Strobilurin fungicide increases the susceptibility of amphibian larvae to trematode infections.

The global rise in fungal pathogens has driven the increased usage of fungicides, yet our understanding of their ecotoxicity remains largely limited to acute toxicity. While such data is critical for projecting the risk of fungicide exposure to individual species, the contamination of natural systems with fungicides also has the potential to alter species interactions within communities including host-parasite relationships. We examined the effects of the fungicide pyraclostrobin on the susceptibility of larval American bullfrogs (Rana catesbeiana) to trematode (echinostome) infections using a controlled laboratory experiment. Following a 2-wk exposure to 0, 1.0, 5.2, or 8.4 µg/L of pyraclostrobin, tadpoles were then exposed to parasites either in the 1) presence (continued/simultaneous exposure) or 2) absence (fungicide-free water) of pyraclostrobin. We found that when exposed to pyraclostrobin during parasite exposure, meta cercariae counts increased 4 to 8 times compared to control tadpoles. Additionally, parasite loads were approximately 2 times higher in tadpoles with continued fungicide exposures compared to tadpoles that were moved to fresh water following fungicide exposure. This research demonstrates that fungicides at environmentally relevant concentrations can indirectly alter host-parasite interactions, which could elevate disease risk. It also underscores the need for studies that expand beyond traditional toxicity experiments to assess the potential community and ecosystem-level implications of environmental contaminants.

Acute aquatic toxicity of two commonly used fungicides to midwestern amphibian larvae.

Fungicide usage has increased globally in response to the rise in fungal pathogens, especially in the agricultural sector. However, research examining the toxicity of fungicides is still limited for many aquatic species. In this study, we examined the acute toxicity of two widely used fungicides, chlorothalonil and pyraclostrobin, on six North American larval amphibian species across multiple families using 96-h LC50 tests. We found that pyraclostrobin was approximately 3.5x more toxic than chlorothalonil; estimated LC50 values ranged from 5-18 µg/L for pyraclostrobin and 15-50 µg/L for chlorothalonil. Comparing across amphibian groups, we found that salamanders were 3x more sensitive to pyraclostrobin than anuran species and equally as sensitive to chlorothalonil. Notably, our estimated LC50 values within the range of the expected environmental concentration for these fungicides suggesting environmental exposures could lead to direct mortality in these species. Given the widespread and increasing usage of fungicides, additional work should be conducted to assess the general risk posed by these chemicals to amphibian and their associated aquatic habitats.

Plastid phylogenomics uncovers multiple species in Medicago truncatula (Fabaceae) germplasm accessions.

Medicago truncatula is a model legume that has been extensively investigated in diverse subdisciplines of plant science. Medicago littoralis can interbreed with M. truncatula and M. italica; these three closely related species form a clade, i.e. TLI clade. Genetic studies have indicated that M. truncatula accessions are heterogeneous but their taxonomic identities have not been verified. To elucidate the phylogenetic position of diverse M. truncatula accessions within the genus, we assembled 54 plastid genomes (plastomes) using publicly available next-generation sequencing data and conducted phylogenetic analyses using maximum likelihood. Five accessions showed high levels of plastid DNA polymorphism. Three of these highly polymorphic accessions contained sequences from both M. truncatula and M. littoralis. Phylogenetic analyses of sequences placed some accessions closer to distantly related species suggesting misidentification of source material. Most accessions were placed within the TLI clade and maximally supported the interrelationships of three subclades. Two Medicago accessions were placed within a M. italica subclade of the TLI clade. Plastomes with a 45-kb (rpl20-ycf1) inversion were placed within the M. littoralis subclade. Our results suggest that the M. truncatula accession genome pool represents more than one species due to possible mistaken identities and gene flow among closely related species.

Unusual presentation of supraventricular tachycardia degenerating into ventricular fibrillation during pregnancy: Aortocaval compression the probable culprit.

Cardiac arrhythmias are common and often benign in pregnancy. However, haemodynamic instability can occur when tachyarrhythmias are accompanied by aortocaval compression, which can lead to loss of cardiac output. We present an atypical case of a pregnant woman with a supraventricular tachyarrhythmia, which degenerated into ventricular fibrillation arrest while supine due to aortocaval compression. Inducible atypical atrioventricular nodal re-entry tachycardia was subsequently detected on electrophysiological study and presumed to be the most likely initial supraventricular tachyarrhythmia.

Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19.

Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.

Multimodal Imaging Characteristics of a Migrating Oropharyngeal-Spinal Foreign Body in a Cat.

A 2.5 yr old female spayed domestic shorthair presented for acute tetraparesis, dull mentation, and fever. MRI and computed tomography identified a thin linear foreign body extending from the caudal nasopharynx through the atlanto-occipital joint and cervicomedullary junction. Signal changes within the musculature were consistent with myositis, edema, and abscessation. Inflammation and edema surrounded the foreign body, and a dorsal cervical myelopathy extended caudally to the level of C6. Computed tomography attenuation values of the foreign body were most consistent with plant material. Euthanasia was performed; postmortem dissection of the soft palate confirmed a plant stem with abscess.

Comparing the clinical and prognostic impact of proximal versus nonproximal lesions in dominant right coronary artery ST-elevation myocardial infarction.

OBJECTIVE: To evaluate the prognostic significance of culprit lesion location in dominant right coronary artery (RCA) ST-elevation myocardial infarction (STEMI). BACKGROUND: In RCA STEMI, proximal culprit lesions have been shown to have higher rates of acute complications such as bradycardia and cardiogenic shock (CS) but data on mortality is limited. METHODS: We retrospectively identified and analyzed data from consecutive patients with a dominant RCA STEMI who underwent either primary or rescue percutaneous coronary intervention (PCI) between January 2003 and December 2016. We compared the rates of sustained ventricular tachycardia (VT), CS, intra-aortic balloon pump (IABP), temporary cardiac pacing (TCP) and death between culprit lesions located proximal and distal to the origin of the last right ventricular (RV) marginal artery >1 mm in diameter. RESULTS: The 939 patients were included; 599 (63.7%) had a proximal lesion and 340 (36.3%) had a nonproximal lesion. The 801 (85.3%) underwent primary PCI and 138 (14.7%) underwent rescue PCI. There was no difference in first medical contact to balloon or fibrinolysis times between the groups; p = .98 and .71. There was no significant difference in the rate of sustained VT (3.0%vs. 3.2%, p = .85) but proximal lesions were more likely to develop CS (10.9%vs. 5.8%, p = .01), require IABP (7.3%vs.2.9%, p < .01) and TCP (6.3%vs. 2.6%, p = .01). Thirty-day mortality was higher for proximal lesions (5.0%vs. 0.9%, p < .01) particularly for those with CS (35.3%vs. 10.0%, p = .05). CONCLUSION: Culprit lesions located proximal to the origin of the last RV marginal artery had a higher rate of acute complications such as CS and mortality.

First-in-Human Phase 1 Study To Assess Safety, Tolerability, and Pharmacokinetics of a Novel Antifungal Drug, VL-2397, in Healthy Adults.

VL-2397 is an antifungal drug with a novel mechanism of action, rapid fungicidal in vitro activity, and potent in vivo activity against Aspergillus fumigatus, including azole-resistant strains. VL2397-101, a phase 1 first-in-human, randomized, double-blind, placebo-controlled dose-escalation study, was conducted in healthy adults to determine the safety, tolerability, and pharmacokinetics (PK) of single and multiple ascending intravenous (i.v.) doses of VL-2397. All dosing cohorts were fully enrolled; all subjects completed the safety follow-up. A safety committee reviewed the safety data for each dosing cohort prior to recommending the initiation of each subsequent cohort. No serious adverse events (SAEs) occurred; the majority of treatment-emergent adverse events (TEAEs) were mild and self-limited. The most common drug-related TEAEs were infusion site reactions. No clinically concerning trends were noted in vital signs, electrocardiograms, physical examinations, or safety laboratory results. Following single infusions of VL-2397, the overall and maximum exposures rose less than proportionally with increasing doses from 3 mg to 1,200 mg as indicated by area under the concentration-time curve over 24 h (AUC24) and maximum concentration (Cmax). No signs of VL-2397 accumulation were observed following i.v. infusions of 300, 600, and 1,200 mg every 24 h (q24h) for 7 days. Renal elimination played a major role in total body clearance, with up to 47% of unmetabolized drug in urine 24 h after administration at single doses of >30 mg. Overall, VL-2397 dosing in the study appeared to be safe and well tolerated in the healthy subjects. The safety profile, consistent PK, and lack of drug accumulation support further development of VL-2397 in patients with invasive aspergillosis.

Real-world use of ticagrelor versus clopidogrel in percutaneous coronary intervention-treated ST-elevation myocardial infarction patients: A single-center registry study.

OBJECTIVES: The primary aim was to investigate the efficacy and safety of dual antiplatelet therapy (DAPT) using ticagrelor (T-DAPT) versus clopidogrel (C-DAPT) in a real-world ST-elevation myocardial infarction (STEMI) population. METHODS: We retrospectively analyzed 655 consecutive patients having primary percutaneous coronary intervention (PCI) for STEMI at Liverpool Hospital, Sydney, Australia (from January 2013 to April 2016). Medical and procedural therapies were at clinician discretion. Patient data were retrieved from hospital records and primary clinicians. RESULTS: T-DAPT (65%) was used more frequently, and in patients with lower mean CRUSADE (Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes with Early Implementation of the American College of Cardiology/American Heart Association Guidelines) score, than C-DAPT (24.6 vs. 32.2; p < 0.0001, respectively). All-cause mortality was 9.0% at 2.7 years follow-up, with fewer deaths for T-DAPT (4.5% vs. 17.2%; p < 0.0001). T-DAPT incurred less BARC (Bleeding Academic Research Consortium) 3-5 major bleeding (5.0% vs. 12.4%; p < 0.0001). Multivariate regression showed that C-DAPT, GRACE (Global Registry of Acute Cardiac Events) score, and renal insufficiency were independently associated with mortality. Intra-aortic balloon pump (IABP) and GRACE score independently predicted BARC 3-5 bleeding. Early DAPT discontinuation (1.7%) and ticagrelor intolerance (7.6%) was rare. Switching DAPT regimen was infrequent (21.7%) and mostly attributed to clinician preference (73.2%). Independent determinants of C-DAPT selection were older age, diabetes, prior PCI, IABP, and higher CRUSADE score. CONCLUSION: Ticagrelor was preferred in low bleeding risk patients, which may have contributed to less BARC 3-5 bleeding and lower mortality for T-DAPT. Thus, bleeding mitigation is a clinical priority when selecting DAPT for PCI-treated STEMI patients. Continuation of initial DAPT regimen was typical, but early switching from clopidogrel to ticagrelor shows willingness to optimize DAPT. Patients with very low CRUSADE scores (<21.5) may be appropriate for switching to a potent P2Y12 inhibitor.

Clinical and Angiographic Predictors of Mortality in Sudden Cardiac Arrest Patients Having Cardiac Catheterisation: A Single Centre Registry.

BACKGROUND: Immediate cardiac catheterisation (CC) is recommended in ST-elevation myocardial infarction (STEMI) following sudden cardiac arrest (SCA). Guidelines advise urgent CC for SCA patients without-STEMI, at clinician discretion. We examined the clinical and angiographic factors predicting mortality in SCA patients having CC. METHODS: Consecutive SCA patients having CC at Liverpool Hospital, Sydney (January 2011-September 2015) were retrospectively analysed. Patient data were retrieved from hospital records, and angiographic SYNTAX scores (SS) were quantified online. Independent predictors of mortality were derived using multivariate logistic analysis. RESULTS: The study cohort comprised 104 SCA patients; mean age 61±12years, and 79% male. Immediate CC (<2hours post-SCA) was performed in 35% overall. Compared to the without-STEMI subgroup, STEMI patients had more ventricular fibrillation (91 vs 50%; p<0.0001), and higher mean peak serum high-sensitivity troponin-T (8.25±14.7 vs 1.97±6.13 ug/L; p=0.006); in the context of higher median SS (18 vs 6.5; p=0.002) and target-lesion SS (tSS, 10 vs 0; p<0.001). Percutaneous coronary intervention (PCI; 75 vs 23%; p<0.0001) and target vessel revascularisation (11 vs 0%; p=0.005) were more frequent for STEMI. All-cause mortality was 39%, at 1.3±1.5years follow-up. Independent mortality predictors were: delayed CC (HR 4.08), serum lactate >7mmol/L (HR 3.47), and tSS (HR 1.05). CONCLUSIONS: Elevated serum lactate, tSS, and delayed CC, were predictive of longer-term mortality in SCA patients having CC. Late CC in patients without-STEMI suggest scope for improvement in real-world systems of care. Closer scrutiny of target lesion complexity may aid prognostication in SCA survivors.

Surveying GPCR solubilisation conditions using surface plasmon resonance.

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins.

The evolution of sexual signal modes and associated sensor morphology in fireflies (Lampyridae, Coleoptera).

Animals employ different sexual signal modes (e.g. visual, acoustic, chemical) in different environments and behavioural contexts. If sensory structures are costly, then evolutionary shifts in primary signal mode should be associated with changes in sensor morphology. Further, sex differences are expected if male and female signalling behaviours differ. Fireflies are known for their light displays, but many species communicate exclusively with pheromones, including species that recently lost their light signals. We performed phylogenetically controlled analyses of male eye and antenna size in 46 North American taxa, and found that light signals are associated with larger eyes and shorter antennae. In addition, following a transition from nocturnal light displays to diurnal pheromones, eye size reductions occur more rapidly than antenna size increases. In agreement with the North American taxa, across 101 worldwide firefly taxa in 32 genera, we found light displays are associated with larger eye and smaller antenna sizes in both males and females. For those taxa with both male and female data, we found sex differences in eye size and, for diurnal species, in antenna size.

Caries outcome following an intensive fluoride varnish treatment regimen for children at high risk for early childhood caries.

PURPOSE: To evaluate the caries relapse rate for a cohort of 2- to 4-year-old children at high risk of early childhood caries when treated with an intensive fluoride varnish (FV) regimen. METHODS: Eighty paediatric patients were recruited. Forty of these patients were high risk and received the FV treatment (three applications within 2 weeks and additional applications at 1 and 3 months) during 2009-2010. Mutans streptococci (MS) levels in the saliva were evaluated during the treatment period. A comparative group of 40 children, selected from an electronic record search at the New York University College of Dentistry to be of similar age, gender, and ethnicity, but not at elevated risk for ECC, received the standard of care (semi-annual FV treatment). Detailed caries examination and treatment records were obtained for all patients from 2009 to 2014. RESULTS: A significant reduction (P < 0.001) in MS levels was observed in the intensive FV treatment group at the 3-month visit compared with baseline. There was no effect of the intensive FV treatment on caries outcome in the anterior teeth, and the overall caries scores were significantly increased on the posterior teeth. CONCLUSION: The intensive FV regimen appears insufficient to prevent caries relapse in children at high risk for caries.

Selective anti-scatter grid removal during coronary angiography and PCI: a simple and safe technique for radiation reduction.

Objectives The aim of this study was to quantify the radiation dose reduction during coronary angiography and percutaneous coronary intervention (PCI) through removal of the anti-scatter grid (ASG), and to assess its impact on image quality in adult patients with a low body mass index (BMI). Methods A phantom with different thicknesses of acrylic was used with a Westmead Test Object to simulate patient sizes and assess image quality. 129 low BMI patients underwent coronary angiography or PCI with or without the ASG in situ. Radiation dose was compared between both patient groups. Results With the same imaging system and a comparable patient population, ASG removal was associated with a 47% reduction in total dose-area product (DAP) (p < 0.001). Peak skin dose was reduced by 54% (p < 0.001). Operator scatter was reduced to a similar degree and was significantly reduced through removal of the ASG. Using an image quality phantom it was demonstrated that image quality remained satisfactory. Conclusions Removal of the ASG is a simple and effective method to significantly reduce radiation dose in coronary angiography and PCI. This was achieved while maintaining adequate diagnostic image quality. Selective removal of the ASG is likely to improve the radiation safety of cardiac angiography and interventions.

Late clinical outcomes for SeQuent please paclitaxel-coated balloons in PCI of instent restenosis and de novo lesions: A single-center, real world registry.

OBJECTIVES: The aims of this study were to evaluate clinical outcomes following PCI using SeQuent Please paclitaxel-coated balloons (PCB) of ISR and denovo lesions (DNL), in all-comer patients at Liverpool Hospital, Sydney, Australia. BACKGROUND: There have been promising results for PCI using drug-coated balloons; however, long-term data for clinical outcomes are lacking. METHODS: Baseline patient demographics, PCI procedural details, and clinical outcomes were collected. The primary endpoint was the incidence of MACE, a composite of cardiac death, myocardial infarction (MI), and clinical-driven target lesion restenosis (TLR). The median follow-up for clinical events was 1.3 [0.6-1.9] years. RESULTS: A total of 188 lesions (n = 147 patients) were treated with PCB, comprising 118 (63%) ISR lesions and 70 (38%) DNL. Patient mean age was 67 ± 11years, 79% were male, and 54% had type 2 diabetes mellitus (DM). MACE was recorded in 17 patients (12%), with cardiac death confirmed in 1 patient (0.7%). MACE was significantly lower for DNL than ISR (1% vs. 15%, P = 0.03), and PCB had favourable TLR for DNL. Cox regression demonstrated that DM (HR 7.17, 0.92-55.6, P = 0.05) and prior CABG (HR 3.22, 1.17-8.83, P = 0.02) were independent predictors of MACE for ISR lesions. CONCLUSIONS: MACE rates were acceptable, with overall low incidence of cardiac death, MI, and TLR, for PCB treatment of ISR and DNL. Independent predictors of poor outcome in the ISR group were DM and prior CABG. The particularly low MACE for the DNL group supports direct PCB as a viable stent-sparing PCI strategy in challenging patients and lesion subsets. © 2016 Wiley Periodicals, Inc.

Influence of Age and Gender on Clinical Outcomes Following Percutaneous Coronary Intervention for Acute Coronary Syndromes.

BACKGROUND: Gender and age are non-modifiable factors influencing clinical outcomes in acute coronary syndromes (ACS). There is evidence that coronary artery disease pathophysiology varies in women. We therefore evaluated the effect of age and gender on clinical outcomes in patients with ACS undergoing percutaneous coronary interventions (PCI). METHODS: Among 3178 (25% female) consecutive ACS patients who underwent PCI at Liverpool Hospital, Sydney from 2003 to 2010, using femoral access in 98% of cases, we determined late events including mortality, myocardial infarction and bleeding according to Bleeding Academic Research Consortium (BARC) criteria. RESULTS: Females compared with males were older (median 68 vs. 60 years; p<0.001), and were more likely to have diabetes (30% vs. 22% p<0.001), hypertension (62% vs. 49%, p<0.001), anaemia (26% vs. 15%, p<0.001), and renal impairment (43% vs. 20%, p<0.001); they were more likely to be non-smokers (19% vs. 30%, p<0.001). Females had less class B2/C lesions (64% vs.68%, p=0.048), but had more calcified lesions (20% vs. 11%, p<0.001), and smaller stent diameters (2.75[2.5-3.0] vs. 3.0[2.75-3.5] mm, p<0.001). Females had higher three-year mortality rates (11% vs. 7.0%, p=0.001), and more type 2-5 BARC bleeding post-PCI (22% vs. 16%, p=0.003). Among patients under 55 years (n=988), mortality and bleeding were higher in females (6.0% vs. 3.0%, p=0.028) and (26% vs. 14%, p=0.001) respectively. There was no effect of gender on mortality or bleeding in patients 55 years and over. However, on multivariable stepwise regression analysis, female gender was not an independent predictor of mortality, but was a significant predictor of bleeding (OR=1.84 [95% CI:1.38-2.45], p<0.001). CONCLUSION: Bleeding and mortality were higher in younger females with ACS who underwent PCI. While females had more post-PCI bleeding events, which were associated with late mortality, gender per se was not an independent predictor for mortality.

Discovery of New Bromodomain Scaffolds by Biosensor Fragment Screening.

The discovery of novel bromodomain inhibitors by fragment screening is complicated by the presence of dimethyl sulfoxide (DMSO), an acetyl-lysine mimetic, that can compromise the detection of low affinity fragments. We demonstrate surface plasmon resonance as a primary fragment screening approach for the discovery of novel bromodomain scaffolds, by describing a protocol to overcome the DMSO interference issue. We describe the discovery of several novel small molecules scaffolds that inhibit the bromodomains PCAF, BRD4, and CREBBP, representing canonical members of three out of the seven subfamilies of bromodomains. High-resolution crystal structures of the complexes of key fragments binding to BRD4(1), CREBBP, and PCAF were determined to provide binding mode data to aid the development of potent and selective inhibitors of PCAF, CREBBP, and BRD4.

Multi-Center Retrospective Evaluation of Screw and Polymethylmethacrylate Constructs for Atlantoaxial Fixation in Dogs.

OBJECTIVE: To evaluate outcome and adverse events following ventral stabilization of the atlantoaxial (AA) joint in dogs with clinical AA subluxation using screw/polymethymethacrylate (PMMA) constructs in a retrospective, multi-center cohort study. STUDY DESIGN: Historical cohort study. ANIMALS: 35 client-owned dogs. METHODS: Medical records from 3 institutions were reviewed to identify dogs with AA subluxation treated with ventral screw and PMMA constructs. Data on signalment, pre- and postoperative neurologic status, imaging performed, and adverse events were retrieved. Neurologic examination data were abstracted to generate a modified Frankel score at admission, discharge, and re-examination. Telephone interview of owners >180 days postoperative was conducted. RESULTS: Thirty-five dogs with AA subluxation treated with ventral screw/PMMA constructs were included. Most dogs were young (median age 1 year), small breed dogs with acute onset of neurologic signs (median duration 22.5 hours). Most dogs were non-ambulatory at the time of admission (median modified Frankel score 3). Adverse events were identified in 15/35 dogs including 9 dogs with major adverse events. Four dogs required a second surgery due to vertebral canal violation (n = 2) or implant failure (n = 2). Re-examination at 4-6 weeks postoperative reported 15/28 dogs with improved neurologic status and 19/28 dogs were ambulatory. Telephone follow-up was available for 23/35 dogs with 23/23 reported as ambulatory (median follow-up 390 days). CONCLUSIONS: Ventral application of screw and PMMA constructs for AA subluxation, as described here, is associated with clinical improvement in the majority of dog. Major adverse events are infrequent and the technique is considered relatively safe.