A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations.

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

Typing of Leishmania isolates from vectors and leporids of the Madrid (Spain) outbreak.

In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 gene, a commonly used marker, were not performed. In the present study, 19 isolates from P. perniciosus (n = 11), hares (n = 5) and rabbits (n = 3) from the outbreak area, all characterized as ITS-Lombardi in previous studies, were analysed by MLEE and hsp70 sequencing. The hsp70 results confirmed that all the analysed strains are L. infantum. However, by MLEE, 4 different zymodemes of L. infantum were identified based on variable mobilities of the NP1 enzyme: MON-34 (NP1100, n = 11), MON-80 (NP1130, n = 6), MON-24 (NP1140, n = 1) and MON-331 (NP1150, n = 1). The relative frequency of these zymodemes does not correspond to their usual occurrence in Spain. Moreover, MON-34 and MON-80 were found in P. perniciosus, hares and rabbits for the first time. These findings continue to provide insights into the outbreak and call for further studies with a higher number of strains.

[Second victim experience and support from health professionals]. / Segunda víctima: experiencia y medidas de apoyo percibidas por los profesionales sanitarios.

INTRODUCTION: The second victims are those professionals who, when faced with a mistake, are traumatized, questioning their professional and personal skills. Knowledge of their experience allows institutions to understand the consequences of error in professionals to carry out care actions for the professionals involved, improving care security. The objective of this study is to characterize the phenomenon of second victims and to know the perceived support measures. METHOD: Cross-sectional descriptive observational quantitative study. Health professionals from five highly complex institutions participated through convenience sampling. The data collection instrument was the Second Victim Experience and Support Tool (SVEST) validated in Argentina, composed of impact dimensions (psychological, physical and professional skills) of support received (colleagues, supervisors, institutional and measures not related to the workplace) and two outcome dimensions (professional abandonment and absenteeism). RESULTS: 1134 professionals participated and 56% said they had made a mistake. The mean STEVS was 2.5, with the psychological impact dimension predominating (x = 3.4). As part of the support measures, professionals perceived greater support from family and friends (not related to work) (x = 3.1). The greater the impact, the lower the perception of support measures (Rho: -0.8, p = 0.047). DISCUSSION: More than half of the professionals refer to being second victim with a significant psychological impact, who, despite working in institutions that have patient safety protocols, received support from nonwork-related environments.

Introducción: Las segundas víctimas son aquellos profesionales que, ante un error, quedan traumatizados, cuestionándose sus habilidades profesionales y personales. El conocimiento de su experiencia permite a las instituciones entender las consecuencias del error en los profesionales para realizar acciones de atención a los profesionales involucrados mejorando la seguridad de atención. El objetivo de este estudio es caracterizar el fenómeno de segundas víctimas y conocer las medidas de apoyo percibidas. Métodos: Estudio cuantitativo observacional descriptivo transversal. Participaron profesionales de la salud de cinco instituciones de alta complejidad a través de un muestreo por conveniencia. El instrumento de recogida de datos fue el Second Victim Experience and Support Tool (SVEST) validado en Argentina, compuesta por dimensiones de impacto (psicológico, físico y de habilidades profesionales) de apoyo recibido (colegas, supervisores, institucional y medidas no relacionadas al ámbito laboral) y dos dimensiones de resultado (abandono profesional y ausentismo). Resultados: Participaron 1134 profesionales. El 56% manifestó haber cometido un error y completó el SVEST. La media del SVEST fue de 2.5 predominando la dimensión de impacto psicológico (x = 3.4). Como parte de las medidas de apoyo los profesionales percibieron mayor apoyo en familiares y amigos (x = 3.1). A mayor impacto menor percepción de las medidas de apoyo (Rho: -0.8, p = 0.047). Discusión: Más de la mitad de los profesionales refieren ser segundas víctimas con un impacto psicológico importante, los cuales, a pesar de trabajar en instituciones que cuentan con protocolos de seguridad del paciente, reciben el apoyo principalmente de entornos no relacionados al trabajo.

Self-care interventions of community-dwelling older adults: a systematic review and meta-analysis.

Introduction: The current notion of "care in old age" should be reconceptualized in the ageing societies of the 21st century. Currently, "being old" means that one is actively involved in their care and has the desire to retain control and independence. Objective: Understand and analyze the efficacy of interventions in the physical and psychological self-care practices of healthy community-dwelling older people. Methodology: Systematic review and meta-analysis. The guidelines of the PRISMA guide were followed. The methodological quality of the studies was checked using Cochrane Effective Practice and Organisation of Care criteria, and the search was performed between 2016 and 2021. Results: Of the 1,866 evaluated, 8 studies met the criteria. The systematic review reveals that self-care interventions focus on physical health-related variables but not on psychological variables. The meta-analysis shows that interventions significantly improve physical health-related variables (care visits, hospital admission, medication, and gait speed). Conclusion: Self-care training programs should include psychological variables to increase health and well-being in healthy older people.

Practices of Self-Care in Healthy Old Age: A Field Study.

Two competing psychological approaches for how to care for oneself to stay healthy in old age have coexisted and dominated the scientific literature. OBJECTIVE: Identify the self-care practices of healthy older adults and establish the relationship between these practices and the cognitive processes involved. METHOD: 105 healthy older people (83.91% women) recorded their self-care practices using the Care Time Test and underwent a cognitive evaluation. RESULTS: The frequency and variety of different activities that participants spent performing on a day of the week where they had the fewest obligations are as follows: nearly 7 h on seven survival activities, 4 h and 30 min on three maintenance of functional independence activities and 1 h on one activity that promoted personal development. Older people who carry out activities in a developmental approach showed better everyday memory (8.63 points) and attention levels (7.00 points) than older people who carry out activities using a conservative approach (memory: 7.43; attention level: 6.40). CONCLUSION: The results evidenced that the frequency and variety of activities that promote personal development are associated with better attention and memory performance.

Influence of Coping and Self-Efficacy in Inflammatory Bowel Disease.

(1) Background: Coping includes the specific cognitive processes and behaviours that the patient uses when faced with the stress of living with a chronic disease. Self-efficacy is the knowledge that individuals have about their abilities and their confidence to face a problem or cope with a situation (disease). The aim of this study was to explore the role of coping and self-efficacy in inflammatory bowel disease. (2) Materials and Methods: A total of 92 participants were included (33 had been diagnosed with Crohn's disease, 23 with ulcerative colitis and 36 were healthy participants). The Coping Strategies Inventory was used to measure which coping strategies were employed, differentiating them as active or passive. The General Self-Efficacy Scale was used to measure self-efficacy. (3) Results: The results indicate that people with inflammatory bowel disease used strategies related to passive coping more than healthy people (mean of 36.39 ± 13.92 vs. 29.77 ± 10.70, p = 0.017). Additionally, people with inflammatory bowel disease used social withdrawal more than healthy participants (mean of 8.30 ± 5.07 vs. 4.47 ± 4.17, p < 0.001). In addition, there are significant differences in emotion-focused engagement coping strategies. People with inflammatory bowel disease used this strategy less than healthy people (mean of 21.77 ± 7.75 vs. 25.03 ± 7.00, p = 0.044). Finally, healthy participants used the emotion-focused disengagement strategy less than those diagnosed with inflammatory bowel disease (mean of 9.81 ± 7.74 vs. 15.61 ± 10.14, p = 0.004). (4) Conclusions: Actions aimed at the development of active coping strategies and patient socialisation must be included in the treatment of inflammatory bowel disease.

Closing the gap to effective gene drive in Aedes aegypti by exploiting germline regulatory elements.

CRISPR/Cas9-based homing gene drives have emerged as a potential new approach to mosquito control. While attempts have been made to develop such systems in Aedes aegypti, none have been able to match the high drive efficiency observed in Anopheles species. Here we generate Ae. aegypti transgenic lines expressing Cas9 using germline-specific regulatory elements and assess their ability to bias inheritance of an sgRNA-expressing element (kmosgRNAs). Four shu-Cas9 and one sds3-Cas9 isolines can significantly bias the inheritance of kmosgRNAs, with sds3G1-Cas9 causing the highest average inheritance of ~86% and ~94% from males and females carrying both elements outcrossed to wild-type, respectively. Our mathematical model demonstrates that sds3G1-Cas9 could enable the spread of the kmosgRNAs element to either reach a higher (by ~15 percentage point) maximum carrier frequency or to achieve similar maximum carrier frequency faster (by 12 generations) when compared to two other established split drive systems.

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias.

CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.

Charged lithium adsorption on pristine and defective silicene: a theoretical study.

We investigated by first principle calculations the adsorption of Liq(q= -1, 0 or +1) on a silicene single layer. Pristine and three different defective silicene configurations with and without Li doping were studied: single vacancy (SV), double vacancy (DV) and Stone-Wales (STW). Structural studies and the adsorption energies of various sites were obtained and compared in order to understand the stability of the Li on the surface. Moreover, electronic structure and charge density difference analysis were performed before and after adsorption at the most stables sites, which showed the presence of a magnetic moment in the undoped SV system, the displacement of the Fermi level produced by Li doping and a charge transfer from Li to the surface. Additionally, quantum capacity (QC) and charge density studies were performed on these systems. This analysis showed that the generation of defects and doping improves the QC of silicene in positive bias, because of the existence of 3p orbital in the zone of the defect. Consequently, the innovative calculations performed in this work of charged lithium doping on silicene can be used for future comparison with experimental studies of this Li-ion battery anode material candidate.

Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source.

The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59-88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.

Opportunistic feeding behaviour and Leishmania infantum detection in Phlebotomus perniciosus females collected in the human leishmaniasis focus of Madrid, Spain (2012-2018).

BACKGROUND: An outbreak of human leishmaniasis due to Leishmania infantum has been registered in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys carried out in the municipalities of Fuenlabrada, Leganés, Getafe and Humanes de Madrid showed that Phlebotomus perniciosus is the only potential vector. In this work, an intensive molecular surveillance was performed in P. perniciosus females captured in the region between 2012 and 2018. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1805 P. perniciosus females were analyzed for Leishmania infection, and 1189 of them also for bloodmeal identification. Eleven different species of vertebrate were detected by amplification and subsequent sequencing of the 359 bp cytb fragment. The most prevalent blood source identified was hare (n = 553, 46.51%), followed by rabbit (n = 262, 21.95%). Less frequent were cat (n = 45, 3.80%), human (n = 34, 2.90%), pig (n = 14, 1.20%), horse (n = 11, 0.93%), sheep (n = 3, 0.25%), rhea (n = 3, 0.25%), partridge (n = 1, 0.09%) and chicken (n = 1, 0.09%). The distribution of the blood meal sources varied between the different locations. Regarding L. infantum detection, PCR amplification of a fragment of kDNA, cpb gene and ITS1 region showed 162 positive specimens (8.97%). The highest infection rate was found in the municipality of Leganés (15.17%). CONCLUSIONS: The results of this molecular survey in P. perniciosus, the only leishmaniasis vector in the outbreak occurred in southwestern Madrid region, showed its opportunistic blood-feeding behaviour, high infection rates and the differences between the different points. This study was an essential part of the intensive surveillance plan in the area and the results obtained have supported the implementation of control measures in the outbreak.

Single Time-Point Study of the Home Environment and Functionality of Older Adults in Spain.

Background: The literature favors discussion on socio-spatial conditions at the macro- (city) and micro- (housing) level that promote healthy aging in place. Objectives: (a) Identify the association between physical and social characteristics of the family home and the functional level and quality of life of older people and (b) provide normative data on adequate/inadequate households based on the Home Observation for Measurement of the Environment (HOME) inventory and the Spanish Informant Questionnaire on Cognitive Decline in the Elderly (S-IQCODE) test. Methods: In total, 79 healthy older adults completed the HOME inventory and the Montreal Cognitive Assessment (MoCA), S-IQCODE, Kessler Psychological Distress Scale (K-10) and ICEpop CAPability measure for Older people (ICECAP-O) tests. A regression model, the effect size and the means of the scores of HOME (adequate/inadequate) test and the cognitive level (optimal/normal) were calculated. Results: The regression model discloses that adequate home scores are associated with cognitive level (odds ratio (OR): 0.955, confidence interval (CI)95%: 0.918-0.955); quality of life (OR: 6.542, CI95%: 1.750-24.457), living with other people (OR: 5.753, CI95%: 1.456-22.733) and level of education (OR: 0.252, CI95%: 0.064-0.991). The normative data between HOME and S-IQCODE scores showed a good adjustment (d = 0.70). Conclusion: There is a significant relationship between the physical environment of the home and personal variables (sociodemographic information, quality of life and cognitive functionality). In addition, from this last variable, the normative data of an adequate/inadequate household for an older person have been established.

Molecular detection and identification of Leishmania DNA and blood meal analysis in Phlebotomus (Larroussius) species.

BACKGROUND: Phlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs. METHODOLOGY AND FINDINGS: A total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively. CONCLUSION: Our results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.

Leishmania sp. detection and blood-feeding behaviour of Sergentomyia minuta collected in the human leishmaniasis focus of southwestern Madrid, Spain (2012-2017).

Phlebotomine sand flies are the only known vectors of Leishmania spp. protozoan which causes leishmaniasis in 98 countries. In Spain, 11 sand fly species are described, but only Phlebotomus perniciosus and Phlebotomus ariasi are proven vectors of the disease. On the other hand, Sergentomyia minuta is one of the most abundant and ubiquitous sand flies in this territory, although scarce information is available about this species. Sand flies from this genus are known for their preference to feed on cold-blooded animals and are traditionally involved in the transmission of reptile Leishmania. However, studies have suggested that Sergentomyia spp. could be implicated in the transmission of human pathogenic Leishmania. This study analyses blood meal preferences and Leishmania sp. infection of S. minuta sand flies from the largest human leishmaniasis outbreak in Europe. Sand flies were collected during entomological surveillance carries out from 2012 to 2017 in the active season of these dipterans, from May to October. Molecular detection of Leishmania spp. showed 68 positive specimens of S. minuta out of 377 (18%). The analysis of blood meal preferences by amplification of 359 bp fragment of cytochrome b gene revealed that blood preference of S. minuta is not only limited to reptiles, but they also feed on mammals, including humans. Results suggest the presence of a Leishmania sp., related to Leishmania tarentolae, cycle in S. minuta from the studied area. Although there is no evidence about its incrimination in the L. infantum transmission more investigation is needed to elucidate the intravectorial cycle of Leishmania spp. in S. minuta sand flies, their feeding behaviour and their potential contribution in Leishmania spp. epidemiology in the country.

MITF Expression Predicts Therapeutic Vulnerability to p300 Inhibition in Human Melanoma.

Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300-MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300-MITF-FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. SIGNIFICANCE: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity.

Reproducibility of BiliCare™ Transcutaneus Bilirrubin Meter in Mexican Newborns.

BACKGROUND: Newborn hyperbilirubinemia is considered a worldwide health problem that demands medical evaluation. Noninvasive transcutaneous bilirubin (TcB) has been used as a screening method with different devices but there has not been any evaluation of reproducibility of the same brand devices. The BiliCare™ system is evaluated to demonstrate consistency between measurements with four different devices. METHODS: 336 TcB measurements were obtained with four BiliCare™ devices in 21 Mexican icteric newborns with a mean postnatal age of 44.1 hours of life and 38 weeks of gestation (33-41). Two measurements were taken in the same ear alternatively at the scaphoid fossa with each device. TcB values were compared between devices. Validity was compared with total serum bilirubin (TB). RESULTS: intraclass correlation coefficient demonstrates a minimum limit in the study of 0.945 and maximum of 0.988 with the same device. Correlations with serum and between devices gave results above 0.932. CONCLUSIONS: BiliCare™ transcutaneous bilirubin measurement instrument has very good intra- and interdevice reproducibility; also correlation of TcS with serum bilirubin gave statistically the same results.

Chromatin Immunoprecipitation in Early Mouse Embryos.

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

Emerging Biomarkers in Cutaneous Melanoma.

Earlier identification of aggressive melanoma remains a goal in the field of melanoma research. With new targeted and immune therapies that have revolutionized the care of patients with melanoma, the ability to predict progression and monitor or predict response to therapy has become the new focus of research into biomarkers in melanoma. In this review, promising biomarkers are highlighted. These biomarkers have been used to diagnose melanoma as well as predict progression to advanced disease and response to therapy. The biomarkers take various forms, including protein expression at the level of tissue, genetic mutations of cancer cells, and detection of circulating DNA. First, a brief description is provided about the conventional tissue markers used to stage melanoma, including tumor depth. Next, protein biomarkers, which provide both diagnostic and prognostic information, are described. This is followed by a discussion of important genetic mutations, microRNA, and epigenetic modifications that can provide therapeutic and prognostic material. Finally, emerging serologic biomarkers are reviewed, including circulating melanoma cells and exosomes. Overall the goal is to identify biomarkers that aid in the earlier identification and improved treatment of aggressive melanoma.

The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration.

The Myogenic Regulatory Factors (MRFs) Myf5, MyoD, myogenin and MRF4 are members of the basic helix-loop-helix family of transcription factors that control the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis. The dynamics of their temporal and spatial expression as well as their biochemical properties have allowed the identification of a precise and hierarchical relationship between the four MRFs. This relationship establishes the myogenic lineage as well as the maintenance of the terminal myogenic phenotype. The application of genome-wide technologies has provided important new information as to how the MRFs function to activate muscle gene expression. Application of combined functional genomics technologies along with single cell lineage tracing strategies will allow a deeper understanding of the mechanisms mediating myogenic determination, cell differentiation and muscle regeneration.

Phlebotomine sand fly survey in the focus of leishmaniasis in Madrid, Spain (2012-2014): seasonal dynamics, Leishmania infantum infection rates and blood meal preferences.

BACKGROUND: An unusual increase of human leishmaniasis cases due to Leishmania infantum is occurring in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys have shown that Phlebotomus perniciosus is the only potential vector. Direct xenodiagnosis in hares (Lepus granatensis) and rabbits (Oryctolagus cuniculus) collected in the focus area proved that they can transmit parasites to colonized P. perniciosus. Isolates were characterized as L. infantum. The aim of the present work was to conduct a comprehensive study of sand flies in the outbreak area, with special emphasis on P. perniciosus. METHODS: Entomological surveys were done from June to October 2012-2014 in 4 stations located close to the affected area. Twenty sticky traps (ST) and two CDC light traps (LT) were monthly placed during two consecutive days in every station. LT were replaced every morning. Sand fly infection rates were determined by dissecting females collected with LT. Molecular procedures applied to study blood meal preferences and to detect L. infantum were performed for a better understanding of the epidemiology of the outbreak. RESULTS: A total of 45,127 specimens belonging to 4 sand fly species were collected: P. perniciosus (75.34%), Sergentomyia minuta (24.65%), Phlebotomus sergenti (0.005%) and Phlebotomus papatasi (0.005%). No Phlebotomus ariasi were captured. From 3203 P. perniciosus female dissected, 117 were infected with flagellates (3.7%). Furthermore, 13.31% and 7.78% of blood-fed and unfed female sand flies, respectively, were found infected with L. infantum by PCR. The highest rates of infected P. perniciosus were detected at the end of the transmission periods. Regarding to blood meal preferences, hares and rabbits were preferred, although human, cat and dog blood were also found. CONCLUSIONS: This entomological study highlights the exceptional nature of the Leishmania outbreak occurring in southwestern Madrid, Spain. It is confirmed that P. perniciosus is the only vector in the affected area, with high densities and infection rates. Rabbits and hares were the main blood meal sources of this species. These results reinforce the need for an extensive and permanent surveillance in this region, and others of similar characteristics, in order to control the vector and regulate the populations of wild reservoirs.