Overall, EXIT 360° appeared to be a usable, learn-to-use, engaging, and creative tool with irrelevant negative effects. Further studies will be conducted to evaluate these aspects in the clinical population.Over the last few decades, several studies have shown the feasibility, acceptability, and efficacy of VR-based instruments in the early evaluation of executive dysfunction (ED) in psychiatric and neurologic conditions. Due to the negative impact of ED in everyday functioning, identifying innovative strategies for evaluating ED allows clinicians to detect executive impairment early and minimize its effects. This work aimed to test the usability and user experience (UX) of EXecutive-functions Innovative Tool 360° (EXIT 360°), a 360°-based tool for assessing ED. Seventy-six healthy subjects underwent an evaluation that involved (1) usability assessment using System Usability Scale and (2) evaluation of UX using the ICT-Sense of Presence and UX Questionnaire. Results showed a satisfactory level of usability (mean = 75.9 ± 12.8), with good scores for usability and learnability. As regards UX, EXIT 360° showed an absence of negative effects (mean = 1.79 ± 0.95) and high scores in ecological validity (mean = 4.32 ± 0.54) and engagement (mean = 3.76 ± 0.56). Moreover, it obtained good scores in efficiency (mean = 1.84 ± 0.84), originality (mean = 2.49 ± 0.71), and attractiveness (mean = 1.93 ± 0.98). Interestingly, demographic characteristics and technological expertise had no impact on the performance ( Over the last few decades, virtual reality (VR) appeared to be a promising tool for assessing executive functions (EFs) , a complex construct that involves several higher-order cognitive and behavioral skills with a key role in daily life and independent functioning ,2. SeverIn this framework, we developed EXecutive-functions Innovative Tool 360° (EXIT 360°), an original 360°-based instrument for a multicomponent, ecologically valid evaluation of executive functioning in the neurologic population . EXIT 36In EXIT 360°, the participants have the main goal to go out of the domestic path in the shortest possible time. To do this, participants have to plan a strategy and overcome seven different steps (subtasks) of increasing complexity, designed to evaluate several components of executive functioning simultaneously and quickly .As widely demonstrated ,17,18, iAs regards the user experience, it is defined as “a person’s perceptions and responses that result from the use or anticipated use of a product, system or service” . Over thFinally, the developers have tried to increase engagement in virtual environments by improving the ecological validity of VR-based tools. Specifically, the VR-based instruments want to offer contexts similar to the real world, allowing patients to live a first-person experience “like in real-life”. Focusing on enjoyment and attractiveness enables the increase of users’ motivation and participation and the decrease of anxiety typical of neuropsychological evaluation.In this study, we evaluated if EXIT 360° was able to meet all these significant characteristics: good usability, absence of side effects, high sense of presence, ecological validity, enjoyment, and attractiveness.Seventy-six healthy subjects were consecutively recruited at IRCCS Fondazione Don Carlo Gnocchi in Milan, according to the following inclusion criteria: (a) age between 18 and 90 years; (b) education ≥5; (c) absence of cognitive impairment as determined by the Montreal Cognitive Assessment test  (MoCA scThe study was approved by the “Fondazione Don Carlo Gnocchi ONLUS” Ethics Committee in Milan. All participants were provided with a complete explanation of the purpose and risk of the study before the preparation of the consent form and written informed consent was obtained based on the revised Declaration of Helsinki (2013).The study consisted of a one-session evaluation that involved three main phases: a pre-task evaluation, followed by the EXIT 360° session, and a post-task evaluation .After signing written informed consent, participants underwent an evaluation of their global cognitive profile through the Montreal Cognitive Assessment (MoCA) test, a sensitive screening tool to exclude the presence of cognitive impairment.After that, the psychologist collected participants’ socio-demographic data  and technological expertise. Specifically, the participants evaluated their perceived level of familiarity and competence with several technologies: tablet, smartphone, computer, and Internet . To test their level of familiarity with technologies, the psychologist administered a 5-point scale (from “never” to “every day”) that evaluated “how often, in the last year, did you use…”; while the competence scale involved a 5-point scale (from “nothing” to “much”) to investigate “how competent do you feel in using…”.After the preliminary screening, all participants underwent an evaluation with EXIT 360°. The psychologist started the administration by inviting participants to sit on a swivel chair and wear the Head Display Mobile (HDM). Before wearing the HDM, the psychologist provided the following general instruction of the task:You will now wear a headset. Inside this viewer, you will see some 360° rooms of a house. To visualize the whole environment, I ask you to turn on yourself; you are sitting on a swivel chair for this reason. Within these environments, you will be asked to perform some tasks”.“In the case of presbyopia, participants could wear their glasses.© software, which allowed for organizing virtual environments and several tasks in a single experience. Specifically, the tool consists of 360° immersive domestic photos as virtual environments in which participants have to perform seven subtasks of increasing complexity: (1) Let’s Start; (2) Unlock the Door; (3) Choose the Person; (4) Turn On the Light; (5) Where Are the Objects?; (6) Solve the Rebus; and (7) Create the Sequence . BrieflOverall, all these tasks are designed to evaluate different components of executive functioning (multicomponent assessment) simultaneously and quickly . The levTo respond to these requests, the subject has to choose between three or more “options”, the one that will allow him to solve the assignment in the best possible way. In the HDM, they see a small white dot/square, a “pointer” that follows their gaze; therefore, when subjects want to answer within the environment, they have just to move their head and position the white dot over the answer for a few seconds and the answer will be selected automatically. In this way, it is possible to collect participants’ answers without learning to use complex tools . Interestingly, to provide the instructions in a standardized way, the instructions received from the participants were previously recorded and inserted within the virtual environments.EXIT 360° evaluation involved a preliminary familiarization phase to familiarize the subjects with the technology and control potential adverse effects . Participants were completely immersed in a neutral 360° environment representing a living room, including a table with a plant, a sofa, various chairs, and objects spread throughout the room. The examiner asked participants to explore the environments freely, saying what they saw in the room. Upon completing the familiarization phase, participants were asked to indicate the possible presence of nausea or malaise. If side effects occurred, the examiner had to stop the test immediately.You are about to enter a house. Your goal is to get out of this house in the shortest time possible. To exit, you will have to complete a path and a series of tasks that you will encounter along your way. Are you ready to start?”. During their evaluation, the subjects had to explore the 360° environment freely, simply through the movement of the head as it happens in real situations [In contrast, subjects were immersed in 360° environments that represented a living room. The experimental session (and time registration) began when the participants heard the following instruction: “tuations . As prevCorrect answer for each subtask and total score;Sub-task reaction time: the time (in seconds) from the start of a sub-task until the participant provided an answer; andTotal reaction time: the time in seconds registered from the first instruction until the participant provided the last correct answer.The following indices were calculated:System Usability Scale (SUS) [The usability of the technological instrument by the le (SUS) , a shortle (SUS) .The user experience through:a.Flow Short Scale  to assess their perceived level of abilities in coping with the task, of challenges, and the perceived challenge-skill balance [three items of the  balance .b.Intrinsic Motivation Inventory (IMI) [four items  from the subscale enjoyment of the ry (IMI)  to evaluc.Sense of Presence Inventory (ICT-SOPI) to evaluate user’s engagement. Precisely, this questionnaire consists of 44 items divided into four subgroups : (1) “spatial physical presence”: feeling of being in a physical space in the virtual environment and having control over it ; (2) engagement: the tendency to feel psychologically and pleasantly involved in the virtual environment ; (3) ecological validity: the tendency to perceive the virtual environment as real ; and (4) negative effects: adverse psychological reactions . Moreover, ICT-SOPI is divided into two parts: thoughts and feelings after experiencing the environment (Part A) or while the user was experiencing the environment (Part B) [forty-four items  from the scale (Part B) .d.User Experience Questionnaire, a 26-item scale  that allows calculating six different domains: (1) attractiveness , (2) perspicuity: easily to learn how to use the product; (3) efficiency (user’s effort to solve tasks); (4) dependability (feeling of control of the interaction); (5) stimulation (motivation to use the product); and (6) novelty: (innovation and creation of product) [product) ,34,35.At the end of the EXIT 360° task evaluation, subjects had to rate:p < 0.05 was considered statistically significant.Descriptive statistics included frequencies, percentages, and median and interquartile range (IQR) for categorical variables and mean and standard deviation (SD) for continuous measures. The normality of data distribution was assessed using the Kolmogorov–Smirnov test. Pearson’s correlation was applied to compare the scores of usability, user experience, and technological experience. Moreover, a one-way ANOVA non-parametric  and Chi-square were calculated to verify possible differences in education and gender in age group. Furthermore, a one-way ANOVA between subjects (post-hoc: Bonferroni test) was conducted to evaluate any significant differences in technological expertise due to age group. Finally, ANCOVA between subjects (covariate: education) was performed to assess possible differences in usability score due to age. All statistical analyses were conducted using Jamovi 1.6.7 software. A statistical threshold of n = 76) were predominantly female (M:F = 28:48) with a mean age of 53.5 years  and age of education nearly 13 . All participants included in the study showed an absence of cognitive impairment (MoCA correct score = 25.9 ± 2.63).2 (6) = 29; p < 0.001), but not in the sex (χ2 (6) = 7.1; p = 0.312).p < 0.05) and 70–79 . Moreover, a significant difference appeared between group 30–39 and 50–59 , 60–69 , and 70–79 .Specifically, results showed a significant difference between group 20–29 and 60–69  and competence  = 22.2; p < 0.001). Specifically, results showed a significant difference in familiarity between group 80–89 and all other age groups, except for 70–79 (p > 0.05). Moreover, a significant difference appeared between groups 70–79 and 20–29 (p < 0.001) and 30–39 (p < 0.001). Regarding the level of competence, results indicated a significant difference between group 80–89 and all other age groups (p < 0.05). Moreover, a significant difference appeared between group 70–79 and respectively 20–29 (p < 0.001), 30–39 (p < 0.001), and 40–49 (p < 0.05). In addition, group 60–69 showed a significant difference with 20–29 (p < 0.001) and 30–39 (p < 0.05) groups. Finally, group 50–59 showed a difference only with 20–29 (p < 0.05).The mean score of the ad hoc 5-point scale for evaluation of perceived level of familiarity with technologies was 3.41 ± 1.8 . Specifically, 25% of subjects reported a low (<3) familiarity with technologies, with 28.9% that showed a good (≥4—at least two or three times a week) familiarity with several technologies. Moreover, the mean score of the ad hoc 5-point competence questionnaire was 3.48 ± 1.11, indicating a score between “little” and “enough”. In particular, 28.9% of participants showed a low (<3) competence with technologies, with 30.03% showing a good (≥4—enough or much) competence with several technologies. Analyzing the possible difference between age groups in levels of competence and familiarity with technologies, data showed a significant difference between many age groups both in familiarity  = 12.6; The mean value of the usability, calculated with the SUS, was 75.9 ± 12.8, indicating a satisfactory level of usability, according to the scale’s score acceptability ranges (cut off = 68) and adjective ratings .Specifically, according to the cut-off score (cut-off = 68), more than 77% of participants showed scores above the cut-off .In addition, according to the adjective rating, 36.8% of subjects evaluated EXIT 360° as “good”, 32.9% as “excellent”, and 27.6% “best imaginable” [Interestingly, participants provided good and promising scores to two main aspects that affect the user experience: usability (mean = 3.07 ± 0.55) and learnability (mean = 2.91 ± 0.738). Specifically, 14.5% and 15.8% of participants showed low scores (<2.5) respectively at usability and learnability.The three items of the Flow Short Scale showed a high score in the perceived level of skill in performing EXIT 360°  and allowed for evaluating the level of challenge of EXIT 360°, also in based on own abilities, such as balance/appropriate .n = 11) of participants reported the presence of negative effects , but only one subject showed relevant side effects. Moreover, 75% showed good scores of spatial presence  and 89.5% indicated a good level of engagement while performing EXIT 360° . Finally, 97.4% of participants gave EXIT 360° a good score in terms of ecological validity , with more than 84% that showed high scores (≥4).The UEQ questionnaire showed positive evaluation (>0.8) in all 26 items. Moreover, the UEQ’s scales can be grouped into pragmatic quality  and hedonic quality . Specifically, EXIT 360° obtained a good score in pragmatic quality (mean = 1.97) and a high score in hedonic quality (mean = 2.31).Finally, the means of each UEQ scale were compared to existing values from a benchmark data set  . Resultsp = 0.503) and education . Moreover, the ANCOVA-between subjects (covariate: education) showed no significant differences of age group in SUS total score  = 1.02; p = 0.419). Interestingly, this analysis also showed the absence of impact of education in the SUS total score (p = 0.159). Furthermore, Pearson’s correlation has underlined the absence of significant correlation between SUS total score and technological expertise measured by the two ad hoc questionnaires of familiarity  and competence . In contrast, Pearson’s correlation showed significant and positive linear correlation between SUS total score and three ICT-SOPI domains, spatial presence , engagement , and ecological validity . Finally, p < 0.001).Pearson’s correlation showed the absence of significant linear correlation between the SUS total score and the demographic characteristics, particularly age , evaluated by SUS, indicating a satisfactory level of usability, according to the scale’s score acceptability ranges and adjective ratings . SpecifiAs regards the user experience, EXIT 360° showed promising results in terms of absence of side effects, high sense of presence, ecological validity, enjoyment, and attractiveness. Firstly, participants showed high scores in the perceived level of skill in performing EXIT 360° and evaluated the subtasks of EXIT 360° as balanced/appropriate with respect to their abilities. Secondly, subjects supported a positive overall impression of the EXIT 360°, evaluating it as enjoyable, attractive, activating, funny, friendly, and not boring. Moreover, EXIT 360° demonstrated a good pragmatic quality as it appeared: (1) efficient, fast, and organized (efficiency); (2) understandable, easy to learn, and clear (perspicuity); and (3) supportive and secure (irrelevant side effects) (dependability). In addition, EXIT 360° showed excellent hedonic quality in terms of stimulation  and novelty . Interestingly, results showed that the scales attractiveness, dependability, stimulation, and novelty obtained excellent evaluation compared to existing values from benchmark data . FinallyFinally, data showed a relationship between usability score and user experience evaluation by ICT-SOPI domains spatial presence, engagement, and ecological validity (positive correlation) and negative effect (negative correlation). This conclusion supported the recent innovative higher-level concept of interaction experience, proposed by Sauer and colleagues, in which both usability and user experience appeared as key elements to provide more benefits in using technological tools .Overall, EXIT 360° allowed an ecologically valid evaluation without relevant sicknesses such as dizziness, headache, sweating, and nausea that lead to unpleasant experiences for the users, affecting their performance and significantly decreasing the test results’ validity . MoreoveThe present work has some limitations. Firstly, the technological device used was entry-level: the 360° mobile-powered devices currently available on the market have much higher quality , which would allow for improving the quality of 360° images, providing a better and more realistic experience. Secondly, it is necessary to evaluate the usability, user experience, and engagement of EXIT 360° with the clinical population. Moreover, to date, EXIT 360° can be considered an initial prototype that needs further validation steps to become a valid and standardized instrument for assessing EFs. For this reason, it will be necessary to evaluate the convergent validity of EXIT 360°, comparing it with the traditional neuropsychological battery for executive functions  and its effectiveness in discriminating between healthy control subjects and patients with executive dysfunctions.Results of the present study are very promising and interesting regarding usability, user experience, and engagement of EXIT 360°. Overall, participants obtained a positive global impression of the tool, evaluating it as usable, learn-to-use, clear, enjoyable, attractive, and friendly. Moreover, EXIT 360° is an efficient, fast, and organized instrument, with excellent hedonic quality in terms of stimulation (exciting and interesting) and originality. Finally, EXIT 360° also appeared to be an engaging and challenging tool with high spatial presence, excellent ecological validity, and irrelevant adverse effects. Interestingly, neither demographic characteristics (age and education) nor technological expertise influenced the encouraging results. Further studies will have to be conducted to also evaluate these aspects in a clinical population.


Trisetacus are widespread parasites of conifers. They usually live in buds, cones, and rarely within needles of Pinaceae (pine family) and Cupressaceae (cypress family). We discovered a new species, Trisetacus indelisn. sp., severely damaging seeds of three North American junipers in the western USA. This species possesses two morphologically different forms of females and has two deletion mutations in the gene cytochrome oxidase subunit I (Cox1). Such mutations are rare in eriophyoids and were previously detected only in two pestiferous species from palms and hazelnut. Our molecular-phylogenetic analyses determine the closest known relatives of the new species and suggest that Old and New World Trisetacus independently transitioned to living in seeds of junipers. Additionally we show that reconstruction of the phylogeny of Eriophyoidea based on one gene, Cox1, produces a poorly-resolved but biologically consistent tree topology to hypothesize the evolution of Eriophyoidea. Overall, our study improves our understanding of the diversity of conifer-inhabiting mites and indicates further needs in investigating the phylogeny of Eriophyoidea.Eriophyoid mites are microscopic herbivores associated with higher plants. Some of them are serious pests due to their ability to vector viruses and cause other damage to host plants. Mites of the genus Trisetacus Keifer are widespread parasites of conifers. A new oligophagous species, T. indelis n. sp., was discovered severely damaging seeds of North American junipers  in the western USA. It has two codon deletions in the mitochondrial gene Cox1 rarely detected in Eriophyoidea and includes distinct morphological dimorphism of females. A phylogenetic analysis based on amino acid alignment of translated Cox1 sequences using a large set of out-groups (a) determined that two North American congeners, T. batonrougei and T. neoquadrisetus, were the closest known relatives of T. indelis n. sp., and (b) indicated that Old and New World seed-inhabiting Trisetacus from junipers do not form a distinct clade, suggesting a possible independent transition to living in seeds of junipers in America and Eurasia by Trisetacus spp. Our analysis produced a new topology consistent with a scenario assuming gradual reduction of prodorsal shield setation in Eriophyoidea and an ancient switch from gymnosperms to other hosts. Additionally, our analysis did not support monophyly of Trisetacus; recovered a new host-specific, moderately supported clade comprising Trisetacus and Nalepellinae  associated with Pinaceae; and questioned the monophyly of Trisetacus associated with Cupressaceae.Eriophyoid mites of the genus  Eriophyoid mites are an ancient group of phytoparasitic acariform mites exclusively associated with higher vascular plants and closely related to soil nematalycid mites ,2,3. Becvi, ve, and sc, c1 and tibial solenidion φI, and morphology of the gnathosoma [Pentasetacus (from Araucaria) and Loboquintus (from Cupressus). Phylogenetic relations between these clades and the history of the colonization of terrestrial plants by eriophyoids remains poorly understood, however conifers were hypothesized as the earliest hosts [Currently accepted classification of Eriophyoidea assumes the presence of three families: Phytoptidae, Eriophyidae and Diptilomiopidae; differing in the number of prodorsal shield setae  are common and widespread arthropod parasites of conifers [Trisetacus belong to the two most diverse gymnosperm families, Pinaceae and Cupressaceae. Currently this genus comprises approximately 60 species; however, this number is expected to increase as more conifer species are investigated. Damage to host plants by Trisetacus can occur in a variety of forms [Trisetacus on various pinacean  and cupressacean  host genera. Along with living in buds, several cryptic species of the complex Trisetacus juniperinus s.l., are also capable of infesting male cones and causing swollen galls on junipers , including a dataset from a previous study [Cox1 amino-acid alignment that was twice as long as that produced by the classical LCO-HCO fragment [During field surveys in mountainous areas of the western USA we observed groves of junipers heavily infested by a new us study . In addifragment .Morphology. Live mites of T. indelis n. sp. were collected in 2021 in the USA from plants using a fine minuten pin and a dissecting microscope, then placed in Eppendorf tubes filled with 96% ethanol. The mites were mounted in modified Berlese medium with iodine [h iodine  and cleah iodine ,14,33. Th iodine . Drawingh iodine , scannedDNA extraction and sequencing. For DNA extraction, 1–3 mite specimens of each species were crushed with a fine pin in a 3 μL drop of distilled water on a cavity well microscope slide. The drop was pipetted into a thin-walled PCR tube with 30 μL of 5% solution of Chelex® 100 Resin  before being heated three times (5 min at 95 °C). The solution above the settled Chelex® granules was used as the DNA template for PCR to amplify a fragment of subunit I of Cox1. Thermal cycling profiles and primers (for PCR and for sequencing) used were as specified by [www.genestudio.com, accessed on 10 February 2022).ified by . After aSequence alignment and molecular phylogenetic analyses. A large set of closely related and distant eriophyoid out-groups sequenced in previous studies were included in the analysis: complete dataset of Nalepellidae [Cox1 gene sequences of Eriophyidae s.l. from Genbank that are longer than 1100 bp, ten sequences of phytoptids, and three sequences of pentasetacids. The tree was rooted with sequences of Nanorchestes KY922356.1 and Gordialycus KY922376.1 [Cox1 sequences was checked with Mega X [Cox1 sequences were translated into amino acid sequences and maximum likelihood analysis was conducted in IQ-tree 2 [pellidae , all Cox922376.1  with theh Mega X . The finQ-tree 2 . For amiQ-tree 2  as impleQ-tree 2 .PROTOGYNE FEMALE , Body vermiform, white and slightly yellowish color 267 (252–278), 69 (63–72) wide at the level of setae c2. Prodorsal shield subrhomboidal or subcordial, 29 (28–33), 51 (48–55) wide, without frontal lobe. Prodorsal shield ornamentation variable, usually comprised of median, two submedian I, two submedian II, and several additional short lines. Median line appears concave, projecting from posterior margin of prodorsal shield up to the base of tubercle vi; in some females median line can be fragmented, very faint and almost imperceptible. Admedian lines absent like in other Trisetacus spp., associated with Cupressaceae [vi , directed up and anteriad; sc 51 (46–57), 26 (24–27) apart, directed up and anterolaterad; distance between tubercles of vi and sc 17 (16–18). Gnathosoma directed obliquely down and forward; palps 25 (24–26). Gnathosomal setae: seta ν 0.5 (0.5–1); pedipalp genual seta d non-bifurcate, 9 (8–10); pedipalp coxal seta ep 3 (3–4). Suboral plate rounded anteriorly, smooth.E n = 10,, Body veessaceae ,30. Submceae [vi A. SubmedLeg I 27 (25–30), tarsus 6 (4–6), u’ 3 (2–3), ft’ 13 (11–16), ft’’ 25 (21–28), ω 9 (9–10) with small spherical knob; empodium 9 (8–10), 9/9-rayed, all rays except terminal pair with 4–6 tiny processes each; tibia 5 (4–6), l’ 6 (3–6); φ 7 (7–9); genu 5 (5–6), l’’ 26 (22–30); femur 8 (8–10), bv 14 (12–15). Leg II 24 (23–26), tarsus 5 (4–5), u’ 3 (2–3), ft’ 8 (7–12), ft’’ 22 (21–27), ω 9–10 with small spherical knob; empodium 9 (8–10), similar to empodium I; tibia 5 (4–5); genu 5 (4–5), l’’ 22 (21–28); femur 7 (7–9), bv 12 (11–15).Coxal plates I smooth, coxal plates II with several short, curved ridges near tubercles of 2a; coxal setae 1b 26 (27–34), 16 (15–16) apart; 1a 37(34–42), 12 (12–14) apart; 2a 52 (50–61), 31 (30–33) apart; all coxal setae are very thin in distal half and break off easily during specimen preparation. Prosternal apodeme inconspicuous; cuticle between tubercles of coxal setae 1a smooth; 2 (2–3) incomplete and 1 complete coxigenital annuli before epigynium. External genitalia. Genital coverflap distally rounded, smooth, with medioposterior indentation; 7 (7–8) long, 27 (26–28) wide; setae 3a 18 (16–22), 17 (17–19) apart. Internal genitalia (n = 4). Spermathecae tear-drop shaped, 12–15 long, 7–9 wide; spermathecal tubes narrow, non-recurved, 30–35 long, 1.5–2 wide without widening; spermathecal process absent; longitudinal bridge 12–15; anterior genital apodeme trapezoidal, distinct; oblique apodeme absent.Opisthosoma dorsally with 76 (73–79) annuli, ventrally with 71 (68–76) annuli between posterior margin of coxae II and caudal lobes. Setal lengths: c1 8 (8–11), c2 45 (40–48), d 37 (32–43), e 15 (11–16), f 40 (38–44); h1 25 (22–27); h2 112 (100–122); 10 (10–11) annuli from rear shield margin to c1; 10 (7–10) annuli from rear shield margin to c2; 12 (11–13) annuli between c2–d; 13 (12–16) annuli between d and e; 31 (30–34) annuli between e and f; 5 (5–6) annuli between f and h2.MALE (n = 4). Body vermiform, 214–266, 55–59 wide, white or slightly yellowish. Prodorsal shield 28–32, 49–52 wide. Leg I 27–29, leg II 25–26, empodium 8/8- or 7/8-rayed. Ornamentation of prodorsal shield similar to that of females. Genital area 11–12 long × 24–25 wide, setae 3a 16–22, 20–21 apart.DEUTOGYNE FEMALE (n = 10). Difference in color is the only qualitative trait distinguishing deutogynes and protogenes. Deutogynes are reddish. Slide-mounted deutogynes are notably shorter and narrower than protogynes (e (27–36 vs. 11–16). Additionally, most of the deutogynes possess fewer empodial rays on legs I and II (8/8 vs 9/9), although some transitional forms of deutogynes with 8/9- and 9/9-rayed empodia were also observed.otogynes ; howeverotogynes I. DeutogType material. Holotype female from slide E4679, paratype females and males from slide series E4680–E4683 collected on 24 February 2021 near Madeline, California , from inside enlarged seeds of Juniperus occidentalis Hook. (Cupressaceae). Type material is deposited in the Acarological Collection of the Zoological Institute of the Russian Academy of Science (ZIN RAS) in Saint-Petersburg and in the collection of eriophyoid mites of J. Amrine .Relation to host. Mites penetrate immature female cones, feed on seed endopsperm, and cause seed deformation; infested seeds may protrude from the cones up to 1–2 mm  from inside enlarged seeds of Juniperus osteosperma Hook.; and from slide series E4689–E4693 collected on 9 February 2021, near Dales, California  from inside enlarged seeds of J. californica Carr. (Cupressaceae). This material has been deposited in the Acarological Collection of ZIN RAS, Saint-Petersburg, Russia.Etymology. The specific epithet, indelis, is an adjective, gender masculine, derived from “indel” and corresponding to the codon deletion mutations present in the Cox1 sequence of this species in comparison to all currently known members of the genus Trisetacus [isetacus .Differential diagnosis.T. indelis n. sp., is morphologically close to T. kirghisorum and T. batonrougei. Main differences between them concerns prodorsal shield ornamentation and shapes of the spermathecal apparatus. In T. batonrougei median and admedian lines are absent [T. kirghisorum three short lines (median and two admedian) [T. indelis n. sp. one long median line are present   and in T present A,B. In Td narrow F. In T.  segment .Cox1 sequences of T. indelis n. sp. were obtained  in comparison to all other known Trisetacus spp. [Cox1 sequences of eriophyoid mites that are currently present in GenBank, codon deletions are present only in the 21 sequences of Phytoptus avellanae (1 codon deletion) and four sequences of Retracrus (2 codon deletions). They are situated in the same region of the Cox1 gene  as in T. indelis n. sp.Seven nearly identical cus spp. . Among aLoboquintus and Pentasetacus) were situated in the unresolved basal part of the tree and clustered with different large eriophyoid clades. The genus Trisetacus was polyphyletic. All members of Trisetacus associated with Pinaceae formed a well-supported clade sister to Nalepellinae , which are also associated with Pinaceae. Trisetacus from Cupressaceae appeared as polyphyletic and comprised several well-supported clades, each of them including mite species associated with different species of the plant genus Juniperus. The mite genus Boczekella was sister to one of the clades of Trisetacus from Cupressaceae. Trisetacus spp., living in seeds of Juniperus spp., did not cluster together. However, all analyzed species  from seeds of American junipers  formed a highly supported clade sister to a clade comprising a complex of cryptic species, viz. Trisetacus juniperinus s.l. + T. bioti s.l. associated with various cupressacean genera . Only one population from this complex (Trisetacus sp. F20 from Crimea) lives in seeds. The species T. quadrisetus, T. kirghisorum, and Trisetacus sp. F20 associated with seeds of the Eurasian junipers J. excelsa, J. foetidissima, and J. communis, were not clustered together although monophyly of the lineage T. quadrisetus + T. kirghisorum was strongly supported. The molecular phylogenetic analysis revealed a monophyletic Eriophyoidea, as expected, comprising three large clades  with poorly resolved relations between them . Two arcA new mite species destroying seeds of North American junipers. In this study, we report on a new Trisetacus species, T. indelis n. sp., associated with junipers native to the western USA. This species is remarkable in several aspects. First, this is the first member of Nalepellidae possessing codon-deletion mutations in the Cox1 gene. Such mutations in Cox1 are rare in Eriophyoidea, currently known in only two other eriophyoid genera (both from Phytoptidae s.str.): Phytoptus and Retracrus [′ direction from the Cox1 start codon, which should be noted in future when designing primers.etracrus ,43. In aCox1 barcodes [Trisetacus, distinct deuterogeny has been reported for T. kirghisorum and T. piceae. However, contrary to all known examples of deuterogeny in Eriophyoidea, in these latter two species both sexes  are subjected to seasonal dimorphism [T. kirghisorum to the clade comprising Trisetacus from Pinaceae (which includes T. piceae) than to the clade comprising Trisetacus from seeds of American junipers revealed in the molecular phylogenetic analysis presented here  growing in mountainous areas of the western USA [Trisetacus species, T. batonrougei and T. quadrisetus, associated with Juniperus scopulorum and J. virginiana, respectively, which have a more eastern distribution in USA than the known hosts of T. indelis n. sp. Because Cox1 is a relatively quickly evolving gene often used for delimiting host races and cryptic species [T. indelis n. sp., from different hosts and localities. The observed striking pairwise sequence similarity and presence of the indicative indels, which are very rare in Cox1 genes of Eriophyoidea, suggest that T. indelis n. sp., may be a recently evolved species, quickly expanding its host range and area of distribution. The single synonymous nucleotide substitution observed in a population of J. osteosperma (isolates ost6 and ost 10) may be a signature of early host-dependent species divergence in the form of a point mutation. Future studies are needed to trace the evolutionary history of T. indelis n. sp., and assess its impact on the seed ecology of its hosts, two of which  are considered weedy in parts of their native ranges [Third, nophages . This sptern USA . Our ana species ,53,54,55e ranges .Implications to the phylogeny of Eriophyoidea. No morphological or multigene molecular phylogenetic analyses to date have resolved the basal phylogeny of Eriophyoidea [Cox1, 16S) are suboptimal for resolving the phylogeny of Eriophyoidea. However, it was noted that in many cases when it was possible to obtain well-resolved multigene phylogenies of different acarine groups, it has often largely reproduced the topology of trees based only on the Cox1 gene [Cox1 amino acid alignments produced an incompletely resolved tree of Eriophyoidea but revealed several new groupings marked by host associations and common morphology.phyoidea ,16,36,57ox1 gene . Our anaTrisetacus indelis n. sp., within a clade of American seed-infesting mites from junipers. Similar to a previous phylogeny of Nalepellidae based on the D1D2 28S fragment [Trisetacus, (b) produces a new moderately supported clade from pinacean hosts , (c) rejects monophyly of Trisetacus from Cupressaceae, (d) suggests a possible independent transition of Trisetacus to living in seeds of New and Old World junipers, and (e) again indicates an uncertain position of Boczekella associated with Laricoideae (Pinaceae) in the basal part of the nalepellid clade. Additionally, our analysis offers a new hypothesis on the basal divergence of Eriophyoidea suggesting (i) gradual loss of prodorsal shield setae , and opisthosomal setae c1 in major eriophyoid lineages—Nalepellidae, Phytoptidae s.str., and Eriophyidae s.l., (ii) gymnosperms as the most likely ancestral hosts of Eriophyoidea, and (iii) pentasetacids as an archaic polyphyletic group comprising “living fossils.” While these hypotheses have been widely discussed in the relevant literature [The analysis presented here unambiguously placed fragment , our curterature ,36,59,60


Prehospital telephone triage stratifies patients into five categories, “need immediate hospital visit by ambulance,” “need to visit a hospital within 1 hour,” “need to visit a hospital within 6 hours,” “need to visit a hospital within 24 hours,” and “do not need a hospital visit” in Japan. However, studies on whether present and past histories cause undertriage are limited in patients triaged as need an early hospital visit. We investigated factors associated with undertriage by comparing patient assessed to be appropriately triaged with those assessed undertriaged.We included all patients classified by telephone triage as need to visit a hospital within 1 h and 6 h who used a single after-hours house call (AHHC) medical service in Tokyo, Japan, between November 1, 2019, and November 31, 2020. After home consultation, AHHC doctors classified patients as grade 1 (treatable with over-the-counter medications), 2 , or 3 (requires ambulance transportation). Patients classified as grade 2 and 3 were defined as appropriately triaged and undertriaged, respectively.We identified 10,742 eligible patients triaged as need to visit a hospital within 1 h and 6 h, including 10,479 (97.6%) appropriately triaged and 263 (2.4%) undertriaged patients. Multivariable logistic regression analyses revealed patients aged 16–64, 65–74, and ≥ 75 years ; those with diabetes mellitus (2.31 [95% CI 1.25–4.26]); those with dementia (2.32 [95% CI 1.05–5.10]); and those with a history of cerebral infarction (1.98 [95% CI 1.01–3.87]) as more likely to be undertriaged.We found that older adults and patients with diabetes mellitus, dementia, or a history of cerebral infarction were at risk of undertriage in patients triaged as need to visit a hospital within 1 h and 6 h, but further studies are needed to validate these findings.The online version contains supplementary material available at 10.1186/s12873-021-00552-x. Prehospital telephone triage systems identify the appropriate care required for patients and determine their need for a hospital visit or an immediate hospital visit by ambulance . TelephoRecently, to reduce overcrowding in emergency departments, many countries have launched after-hours house call (AHHC) medical services or out-of-hours services  because Similarly, a private AHHC service was established in Tokyo, Japan. This service provides consultations through telephone triages using developed telephone triage protocols. These protocols classify patients into five categories: “need immediate hospital visit by ambulance (red),” “need to visit a hospital within 1 hour (orange),” “need to visit a hospital within 6 hours (yellow),” “need to visit a hospital within 24 hours (green),” and “do not need a hospital visit (white).” The AHHC or out-of-hours medical services target patients triaged as “need to visit a hospital within 1 hour (orange)” and “need to visit a hospital within 6 hours (yellow).”Previous telephone triage studies on AHHC medical services have explored the safety, efficiency, or quality of records documented by the nurse or doctor during telephone triage –11. HoweWe aimed to investigate risk factors for undertriaging by comparing patients who are classified as need an early hospital visit from the appropriately triaged and undertriaged groups using records from a single AHHC medical service.This study had a retrospective design and used anonymized data from medical records of patients who used the AHHC medical service from November 1, 2019, to November 31, 2020. The study design was reviewed and approved by the Research Ethics Committee of the University of Tsukuba .A well-used telephone triage protocol developed by the Fire and Disaster Management Agency functions as follows: when a patient calls an emergency telephone consultation service, an operator classifies the patient into one of the five categories  based on a telephone triage protocol , 12, 13.We used a private AHHC medical service in Tokyo, established by Fast Doctor Ltd.  in 2016, which sends doctors directly to the residence of patients who need a hospital visit. The company operates 7 days a week outside of regular hospital visiting hours (18:00 to 06:00 on weekdays and Saturdays and 24 h on Sundays and holidays).When a patient calls the AHHC service, operators (trained telephone triage nurses) perform the telephone triage using the telephone triage protocol. The operators determine the following: whether the patient is to remain at home (white), whether an AHHC doctor’s visit to the patient’s residence is needed (orange and yellow), or whether they need an ambulance (red). The operators call an ambulance for patients triaged as red and opt to provide the patient with information about nearby clinics or a primary hospital for patients triaged as green. The trained telephone triage nurses can consult a doctor beside the AHHC medical service call center.After the AHHC medical service doctors conduct home visits for patients, these doctors classify patients into three categories: grade 1 (can be treated using over-the-counter medications), grade 2 , or grade 3 (require ambulance transportation) –16.The doctors affiliated to the AHHC medical service are specialized attending doctors working at a university hospital and aged approximately 35 years. The telephone triage nurses are trained to use the telephone triage protocol in the AHHC medical service.Figure We included all patients who had used the AHHC medical services between November 1, 2019, and November 31, 2020, and excluded those with missing records of age, sex, telephone triage categories, or doctor grade assessments after consultations.The study used anonymized data from medical records of patients using the AHHC medical service (Fast Doctor Ltd.).Data on the following variables of patients were extracted from medical records: sex, age, comorbidities , telephone triage categories, protocols, doctor assessments after consultations, vital signs, and time from patients’ phone call to doctors’ consultations.t-test or the Wilcoxon-Mann-Whitney test was performed to compare continuous variables , as appropriate. Second, we conducted a multivariable logistic regression analysis to identify factors associated with undertriage. Third, we conducted sensitivity analyses to exclude patients aged 0–15 years because the protocol used for these patients is different from that used for patients aged > 15 years. All statistical analyses were performed using JMP 16.0 statistical software . The significance threshold was set at P of < 0.05.First, we compared baseline characteristics between the appropriately triaged and undertriaged groups. Pearson’s chi-square test or Fisher’s exact test was performed to compare categorical variables , and Student’s A total of 22,008 patients consulted the AHHC medical service between November 1, 2019, and November 31, 2020. We excluded 2249 patients owing to the lack of data on telephone triage, leaving a total of 19,759 patients classified as yellow or orange. After AHHC doctor consultation, 9017 (45.6%), 10,479 (53.0%), and 263 (1.3%) were classified as grade 1, grade 2 (appropriately triaged), and grade 3 (undertriaged), respectively  and undertriaged (263 patients) groups. The patient characteristics are shown in Table The proportions of patients aged 65–74 and ≥ 75 years were higher in the undertriaged group than in the appropriately triaged group. The proportion of patients with hypertension, diabetes mellitus, hyperlipidemia, gout, heart failure, cerebral infarction, cancer, and dementia was higher in the undertriaged group than in the appropriately triaged group.The results of the multivariable logistic regression analyses are shown in Table In this study, we found that 1) undertriage occurred in 1.3% of patients who were classified by the telephone triage as “hospital visit required” and 2) patients with old age, diabetes mellitus, dementia, and a history of cerebral infarction were at risk of undertriage.Securing a safe and efficient telephone triage is challenging as a balance between an acceptable low level of overtriage and a minimal level of undertriage needs to be maintained for high patient safety . Thus, iWe found that the proportion of undertriage was < 2%. A previous systematic review showed that telephone undertriage occurred in 10% of AHHC medical service cases . MeanwhiWe found that old age, diabetes mellitus, dementia, and a history of cerebral infarction were associated with undertriage. A previous study conducted at a trauma center showed that age ≥ 65 years , female The telephone triage protocols are used to carefully record and document the patient’s medical history, including hypertension, diabetes mellitus, myocardial infarction, heart failure, liver disease, chronic lung disease, and collagen disease, as well as age ≥ 65 years and the AHHC medical service used in the telephone protocols. However, age ≥ 75 years and history of cerebral infarction are not factors incorporated in the telephone triage protocol. In Japan, the population is rapidly aging, leading to an increased incidence of dementia and cerebral infarction ; thus, tThis study has several limitations. First, the study included a relatively small number of undertriaged patients; thus, we could not stratify and analyze each telephone triage level and protocol (Supplementary File Telephone triage in an AHHC should be carefully performed for patients with advanced age, dementia, and a history of cerebral infarction. Further studies are warranted to evaluate other telephone triage protocols.Additional file 1: File 1. All patients classified as grades 1, 2, and 3Additional file 2: File 2. Protocol causing undertriage


Plant pathogenic fungi are responsible for many serious diseases that affect agricultural productions both pre- and post-harvest. In this respect, the losses of products along the post-harvest chains  determine strong impactful consequences, especially in agriculture-based-economy countries ,2,3. To In order to satisfy the growing request for high-quality and, at the same time, safe and eco-friendly products, throughout the past two decades, the research field strongly focused on the investigation of the potentialities of alternative means to synthetic fungicides to control plant diseases; these include antagonistic microorganisms or derivatives thereof, natural biostimulants ,9,10, asWith the perspective of reducing environmental pollution and related consequences for human health, nowadays, the scientific research is also strongly focused on valorizing wastes, especially those largely generated by processing industries . Within Shrimp are, overall, considered a high-value aquaculture product , not onlShrimp wastes as such have been used for feeding in veterinary practice and aquaculture  as well The aforementioned products arising from shrimp wastes represent, therefore, a precious asset in several fields of application; however, it is an accepted fact that their processing generates highly impactful new wastes, which in turn contribute to environmental pollution and, consequently, negatively affect human health.in vitro and in vivo control of major fungal and oomycete pathogens of the genera Alternaria, Colletotrichum, Fusarium, Penicillium, Plenodomus and Phytophthora.The investigation of the potentialities of new products arising from a minimal and sustainable processing of shrimp wastes stands, therefore, as an essential challenge for scientific research. Considering that plant pathology is strongly focused on finding eco-friendly strategies for controlling plant pathogens and related diseases, the present study evaluated the effectiveness of new substances obtained by the minimal processing of shrimp wastes in the in vitro by an agar diffusion test toward several fungal and oomycete pathogens. “Dry-powder”, “EtOAc-extract” and “MetOH-extract” did not demonstrate any inhibitory effect in the mycelial growth of all pathogens under study (data not shown); therefore, they were not further tested. “Nitric-extract” was the only extract that negatively affected the mycelial growth of all pathogens; the diameter of the inhibition halos consequently observed at each concentration was, therefore, recorded at the end of the incubation period  and minimal fungicidal concentration (MFC). The in vivo effectiveness of the selected substance in the control of post-harvest infections of fruits by Penicillium spp. was finally tested.In this study, wastes from the shrimp species Parapenaeus longirostris were processed to obtain four substances: (i) “Water-extract”, (ii) “EtOAc-extract”, (iii) “MetOH-extract” and (iv) “Nitric-extract”. All these extracts were analyzed, to determine their composition in metabolites and phenolic compounds, by HPLC-ESI-MS/TOF. Then, the antifungal activity of the “dry-powder”, “EtOAc-extract”, “MetOH-extract” and “Nitric-extract” was preliminarily tested riod see a–o. The The metabolites detected by HPLC-ESI-MS/TOF in the analyzed substances are presented, as a heat map, in The most important phenolic acids detected by HPLC-ESI-MS/TOF in the substances analyzed are presented in in vitro preliminary tests evidenced an inhibitory effect on the growth of the pathogens examined only for the waste shrimp extracted with nitric acid, named “Nitric-extract”. Additionally, none of the control solutions (each solvent used for the preparation of the respective extract) inhibited mycelial growth. In the agar diffusion test, “Nitric-extract” at concentrations of 100, 75 and 50% showed an inhibitory effect on all strains of fungal and oomycete pathogens, while at concentration of 25%, an inhibitory effect was still observed only on Ph. nicotianae T2.C-M1A, F. sacchari CBS 145949, A. alternata 646, P. digitatum P1PP0, P. commune CECT 20767, C. gloeosporioides C2, F. proliferatum CBS 145950, Pl. tracheiphilus Pt2 and Ph. nicotianae T3-B-K1A, in order of significance , on Ph. nicotianae T2.C-M1A as well as on three typically post-harvest pathogens, i.e., F. sacchari CBS 145949, A. alternata 646 and P. digitatum P1PP0.Results from the ificance . The diaificance . Signifiificance . At the P. expansum CECT 2278 and F. saccari CBS 145949, while the lowest (2%) were recorded for C. gloeosporioides C2, Ph. nicotianae T3-B-K1A and Pl. tracheiphilus Pt 2. Values of MFC were the same as MIC for the majority of the strains. Only for strains P. commune CECT 20767, A. alternata 646, Ph. nicotianae T3-B-K1A and Ph. citrophthora Ax1Ar, MFC was higher than MIC, indicating that for these four strains, MIC exerted only a fungistatic effect.To further test the inhibitory activity of “Nitric-extract” on the growth of pathogens, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were determined, and results are summarized in in vivo on citrus (oranges and lemons) and apple fruits artificially infected by P. digitatum and P. expansum, respectively. Results are summarized below.The antifungal activity of “Nitric-extract” was finally tested P. digitatum P1PP0 of oranges, all concentrations of “Nitric-extract” significantly reduced rot severity compared to the water control (treatment ID01) (Three days post inoculation with nt ID01) . HoweverFive days after inoculation , all conP. digitatum P1PP0 in lemons  and in vivo antifungal activity.This study evaluated, for the first time, the potentialities of minimally processed shrimp wastes in the kinetics , which aCandida and Aspergillus species [Lactobacillus paracasei [Staphylococcus aureus [Pseudomonas aeruginosa-bioconverted oil extract of docosahexaenoic acid was effective against the mycelial growth of several plant pathogens, including Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, F. solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum [Results from the chemical analysis showed that all substances extracted from the shrimp waste were miscellaneous mixtures of a conspicuous number of metabolites and phenolic compounds. Interestingly, a high relative abundance of the 2-Hydroxyisocaproic, 3-(4-Hydroxyphenyl) propionic and 4-Aminobenzoic acids in “MetOH-extract”, and of docosahexaenoic acid in “EtOAc-extract” were reported. Various studies reported fungicidal activity for these molecules when tested as pure substances; 2-hydroxyisocaproic acid was effective against  species ; 3- in determining the  extract . Example extract  observed extract  found thin vitro preliminary test together with those from MIC and MFC tests overall demonstrated that the pathogens mostly affected by “Nitric-extract” were Pl. tracheiphilus Pt 2, C. gloeosporioides C2 and Ph. Nicotianae—both tested isolates. Plenodomus tracheiphilus is the causal agent of ‘mal secco, one of the most destructive diseases affecting lemon trees [Pl. tracheiphilus-infections. However, many copper-based treatments are not cost effective in commercial lemon groves, and also represent a significant source of environmental pollution [C. gloeosporioides, the causative agent of anthracnoses in several fruits and vegetables [Phytophthora nicotianae is very likely the most widespread and destructive Phytophthora species worldwide, affecting a very wide host range of more than 255 plant species [Pl. tracheiphilus, C. gloeosporioides and Ph. nicotianae, but all pathogens tested in the present study. To this aim, further investigations are needed to evaluate the phytotoxicity, if any, of the extract, its attitude to systemic translocation, which is of particular relevance in the case of tracheomycoses, such as ‘mal secco’ caused by Pl. tracheiphilus, as well as the most effective method of application, e.g., by drenching, spraying or incorporation into fruit coatings, which also depends on the type of disease.Results from the on trees . Becauseon trees . It is collution . Anothergetables  as well getables . Phytoph species ,56,57. Cin vivo against molds caused by Penicillium species in orange, lemon and apple fruits, which are the most economically important post-harvest diseases affecting these fruits [Penicillium, several other synthetic fungicides, including azoxystrobin, fludioxonil, cyprodinil and pyrimethanil, have been proposed as alternatives for the chemical control of these post-harvest fruit diseases [As a preliminary step towards the application of “Nitric-extract” to control plant diseases, its effectiveness was tested e fruits ,59. Poste fruits ,61. Morediseases ,61,62,63diseases ,67,68,69Penicillium species [in vivo control of Penicillium spp. using a natural substance that is derived from minimum waste treatment.There is boundless literature evaluating the efficacy of alternative strategies to the use of conventional synthetic fungicides for the control of postharvest molds of  species ,76,77,783 in water solution , although, in vitro, they were not effective in inhibiting the mycelial growth of all pathogens included in this study. As already observed for other inorganic salts [in vivo effectiveness of NaNO3 was not the consequence of direct antifungal activity, but the possible result of the triggering of defense mechanisms in fruits. Further tests are ongoing to verify this hypothesis.Overall, treatments with “Nitric-extract” at the highest concentrations were the most effective in positively affecting the reduction of rot severity in all tested fruits. Additionally, an interesting weak positive effect was also observed in all control treatments, including NaNOic salts , it cannPenicillium spp., although the effective dose was much higher than that of traditional synthetic fungicides [The results from the treatments with “Nitric-extract” demonstrated that three days post-treatment, “Nitric-extract” as such determined a significant reduction of rot severity over any other treatment in all fruits . Additionally, “Nitric-extract” at 75% significantly reduced rot severity in lemon and apples over controls; “Nitric-extract” at 50% had a significant effect over controls only on apples; the concentration of 25% was as effective as the controls in all fruits. Five days post-treatment, “Nitric-extract” as such still maintained significant effects in reduction of rots only in lemons and apples; “Nitric-extract” at 75% demonstrated significant reduction of rot severity only in lemons; finally, “Nitric-extract” at 50% and at 25% were as effective as the controls in all tested fruits. Overall, the results showed an interesting performance of “Nitric-extract” in controlling postharvest mold caused by ngicides , its usein vitro against a wide range of fungal and oomycete pathogens by the nitric extract appears promising and could be exploited in the context of new strategies for the management of plant diseases caused by these pathogens. In vivo preliminary results suggest a possible use of nitric extract for post-harvest treatments against citrus and apple molds caused by Penicillium species. To this aim, and to optimize the efficacy of treatments, next steps will be to define the methods and times of application. In this study, nitric extracts were applied to fruits 24 h after inoculation with the pathogen, indicating curative efficacy. However, an additional aspect that would merit further investigation is whether nitric extract, like other natural substances, is able to elicit plant defense mechanisms against infections by pathogens. In this case, the treatment of fruits with this extract might also have preventive efficacy against infections by molds. Regarding this, it cannot be ruled out that the other shrimp waste extracts, which, in preliminary in vitro tests did not show inhibitory activity on the mycelium growth, may also be effective in vivo acting as resistance elicitors. Last but not least, a prerequisite for the use of nitric extracts of shrimp waste to prevent post-harvest molds is to evaluate if the treatment leaves unpleasant odors on the fruits. A sensory analysis using an electron nose is planned to clarify this aspect. Although the effective dose of nitric shrimp waste extract is far higher than the label dose of synthetic fungicides used to control post-harvest fruit diseases, this extract, as a natural substance, could be an interesting alternative to traditional post-harvest chemical treatments, as it is more eco-friendly and far less toxic than synthetic fungicides.This study is part of a research program aimed at exploring the antifungal activity of extracts obtained from minimally processed shrimp wastes and their possible application in agriculture. The antifungal activity shown Parapenaeus longirostris, common name deep-water rose shrimp, was collected in a local fish market in Catania , in February 2021. Shrimp waste was kept on ice until processed in the laboratory and firstly, washed with distilled water; then, dried in an oven at 30 °C for a week. The dried sample was powdered and homogenized. Then, 10 g of shrimp waste powder was packed in plastic food bags labelled “dry-powder” and stored at −20 °C until further use; 10 g of shrimp waste powder was processed to extraction (20 min long sonication by means of ArgoLab DU-100) with (i) 50 mL of ethyl acetate (EtOAc from Aldrich) or (ii) 50 mL of methanol (MetOH from Aldrich). Then, the supernatant was carefully collected and transferred into a clean beaker. The powder was then re-subjected to the described procedure for a total of three times. The 150 mL of supernatant were firstly filtrated and then evaporated under vacuum at the temperature of 40 °C until a crude extract was obtained. The crude extracts, representing the “EtOAc-extract” and “MetOH-extract”, were stored at −20 °C until further use. (iii) To obtain “Nitric-extract”, 20 mL of nitric acid  was added to 5 g of shrimp waste powder; the mixture was then stirred for 1 h at 150 rpm and then the acid was neutralized by adding 80 mL of NaOH (0.10 g/mL). pH was verified to be around pH 5. To obtain the “water-extract”, 25 mL of water with 1% of acetic acid were added to 5 g of shrimp waste powder; the mixture was homogenized by vortexing and ultrasonication. The liquid extracts were then filtrated and stored at −20 °C until further use.Around 5 kg of shrimp waste  of the species The differential analysis of the metabolites contained in the four substances tested was carried out by HPLC-ESI-MS-TOF. Before the analysis, each sample was subjected to specific pretreatments. In particular, “EtOAc-extract” and “MetOH-extract” were dissolved in a methanol solution at 1% of acetic acid. Finally, “Water-extract” and “Nitric-extract” were mixed to acidified water. Each sample was finally filtered with 0.22 µm filter and then analyzed using an UPLC  coupled with a quadrupole time of flight mass spectrometer (Agilent 6546 LC/Q-TOF) operating in positive and negative ionization mode. Chromatographic separation was performed with an Agilent Zorbax RRHD SB-C18, 2.1 mm × 50 mm, 1.8 µm column. Mobile phase A was composed of Milli-Q water and acetonitrile was used for mobile phase B (both phases were acidified with 0.1% formic acid), with gradient elution, as follows: 0 min, 2% B; 22 min 95% B; 25 min, 5% B. The column was equilibrated for 3 min before every analysis. The flow rate was 0.4 mL/min, and 5 μL of sample was injected. Dual AJS ESI source conditions were as follows: gas temperature: 325 °C; gas flow: 10 L/min; nebulizer pressure: 40 psig; sheath gas temperature: 295 °C; sheath gas flow: 12 L/min; capillary voltage: 4000 V; nozzle voltage: 500 V; Fragmentor: 120 V; skimmer: 70 V; product ion scan range: 100–1500 Da; MS scan rate: 5 spectra/s; MS/MS scan rate: 3 spectra/s; maximum precursors per cycle: 2; and collision energy: 10, 20, 40 eV. The analysis of the metabolites was carried out in triplicate. Untargeted LC/Q-TOF based metabolomics approach was used to identify the metabolic profiling of shrimp waste extracts. Integration, data elaboration and identification of metabolites were managed using MassHunter Qualitative Analysis software B.08.00 and library PCDL Manager B.08.00.Penicillium spp. ; three Phytophthora spp. ; Plenodomus tracheiphilus strain Pt2; two Alternaria species ; three Colletotrichum species ; two Fusarium species (F. proliferatum strain CBS 145950 and F. sacchari strain 145949). All strains were from the collection of the laboratory of Molecular Plant Pathology of the Di3A .Fungal and oomycete strains were included in this study. Most of them had been previously characterized ,55,79,80The antifungal activity of the “dry-powder”, “EtOAc-extract”, “MetOH-extract” and “Nitric-extract” were preliminarily checked in order to select, among them, the most promising one to be used in further tests.For testing the effectiveness of the “dry-powder” in affecting mycelial growth, 16 g of shrimp waste powder were homogenized with 1 L of autoclaved PDA and poured in 90 mm Petri dishes. For each pathogen, a mycelial plug (diameter 3 mm) from a 7-day-old culture grown on PDA at 25 °C was transferred in the center of a “dry-powder”—amended PDA plate; control cultures of each pathogen, obtained by subcultures in “dry-powder”—non amended PDA plates, were included in the test. The plates were incubated at room temperature (20 ± 2 °C) for three days  or for 15 days (for oomycete pathogens). At the end of the incubation period, no negative effects were observed in mycelial growth compared with controls for any of the pathogens. The “dry-powder” was not further tested.The effect of “EtOAc-extract”, “MetOH-extract” and “Nitric-extract” on the mycelial growth of the pathogens was tested at different concentrations. To this purpose, “EtOAc-extract” and “MetOH-extract” were separately diluted in 1% dimethyl sulfoxide to obtain, for each substance, four solutions at the following concentrations 10, 25, 50 and 100 mg/mL; “Nitric-extract” was diluted in water to obtain the following concentrations: 25, 50, 75 and 100%.4 conidia/mL) were homogeneously spread on the surface of a PDA plate; by using a cork borer, five wells  were then realized on the PDA plate; then, 60 µL of each concentration of the substance were pipetted into the respective well; the plates were finally incubated at 25 °C for three days. For the oomycete pathogens (Phytophthora spp.), the influence of “EtOAc-extract”, “MetOH-extract” and “Nitric-extract” was tested separately as follows: for each Phytophthora strain, a mycelial plug (diameter 3 mm) from a 7-day-old culture grown on PDA at 25 °C was transferred in the center of a PDA plate and surrounded by 5 wells at a distance of 3 cm from the plug; then, 60 µL of each concentration of the substance tested were pipetted into the respective well. The plates were then incubated at 25 °C for 15 days.“EtOAc-extract”, “MetOH-extract”, “Nitric-extract” and each fungal pathogen were tested separately in a 90 mm PDA plate as it follows: 500 µL of a suspension of conidia of the fungal pathogen  to obtain 10 serial dilutions (1 mL each) of the substance tested . Then, the tubes were incubated at 25 °C for 3 days.The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) are dilution end points of a substance which completely inhibits the growth or kills the fungi tested; both are widely used in routine tests of substances with antimicrobial activity ,81. The After the incubation period, the MIC was the lowest concentration where no cloudiness was visible in the tubes, which means that no pathogen growth was observed. The determination of MFC was an additional step of the MIC test. The MFC is defined as the lowest concentration of a substance required to kill a fungal pathogen corresponding to no visible subculture growth on an unamended culture medium in environmental conditions favorable to the growth. In the present study, the evaluation of the MFC was carried out by transferring 10 μL from each of the wells where solution cloudiness was not observed into PDA medium. The inoculated plates were incubated at 25 °C for 3 days. The MFC for each pathogen was represented by the plated concentration that did not lead to any mycelial growth after the incubation period.in vivo against infections caused by P. digitatum and P. expansum on citrus (oranges and lemons) and apple fruits, respectively.The antifungal activity of the “Nitric-extract” was evaluated For the test, “Nitric-extract” was tested in all fruits  as such (ID02) or as three serial dilutions in sterilized distilled water (sdw) . In addition, ID03, ID05, ID07 and ID09 were the respective controls.3 at the concentration 0.002 mol/mL (0.17 g/mL); (iii) NaNO3 0.17 g/mL diluted in sdw at 75%—ID05; (iv) NaNO3 0.17 g/mL diluted in sdw at 50%—ID07; NaNO3 0.17 g/mL diluted in sdw at 25%—ID09  water (ID01); (ii) a solution of nitric acid  and sodium hydroxide  at the ratio 1:4—they are the solvent and base used for the preparation of “Nitric-extract”, respectively, which leads to a water solution of NaNO25%—ID09 .Citrus × sinensis) cv. Valencia and lemons (Citrus × limon) cv. Femminello Siracusano, while apples  were of the cv. Braeburn. Before the tests, all fruits were preliminarily surface-disinfected by dipping in 1% NaClO (NaClO 0.5% for apples) for 2 min, rinsing under tap water and air-drying at room temperature (20 ± 2 °C).All fruits used in this test came from organic crops. Citrus fruits were mature oranges  was pipetted into each wound. Inoculated fruits were incubated in a plastic container at 20 °C and 80% RH (relative humidity) for 24 h. For all fruit , the treatment with “Nitric-extract” as such or as a dilution was carried out as follows: after the incubation period, at each inoculation point, 20 µL of the substance was placed into the wound; overall, 3 fruits per treatment were used. An additional control group, represented by 3 fruits wounded as above, received 20 µL of sterile distilled water (sdw) per wound. The experiment was repeated another two times, with similar results. Analysis of variance did not reveal any differences among the experiments (F not significant); therefore, only the results of a single experiment are reported here.Surface-disinfected fruits  were wounded with a 2 mm-diameter plastic tip at four points along the equatorial surface; then, 10 µL of conidial suspension  (accessed on 9 November 2021). In order to normalize the distributions, data were transformed in square-root values, but untransformed values are reported in the respective graphs. Tukey’s HSD (honestly significant difference) post-hoc test was applied to evidence significant statistical differences (p ≤ 0.05).All data were subjected to one-way analysis of variance (ANOVA) using the R software (


Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance.Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co–)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-μs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of  Furthermore, analysis of this library revealed that polar substitutions at buried positions are beneficial for tolerance and that substitutions to chemically different amino acids are most likely beneficial BsLipA in aIL further highlighted the wide range of effects aIL have on BsLipA, which subsequently can be exploited to increase tolerance to IL and aIL via substitutions For the enzyme BsLipA by all-atom molecular dynamics (MD) simulations of in total 130 μs length and subsequent analyses of structural stability  with As an outstanding result, we present a potentially underestimated, indirect mechanism of aIL-induced enzyme inhibition. It originates from specific aIL interaction sites on the enzyme surface and disturbs the integrity of the core enzyme structure via percolation effects along pathways of perturbed interactions that underlie changes in local structural stability. This result provides a so far overlooked foundation to guide the rational design of enzyme variants with improved aIL resistance.22.1versus polarizable ones for studying the effects of aIL on proteins. For details on the analysis, see To investigate whether the chosen force field/water model and partial charge combination for aIL adequately reproduce experimental data, we computed the system density A and theBsLipA and the dynamical properties of IL and salt ions around the protein. The RMS average correlation (RAC) BsLipA at different time intervals τ within a single trajectory . As to anions, [TfO−] binds strongly to a wide range of residues, including aliphatic, polar, and charged ones, with positively charged residues showing the highest occupancy (59% for K23 in 0.7 M [BMIM/TfO]). Halogen anions showed lower binding frequencies of 13–21% for binding to charged residues. Note that the extensive simulation times used here allowed computing binding frequencies and interaction patterns for sufficiently long timescales to reach convergence for most interactions . By contrast, simulation times as long as 500 ns used previously were insufficient to yield converged results even for the strongest binding sites of [BMIM+] Next, we computed the relative occupancy of ions at all KD   ). Howevetive ΔG0 .Table 1TBsLipA that go beyond pure concentration effects and that IL ions replace water molecules at binding regions more strongly than inorganic ions do. On average, at a 1 M standard state, ΔG0 of IL ion binding to residues are positive, as are those of inorganic ions. Still, more preferred residue types can be identified . For interactions to specific residues negative ΔG0 are found, such as to W42 for [BMIM+] or K23 for [TfO−].To conclude, our results demonstrate that IL ions show preferential interactions with 2.3BsLipA structure and dynamics. The root-mean-square deviation (RMSD) of backbone atoms, a measure for structural similarity with respect to the crystal structure, is < 1.5 Å in all solvents and statistically not significantly different from MD simulations in pure water  of backbone atoms, a measure for atomic mobility, revealed only a few regions with values > 1.0 Å B; these olutions  of all rolutions A, the avolutions B, or theolutions C, did no2.4BsLipA residues perturb the structural stability (rigidity and flexibility) of BsLipA, we used the rigidity theory- and ensemble-based Constraint Network Analysis (CNA) ij,rcneighbor that displays at which stage of the unfolding simulation a rigid contact i,jR between two residues i and j at most 5 Å apart is lost; lower values of the cutoff energy Ecut display a higher local stability.To investigate how specific interactions of IL ions with iE,CNA , β3(D72-A75), β4(N98-L102), β5(L124-Y129), the α-helices α7 (N106-T109), α10 (S163-N174) and the residues H10-F19 (including part of α1), H76-G79, and S130-Y139.Analysis of the residue-wise extent of the structural stabilization and destabilization with regard to water, Δ,CNA Eq. , showed in water A, B, witin water A–D. ThisECNA Eq. , which hH3,ECNA = −0.07 to 0.39 kcal mol−1) and N4  is likely connected to the strong stabilization of K35  and its neighboring residues D34 , L36 , and Y37 , which arise from a redistribution of noncovalent interactions (see below). In [BMIM/I] and [BMIM/TfO], one additional major residue patch became destabilized   F. IntereBsLipA structural stability, but only to small global changes in structural stability, in line with the results from the previous section. Due to rigidity percolation To conclude, these results show that incubation in aIL leads to local decreases and increases of 2.5BsLipA residues. Here, changes in hydrogen bonds  are exemplarily described. In all aIL, BsLipA showed a significantly increased average amount of intramolecular hydrogen bonds  . These results indicate that interactions of IL ions induce changes in the overall hydrogen bond network.To understand the origin of the changes in structural stability in aIL at the molecular level, we analyzed changes in noncovalent interactions between  t-test) A. None op < 0.05, two-sided independent Student’s t-test) altered interactions in 0.6 M [BMIM/Cl] and 0.6 M [K/Cl] to 33 in [BMIM/TfO]. The number of significantly altered interactions showed a significant and strong correlation with the concentration , suggesting that the interactions were influenced by IL ions (We then computed the changes in the average occurrence frequency for each H-bond with respect to water. Considering only H-bonds with absolute deviations > 5% resulted in a range of seven significantly ( IL ions .BsLipA structure will be discussed now. These interaction sites are I) the E65Oε-Y37Oη-D34O interaction, which was significantly perturbed in all 12 solvents, and II) the residue patch around L108, which was significantly perturbed in 0.7 M [BMIM/TfO].Further analyses revealed that these changes subsequently induced conformational changes that were transferred to adjacent residues in an attenuated manner via perturbation pathways (PP). As a result, we observed multiple neighboring residues of interaction sites of aIL that showed significantly altered hydrogen bond frequencies and interaction distances A/B. Two BsLipA in all solvents with D34, Y37, and E65 being the key contributors was observed  and the catalytic site (PP2).2.6GiP)  Eq.  for intrGiP of 4.0 . A potential resistance mechanism of the D34K variant that showed substantially increased resistance to all aIL (2.9-fold to 4.1-fold) is shown in +] molecules around the residue patch in contrast to forming stabilizing intramolecular interactions, such as salt bridges or charge-assisted hydrogen bonds, preventing potential conformational changes that are introduced upon binding of [BMIM+] to D34 and its adjacent residues. The substitution was predicted by FoldX Gunf compared to BsLipAwildtype , indicating that the significant increase in aIL resistance is not induced by an increase in thermodynamic stability of the BsLipA variant but instead by effects based on changes in the overall aIL-protein interactions.In PP1, D34 is one of the residues with the highest increase in tolerance upon substitution towards all tested aIL (2.4–4.1-fold) when substituted with positively charged residues. For PP1 involving the solvent-accessible residues H3, D34, K35, Y37, and E65, a cation-induced structural reorganization was suspected. Therefore, we excluded the positively charged residue K35, which already should be beneficial for reducing cation binding, and residue H3, as it is part of a π-stacking interaction with W31. Considering only substitutions at the remaining positions resulted in an average GiP of 2.9 for 0.7 M [BMIM/TfO]. Interestingly, for these substitutions, all other libraries also revealed an increased GiP of 4.0, 3.6, and 3.2 in 0.9 M [BMIM/Br], 1.2 M [BMIM/Cl], and 0.6 M [BMIM/I], respectively, thus potentially highlighting a general beneficial substitution site of the BsLipA structure. A potential resistance mechanism of the T109E variant that showed substantially increased resistance to all aIL (1.9-fold to 2.4-fold) is shown in –] molecules around the residue patch. Notably, the substitution was predicted by FoldX Gunf compared to BsLipAwildtype , indicating that beneficial substitutions could be missed when relying on single evaluation criteria, such as ΔΔGunfIn PP2, which involved the solvent-accessible residues L108 and T109, an anion-induced structural reorganization was suspected. Considering only substitutions at these positions resulted in a GiP for the improved residual activity of BsLipA. Therefore, positions in such pathways are valuable targets for introducing focused substitutions that counteract the proposed mechanisms of aIL-induced enzyme inactivation.To conclude, substitutions at residues that are part of a perturbation pathway yield pronounced 3BsLipA surface residues and induce changes in the local protein stability through a redistribution of intramolecular interactions. Such changes percolate over neighboring residues to distant regions of the protein and may affect the integrity of the protein core and the catalytic site. Finally, by data mining an experimental complete SSM library of BsLipA, we show that residues that are part of a PP yield pronounced GiP for the improved residual activity of BsLipA.In this study, we showed that aIL ions preferentially interact with specific BsLipA over time, II) the evolution of binding free energies of IL ions to BsLipA residues over time, and III) the backbone RAC of BsLipA , which were found to be negative at 1 M standard state only for ∼ 2–8% of all surface residues. Mostly large and weakly hydrated organic ions interacted strongly with BsLipA. These results suggest that qualitative inspection of spatial distributions of solvent molecules (including aIL ions) as done previously +] molecules at the catalytic site cleft and may affect enzyme activity in this aIL similarly to the crowding of [BMIM+] molecules observed for the I12F variant +], as does W compared to F and Y  conformation BsLipA activity in that substitutions at residues that are part of a PP yield pronounced GiP for an improved residual activity of BsLipA  simulations of BsLipA in respective solvents used for evaluating the SSM library BsLipA in the respective solvents are listed in To investigate the interactions of +] or [K+] cations and [Cl−] or [I−] anions in the lowest and highest concentrations (0.6 M and 1.2 M). A solution of 0.6 M [Na/Cl] mimics natural seawater of ∼ 0.67 M To test for ion- and concentration-specific effects, the range of solvents was extended to salt solutions containing  ions were added as counterions. The systems were then solvated using the OPC water model Packmol BsLipA, atomic partial charges and force field parameters were taken from the Amber ff14SB force field For 4.2−1 Å−2 were applied to all protein atoms for 2500 cycles (500 cycles steepest descent (SD) followed by 2000 cycles conjugate gradient (CG) minimization). Second, the harmonic restraints were reduced to a force constant of 1 kcal mol−1 Å−2 for 10,000 cycles (2000 cycles SD and 8000 cycles CG minimization). Third, 1000 cycles SD and 4000 cycles CG minimization without any restraints were performed.The simulations were performed following ref. −1 Å−2 were applied on protein atoms, and a time step of 2 fs was used. The temperature was then raised from 100 K to 300 K over 50 ps of isobaric-isothermal (NPT) MD simulations. Subsequently, the density was adapted to 1 g cm−3 over 200 ps of NPT–MD simulations. Finally, the harmonic restraints were reduced to 0 kcal mol−1 Å−2 over the course of six NVT–MD simulations with a length of 50 ps each. In all MD simulations, the particle mesh Ewald (PME) method τ) of 0.5 ps for heat bath-coupling to keep the system temperature at the target temperature of 300.0 K during the simulations. The SHAKE In the subsequent thermalization, the system was first heated from 0 K to 100 K over 50 ps in a canonical (NVT) MD simulation. Harmonic restraints of 1 kcal molThe production simulations were performed in the NPT ensemble at 300.0 K for 2.0 µs with a time step of 4 fs using the hydrogen mass repartitioning (HMR) method 4.3cpptrajBsLipA; VIII) the analysis of hydrogen bonds to describe the intramolecular polar interaction network; IX) dihedral angles of the catalytic site residue S77 to describe the conformational state of the catalytic site; X) intramolecular distances of functional side chain groups , dissociation constants and absolute binding free energies were computed for each individual protein residue for both solvent cation and anion, respectively To determine the system-specific correction factor nWATaIL is the number of water molecules in the aIL system, and nWATWAT is the number of water molecules in the system with pure water.where 4.6BsLipA residues, we computed the distances between the center of mass of every ion to the center of mass of functional groups of every BsLipA residue (BsLipA residue using the center of mass (COM) as defined in +] and [TfO−], the N2 or C1 atoms were used to represent the COM, respectively. The trajectories of the aIL with experimental conditions were used to calculate the anion RDFs. For the calculation of cation RDFs, the systems of [BMIM/Cl], [Na/Cl], and [K/Cl] at a concentration of 0.6 M were used. All RDFs were normalized according to the average system density of the input frames. The radial distribution functions of ions at the protein surface  with C0 as the standard concentration of 1 M were then computed according to Eq. Binding free energies  to one. For the analysis of the hydrogen bonds involved in the catalytic site network, looser distance and angle cutoffs for hydrogen bonds of 3.5 Å and 120° were used.To investigate changes in the intramolecular hydrogen bond network of 4.9et al.EHB is computed by a modified version of the potential by Mayo et al.T), hydrogen bonds with an energy EHB > Ecut(σ) are removed from the network at temperature T. In this study, the thermal unfolding simulation was carried out by decreasing Ecut from −0.1 to −6.0 kcal mol−1 with a step size of 0.1 kcal mol−1. Ecut can be converted to a temperature T using the linear equation introduced by Radestock et al. ECNA=4.10The residual activity (RA) of variants was calculated following Eq. :(6)ResidaIL ≥ RAbuffer + 3 σaIL, with RAaIL and RAbuffer being the residual activity of the variant in aIL and buffer, respectively and σaIL being the true standard deviation of the assay aIL was chosen because it corresponds to a p-value below 0.01. For a detailed description of how cultivation, purification, and activity assays were performed, please see ref. where EV indicates the noise measured for the empty vector. The tolerance was considered significantly improved, when RA4.11BsLipA variants, we first calculated the precision P P=VarGiP was calculated by dividing the precision over a subset of residues considered by the precision over the whole library, which is equivalent to the result due to random mutagenesis (Eq. The esis Eq. .(8)GiP=PThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Induction of ovarian stimulation by use of the gonadotropin-releasing hormone agonist (GnRHa) long protocol in the luteal phase is a common practice and results in stable pregnancy and live births; it is often used in patients with normal ovarian function. Some patients with normal ovulation may be pregnant before ovulation induction, which can be easily confirmed by asking the patient about cessation of menstruation. However, some pregnancy complications may cause vaginal bleeding along with normal menstrual blood loss; in such a situation, hormone levels can often mirror that seen in pituitary down-regulation and the value of β-HCG may be less than 5 mIU/mL. Under these conditions, the physician might start the cycle of ovarian stimulation. During ovarian stimulation, the increase in β-HCG can cause premature luteinization and follicle maturation disorder, and poor embryo quality, which can easily be overlooked. In this study, we report a case of pregnancy at the end of controlled ovarian stimulation induced by GnRHa long protocol in the luteal phase, followed by follicle maturation disorder and poor embryo quality. This case provided a reference and served as a cautionary note that could perhaps obviate occurrence of similar cases.A 30-year-old woman with a diagnosis of unexplained infertility was scheduled for in vitro fertilization embryo culture (IVF) at our clinic. Pregnancy was confirmed at the end of controlled ovarian stimulation, which was followed by follicular maturation disorder and poor embryo quality.The patient with a diagnosis of unexplained infertility was scheduled for IVF at our clinic.Oocyte retrieval was still arranged for her after confirmation of pregnancy. As per the β-HCG level and the trans-vaginal ultrasound examination findings, we considered 2 possibilities: an adverse intrauterine pregnancy or extra-uterine pregnancy. Therefore, we decided to terminate the pregnancy; hence, 50 mg/d of mifepristone was given for 2 days, combined with 200 μg misoprostol.Elevated β-HCG level had an adverse effect on maturation and fertilization of oocytes, and even embryo quality.Once pregnancy is confirmed, ovulation induction should be terminated as soon as possible. Some patients with normal ovulation may be pregnant before the induction of ovulation; this can be easily confirmed by cessation of menstruation. However, some complications in pregnancy can cause vaginal bleeding along with loss of normal amount of menstrual blood; in such a situation, hormone levels can often mirror that seen in pituitary down-regulation. Under this condition, the physician might start the cycle of ovarian stimulation. During stimulation, the rise in β-HCG can cause premature luteinization of follicles, hinder maturation of follicles, and lead to poor embryo quality, which can easily be overlooked by the treating physician. Herein, we report a case of pregnancy at the end of controlled ovarian stimulation induced by GnRHa long protocol in the luteal phase, which was followed by follicle maturation disorder and poor embryo quality, thereby providing a reference that can serve as a cautionary signal helping perhaps obviate occurrence of similar cases.2P value increased to 9.64 ng/mL, LH was <0.2 IU/L, and E2 was 5446 pg/mL. Because the serum P was higher than before, β-HCG was detected at a level of 275.1 mIU/mL. Induction with 4000 IU of HCG  was done and oocyte retrieval was performed 36 hours after HCG induction. Routine luteal support  was given after oocyte retrieval.A 30-year-old woman  with a diagnosis of unexplained infertility was scheduled for in vitro fertilization embryo culture (IVF) at our clinic. She failed to conceive after 2 cycles of artificial insemination at our clinic. Her menstrual cycle was 30 to 35 days, lasting 3 to 4 days. Her basal serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were 7.21 IU/L, 3.17 IU/L, and <20 pg/mL, respectively. The numbers of antral follicles were 8 per ovary. In the mid-luteal phase, the patient was administered 0.1 mg/d of short-acting GnRHa  for pituitary down-regulation in order to initiate IVF. After 14 days, serum FSH was 3.05 IU/L, LH was 0.63 IU/L, progesterone (P) was 1.23 ng/mL, and E2 was 35.0 pg/mL; the value of β-HCG was less than 5 mIU/mL (Table  as 7CIII was frozen; another 2 embryos were grade IV at the four-cell stage and continued to be cultured to day 5, but no blastocysts were obtained (Fig. On the first day after oocyte retrieval, 4 oocytes had 2 pro-nuclei, one had 3 pro-nuclei, 3 were metaphase I oocytes, 2 were germinal vesicle oocytes, and one had a giant polar body. On day 3 of insemination, 1 embryo evaluated by Gardner's criteriaOn the first day after egg retrieval, the serum β-HCG was 290.4 mIU/mL, and on trans-vaginal ultrasound examination, the gestational sac was not seen inside or outside of the uterus. However, after 2 days, β-HCG was only 288.2 mIU/mL. As per the level of β-HCG, there were currently 2 possibilities: an adverse intrauterine pregnancy or extra-uterine pregnancy. Therefore, we decided to terminate the pregnancy; 50 mg/d of mifepristone was given for 2 days, combined with 200 μg misoprostol. β-HCG was checked again and was found to be decreased to 0.7 mIU/mL after 10 days. The ethics committee of Hangzhou Women's Hospital approved this study. In addition, the patient has signed the consent form for publication of this case.3 Hillier et al proposed the existence of a therapeutic window for LH during ovarian stimulation. According to this, there is not only a threshold requirement for LH to guarantee follicular development but also a ceiling level beyond which LH might inhibit the proliferation of granular cells and induce luteinization of follicles before ovulation, resulting in impaired oocyte and embryo quality.–5 Some studies have suggested that LH supplementation has a beneficial effect on the maturation and fertilization of oocytes in patients who are undergoing pituitary down-regulation with significant serum LH suppression.–8 However, it has been consistently reported that high LH levels during the follicular phase are associated with poor oocyte and embryo quality, with a negative impact on IVF outcome.,10 Nonetheless, there is no consensus about the specific threshold of the LH window during follicular development.LH is essential for providing the androgen substrate for estrogen synthesis, and an LH surge is required for final follicular maturation and ovulation. Moreover, HCG has a slower plasma metabolic clearance and may be more effective than LH. In our case, there was continuous secretion of endogenous β-HCG from trophoblastic tissue. Therefore, the high serum HCG level which was equivalent to high level of LH had an adverse effect on the maturation and fertilization of oocytes, and even embryo quality. Therefore, once pregnancy is confirmed, ovulation induction should be terminated as soon as possible.In assisted reproduction, because of its structural similarity and similarity in biological action with LH, HCG acts on the same receptor and has been used to mimic the endogenous LH surge.The authors are grateful to the doctors and staff who were involved in this work.Conceptualization: Zhenyun Lin.Data curation: Chong Wang.Writing – original draft: Meiyan Jiang.Writing – review & editing: Xiaoyang Fei.