Dysregulation of RNA-Exosome machinery is directly linked to major cancer hallmarks in prostate cancer: Oncogenic role of PABPN1.

Novel biomarkers and therapeutic strategies for prostate-cancer (PCa) are required to overcome its lethal progression. The dysregulation/implication of the RNA-Exosome-complex (REC; cellular machinery controlling the 3'-5'processing/degradation of most RNAs) in different cancer-types, including PCa, is poorly known. Herein, different cellular/molecular/preclinical approaches with human PCa-samples (tissues and/or plasma of 7 independent cohorts), and in-vitro/in-vivo PCa-models were used to comprehensively characterize the REC-profile and explore its role in PCa. Moreover, isoginkgetin (REC-inhibitor) effects were evaluated on PCa-cells. We demonstrated a specific dysregulation of the REC-components in PCa-tissues, identifying the Poly(A)-Binding-Protein-Nuclear 1 (PABPN1) factor as a critical regulator of major cancer hallmarks. PABPN1 is consistently overexpressed in different human PCa-cohorts and associated with poor-progression, invasion and metastasis. PABPN1 silencing decreased relevant cancer hallmarks in multiple PCa-models (proliferation/migration/tumourspheres/colonies, etc.) through the modulation of key cancer-related lncRNAs (PCA3/FALEC/DLEU2) and mRNAs (CDK2/CDK6/CDKN1A). Plasma PABPN1 levels were altered in patients with metastatic and tumour-relapse. Finally, pharmacological inhibition of REC-activity drastically inhibited PCa-cell aggressiveness. Altogether, the REC is drastically dysregulated in PCa, wherein this novel molecular event/mechanism, especially PABPN1 alteration, may be potentially exploited as a novel prognostic and therapeutic tool for PCa.

Development and In-House Validation of an Enzyme-Linked Immunosorbent Assay and a Lateral Flow Immunoassay for the Dosage of Tenofovir in Human Saliva.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody-antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4-10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.

ICT training for educators of Granada for working with people with autism.

Background: Innovative methodologies based on Information and Communication Technology (ICT) are a tested and motivating option for working with people with autism. Their use, however, should not be indiscriminatory and arbitrary, but didactic and appropriate. Objective: We aimed to discover the training in ICT they had, its frequency of use, and the types of digital resources that they used. Methods: We administered the questionnaire, "Demands and Potentials of ICT and Apps for Assisting People with Autism" to 310 educators in the city of Granada (Spain). The participants belonged to schools and associations that worked with people with autism. Adopting a quantitative-type study, we carried out descriptive analyses (frequencies, mean, mode, and standard deviation). Having confirmed that the data did not follow a normal distribution (Kolmogorov-Smirnov test for samples of >50 participants), we carried out non-parametric inferential and intrafactorial correlation analyses. We also calculated the effect size. Results: The educators revealed that they had ICT training for working with people with autism, but not enough. This suggests that there is still a need to improve the digital competence of these professionals. Strong, direct and significant correlations were found between ICT training and the frequency with which they were used. There were also statistically significant differences according to sex, gender, age, type of institution, and type of educator. The educators who worked as Therapeutic Pedagogy teachers and those who worked in Special Education schools were shown to be more competent than the rest in educational technology applied to people with autism. Conclusions: The results, which were not wholly positive since ICT training should be better and have achieved higher scores, revealed that there is a foundation in ICT education but it needs to be heightened and improved with greater knowledge and more practical experience.

Opinions, training and requirements regarding ICT of educators in Florence and Granada for students with functional diversity.

Information and communication technology (ICT) is a means of learning for people with functional diversity (FD), in the context of both formal and non-formal education. These digital options favor their inclusion, participation and motivation, so having competent professionals in the field of educational technology is crucial for the full development of this population. Within this framework, the opinion, training and requirements regarding ICT of 809 educators in Florence (Italy) and Granada (Spain) were analyzed, following a quantitative study, with a non-experimental, descriptive, cross-sectional and comparative design. Descriptive statistics (mean, standard deviation and mode) and frequencies were used. After checking the normality of the data (Kolmogórov-Smirnov test), inferential analyses were performed, applying Student's t- test and one-factor ANOVA, calculating the effect size (Cohen's d and eta squared). Statistically significant differences were found according to city of origin, gender, and years of experience with students with FD. The results point to the need for different educators to have sufficient technological training, as despite being aware of the functionalities of ICT and having a favorable opinion of them, resources and knowledge about their use fall short of what is desirable.

Correction: Addante-Moya et al. Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development. Toxins 2021, 13, 883.

In the original publication [...].

Alternative splicing in bladder cancer: potential strategies for cancer diagnosis, prognosis, and treatment.

Bladder cancer is the most common malignancy of the urinary tract worldwide. The therapeutic options to tackle this disease comprise surgery, intravesical or systemic chemotherapy, and immunotherapy. Unfortunately, a wide number of patients ultimately become resistant to these treatments and develop aggressive metastatic disease, presenting a poor prognosis. Therefore, the identification of novel therapeutic approaches to tackle this devastating pathology is urgently needed. However, a significant limitation is that the progression and drug response of bladder cancer is strongly associated with its intrinsic molecular heterogeneity. In this sense, RNA splicing is recently gaining importance as a critical hallmark of cancer since can have a significant clinical value. In fact, a profound dysregulation of the splicing process has been reported in bladder cancer, especially in the expression of certain key splicing variants and circular RNAs with a potential clinical value as diagnostic/prognostic biomarkers or therapeutic targets in this pathology. Indeed, some authors have already evidenced a profound antitumor effect by targeting some splicing factors (e.g., PTBP1), mRNA splicing variants (e.g., PKM2, HYAL4-v1), and circular RNAs (e.g., circITCH, circMYLK), which illustrates new possibilities to significantly improve the management of this pathology. This review represents the first detailed overview of the splicing process and its alterations in bladder cancer, and highlights opportunities for the development of novel diagnostic/prognostic biomarkers and their clinical potential for the treatment of this devastating cancer type. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease.

Vegetation Cover Management and Landscape Plant Species Composition Influence the Chrysopidae Community in the Olive Agroecosystem.

Habitat manipulation through the promotion of semi-natural habitats such as cover and patch vegetation is a possible means of offsetting the negative impacts of the agricultural practices. A baseline situation is crucial before any successful habitat manipulation is attempted. We studied the effects that current vegetation cover management practices have on plant composition and the potential attraction that the plant families from the semi-natural habitats could have on the Chrysopidae community, a key pest control agent, in five olive farms in Granada (Spain). Vegetation cover was assessed using a point quadrat methodology in eight transects per farm. In addition, the patch vegetation was characterized with 60 transects using a line intercept methodology. The woody patch vegetation and olive tree canopies were vacuumed using a field aspirator to collect adult Chrysopidae. In the cover vegetation we observed great variability in both the richness and diversity of plant communities caused by the vegetation cover management techniques and the transect position (in the middle of the rows or beneath the tree canopy). The plant families with the greatest plant cover were the Asteraceae and Fabaceae, where Asteraceae was favoured by tillage and Fabaceae by grazing, while in the patch vegetation, the predominant families were the Rosaceae and Fagaceae. Our results indicate that the genus Chrysoperla was mostly correlated with the Plantaginaceae, Brassicaceae and Asteraceae plant families in the cover vegetation, and with the Caryophyllaceae and Rosaceae families in the patch vegetation. The genera Apertochrysa and Pseudomallada were associated with the families Malvaceae and Poaceae in the cover vegetation, and with the families Cupressaceae, Poaceae and Pinaceae in the patch vegetation. Our study shows to the farmers the possibilities of vegetation cover management to select plant families for the cover vegetation.

Phytosociological analysis of the mesophilic forest of the San Pedro volcano (Guatemala).

From a standpoint of phytosociological research, little is known about the phytocoenosis found on the volcanoes of Central America. This paper analyses the distribution of the vegetation on the volcano of San Pedro in terms of its species-richness, composition, structure and abundance, and the possible relationships between these components and the changes in elevation and orientation that occur there. We divided the study area into three altitudinal belts between 2,400 and 3,020 m a.s.l. where carried out 36 inventories, each one in an area of 0.1 ha. We then applied multivariate analysis to classify and order the data in the matrix obtained from the frequency of the sampled plants. Our results lead us to propose two mixed cloud-forest associations within the class Alnetea acuminatae. The first, Saurauio oreophilae-Alnetum acuminatae ass. nova, is found on the more humid western side, while the second, Adianto andicolae-Quercetum peduncularis ass. nova, appears in sunnier and less shady sites, mainly on the east face. As part of this latter association, we also identified the new subassociation festucetosum amplissimae subass. nova. These syntaxa are part of the alliance Oreopanacion xalapensis all. nova, which we have created to embrace the mesophytic montane forests dominated by broad-leaved species.

Design of a novel hapten and development of a sensitive monoclonal immunoassay for dicamba analysis in environmental water samples.

Weed resistance to glyphosate has been a driving force behind the increased use of alternative herbicides in agriculture. Recently, dicamba-tolerant recombinant plants were introduced to the market, which may result in residues of this agrochemical contaminating environmental waters. Given that restrictions on the use of dicamba have consequently been established by regulatory agencies, it is therefore also desirable to conduct extensive controls on dicamba residues. Immunoassays are currently the most powerful bioanalytical technology for the rapid monitoring of chemical residues and contaminants. In the present study, a novel hapten was designed maintaining unaltered all the antigenic moieties of the target molecule, and this was used to generate high-affinity monoclonal antibodies against dicamba for the first time. Additionally, a collection of haptens with different linker composition or linker tethering site was synthesized and conjugated to proteins. Using these novel immunoreagents, a direct competitive enzyme-linked immunosorbent assay with a limit of detection for dicamba of 0.24 ng/mL was developed and validated. Analysis of water samples from different origins afforded recovery values between 90 % and 120 %, and coefficients of variation below 20 % were obtained. These results indicate that the developed immunochemical assay is suitable for the rapid determination of dicamba residues in environmental water samples.

Rapid Immunochemical Methods for Anatoxin-a Monitoring in Environmental Water Samples.

Algal blooms that contaminate freshwater resources with cyanotoxins constitute, nowadays, a global concern. To deal with this problem, a variety of analytical methods, including immunochemical assays, are available for the main algal toxins, for example, microcystins, nodularins, and saxitoxins, with the remarkable exception of anatoxin-a. Now, for the first time, highly sensitive, enantioselective immunoassays for anatoxin-a have been validated using homemade monoclonal antibodies. Two competitive enzyme-linked immunosorbent assays were developed in different formats, with detection limits for (+)-anatoxin-a of 0.1 ng/mL. Excellent recovery values between 82 and 117%, and coefficients of variation below 20%, were observed using environmental water samples fortified between 0.5 and 500 ng/mL. In addition, a lateral-flow immunochromatographic assay was optimized for visual and instrumental reading of results. This test showed a visual detection limit for (+)-anatoxin-a of 4 ng/mL. Performance with a reader was validated in accordance with the European guidelines for semiquantitative rapid methods for small chemical contaminants. Thus, at a screening target concentration of 2 ng/mL, the probability of a blank sample to be classified as "suspect" was as low as 0.2%. Finally, the optimized direct enzyme immunoassay was validated by comparison with high-performance liquid chromatography-tandem mass spectroscopy data and showed a good correlation (r = 0.995) with a slope of 0.94. Moreover, environmental water samples containing more than 2 ng/mL of anatoxin-a were detected by the developed dipstick assay. These results provide supplementary and complementary strategies for monitoring the presence of anatoxin-a in water.

Functionality of Apps for People with Autism: Comparison between Educators from Florence and Granada.

BACKGROUND: Studies on the potential of smartphone apps for people with autism are currently increasing in number, given the large digital supply available and the benefits they offer. We analyzed the opinion of educators from Florence (Italy) and Granada (Spain) regarding the benefits and applicability of apps, frequency of their use, and the type of apps used for people with autism. METHODS: The study involved 1261 professionals, of whom 286 worked with apps, using a non-experimental quantitative design, descriptive and frequency statistics, parametric inferential analyses (Student's t and one-factor ANOVA), and calculation of the effect size (Cohen's d and eta squared) and intrafactorial correlations. RESULTS: Statistically significant differences were observed in respect of city, sex, age, years of experience, place of work, and type of teacher. The teachers from Granada found more benefits and applicability in apps, and revealed a slightly higher usage than those from Florence. CONCLUSIONS: It is an arduous but worthy task for professionals from schools and associations that work with people with autism to acquire the necessary knowledge to apply methodologies based on information and communication technology (ICT), as this will help achieve the integrated development of people with different capabilities.

Alternative Hapten Design for Zearalenone Immunoreagent Generation.

Appropriate hapten design and synthesis have been identified as critical steps to generate high-performance immunoreagents and to develop sensitive and selective immunoanalytical methods. Antibodies and immunoassays for the major mycotoxin zearalenone have been reported and marketed. However, zearalenone haptens have mostly been prepared by the oxime active ester technique, and hapten characterization has generally been poor or non-existent. In the present study, novel haptens of zearalenone with longer linkers and with alternative tethering sites have been designed for immunizing and assay conjugate preparation. All of these molecules were purified and spectroscopically verified, and a structure-activity relationship evaluation was carried out. This approach revealed that the hapten with the linker at the carbonyl group generated antibodies with a higher affinity than the hapten functionalized at the phenyl moiety. Antibodies produced with the latter hapten, on the other hand, showed lower cross-reactivity values to the major zearalenone metabolites. Finally, similar immunoassay sensitivity was achieved with all of the antibodies when heterologous haptens were employed. Furthermore, by altering the structure of the competing antigen, the immunoassay selectivity was modified. These results demonstrate that immunochemical methods for zearalenone rapid analysis can still be improved in terms of sensitivity and selectivity.

What students with intellectual disabilities know about writing planning.

BACKGROUND: Studies on the writing of students with intellectual disabilities have been scarce and unrepresentative. Therefore, the purpose of this research was to analyse this group of students' abilities to plan their texts METHOD: A collective case study was carried out, with an eminently qualitative approach, although aided by an initial quantitative analysis. Fifteen students with unspecified intellectual disabilities were interviewed using open-ended questions. For data interpretation, content analysis and quantification of responses were used. RESULTS: The results revealed the abilities and limitations of these students regarding the knowledge and use of one of the key processes of written expression (writing planning). CONCLUSION: The cognitive operations of planning, in which these students admitted the greatest problems, were the ordering and recording of ideas, and textual structuring.

Chemical strategies for triggering the immune response to the mycotoxin patulin.

Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.

Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development.

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these Alternaria mycotoxins.

Spindle Dynamics during Meiotic Development of the Fungus Podospora anserina Requires the Endoplasmic Reticulum-Shaping Protein RTN1.

The endoplasmic reticulum (ER) is an elaborate organelle composed of distinct structural and functional domains. ER structure and dynamics involve membrane-shaping proteins of the reticulon and Yop1/DP1 families, which promote membrane curvature and regulate ER shaping and remodeling. Here, we analyzed the function of the reticulon (RTN1) and Yop1 proteins (YOP1 and YOP2) of the model fungus Podospora anserina and their contribution to sexual development. We found that RTN1 and YOP2 localize to the peripheral ER and are enriched in the dynamic apical ER domains of the polarized growing hyphal region. We discovered that the formation of these domains is diminished in the absence of RTN1 or YOP2 and abolished in the absence of YOP1 and that hyphal growth is moderately reduced when YOP1 is deleted in combination with RTN1 and/or YOP2. In addition, we found that RTN1 associates with the Spitzenkörper. Moreover, RTN1 localization is regulated during meiotic development, where it accumulates at the apex of growing asci (meiocytes) during their differentiation and at their middle region during the subsequent meiotic progression. Furthermore, we discovered that loss of RTN1 affects ascospore (meiotic spore) formation, in a process that does not involve YOP1 or YOP2. Finally, we show that the defects in ascospore formation of rtn1 mutants are associated with defective nuclear segregation and spindle dynamics throughout meiotic development. Our results show that sexual development in P. anserina involves a developmental remodeling of the ER that implicates the reticulon RTN1, which is required for meiotic nucleus segregation. IMPORTANCE Meiosis consists of a reductional cell division, which allows ploidy maintenance during sexual reproduction and which provides the potential for genetic recombination, producing genetic variation. Meiosis constitutes a process of foremost importance for eukaryotic evolution. Proper partitioning of nuclei during this process relies on accurate functioning and positioning of the spindle, the microtubule cytoskeletal apparatus that conducts chromosome segregation. In this research, we show that in the model fungus Podospora anserina this process requires a protein involved in structuring the endoplasmic reticulum (ER)-the reticulon RTN1. The ER is a complex organelle composed of distinct structural domains, including different peripheral domains and the nuclear envelope. Our findings suggest that spindle dynamics during meiosis relies on remodeling of the ER membrane, which involves the activity of RTN1. Our research discloses that the proteins implicated in shaping the ER are main contributors to the regulation of nuclear dynamics during the sexual cycle.

Optical-component-only adaptive optics.

This Letter introduces a technique for performing binary adaptive optics, which is carried out by optical components only, without the help of any electronic or optoelectronic device. In this technique, the interferogram produced by a point diffraction interferometer modulates a light-driven crystal. The modulated light-driven crystal may produce pupil-plane only-phase or only-amplitude binary masks to mitigate phase aberrations. The capability of working unsupported makes it suitable for application in hard-to-reach or hazardous locations such as satellites, underwater, or contaminated places. The Letter includes an experimental validation where the ability of the technique to produce pupil amplitude masking is confirmed.

Mechanosynthesis and Thermoelectric Properties of Fe, Zn, and Cd-Doped P-Type Tetrahedrite: Cu12-xMxSb4S13.

This contribution deals with the mechanochemical synthesis, characterization, and thermoelectric properties of tetrahedrite-based materials, Cu12-xMxSb4S13 (M = Fe2+, Zn2+, Cd2+; x = 0, 1.5, 2). High-energy mechanical milling allows obtaining pristine and substituted tetrahedrites, after short milling under ambient conditions, of stoichiometric mixtures of the corresponding commercially available binary sulfides, i.e., Cu2S, CuS, Sb2S3, and MS (M = Fe2+, Zn2+, Cd2+). All the target materials but those containing Cd were obtained as single-phase products; some admixture of a hydrated cadmium sulfate was also identified by XRD as a by-product when synthesizing Cu10Cd2Sb4S13. The as-obtained products were thermally stable when firing in argon up to a temperature of 350-400 °C. Overall, the substitution of Cu(II) by Fe(II), Zn(II), or Cd(II) reduces tetrahedrites' thermal and electrical conductivities but increases the Seebeck coefficient. Unfortunately, the values of the thermoelectric figure of merit obtained in this study are in general lower than those found in the literature for similar samples obtained by other powder processing methods; slight compositional changes, undetected secondary phases, and/or deficient sintering might account for some of these discrepancies.

Immunochemical method for penthiopyrad detection through thermodynamic and kinetic characterization of monoclonal antibodies.

Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained KD values from antibody-coated assays were quite close to the corresponding KD values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The KD value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL-1) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL-1. An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation.

Enzyme and lateral flow monoclonal antibody-based immunoassays to simultaneously determine spirotetramat and spirotetramat-enol in foodstuffs.

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of spirotetramat and spirotetramat-enol of 0.1 µg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.