Improving the Implementation of Advance Directives in Spain.

Since 2002, legislation in Spain has allowed for the creation and documentation of end-of-life decisionmaking. Over the intervening years, the actual implementation of such documents is very low. Through extensive analysis of the literature, this article explores the current status of the use of and attitudes toward advance directives in Spain and then proposes strategies for improvement in their implementation.

Triage during the COVID-19 epidemic in Spain: better and worse ethical arguments.

The COVID-19 pandemic has generated an imbalance between the clinical needs of the population and the effective availability of advanced life support (ALS) resources. Triage protocols have thus become necessary. Triage decisions in situations of scarce resources were not extraordinary in the pre-COVID-19 era; these protocols abounded in the context of organ transplantation. However, this prior experience was not considered during the COVID-19 outbreak in Spain. Lacking national guidance or public coordination, each hospital has been forced to put forth independent and autonomous triage protocols, most of which were, nonetheless, based on common ethical principles and clinical criteria. However, controversial, non-clinical criteria have also been defended by Spanish scientific societies and public institutions, including setting an age cut-off value for unilaterally withholding ALS, using 'social utility' criteria, prioritising healthcare professionals or using 'first come, first served' policies. This paper describes the most common triage criteria used in the Spanish context during the COVID-19 epidemic. We will highlight our missed opportunities by comparing these criteria to those used in organ transplantation protocols. The problems posed by subjective, non-clinical criteria will also be discussed. We hope that this critical review might be of use to countries at earlier stages of the epidemic while we learn from our mistakes.

Why have Advance Directives failed in Spain?

BACKGROUND: In Spain, there has been great effort by lawmakers to put Advance Directives (ADs) into practice since 2002. At the same time, the field of bioethics has been on the rise, a discipline that has spurred debate on the right of patients to exercise their autonomy. Despite all this, the implementation of ADs can be said to have failed in Spain, because its prevalence is very low, there is a great lack of knowledge about them and they have very little impact on clinical decisions. The purpose of this article is to analyze and discuss the main reasons for the failure of ADs in Spain. MAIN BODY: The main reasons why ADs have no impact on clinical practice in Spain have been fundamentally four: (1) the training of health professionals about the end of life and AD is lacking; (2) there has been no public process to increase awareness about AD, and therefore people (with the exception of specific highly sensitized groups) know little about them; (3) the bureaucratic procedure to document and implement ADs is excessively complex and cumbersome, creating a significant barrier to their application; (4) in Spain, the remnants of a paternalistic medical culture continue to exist, which causes shared decision-making to be difficult. CONCLUSION: Due to the four reasons mentioned above, AD have not been a useful tool to help honor patients' autonomous decisions about their future care and, therefore, they have not achieved their objective. However, despite the difficulties and problems identified, it has also been observed that health care professionals and the Spanish public have a very positive view of AD. Having identified the problems which have kept AD from being successful, strategies must be developed to help improve their implementation into the future.

Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.

The replication machinery of bacteriophage Φ29 is a paradigm for protein-primed replication and it holds great potential for applied purposes. To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin. We have observed that the strength of the origin is determined by a combination of factors. The strongest origin (30-fold respect to wt) has the sequence CCC at the 3' end of the template strand, AAA at the 5' end of the non-template strand and 6 nucleotides as optimal unpairing at the end of the origin. We also show that the presence of a correctly positioned displaced strand is important because origins with 5' or 3' ssDNA regions have very low activity. Most of the effect of the improved origins takes place at the passage between the terminal protein-primed and the DNA-primed modes of replication by the DNA polymerase suggesting the existence of a thermodynamic barrier at that point. We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.

Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Phi29.

The DNA amplification performed by terminal protein-primed replication systems has not yet been developed for its general use to produce high amounts of DNA linked to terminal protein (TP). Here we present a method to amplify in vitro heterologous DNAs using the Φ29 DNA replication machinery and producing DNA with TP covalently attached to the 5' end. The amplification requires four Φ29 proteins, DNA polymerase, TP, single-stranded DNA binding protein and double-stranded DNA binding protein (p6). The DNA to be amplified is inserted between two sequences that are the Φ29 DNA replication origins, consisting of 191 and 194 bp from the left and right ends of the phage genome, respectively. The replication origins do not need to have TP covalently attached beforehand to be functional in amplification and they can be joined to the DNA to be amplified by cloning or ligation. The facts that two functional origins were required at the ends of a linear template DNA and that the kinetics of DNA synthesis was very similar to that obtained using the TP-containing Φ29 genome as template support the proposal that genuine amplification is taking place. Amplification factors of 30-fold have been obtained. Possible applications of DNAs produced by this method are discussed.

Targeting EGFR in Lung Cancer: Current Standards and Developments.

Lung cancer is the second most common malignant tumor and the leading cause of cancer death. Epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) is a distinct subtype of lung cancer comprising approximately 15-40% of non-squamous tumors. The development of first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) has been a significant step forward in the treatment of patients with EGFR-mutant tumors, and over the last few years has been the therapy of choice in the initial management of patients with activating mutations in EGFR, with some differences in efficacy and toxicity profile. Up to 50% of patients treated with first- and second-generation TKIs develop an EGFR exon 20 T790M mutation at the time of progression. In this context, osimertinib has shown a great benefit in terms of progression-free survival (PFS) in the second-line setting, including central nervous system metastasis control. The FLAURA trial, which compared osimertinib to first-generation inhibitors as first-line therapy, showed a clear PFS advantage for osimertinib and a trend towards an increased overall survival (OS) assessed by investigator review. Although T790M mutation is the most common mechanism of resistance to first- and second-generation EGFR TKIs, other EGFR-dependent and -independent mechanisms have been described, such as HER2 and MET amplifications or BRAF and MEK mutations. Some mechanisms of resistance to osimertinib and other third-generation TKIs have also been described. Several fourth-generation TKIs, targeted drug combinations and immunotherapy strategies are under investigation to overcome resistance to EGFR TKIs in order to improve EGFR-mutant NSCLC patient outcomes.

Engineering Permissive Insertion Sites in the Bacteriophage Phi29 DNA-Linked Terminal Protein.

Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5' ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to the interior of the cell, to the nucleus, or even to subcellular compartments. In order to be able to insert different peptide modules on the TP sequence to endow it with desired functions and/or eliminate unwanted regions of the protein, we have carried out a transposition screening to detect insertion-permissive points on the sequence of the TP. We report the functional characterization of 12 insertion mutants of the TP, and the identification of one site at position 38 that allows the insertion of peptides up to 17 amino acids in length while maintaining the ability of the TP to support DNA amplification in vitro. A protein with one insertion at that position containing a cysteine residue, a linker, and a thrombin recognition site was purified and its amplification activity was optimized.