Young people are the most likely to accept the use of new technology and least likely to understand the risks associated with cell phone use while driving (CPWD; defined here as talking on the phone only) and texting while driving (TextWD). Due to inexperience, young drivers are the most at risk when using cell phones while driving and therefore should be the target of the majority of prevention strategies. The intent of this review is to determine factors that influence young drivers to engage in CPWD and TextWD and suggest a basis for prevention campaigns and strategies that can effectively prevent current and future generations from using cell phones while driving. METHODS: We conducted a search for original articles in PubMed, the Cochrane Library, and Web of Science. All abstracts were reviewed and for those that met the inclusion criteria, full articles were obtained and assessed. RESULTS: Four hundred and twelve articles were identified in the search and, of those, 37 full-text articles were obtained. A total of 29 articles about the frequency of CPWD and the psychological effects influencing young driver's tendency to engage in CPWD were included. There was a high frequency of both CPWD and TextWD despite a high perceived risk of both behaviors. This discrepancy was explained by a high perceived controllability, the effect of social norms, call importance, and lack of effective law enforcement. The intervention strategies reviewed were also found to be ineffective over the long term. CONCLUSIONS: The systematic review reveals that young drivers are an at-risk group for distracted driving. We propose preventative strategies based on identifying factors that influence drivers to engage in CPWD and TextWD as well as by reviewing strategies found in the reviewed articles. Further research is necessary to determine the effectiveness of these proposed strategies.

Identification and analysis of two novel sites of rat GnRH receptor gene promoter activity: the pineal gland and retina.

BACKGROUND AND AIMS: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary. METHODS: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created. RESULTS: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context. CONCLUSION: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.

Reporter transgenic mouse models highlight the dual endocrine and neural facet of GnRH receptor function.

In the pituitary of mammals, the GnRH receptor (GnRHR) plays crucial roles in the neuroendocrine control of reproductive function. This receptor is specifically expressed by the gonadotrope cells scattered among the five other endocrine cell types constituting the anterior pituitary; it is also expressed in other organs, such as the gonads and brain where its function is not well defined. To gain insight into GnRHR function, distribution, and regulation, several transgenic approaches have been developed using a range of reporter genes under the control of the mouse, rat, or ovine GnRHR gene (Gnrhr) promoters. Comprehensive reviews of the literature, together with recent results obtained in our laboratory, illustrate how these transgenic models highlight the endocrine as well as the neural facet of GnRHR function. In this review, the endocrine aspect will be discussed with regard to the pituitary and gonad function, whereas the neural aspect will be discussed with regard to hippocampal formation and the oculomotor pathway, the latter constituting an unpreviously described site of Gnrhr promoter activity. These approaches should help elucidate the properties of the mammalian GnRH system.

GnRH receptor gene expression in the developing rat hippocampus: transcriptional regulation and potential roles in neuronal plasticity.

In the pituitary of mammals, the GnRH receptor (GnRHR) plays a primary role in the control of reproductive function. It is further expressed in the hippocampus, where its function, however, is not well defined. By quantitative RT-PCR analyses, we demonstrate herein that the onset of GnRHR gene (Gnrhr) expression in the rat hippocampus was unexpectedly delayed as compared to the pituitary and only occurred after birth. Using a previously described transgenic mouse model bearing the human placental alkaline phosphatase reporter gene under the control of the rat Gnrhr promoter, we established a positive correlation between the temporal pattern of Gnrhr mRNA levels and promoter activity in the hippocampal formation. The gradual appearance of human placental alkaline phosphatase transgene expression occurred simultaneously in the hippocampus and interconnected structures such as the lateral septum and the amygdala, coinciding with the establishment of hippocampo-septal projections. Analysis of transcription factors together with transient transfection assays in hippocampal neurons indicated that the combinatorial code governing the hippocampus-specific expression of the Gnrhr is distinct from the pituitary, likely involving transactivating factors such as NUR77, cyclic AMP response element binding protein, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene homolog. A silencing transcription factor acting via the -3255/-1135 promoter region of the Gnrhr may be responsible for the transcriptional repression observed around birth. Finally, GnRH directly stimulated via activation of its receptor the expression of several marker genes of neuronal plasticity such as Egr1, synaptophysin, and spinophilin in hippocampal primary cultures, suggesting a role for GnRHR in neuronal plasticity. Further characterization of these mechanisms may help unravel important functions of GnRH/GnRHR signaling in the brain.

Cognitive processing specificity of anxious apprehension: impact on distress and performance during speech exposure.

The present study examined the impact of different modes of processing anxious apprehension on subsequent anxiety and performance in a stressful speech task. Participants were informed that they would have to give a speech on a difficult topic while being videotaped and evaluated on their performance. They were then randomly assigned to one of three conditions. In a specific processing condition, they were encouraged to explore in detail all the specific aspects (thoughts, emotions, sensations) they experienced while anticipating giving the speech; in a general processing condition, they had to focus on the generic aspects that they would typically experience during anxious anticipation; and in a control, no-processing condition, participants were distracted. Results revealed that at the end of the speech, participants in the specific processing condition reported less anxiety than those in the two other conditions. They were also evaluated by judges to have performed better than those in the control condition, who in turn did better than those in the general processing condition.

In utero and exo utero surgery on rodent embryos.

Mammalian development has been best characterized using the mouse model. Direct intervention of the postimplantation mouse embryo in utero represents one of many experimental approaches that can be used to probe mammalian embryogenesis. Experimental access to the mouse embryo is difficult, but techniques have been developed to circumvent some of the challenges of operating on the embryo in vivo. Experimental studies have been carried out on postimplantation stage embryos from E8.5 to term, so much of the gestational period is accessible for experimentation. One approach that has helped to enhance embryo accessibility was the development of surgical techniques based on the finding that embryonic development continued normally exo utero. Exo utero development refers to the surgically created condition in which the embryo develops outside of the uterine cavity, yet within the female abdominal cavity and attached, via the placenta, to the uterus. Using this approach it is feasible to carry out precise surgical manipulations of the mouse embryo without compromising embryo viability associated with postsurgery uterine contractions. In this chapter we review technical aspects of both in utero and exo utero surgical approaches and how these surgeries are used in conjunction with other experimental applications.

[An ambiguous role of steroidogenic factor 1 in the rat GnRH receptor gene expression. Lessons from transgenic mice]. / Le rôle ambigu du facteur de transcription SF-1 dans l'expression génique du récepteur de la GnRH. Leçons de la transgenèse.

Because the GnRH receptor plays a paramount role within the reproductive axis, the understanding of the molecular apparatus that governs the tissue-specific expression and regulation of this gene must lead to a better knowledge of the physiology and the physiopathology of the gonadotrope function. To elucidate these mechanisms, we have used two complementary in vivo and in vitro approaches. Firstly, we have isolated the pituitary promoter of the rat GnRH receptor gene and investigated its activity using transient transfection into two gonadotrope-derived cell lines, the alphaT3-1 and the LbetaT2 cell lines. We have thus defined a primary set of transcription factors involved in the tissue-specific expression of the GnRH receptor gene. These include the steroidogenic factor-1 (SF-1) which plays a decisive role while functionally interacting with proteins related to the GATA and LIM homeodomain families of transcription factors. In addition, we highlighted the critical implication of SF-1 and its functional interaction with a CREB-related factor in the stimulatory action of PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) on promoter activity. These results have led us to analyze the activity of this promoter by transgenesis in the mouse using human placental alkaline phosphatase as a reporter gene. In agreement with the in vitro data, the pituitary promoter was found to confer gonadotrope-specific activity in the pituitary. It was also able to direct transgene expression in several areas of the central nervous system known to express the endogenous GnRH receptor, in particular in the hippocampo-septal complex. Some of these tissue do not express SF-1, suggesting that, in vivo, its role would not be as decisive as suggested by the in vitro experiments. Surprisingly, during pituitary ontogenesis, the transgene is expressed as early as E 13.5 whereas SF-1 is not yet present in the pituitary. Thus, in vivo, SF-1 would not be necessary for the activation of the GnRH receptor gene during the early developmental stages in the pituitary. These results are consistent with data obtained following general or pituitary-specific knockout of the gene encoding SF-1, suggesting that the GnRH receptor is expressed despite the absence of this factor. Identifying the factors responsible for the activation of the GnRH receptor gene at these early developmental stages should make it possible to refine the role of SF-1, not only in gene regulation but more generally, in the physiology and the physiopathology of the gonadotrope function.

The promoter of the rat gonadotropin-releasing hormone receptor gene directs the expression of the human placental alkaline phosphatase reporter gene in gonadotrope cells in the anterior pituitary gland as well as in multiple extrapituitary tissues.

Previous studies dealing with the mechanisms underlying the tissue-specific and regulated expression of the GnRH receptor (GnRH-R) gene led us to define several cis-acting regulatory sequences in the rat GnRH-R gene promoter. These include functional sites for steroidogenic factor 1, activator protein 1, and motifs related to GATA and LIM homeodomain response elements as demonstrated primarily in transient transfection assays in mouse gonadotrope-derived cell lines. To understand these mechanisms in more depth, we generated transgenic mice bearing the 3.3-kb rat GnRH-R promoter linked to the human placental alkaline phosphatase reporter gene. Here we show that the rat GnRH-R promoter drives the expression of the reporter gene in pituitary cells expressing the LHbeta and/or FSHbeta subunit but not in TSHbeta- or GH-positive cells. Furthermore, the spatial and temporal pattern of the transgene expression during the development of the pituitary was compatible with that characterizing the emergence of the gonadotrope lineage. In particular, transgene expression is colocalized with the expression of the glycoprotein hormone alpha-subunit at embryonic day 13.5 and with that of steroidogenic factor 1 at later stages of pituitary development. Transgene expression was also found in specific brain areas, such as the lateral septum and the hippocampus. A single promoter is thus capable of directing transcription in highly diverse tissues, raising the question of the different combinations of transcription factors that lead to such a multiple, but nevertheless cell-specific, expressions of the GnRH-R gene.

Mouse muscle identity: the position-dependent and fast fiber-specific expression of a transgene in limb muscles is methylation-independent and cell-autonomous.

We previously characterised transgenic mice in which fast-muscle-specific regulatory sequences from the human aldolase A pM promoter drive the chloramphenicol acetyltransferase gene expression. Mutation of a NF1/MEF2 binding site (M2 motif) in this promoter does not affect fibre-type specificity of the transgene but modifies its expression in a subset of fast-twitch fibres at the limb level, preferentially affecting distal limb muscles. We investigated the molecular and cellular bases of this peculiar expression pattern that provided an adequate model to characterise the mechanisms responsible for muscle positional information. By direct electrotransfer of mutated M2 construct in adult muscle, we demonstrate that positional differences in mutated M2 transgene expression are not observed when the transgene is not integrated into chromatin. Also, this transgene expression pattern does not seem to be correlated with the extent of CpG methylation in its promoter sequence. Finally, we show that positional values reflected by CAT levels are maintained in primary cultures established from different adult limb muscles, as well as in heterotopically transplanted muscles. Our results suggest that mutation of the M2 site contributes to reveal a molecular memory of fibre fate that would be set up on pM promoter during development and persist into adulthood possibly through a chromatin imprint maintained in satellite cells associated with various limb muscles.

Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters.

Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.