Environmental impact assessment of the electrokinetic adsorption barriers to remove different herbicides from agricultural soils.

In this study, the sustainability of the electrokinetic remediation soil flushing (EKSFs) process integrated without and with adsorption barriers (EKABs) have been evaluated for the treatment of four soils contaminated with Atrazine, Oxyfluorfen, Chlorosulfuron and 2,4-D. To this purpose, the environmental effects of both procedures (EKSFs and EKABs) have been determined through a life cycle assessment (LCA). SimaPro 9.3.0.3 was used as software tool and Ecoinvent 3.3 as data base to carry out the inventory of the equipment of each remediation setup based on experimental measurements. The environmental burden was quantified using the AWARE, USEtox, IPPC, and ReCiPe methods into 3 Endpoint impact categories (and damage to human health, ecosystem and resources) and 7 Midpoints impact categories (water footprint, global warming potential, ozone depletion, human toxicity (cancer and human non-cancer), freshwater ecotoxicity and terrestrial ecotoxicity). In general terms, the energy applied to treatment (using the Spanish energy mix) was the parameter with the greatest influence on the carbon footprint, ozone layer depletion and water footprint accounting for around 70 % of the overall impact contribution. On the other hand, from the point of view of human toxicity and freshwater ecotoxicity of soil treatments with 32 mg kg-1 of the different pesticides, the EKSF treatment is recommended for soils with Chlorosulfuron. In this case, the carbon footprint and water footprint reached values around 0.36 kg of CO2 and 114 L of water per kg of dry soil, respectively. Finally, a sensitivity analysis was performed assuming different scenarios.

TET enzymes and 5hmC epigenetic mark: new key players in carcinogenesis and progression in gynecological cancers.

DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine (5mC). In general, DNA methylation in cancer is associated with the repression of the expression of tumor suppressor genes (TSG) and the demethylation with the overexpression of oncogenes. DNA methylation was considered a stable modification for a long time, but in 2009, it was reported that DNA methylation is a dynamic modification. The Ten-Eleven-Translocations (TET) enzymes include TET1, TET2, and TET3 and participate in DNA demethylation through the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC). The 5hmC oxidates to 5-formylcytosine (5fC) and 5-carboxylcitosine (5caC), which are replaced by unmodified cytosines via Thymine-DNA Glycosylase (TDG). Several studies have shown that the expression of TET proteins and 5hmC levels are deregulated in gynecological cancers, such as cervical (CC), endometrial (EC), and ovarian (OC) cancers. In addition, the molecular mechanisms involved in this deregulation have been reported, as well as their potential role as biomarkers in these types of cancers. This review shows the state-of-art TET enzymes and the 5hmC epigenetic mark in CC, EC, and OC.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Clinic-pathological agreement in the diagnosis of conjunctival tumours: Analysis of 195 cases.

PURPOSE: The present study aims to assess the agreement between clinical and pathological diagnosis in conjunctival tumours in a specialist ocular oncology unit. METHODS: retrospective study of consecutive patients with conjunctival tumours diagnosed at the Ocular Oncology Unit of the University Hospital of Valladolid was performed from 1992 to 2017. Tumours were classified according to their origin (epithelial, melanocytic, lymphoid and others) and degree of malignancy (benign, premalignant, malignant). A biopsy was performed in cases of symptomatic or growing lesions. Cohen´s kappa (κ) statistics was used as an indicator of agreement between clinical and pathological diagnosis. RESULTS: Of 462 consecutive patients, a biopsy was required in 195 (42.2%). The agreement with the pathological diagnosis was successful in 154 (79.0%) cases. Analysis according to the grade of malignancy showed the lowest rate of agreement among benign (n = 83; 91.6%) and premalignant (n = 62; 90.3%) lesions, with a total agreement in malignant lesions (n = 50; 100%); the Cohen´s kappa coefficient (κ) was 0.90. The highest rates of concordance were found in epithelial, melanocytic and soft tissue lesions with κ values of 1, 0.8 and 1 respectively. The worst rate of concordance was found in lymphoid lesions with a κ value of 0.3. CONCLUSION: Most of the conjunctival tumours were correctly identified clinically; benign and malignant lesions showed the highest rate of accuracy; however, premalignant tumours can hide micro-invasive diseases that can go unnoticed on clinical examination. The biopsy is essential for accurate diagnosis and treatment.

[Impact of COVID-19 on a rare disease (uveal melanoma) in a national reference unit of intraocular tumors in Spain]. / Impacto del COVID-19 en una enfermedad rara (melanoma uveal) en una unidad de referencia nacional de tumores intraoculares en España.

Objective: To analyze the impact of the COVID-19 pandemic on the diagnosis and management of uveal melanoma (a tumor included in the Orphanet catalog of rare diseases) in a Spanish national reference unit for intraocular tumors during the first year of the pandemic. Material and methods: An observational retrospective study of patients with uveal melanoma in the National Reference Unit for Adult Intraocular Tumors of the Hospital Clínico Universitario de Valladolid (Spain) was performed, analyzing the pre- and post-COVID-19 periods: from March 15, 2019 to March 15, 2020 and from March 16, 2020 to March 16, 2021. Demographic data, diagnostic delay, tumor size, extraocular extension, treatment and evolution were collected. A multivariable logistic regression model was used to identify factors that were associated with the variable: enucleation. Results: Eighty-two patients with uveal melanoma were included, of which 42(51.21%) belonged to the pre-COVID-19 period and 40(40.78%) to the post-COVID-19 period. An increase in tumor size at diagnosis and in the number of enucleations was observed during the post-COVID-19 period (p < 0.05). Multivariable logistic regression demonstrated that both medium-large tumor size and patients diagnosed in the post-COVID-19 period were independently related to an increased risk of enucleation (OR 250, 95%CI, 27.69-2256.37; p < 0.01 and OR 10; 95% CI,1.10-90.25; p = 0.04, respectively). Conclusions: The increase in tumor size observed in uveal melanomas diagnosed during the first year of the COVID-19 pandemic may have favored the increase in the number of enucleations performed during that period.

Plasma rich in growth factors membrane as a macular hole treatment in a vitrectomized patient due to rhegmatogenous retinal detachment.

The formation of a macular hole after vitrectomy due to rhegmatogenous retinal detachment is a rare complication. Although there are different surgical options in the treatment of these macular holes with favorable outcomes, it has been shown that the history of macula-off retinal detachment is the most important risk factor related to the need for multiple interventions to close these macular holes, therefore special attention should be paid in the management of these patients. We present the case of a patient with macula-off rhegmatogenous retinal detachment who required treatment with cataract surgery with intraocular lens implant and pars plana vitrectomy. Four years after the primary surgery, she presented a large macular hole, and was treated with membrane of plasm rich in growth factors with closure of the macular hole and visual improvement without recurrence 12 months after surgery.

Initial results of endourological treatment of renoureteral lithiasis by means of flexible ureteroscopy and percutaneous surgery in an outpatient procedure in a tertiary care center.

OBJECTIVE: Urinary lithiasis is a prevalent disease with a high socioeconomic impact, where endourological surgery has shown excellent results with minimal complications. For its part, outpatient surgery is an efficient, safe and quality care model. We present our experience in the outpatient endourological treatment of lithiasis and a review of the main series. MATERIAL AND METHODS: Prospective analysis of 85 flexible or percutaneous procedures for the treatment of lithiasis, carried out in our center between January 2021 and April 2022. The main objective was to analyze the rate of unplanned admission and the success and incidence of complications as secondary objectives. The patients were selected following the inclusion criteria of the care process. RESULTS: The mean age was 56 ± 14 years. Urine culture was positive in 13.9% of the patients, 38% had a pre-surgical double-J catheter. Median stone surface was 55 mm 2 (961 ± 323 Hounsfield Units). 73 flexible and 12 percutaneous procedures were performed. 8 patients required immediate unplanned admission and another 2 during the first month. 94% were stone-free at the third month. No intraoperative complications were detected, although 16.5% of the patients presented some type of postoperative complication. CONCLUSION: In our experience, with a strict selection of patients and following a care process with multidisciplinary participation, endourological procedures are feasible and safe in the outpatient setting. Periodic monitoring of the results is essential for the sake of a constant improvement of the process.

Impact of COVID-19 on a rare disease (uveal melanoma) in a national reference unit of intraocular tumors in Spain.

OBJECTIVE: To analyse the impact of the COVID-19 pandemic on the diagnosis and management of uveal melanoma (a tumour included in the Orphanet catalogue of rare diseases) in a Spanish national reference unit for intraocular tumours during the first year of the pandemic. METHOD: An observational retrospective study of patients with uveal melanoma in the National Reference Unit for Adult Intraocular Tumors of the Hospital Clínico Universitario de Valladolid (Spain) was performed, analysing the pre- and post-COVID-19 periods: from March 15, 2019 to March 15, 2020 and from March 16, 2020 to March 16, 2021. Demographic data, diagnostic delay, tumour size, extraocular extension, treatment and evolution were collected. A multivariable logistic regression model was used to identify factors that were associated with the variable: enucleation. RESULTS: Eighty-two patients with uveal melanoma were included, of which 42 (51.21%) belonged to the pre-COVID-19 period and 40(40.78%) to the post-COVID-19 period. An increase in tumour size at diagnosis and in the number of enucleations was observed during the post-COVID-19 period (p < 0.05). Multivariable logistic regression demonstrated that both medium-large tumour size and patients diagnosed in the post-COVID-19 period were independently related to an increased risk of enucleation (OR 250, 95%CI, 27.69-2256.37; p < 0.01 and OR 10; 95%CI, 1.10-90.25; p = 0.04, respectively). CONCLUSIONS: The increase in tumour size observed in uveal melanomas diagnosed during the first year of the COVID-19 pandemic may have favored the increase in the number of enucleations performed during that period.

Alternatives to classic solvents for the isolation of bioactive compounds from Chrysochromulina rotalis.

This paper demonstrates a sequential partitioning method for isolating bioactive compounds from Chrysochromulina rotalis using a polarity gradient, replacing classic and hazardous solvents with greener alternatives. Seventeen solvents were evaluated based on their Hansen solubility parameters and for having a similar polarity to the solvents they would replace, four of which were selected as substitutes in the classic fractionation process. Considering the fatty acid and carotenoid recovery yields obtained for each of the solvents, it has been proposed to replace hexane (HEX), toluene (TOL), dichloromethane (DCM) and n-butanol (BUT) with cyclohexane, chlorobenzene, isobutyl acetate and isoamyl alcohol, respectively. In addition, cytotoxic activity was observed when the TOL and DCM solvent extracts were tested against tumour cell lines, demonstrating the antiproliferative potential of compounds containing, for example, fucoxanthin, fatty acids, peptides, isoflavonoids or terpenes, among others.

EMMPRIN is an emerging protein capable of regulating cancer hallmarks.

EMMPRIN, also known as Basigin or CD147, is a transmembrane glycoprotein member of the immunoglobulin superfamily. It is expressed basally in cells that regulate physiological processes of the cardiovascular, nervous, and immune systems. However, EMMPRIN is also capable of interacting with different proteins, like VEGFR, SMAD4, Integrin, MCT, CyPA, GLUT1, CAIV, Annexin II, Cav-1, CAML, etc., and regulating signaling pathways that stimulate the cell processes of proliferation, apoptosis, metabolism, adhesion, invasion, migration, metastasis, tumor immune response, and angiogenesis processes, which favors the development of different types of cancer. EMMPRIN is the first protein reported that favors cancer development due to its ability to interact with extracellular, intracellular, and membrane proteins. In conclusion, EMMPRIN regulates several proteins associated with the development of tumor processes. Therefore, blocking the expression of EMMPRIN can be a therapeutic target, and the analysis of its expression can be used as an important biomarker in cancer.

PACAP-PAC1R modulates fear extinction via the ventromedial hypothalamus.

Exposure to traumatic stress can lead to fear dysregulation, which has been associated with posttraumatic stress disorder (PTSD). Previous work showed that a polymorphism in the PACAP-PAC1R (pituitary adenylate cyclase-activating polypeptide) system is associated with PTSD risk in women, and PACAP (ADCYAP1)-PAC1R (ADCYAP1R1) are highly expressed in the hypothalamus. Here, we show that female mice subjected to acute stress immobilization (IMO) have fear extinction impairments related to Adcyap1 and Adcyap1r1 mRNA upregulation in the hypothalamus, PACAP-c-Fos downregulation in the Medial Amygdala (MeA), and PACAP-FosB/ΔFosB upregulation in the Ventromedial Hypothalamus dorsomedial part (VMHdm). DREADD-mediated inhibition of MeA neurons projecting to the VMHdm during IMO rescues both PACAP upregulation in VMHdm and the fear extinction impairment. We also found that women with the risk genotype of ADCYAP1R1 rs2267735 polymorphism have impaired fear extinction.

Experimental study of the quantification of indocyanine green fluorescence in ischemic and non-ischemic anastomoses, using the SERGREEN software program.

Tissue ischemia is a key risk factor in anastomotic leak (AL). Indocyanine green (ICG) is widely used in colorectal surgery to define the segments with the best vascularization. In an experimental model, we present a new system for quantifying ICG fluorescence intensity, the SERGREEN software. Controlled experimental study with eight pigs. In the initial control stage, ICG fluorescence intensity was analyzed at the level of two anastomoses, in the right and in the left colon. Control images of the two segments were taken after ICG administration. The images were processed with the SERGREEN program. Then, in the experimental ischemia stage, the inferior mesenteric artery was sectioned at the level of the anastomosis of the left colon. Fifteen minutes after the section, sequential images of the two anastomoses were taken every 30 min for the following 2 h. At the control stage, the mean scores were 134.2 (95% CI 116.3-152.2) for the right colon and 147 (95% CI 134.7-159.3) for the left colon (p = 0.174) (Scale RGB-Red, Green, Blue). The right colon remained stable throughout the experiment. In the left colon, intensity fell by 47.9 points with respect to the pre-ischemia value (p < 0.01). After the first post-ischemia determination, the values of the ischemic left colon remained stable throughout the experiment. The relative decrease in ICG fluorescence intensity of the ischemic left colon was 32.6%. The SERGREEN program quantifies ICG fluorescence intensity in normal and ischemic situations and detects differences between them. A reduction in ICG fluorescence intensity of 32.6% or more was correlated with complete tissue ischemia.

Genetic variants of antioxidant enzymes and environmental exposures as molecular biomarkers associated with the risk and aggressiveness of bladder cancer.

Bladder cancer (BC) is one of the top 10 most common tumours worldwide; however, no molecular markers are currently available for tumour management and follow-up. BC could benefit from molecular biomarkers in environmental disease, which provide mechanistic understanding of individual susceptibility to exposure-related cancers and allow characterizing genetic alterations in the molecular pathway for malignancy. This case-control study performed a molecular analysis in 99 BC and 125 controls. Buccal swabs were collected to assess SNPs in eleven genes coding for xenobiotic detoxification enzymes, cellular antioxidant defences, and hormone synthesis and signalling (NAT2 (rs1801280), GPX1 (rs1050450 and rs17650792), TXNRD1 (rs7310505), PRDX3 (rs3740562), PON1 (rs662), SOD1 (rs10432782), SOD2 (rs4880), CAT (rs1001179), CYP17A1 (rs743572) and ESR1 (rs746432)). A structured questionnaire was administered to study participants to assess environmental and dietary chemical exposures. Several miRNAs associated with BC and detoxification/antioxidant pathways were analysed in a subsample of the study population, including miR-93-5p, miR-221-3p, miR-126, miR-27a-3p, miR-193b, and miR-193a-5p. Levels of selected environmental pollutants (polycyclic aromatic hydrocarbons and endocrine disrupting chemicals) were determined in urine from a subsample of BC cases and controls. We found that CYP17A1, CAT, SOD1, ESR1, PON1, and GPX1 (rs17650792) were associated with BC risk. Furthermore, exposure to smoke and/or dust, and alcohol intake were identified as risk factors for BC. Increased urinary levels of benzo[a]pyrene and bisphenol A were observed in BC patients relative to controls, along with an increased expression of miR-193b, miR-27a and miR-93-5p in BC. Nevertheless, further studies with a larger sample size are warranted to confirm these exploratory results. This study also shows that the combination of genetic markers (PON1 and CYP17A1) and miRNA (miR-221-3p and miR-93-5p) open a new scenario in the use of non-invasive biomarkers in the stratification of BC to guide personalized medicine, which is extremely urged in the current clinical setting.

Recommendations of the Society of Thoracic Surgery and the Section of Cardiothoracic and Vascular Surgery of the Spanish Society of Anesthesia, Resuscitation and Pain Therapy, for patients undergoing lung surgery included in an intensified recovery program.

In recent years, multidisciplinary programs have been implemented that include different actions during the pre, intra and postoperative period, aimed at reducing perioperative stress and therefore improving the results of patients undergoing surgical interventions. Initially, these programs were developed for colorectal surgery and from there they have been extended to other surgeries. Thoracic surgery, considered highly complex, like other surgeries with a high postoperative morbidity and mortality rate, may be one of the specialties that most benefit from the implementation of these programs. This review presents the recommendations made by different specialties involved in the perioperative care of patients who require resection of a lung tumor. Meta-analyzes, systematic reviews, randomized and non-randomized controlled studies, and retrospective studies conducted in patients undergoing this type of intervention have been taken into account in preparing the recommendations presented in this guide. The GRADE scale has been used to classify the recommendations, assessing on the one hand the level of evidence published on each specific aspect and, on the other hand, the strength of the recommendation with which the authors propose its application. The recommendations considered most important for this type of surgery are those that refer to pre-habilitation, minimization of surgical aggression, excellence in the management of perioperative pain and postoperative care aimed at providing rapid postoperative rehabilitation.

Impact of aging on the incidence and mortality of urological cancers: 20-year projection in Spain.

INTRODUCTION: In 2020, 282,421 new cases of cancer were diagnosed in Spain, and urological neoplasms (UN) were among the most frequent ones. Cancer treatment in eldery people is challenging due to fragility and comorbidities of these patients. To meet the needs of treating UN in the eldery, it is necessary to optimize healthcare resources, for which a deep analysis of cancer registries becomes mandatory. The objective of this work was to provide a detailed analysis of the incidence and mortality of UN in Spanish people over 65 years old in the last year 2020, as well as the estimates for the year 2040. MATERIAL AND METHODS: Incidence and mortality estimates were obtained from the GLOBOCAN database. The urological neoplasms that were included were: testicle, bladder, penis, kidney and prostate. RESULTS: In 2020, 63,278 cases of UN were diagnosed in Spain. Most UN were much more frequent among patients >65 years old, except for testicular cancers. For the year 2040, an incidence increase of 41.5% is estimated, reaching 89,507 new cases of UN per year, with approximately 3 out of 4 patients being over 65 years old. Deaths in people over 65 will increase by 60.15% in 2040. CONCLUSION: In the next two decades, it is expected that new cases UN in people over 65 years will increase above 50%. For Healthcare systems to face it, greater financial and human resources, as well as multidisciplinary teams with experience and geriatric training will be necessary.

Real-life experience of hepatitis C treatment in a Spanish prison.

OBJECTIVE: Hepatitis C virus (HCV) infection is a major public health problem that causes multiple comorbidities. People in prisons who inject intravenous drugs are at increased risk of HCV infection, and HCV infection is 15-fold more prevalent among prisoners compared with the community. The objective of this study was to analyse the clinical and epidemiological characteristics of residents of a Spanish prison with HCV infection who received antiviral treatment. METHODS: An observational, descriptive and retrospective study was performed. All patients with HCV infection diagnosed or followed up in an Infectious Diseases attached to a penitentiary were included in this study. RESULTS: Of 81 patients analysed, sixty-nine (83.1%) patients were male. The mean age was 50.1 (SD8.8) years, and 70% of the inmates had a history of injection drug use. Coinfection with HIV was detected in 30%. In up to 25% of the sample, there were data on chronic liver disease in the degree of liver cirrhosis. The diagnosis of HCV infection had been made more than 15 years earlier in 28% of those studied. Decompensations from liver disease, hepatocellular carcinoma, or hospital admissions were exceptional. Most of the inmates with HCV accepted treatment, and approximately 94% of the patients who completed treatment achieved a sustained virological response without interactions or complications of interest. CONCLUSIONS: The availability of direct-acting antivirals and their exceptional side effects constitute an opportunity to reduce the burden of HCV infection in Spain, particularly in these high-risk populations.

Anaesthetic-surgical guide in the treatment of ascending aorta and surgery of the ascending aorta and aortic arch. Consensus document of the Spanish Society of Cardiovascular and Endovascular Surgery and the Sociedad of Anaesthesiology, Resuscitation and Pain Therapy.

Aortic pathology is always a challenge for the clinician, and must be diagnosed and treated by a multidisciplinary team due to the technical and technological complexity of the resources used. Ongoing efforts to implement a systematic, protocolized approach involving "Aortic teams" made up of cardiologists, cardiac surgeons, vascular surgeons, anaesthesiologists and radiologists, among others are now leading to improved outcomes. The aim of this consensus document drawn up by the Aortic working groups of the Spanish Society of Anaesthesiology, Resuscitation and Pain Therapy (SEDAR) and the Spanish Society of Thoracic and Cardiovascular Surgery (SECTCV) is to disseminate a set of working protocols. The latest consensus document of the European Association for Cardio-Thoracic Surgery (EACTS) and the European Society for Vascular Surgery (ESVS) define the concept of "AORTIC TEAM"(1). The aortic team should be closely involved from diagnosis to treatment and finally follow-up, and should be formed of cardiac and vascular surgeons working together with anaesthesiologists, cardiologists, radiologists and geneticists. Treatment of aortic pathologies should be centralised in large centres, because this is the only way to effectively understand the natural course of the disease, provide the entire range of treatment options under one umbrella and treat potential complications. A streamlined emergent care pathway (24/7 availability), adequate transportation and transfer capabilities, as well as rapid activation of the multidisciplinary team must be available. In light of the complexity and constant evolution of therapeutic options, we present this first version of the Anaesthesiology and surgical guidelines for surgery of the ascending aorta and aortic arch. Some questions will no doubt remain unanswered, and future versions will include new techniques that, though implemented in some centres, are still not widely recommended.