Screening Mutants by Single Fly Genomic PCR.

Imprecise P-element excision or FRT-mediated recombination is routinely performed to mutagenize a gene of interest. It is, however, tedious to maintain all independent and individual excised mutant fly lines before the presence of a mutation is confirmed. Here, we provide a method to detect and confirm the presence of a mutation, as and when mutant flies are generated. By allowing for the maintenance and expansion of only the confirmed mutant lines, this protocol will help to save time, money, and space.

CG7379 and ING1 suppress cancer cell invasion by maintaining cell-cell junction integrity.

Approximately 90% of cancer-related deaths can be attributed to a tumour's ability to spread. We have identified CG7379, the fly orthologue of human ING1, as a potent invasion suppressor. ING1 is a type II tumour suppressor with well-established roles in the transcriptional regulation of genes that control cell proliferation, response to DNA damage, oncogene-induced senescence and apoptosis. Recent work suggests a possible role for ING1 in cancer cell invasion and metastasis, but the molecular mechanism underlying this observation is lacking. Our results show that reduced expression of CG7379 promotes invasion in vivo in Drosophila, reduces the junctional localization of several adherens and septate junction components, and severely disrupts cell-cell junction architecture. Similarly, ING1 knockdown significantly enhances invasion in vitro and disrupts E-cadherin distribution at cell-cell junctions. A transcriptome analysis reveals that loss of ING1 affects the expression of several junctional and cytoskeletal modulators, confirming ING1 as an invasion suppressor and a key regulator of cell-cell junction integrity.

Generation and live imaging of tumors with specific genotypes in the living fly pupa.

This protocol describes the step-by-step generation of tumors with specific genotypes on the dorsal thorax epithelium of the fly. This in vivo system allows the imaging of tumor cell morphology and behavior in high spatial and temporal resolution. Phenotypes such as cell invasion, cell division, and tumor size can be quantified and compared to specific controls or to the neighboring wild-type tissue. Thus, this model allows the study of conserved genes that enhance or suppress epithelial tumor progression. For complete details on the use and execution of this protocol, please refer to Canales Coutiño et al. (2020).

In vivo Evaluation of a Newly Synthesized Acetylcholinesterase Inhibitor in a Transgenic Drosophila Model of Alzheimer's Disease.

Alzheimer's disease is a neurodegenerative disease characterized by disrupted memory, learning functions, reduced life expectancy, and locomotor dysfunction, as a result of the accumulation and aggregation of amyloid peptides that cause neuronal damage in neuronal circuits. In the current study, we exploited a transgenic Drosophila melanogaster line, expressing amyloid-ß peptides to investigate the efficacy of a newly synthesized acetylcholinesterase inhibitor, named XJP-1, as a potential AD therapy. Behavioral assays and confocal microscopy were used to characterize the drug effect on AD symptomatology and amyloid peptide deposition. The symptomatology induced in this particular transgenic model recapitulates the scenario observed in human AD patients, showing a shortened lifespan and reduced locomotor functions, along with a significant accumulation of amyloid plaques in the brain. XJP-1 treatment resulted in a significant improvement of AD symptoms and a reduction of amyloid plaques by diminishing the amyloid aggregation rate. In comparison with clinically effective AD drugs, our results demonstrated that XJP-1 has similar effects on AD symptomatology, but at 10 times lower drug concentration than donepezil. It also showed an earlier beneficial effect on the reduction of amyloid plaques at 10 days after drug treatment, as observed for donepezil at 20 days, while the other drugs tested have no such effect. As a novel and potent AChE inhibitor, our study demonstrates that inhibition of the enzyme AChE by XJP-1 treatment improves the amyloid-induced symptomatology in Drosophila, by reducing the number of amyloid plaques within the fruit fly CNS. Thus, compound XJP-1 has the therapeutic potential to be further investigated for the treatment of AD.

European Resuscitation Council Guidelines 2021: Systems saving lives.

The European Resuscitation Council (ERC) has produced these Systems Saving Lives guidelines, which are based on the 2020 International Consensus on Cardiopulmonary Resuscitation Science with Treatment Recommendations. The topics covered include chain of survival, measuring performance of resuscitation, social media and smartphones apps for engaging community, European Restart a Heart Day, World Restart a Heart, KIDS SAVE LIVES campaign, lower-resource setting, European Resuscitation Academy and Global Resuscitation Alliance, early warning scores, rapid response systems, and medical emergency team, cardiac arrest centres and role of dispatcher.

Education, Implementation, and Teams: 2020 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations.

For this 2020 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations, the Education, Implementation, and Teams Task Force applied the population, intervention, comparator, outcome, study design, time frame format and performed 15 systematic reviews, applying the Grading of Recommendations, Assessment, Development, and Evaluation guidance. Furthermore, 4 scoping reviews and 7 evidence updates assessed any new evidence to determine if a change in any existing treatment recommendation was required. The topics covered included training for the treatment of opioid overdose; basic life support, including automated external defibrillator training; measuring implementation and performance in communities, and cardiac arrest centers; advanced life support training, including team and leadership training and rapid response teams; measuring cardiopulmonary resuscitation performance, feedback devices, and debriefing; and the use of social media to improve cardiopulmonary resuscitation application.

Up to 206 Million People Reached and Over 5.4 Million Trained in Cardiopulmonary Resuscitation Worldwide: The 2019 International Liaison Committee on Resuscitation World Restart a Heart Initiative.

Sudden out-of-hospital cardiac arrest is the third leading cause of death in industrialized nations. Many of these lives could be saved if bystander cardiopulmonary resuscitation rates were better. "All citizens of the world can save a life-CHECK-CALL-COMPRESS." With these words, the International Liaison Committee on Resuscitation launched the 2019 global "World Restart a Heart" initiative to increase public awareness and improve the rates of bystander cardiopulmonary resuscitation and overall survival for millions of victims of cardiac arrest globally. All participating organizations were asked to train and to report the numbers of people trained and reached. Overall, social media impact and awareness reached up to 206 million people, and >5.4 million people were trained in cardiopulmonary resuscitation worldwide in 2019. Tool kits and information packs were circulated to 194 countries worldwide. Our simple and unified global message, "CHECK-CALL-COMPRESS," will save hundreds of thousands of lives worldwide and will further enable many policy makers around the world to take immediate and sustainable action in this most important healthcare issue and initiative.

A Genetic Analysis of Tumor Progression in Drosophila Identifies the Cohesin Complex as a Suppressor of Individual and Collective Cell Invasion.

Metastasis is the leading cause of death for patients with cancer. Consequently it is imperative that we improve our understanding of the molecular mechanisms that underlie progression of tumor growth toward malignancy. Advances in genome characterization technologies have been very successful in identifying commonly mutated or misregulated genes in a variety of human cancers. However, the difficulty in evaluating whether these candidates drive tumor progression remains a major challenge. Using the genetic amenability of Drosophila melanogaster we generated tumors with specific genotypes in the living animal and carried out a detailed systematic loss-of-function analysis to identify conserved genes that enhance or suppress epithelial tumor progression. This enabled the discovery of functional cooperative regulators of invasion and the establishment of a network of conserved invasion suppressors. This includes constituents of the cohesin complex, whose loss of function either promotes individual or collective cell invasion, depending on the severity of effect on cohesin complex function.

The multifarious regulation of the apical junctional complex.

Epithelial cells form highly organized polarized sheets with characteristic cell morphologies and tissue architecture. Cell-cell adhesion and intercellular communication are prerequisites of such cohesive sheets of cells, and cell connectivity is mediated through several junctional assemblies, namely desmosomes, adherens, tight and gap junctions. These cell-cell junctions form signalling hubs that not only mediate cell-cell adhesion but impact on multiple aspects of cell behaviour, helping to coordinate epithelial cell shape, polarity and function. This review will focus on the tight and adherens junctions, constituents of the apical junctional complex, and aims to provide a comprehensive overview of the complex signalling that underlies junction assembly, integrity and plasticity.

An apicobasal gradient of Rac activity determines protrusion form and position.

Each cell within a polarized epithelial sheet must align and correctly position a wide range of subcellular structures, including actin-based dynamic protrusions. Using in vivo inducible transgenes that can sense or modify Rac activity, we demonstrate an apicobasal gradient of Rac activity that is required to correctly form and position distinct classes of dynamic protrusion along the apicobasal axis of the cell. We show that we can modify the Rac activity gradient in genetic mutants for specific polarity proteins, with consequent changes in protrusion form and position and additionally show, using photoactivatable Rac transgenes, that it is the level of Rac activity that determines protrusion form. Thus, we demonstrate a mechanism by which polarity proteins can spatially regulate Rac activity and the actin cytoskeleton to ensure correct epithelial cell shape and prevent epithelial-to-mesenchymal transitions.

Time delays to reach dispatch centres in different regions in Europe. Are we losing the window of opportunity? - The EUROCALL study.

AIM: In out of hospital cardiac arrest (OHCA) the start of Cardiopulmonary Resuscitation (CPR) by a single rescuer may be delayed considerably if the total time (TT) to connect the telephone call to the Emergency Medical Communication Centre (EMCC) is prolonged. EUROCALL investigated the TT-EMCC and its components using different calling procedures. METHODS: This prospective, multicentre, randomised study was performed in April 2013. Telephone calls were randomly allocated to time of call, and to those connecting directly to the EMCC (1-step procedure) and those diverted before connecting to the EMCC (2-step procedure). RESULTS: Twenty-one EMCCs from 11 countries participated in the study. Time to first ringtone was similar between 1-step 3.7s (IQR 1.0-5.2) and 2-step calls 4.0s (IQR 2.4-5.2). For the 1878 1-step calls, the median TT-EMCC was 11.7s (IQR 8.7-18.5). For the 1550 2-step calls, the median time from first ringtone to first call-taker was 7s (IQR 4.6-11.9) and from first call-taker to EMCC was 18.7s (IQR 13.4-29.9). Median TT-EMCC was 33.2 s (IQR 24.7-46.1) and was significantly longer than the TT-EMCC observed with the 1-step procedure (P<0.0001). Significant differences existed among participating regions between and within different countries both for 1-step and 2-step procedures. CONCLUSION: TT-EMCC was significantly shorter in a 1-step procedure compared to a 2-step procedure. Regional differences existed between countries but also within countries. This may be relevant in cases of OHCA and other situations where patient outcome is critically time-dependent.

Glycosylated Notch and Cancer.

Glycosylation is one of the key components influencing several signaling pathways implicated in cell survival and growth. The Notch signaling pathway plays a pivotal role in numerous cell fate specifications during metazoan development. Both Notch and its ligands are repeatedly glycosylated by the addition of sugar moieties, such as O-fucose, O-glucose, or O-xylose, to bring about structural and functional changes. Disruption to glycosylation processes of Notch proteins result in developmental disorders and disease, including cancer. This review summarizes the importance and recent updates on the role of glycosylated Notch proteins in tumorigenesis and tumor metastasis.

Combination of chest compressions and interposed abdominal compressions in a swine model of ventricular fibrillation.

PURPOSE: The aim of this study was to investigate the effects of the combination of chest compressions and interposed abdominal compressions (IAC-CPR) in a swine model of ventricular fibrillation (VF). METHODS: Twenty healthy female Landrace-Large White pigs were the study subjects. At the end of the eighth minute of VF, animals in the control group (Group A) received chest compressions at a rate of 100/min, while animals in the experimental group received chest compressions and simultaneous interposed abdominal compressions (CC-IAC - Group B), both at a rate of 100/min. The primary end point of the experiment was return of spontaneous circulation (ROSC). Secondary outcomes were 48-h survival rate and 48-h neurologic outcome. RESULTS: Six animals (60%) from Group A and 9 animals (90%) from Group B achieved ROSC (P=.121). There was a statistically significant difference in systolic aortic pressure, mean aortic pressure, right atrial pressures, and end-tidal carbon dioxide (ETCO2) between the two groups during the first cycle of CPR, while during the second cycle, diastolic aortic pressure was significantly higher in Group B. Coronary perfusion pressure (CPP) values in group B were significantly higher compared with those in Group A during the first and second cycle of CPR. Neurologic examination was statistically significantly better in Group B (75.00±10.00 vs. 90.00±10.00, P=.037). CONCLUSION: ROSC did not differ statistically significant in the IAC-CPR compared to the CPR group only, while CPP was significantly higher in IAC-CPR-treated animals.

Attitudes of healthcare professionals involved in cardiology practice towards key points of contemporary guidelines on resuscitation.

Hellenic J Cardiol. 2014; 55: 378-385. At the request of the authors, the name of the second author of this Original Research article has been changed from Athanasios Patialakas to Athanasios Patialiakas.

The interdependence of the Rho GTPases and apicobasal cell polarity.

Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease.

Attitudes of healthcare professionals involved in cardiology practice towards key points of contemporary guidelines on resuscitation.

INTRODUCTION: Cardiopulmonary resuscitation (CPR) is not always executed in compliance with contemporary guidelines and the quality of CPR may differ among hospitals within the same country or among categories of healthcare providers and medical specialties. The aim of this study was to assess attitudes of cardiology healthcare professionals towards CPR guidelines. METHODS: An anonymous questionnaire was posted online during 2009. Responders were asked about their age, gender, occupation, and training/experience in CPR. Responders' attitudes towards CPR were assessed using 7 questions regarding the accuracy of their opinions about the automated external defibrillator, public defibrillation programs, CPR performance, and therapeutic hypothermia. A score (0 to 7) was formed by assigning grade 1 to answers that accorded with European Resuscitation Council (ERC) guidelines and grade 0 to all other answers. The reliability analysis for this score yielded a Cronbach's alpha of 0.78. RESULTS: There were 544 responders (158 females), median age 34 years (30, 40). Median score was 5 (3, 6). Attending an ERC resuscitation course (beta=0.33, SE beta=0.05, p<0.001), age (beta=-0.15 SE beta=0.05, p=0.002), involvement in >10 CPRs /year (beta=0.19, SE beta=0.05, p<0.001), and being a physician (beta=0.17, SE beta=0.05, p=0.001) were all independent predictors of score. Attendance at an ERC course (OR: 2.7 [1.5 to 4.7]), being a physician (OR: 2 [1.3 to 5]) and involvement in >10 CPRs /year (OR: 1.7 [1.1 to 2.7]) were also independent predictors for attitudes that accorded with contemporary guidelines regarding therapeutic hypothermia. CONCLUSIONS: Attending an ERC resuscitation course, frequent involvement in CPR attempts, younger age, and being a physician were all independent predictors for more positive attitudes towards the guidelines. These factors, with the exception of age, were also associated with positive attitudes towards the implementation of therapeutic hypothermia.

Systematic review of the mechanisms driving effective blood flow during adult CPR.

BACKGROUND: High quality chest compressions is the most significant factor related to improved short-term and long-term outcome in cardiac arrest. However, considerable controversy exists over the mechanisms involved in driving blood flow. OBJECTIVES: The aim of this systematic review is to elucidate major mechanisms involved in effective compression-mediated blood flow during adult cardiopulmonary resuscitation (CPR). DESIGN AND SETTING: Systematic review of studies identified from the bibliographic databases of PubMed/Medline, Cochrane, and Scopus. SELECTION CRITERIA: All human and animal studies including information on the responsible mechanisms of compression-related blood flow. DATA COLLECTION AND ANALYSIS: Two reviewers (MG, TX) independently screened all potentially relevant titles and abstracts for eligibility, by using a standardized data-worksheet. MAIN RESULTS: Forty seven studies met the inclusion criteria. Because of the heterogeneity in outcome measures, quantitative synthesis of evidence was not feasible. Evidence was critically synthesized in order to answer the review questions, taking into account study heterogeneity and validity. The number of included studies per category is as follows: blood flow during chest compression, nine studies; blood flow during chest decompression, six studies; effect of chest compression on cerebral blood flow, eight studies; active compression-decompression CPR, 14 studies; and effect of ventilation on compression-related blood flow, 13 studies. CONCLUSION: The evidence so far is inconclusive regarding the major responsible mechanism in compression-related blood flow. Although both 'cardiac pump' and 'thoracic pump' have a key role, the effect of each mechanism is highly depended on other resuscitation parameters, such as positive pressure ventilation and compression depth.

Comparative analysis of two venipuncture learning methods on nursing students.

INTRODUCTION: Venus cannulation is one of the main nursing actions. For the safety of patients and nurses, this skill is important to be performed in a proper and responsible way. Thus, the need for adequate education on this skill is essential. STUDY OBJECTIVE: Comparison between the 4-stage (demonstration, deconstruction, comprehension, performance) learning method and the simplified 2-stage method (deconstruction, performance) on venous cannulation. METHODS: The study took place from November 2011 to March 2012. In total, 117 students of the Nursing Department of Technological Educational Institute of Athens were randomized into two education groups of the 2- (Group A) and 4- (Group B) stage method respectively. Students of both groups were assessed during training and 45days later for skill retention. For the assessment of all students, a check list with the steps required to venous cannulation was used. The time to complete the process was also assessed. RESULTS: Group A included 54 students and Group B 50 students. During initial training, the average time to complete the process without any errors was lower for Group A compared to Group B (1.77±0.12min vs. 2.33±0.13min, p=0.006). Regarding the appropriate steps for venous cannulation, Group A made fewer errors compared to Group B (0.95±0.95 vs. 1.38±1.10, p=0.021). In addition, Group A omitted more steps than Group B (0.29±0.06 vs. 0.11±0.05, p=0.042). During skill retention assessment, no significant difference was observed in the number of incorrect steps or in the number of omitted steps between groups. CONCLUSION: According to our findings, the 4-stage method, is associated with less omitted steps, while the 2-stage method, is associated with less time required to complete the procedure and less errors during performance, at initial stages. However, skill retention does not seem to be associated with the type of the educational methodology.

ERC initiatives to reduce the burden of cardiac arrest: the European Cardiac Arrest Awareness Day.

The rate of survival from out-of-hospital cardiac arrest in Europe remains unacceptably low and could be increased by better bystander cardiopulmonary resuscitation (CPR) rates. The European Resuscitation Council has announced that there will be a European Cardiac Arrest Awareness Day every year on the 16th of October. This is to coincide with the goals of the Written Declaration passed by the European Parliament in June 2012 that emphasised the importance of equal access to CPR and automated external defibrillator (AED) training. The topic of this year's Awareness Day is 'Children Saving Lives' and it is hoped that all national resuscitation councils will promote awareness of the benefits of training all children in CPR and AED use and lobby for legislative change to ensure that all children receive this training. Children are not just the adults of tomorrow - they are the lifesavers of today and tomorrow.