I2 statistic and Cochran’s Q test were used to determine heterogeneity. The protocol was registered in PROSPERO (CRD42020213391). Seven randomised controlled trials were included with a total of 1760 patients. There was no significant difference in pain scores comparing IN ketamine with IV analgesics or placebo at 5 , 15 , 25 , 30 , and 60  minutes. There was also no significant difference in the need for rescue analgesics between IN ketamine and IV analgesics . Only mild adverse effects were observed in patients who received IN ketamine. Our results suggest that IN ketamine is non-inferior to IV analgesics and may have a role in acute pain management among adults in the ED.Due to overcrowding, personnel shortages, or problematic intravenous (IV) cannulation, acute pain management is often sub-optimal in emergency departments (EDs). The objective of this systematic review and meta-analysis was to evaluate the efficacy and safety of intranasal (IN) ketamine for adult acute pain in the emergency setting. We searched and identified studies up to 21 May 2021 via PubMed, Scopus, Web of Science, Cochrane Database, and Google Scholar. The random-effects model with 95% confidence intervals (CIs) was used to estimate mean differences (MDs) and odds ratios (ORs). The  Acute pain is one of the most frequent presentations to the emergency departments (ED). Between 2000 and 2010, pain was the leading complaint in 45.4% of the total ED visits, and patients reported pain as a symptom twice as high as those recorded by healthcare professionals (40% vs. 20%) . InadequEmergency staff shortage, limited beds, and inadequate patient monitoring are the critical management issues in overcrowded ED, which necessitate the use of analgesics that are easier to deliver, faster and with fewer adverse effects . In the The opioid abuse and addiction problems are rapidly growing in the United States (U.S.). In 2017, approximately 47,000 people died from opioid overdose in the U.S. . Opioids2+ channel pore in the central nervous system (CNS) and spinal cord. It also reduces the presynaptic release of excitatory neurotransmitter (glutamate). Furthermore, ketamine and its metabolites have a ten-fold lower affinity for opioid receptors  than the NMDA receptor and antagonistic activity for nicotinic, muscarinic, and monoaminergic receptors [Ketamine has been extensively used for procedural sedation and as an induction agent for rapid sequence intubation (RSI) since its introduction to medicine in the 1960s due to its safe hemodynamic profile . The aneeceptors .Ketamine’s effects are dose-dependent, where the analgesic dosage for pain relief without sedation is about 10–30% of the dissociative sedative dosage. The typical ketamine analgesic dose without sedation is 0.1 to 0.3 mg/kg IV, 0.5 mg/kg intramuscular (IM) and 1 mg/kg intranasal (IN) as compared to 1 to 2 mg/kg IV, 3 to 4 mg/kg IM and 6 to 9 mg/kg IN for the ketamine dissociative sedation dose ,16,17. IKetamine is extensively utilised in adult populations as an adjuvant to opioid pain therapy to reduce opioid use. In addition, ketamine is being studied as a first-line analgesic treatment for acute pain to minimise opioid consumption . Recent We conducted an SRMA to assess the efficacy and safety of IN ketamine as an analgesic agent for acute pain management among adults in ED. This SRMA was conducted in accordance with the protocol registered with PROSPERO (CRD42020213391). The methodology and reporting were constructed based on the updated guideline of the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guideline .Studies published from inception to 21 May 2021 were searched and identified via electronic databases of PubMed, Scopus, Google Scholar, Web of Science, and Cochrane Library. Only RCTs comparing IN ketamine to IV analgesics  or IN placebo in acute pain management were included. The SRMA’s inclusion criteria were as follows: (1) RCT; (2) population: adults (age ≥ 15 years old) who presented to ED with acute pain with numeric rating scale (NRS) or visual analogue scale (VAS) ≥ 5; (3) intervention: IN ketamine only; (4) comparison: IV morphine, IV fentanyl, IV ketamine or IN placebo; (5) outcomes: pain score reduction, the requirement of rescue analgesia and adverse events. We restricted the publications to the English language only. Ketamine’s analgesic effects on perioperative pain  following anesthesia, cancer-related pain, and other non-acute pain were eliminated from the study. Studies performed in non-ED settings, such as postoperative or prehospital, were also excluded. We excluded studies where IN ketamine was given as a combination therapy with other analgesics such as inhaled nitrous oxide or IV morphine. Studies that used higher dosage for procedural sedation or IV, IM, oral, or topical routes as ketamine intervention were not considered. Our SRMA eliminated studies that compared IN ketamine to analgesics administered orally, topically, IM, or IN. The titles and abstracts were screened, and eligible RCTs were selected by two authors independently (Y.S.S. and M.A.I.). We gathered full-text copies of the relevant articles to determine if they met the inclusion criteria. Discrepancies between the authors were resolved through discussions.Our primary outcome was to evaluate the analgesic efficacy of IN ketamine in pain score reduction using validated pain scales (NRS or VAS) from baseline to post-intervention and the requirement for rescue analgesics. The following time points were included: 5, 10, 15, 20, 25, 30, and 60 min. We analysed the prevalence of the adverse events, such as dizziness, nausea, vomiting, agitation, difficulty in concentration, confusion, emergence phenomenon, dry mouth, fatigue, disorientation, drowsiness, euphoria, headache, and hypotension as the secondary outcomes for evaluating the safety of IN ketamine.The data were extracted independently by two authors (Y.S.S. and M.A.I.) from the full-text reports and We calculated the mean differences (MDs) with associated 95% confidence intervals (CIs) for the continuous data of pain score reduction at 5, 10, 15, 20, 25, 30, and 60 min of post-intervention for IN ketamine versus IV analgesics  or placebo. Subgroup analyses were carried out based on the comparative drugs. We computed the odds ratios (ORs) with associated 95% CIs for rescue analgesia and adverse events for the dichotomous data. We also calculated the prevalence of adverse events with associated 95% CIs. We attempted to contact the authors for the unreported outcomes of interest, such as MD or standard deviations. We used the Review Manager (RevMan) calculator to impute missing data when we could not contact the authors as recommended by the Cochrane handbook . We alsoI2 statistic was used to assess trial heterogeneity (I2 > 75% indicating significant heterogeneity). The Cochran’s Q test was employed to assess the significance of the heterogeneity test; a p-value of 0.05 indicated significant heterogeneity [The random-effects model was used in all meta-analyses, and the ogeneity ,30. We pn = 4), review articles (n = 42), case reports (n = 16), duplicate studies (n = 207)), another 512 studies were excluded based on their abstracts as they did not comply with the objective and inclusion criteria of this study. Finally, seven RCTs [Our search via the electronic databases identified 788 potentially relevant studies. After removing 269 studies (non-human studies (dies n = 7), anothven RCTs ,33,34,35The seven included trials involving 1760 participants; 883 participants being randomised to receive IN ketamine, whereas 877 participants being randomised to receive IV analgesics  or placebo. All trials were double-blinded RCTs with mean ages ranging from 30.4 to 40.3 years. Five trials were conducted in Iran ,32,33,34The detailed quality assessment of the included studies is shown in p = 0.26), 15 , 25 , 30 , and 60 min  (p = 0.27) and 30 min .Comparing IN ketamine with control group , we found no statistically significant differences in pain scores at 5  . A poolep = 0.35, I2 = 70%; Three trials investigated the need for rescue analgesics ,32,33. Wp < 0.0001), difficulty concentrating , confusion , and disorientation  were significantly higher than that of the control group. For hypotension, the odds were significantly lower in the IN ketamine group than the control group ; besides, for other adverse events, there was no significant difference between IN ketamine and the control group  were observed estimating pain score and rescue analgesic requirement compared to the main findings .Ketamine has been used globally as an anesthetic agent for more than 50 years and is widely used in ED for procedural sedation and as a RSI induction agent ,37. MultOne of the common reasons for delays in pain management at EDs is due to difficult IV access. A prospective cohort study by Witting found that 39% of adult patients in ED had a failed first attempt of IV access, and 22% of them also had a failed second attempt . AlthougIn our SRMA, IN ketamine was found to be non-inferior compared to IV analgesics as the analyses showed no significant differences in pain scores at 5, 15, 25, 30, and 60 min post-intervention among adults in the ED. Interestingly, we found better pain reduction with IV analgesics at 10 and 20 min post-intervention. Such inconsistent results in our analysis were primarily influenced by the trial of Parvizrad et al., which reported IN ketamine only achieved equal analgesic effect as IV ketamine at 30 min post-intervention in a population of acute traumatic pain . CompariOn pooled analysis comparing IN ketamine with IV morphine, we found no statistically significant difference in pain reduction at 10, 15, 25, 30, and 60 min. However, we noticed IV morphine is more efficient than IN ketamine in pain reduction at 5 min. This is possibly due to a delayed onset of IN ketamine compared with IV morphine in pain reduction. It is consistent with a systematic review by Poonai et al. , which rReduced use of opioids as rescue analgesia is desirable due to the current opioid crisis in the United States . In our The safety of analgesics in EDs is as crucial as their efficacy. Therefore, analgesics with less adverse effects for pain control are desired, especially in EDs due to staff shortage, limited beds and suboptimal patient monitoring in overcrowded EDs . No lifeIn our current study, the common non-severe and transient adverse events associated with IN ketamine were dizziness, nausea, difficulty concentrating, confusion, emergence phenomenon, and dry mouth. This is in line with a systematic review of side effects associated with ketamine use in depression, where psychotomimetic side effects, neurological or cognitive effects, and other side effects related to the gastrointestinal, ocular, respiratory, and urological systems were found in 42%, 23%, and 23% of the studies, respectively . DizzineAn SRMA by Balzer et al. compared IV LDK and IV morphine for acute pain control in the ED showed equivalent analgesic efficacy within 60 min of administration with similar safety profiles . This isThere are several strengths noted in our study. Firstly, this SRMA only included RCTs representing a high level of evidence. An exhaustive search of the literature was performed via multiple databases to ensure the thorough identification of relevant studies. The stringent exclusion criteria of our study avoided confounding factors such as population with altered pain physiology and altered drugs metabolisms  which would affect the accuracy of our results. Based on the quality assessment, 71.4% of the included studies were high-quality (low-risk of bias), ensuring reliable results. Recently, Li et al.  publisheWe were aware of few limitations in our SRMA. Firstly, only seven trials fulfilled our inclusion criteria. As a result, more clinical trials are needed to assess the efficacy and safety of IN ketamine for acute pain management among adults in ED. Secondly, some of the data might be less precise as we used WebPlotDigitizer Software to extract data from figures and RevMan calculator to impute the missing data as recommended by the Cochrane handbook as we failed to contact the authors. Thirdly, based on the JBI critical appraisal tool, we had 71.4% high-quality (low-risk of bias), 28.6% moderate-quality (28.6%), and none of the studies was low-quality. However, this tool does not thoroughly assess the studies quality . Finally, heterogeneity among studies was significant, probably due to limited trials with variations in populations and different control groups.In conclusion, this SRMA revealed that IN ketamine is non-inferior compared to IV analgesics for acute pain management among adults at 5, 15, 25, 30, and 60 min post-intervention in the ED setting and do not require higher rescue analgesics. The favorable safety profile of IN ketamine as no reported life-threatening adverse event also encourages IN ketamine as a pain reliever in the EDs. However, insufficient evidence proves that IN ketamine is superior to IV analgesics in acute pain management. Therefore, more RCTs are required to evaluate further the efficacy and safety of IN ketamine in managing acute pain in the EDs.


In the last two decades, kisspeptin (Kiss) has been identified as an important player in the regulation of reproduction and other physiological functions in vertebrates, including several fish species. To date, two ligands  and three kisspeptin receptors  have been identified in teleosts, likely due to whole-genome duplication and loss of genes that occurred early in teleost evolution. Recent results in zebrafish and medaka mutants have challenged the notion that the kisspeptin system is essential for reproduction in fish, in marked contrast to the situation in mammals. In this context, this review focuses on the role of kisspeptins at three levels of the reproductive, brain-pituitary-gonadal (BPG) axis in fish. In addition, this review compiled information on factors controlling the Kiss/Kissr system, such as photoperiod, temperature, nutritional status, sex steroids, neuropeptides, and others. In this article, we summarize the available information on the molecular diversity and evolution, tissue expression and neuroanatomical distribution, functional significance, signaling pathways, and gene regulation of Kiss and Kissr in teleost fishes. Of particular note are recent advances in understanding flatfish kisspeptin systems, which require further study to reveal their structural and functional diversity. GPR54 and now as kissr2) was isolated in the Nile tilapia, Oreochromis niloticus  of a kisspeptin receptor  and three kissr types  reported to date  has been detected in Pleuronectiformes, such as oglossus ; both kigalensis , 7, halfmilaevis , and Japlivaceus . Of note to date .Danio rerio) and medaka (Oryzias latipes) kiss and/or kissr knockouts display normal reproduction  neurons , 11, 12.oduction , 22, 23.eproduce . Howevereproduce , suggesteproduce , 24, 25.The aim of this review is to examine the entire literature on the kisspeptin system in teleost fishes, with particular emphasis on diversity and evolution, central and peripheral distribution, physiological effects on reproduction, intracellular signaling pathways and regulatory mechanisms.Tetraodon nigroviridis), Japanese pufferfish (Fugu rubripes) and medaka  in length, with a major cleavage product of 54 aa  and three shorter peptides of 14, 13 and 10 amino acids in length. All these peptides bind to their cognate G protein-coupled receptor today known as kisspeptin receptor . It was  species , 34–36. kissr to be characterized in fish was found in the Nile tilapia    , fugu (Takifugu niphobles), and spotted pufferfish (kissr3 by PCR in Pleuronectiformes and suggested that kissr3 may have been lost during evolution of this order (Kisspeptin receptors are membrane receptors that belong to the superfamily of G protein-coupled receptors (GPCRs) . These r tilapia . Soon afcanadum) , grey muephalus) , fatheadromelas)  and two romelas)  and Atlaromelas) . The zebromelas)  to identr et al. . Since tr et al. , 42, 43.fferfish . Mechalyfferfish , 6 also is order .Odontesthes bonariensis  will allow us to clarify this situation. However, future studies are needed to clarify the pleiotropic role of kisspeptins not only in Pleuronectiforms but also in other teleost species.While studies in mammals have shown that kisspeptin has pleiotropic effects , 61, the to date . Howeverkissr2 and/or kissr3 was observed in Senegalese sole , growth hormone (gh) and prolactin (prl) in goldfish (Carassius auratus) pituitary cells (lhb expression in a dose-dependent manner without affecting follicle-stimulating hormone b (fshb) mRNA levels when acting on pituitary cells  (Astatotilapia burtoni (Dicentrarchus labrax)  with a distribution like Gnrh3 nerve terminals   and the  burtoni . Convers burtoni  and Euro labrax) , the pre labrax)  and Kisserminals , supportEpinephelus coioides, intraperitoneal (ip) injection of Kiss2 leads to upregulation of hypothalamic expression of gnrh1 , depending on the mode of administration. Subcutaneous and slow release of Kiss2 increases gnrh1 expression  is also a target for the central effects of kisspeptin in fish. In hypothalamic explants of half-smooth tongue sole, Kiss2 exerts a stimulatory effect on A levels . To our in vitro studies indicate a direct stimulatory effect of kisspeptin on gonadotropins in different species. For example, Kiss1 significantly triggers Lh release from primary pituitary cell cultures of goldfish  and pomcb   . Plasma hrysops) . Furtherhrysops) .2 in females are significantly increased in immature Nile tilapia treated with Kiss2, and Kiss2 apparently accelerates the process of spermatogenesis  and 11-KT in male European sea bass, causing an increase in cumulative milt, sperm density and sperm motility parameters . Similarogenesis . Recentlogenesis . All theClarias batrachus) and it was suggested that it could locally regulate gonadal steroidogenesis , and hepatic insulin growth factor-1 (igf-1), along with higher levels of plasma Gh, Igf-1, and melatonin in the cinnamon clownfish (Amphiprion melanopus), suggesting a role in controlling growth in this species  in the LD condition is larger than that in the short-day (SD) condition, whereas the kiss2 neurons located in the Nucleus recessus lateralis (NRL) are not altered  breeder, the number of  altered , 132. OnD regime . In zebrhe brain , 135. Sisea bass , while asea bass  and in ta cyanea . Furtherelopment . Taken tkiss1 mRNA levels in the whole brain, as well as kissr3 mRNA levels in the habenula and the brain region containing nucleus of the medial longitudinal fascicle, the oculomotor nucleus, and the interpeduncular nucleus. However, kiss2 mRNA levels in the whole brain and kissr2 mRNA levels in the caudal zone of the periventricular hypothalamus and the posterior tuberal nucleus is significantly decreased when exposed to both low and high temperatures. Interestingly, kissr2 mRNA levels in the nucleus of the medial longitudinal fascicle, the oculomotor nucleus, and interpeduncular nucleus show an increase when animals were exposed to low temperatures compared with the normal rearing temperature for this species, 27°C. These results suggest that the kiss1/kissr3 system is activated by low temperatures, whereas the kiss2/kissr2 system is inhibited by both low and high temperatures, suggesting that these two kisspeptin systems may be involved in different aspects of zebrafish physiology  and kissr2 (kissr2_v1 and kissr2_v2) are identified in pejerrey, and fasting also increases hypothalamic kiss2 and kissr2_v1 mRNA levels, whereas kissr2_v2 shows no expression in the hypothalamus  results in an increase in mRNA levels of sea bass . Overallsea bass –148. Howkiss1 neurons in the NVT, but not in the nucleus preopticus periventricularis (NPPv), is significantly reduced after ovariectomy (OVX) compared with the sham-operated group, and basal levels are restored after E2 treatment  is expressed together with kiss1 in NVT neurons, suggesting that these neurons are involved in the positive feedback regulation of the BPG axis in this species  at mid-gonadal development of male striped bass. In contrast, pubertal males responds to T replacement by up-regulation of kiss1 and kiss2, whereas no changes are observed in juvenile and recrudescent males, suggesting a differential and gonadal stage-dependent role of T in regulating mRNA levels of kiss1 and kiss2 , sex hormone levels (E2 and T), and accelerates the onset of puberty in largemouth bass, Micropterus salmoides  are observed after administration of LPXRFa-1 in the same studies  injection of LPXRFa-2 suppresses pression . Howeverer genes . On the  studies , 162. Inin vitro . Similarese sole , and non grouper .6, Pro9Net]-mGnRHa, has a stimulatory effect on the expression of kiss2 in European sea bass pituitary cell cultures but has no effect on the mRNA levels of kissr2 and kissr3  play an important role in the control of growth, morphogenesis, metabolism, and reproduction in several species, including fish  without affecting mRNA levels of kissr3  can also affect reproductive regulation, in part by affecting kisspeptins system, which is a clear example of neuroendocrine disruption . For exaf kissr3 . In addid kissr2 . In adulmidbrain . Similargoldfish . All thekiss2 and kissr2 in the brain of female Japanese flounder , an industrially produced synthetic hydrazine compound, significantly downregulates mRNA expression of flounder . An inhiflounder . Moreove altered . Moreove by EDCs , 173.kissr in the entire brain of mouthbrooding cichlids, with higher mRNA levels of kissr2 observed in high-status territorial males compared to non-territorial males  PICT-2015-2783 to GMS. and PICT-2017- 2839 to ASM, the National Natural Science Foundation of China 32072949 and the Laboratory for Marine Fisheries and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao) ZZ-B06 to BW, who also received a scholarship supported by the China Scholarship Council .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: Phenotype I hypersensitivity reactions are the most commonly reported drug reactions; however, precision medicine has made it possible to characterize new phenotypes. A recent communication proposed the existence of a “converter phenotype,” which would affect patients who present non-immediate hypersensitivity reactions and in subsequent exposures develop immediate hypersensitivity reactions. This study aimed to describe the clinical characteristics of converter phenotype reactions and their evolution during desensitization to chemotherapeutic drugs and monoclonal antibodies.Methods: We retrospectively reviewed our database of patients undergoing desensitization to chemotherapy or biological agents and selected those with a converter phenotype. Demographic and clinical characteristics of the patients, the results of skin tests, tryptase and IL-6 levels, and desensitization outcomes were assessed.Results: Of 116 patients evaluated, 12 (10.3%) were identified as having a converter phenotype. The median interval between drug exposure and reaction was 90.6 h (range 8-288 h). After the conversion, phenotype I was the most frequent (58.3%), followed by cytokine release reactions (33.3%). Fifty-one desensitizations were undertaken and all treatments completed, with 10 (19.6%) breakthrough reactions. No new changes in the phenotype were detected.Conclusions: The symptoms of non-immediate drug hypersensitivity reactions may indicate the need for an early allergological evaluation to assess the risk of future immediate drug reactions. Clinical characteristics, skin test results, and biomarkers can help predict responses to rapid drug desensitization, guiding clinicians on how to optimize therapy delivery while maintaining patient safety. Phenotype I (PhI) reactions are the most commonly reported kinds of drug hypersensitivity reactions (DHR) according to the Gell and Coombs' classic description; however, precision medicine has brought to light other types that had no place into this classification, such as cytokine release reactions . Gell anNIDHR occur any time as from 1 h after the initial drug administration, frequently appearing after many days of treatment , and incIn 2018, preliminary clinical descriptions about a new phenotype of DHRs to taxanes emerged . The so-Previous publications have demonstrated the benefits of rapid drug desensitization for treating several phenotypes, including PhI, cytokine release reactions (CRRs), mixed phenotypes (MxPh) and NIDHRs , 7–11. NThe pathophysiological mechanisms that induce allergen-specific and temporal immunotolerance in PhI reactions include the inhibition of both the early and late response of the mast cells , 13–15. On the other hand, the mechanisms for achieving tolerance during desensitizations in NIDHRs are poorly understood; previous studies have pointed to the activation of T lymphocytes as the main endotype , 21. In Recent analysis of cytokines during both IDHR and NIDHR shows substantial increases in IL-10 after desensitization, related to the action of regulatory T lymphocytes and regulatory B lymphocytes, meaning that tolerance induced by desensitization implies the modulation of drug-specific response by regulatory mechanisms .Little is known about the mechanisms involved in CPh reactions. However, a previous series of cases presented in poster form on the European Academy of Allergy and Clinical Immunology Annual Congress, suggested that desensitization could be beneficial in these patients, potentially affecting the switch from NIDHR to IDHR .This study aimed to describe the clinical characteristics of CPh reactions and their evolution during desensitization, not only with taxanes but also with other chemotherapeutic drugs and monoclonal antibodies (MAb) that have not yet been associated with this phenotype in the literature.We retrospectively analyzed the medical records of all patients who underwent desensitization to chemotherapeutic or biological drugs at the Alicante General University Hospital between January 2019 and December 2020. Only patients who presented symptoms compatible with CPh were included in the analysis. Patients with non-immediate, severe skin reactions with systemic symptoms (SCARS) were excluded. The Hospital Ethics Committee approved the study protocol.We considered a CPh in patients who experienced an IDHR to chemotherapy or MAbs after having experienced a NIDHR on the previous administration of that same drug . DHRs weThus, the PhI phenotype involves type I reactions, with or without non-specific symptoms; the CRR phenotype implies CRR symptoms, with or without unspecific symptoms; the MxPh involves any combination of type I and/or CRR symptoms, with or without unspecific symptoms; and EPh involves only non-specific symptoms.Patient age, sex, diagnosis, history of atopy, previous lifetime exposures to the culprit drug before the initial delayed reaction, interval of the delayed reaction, delayed symptoms, exposures to the culprit drug at the moment of the immediate reaction, grade of the immediate reaction classified according to Brown's grading system , resultsAtopy was defined as a confirmed history of at least one of the following: allergic rhinitis/asthma, food allergies, contact dermatitis, acute urticaria with an allergic cause, or allergy to Hymenoptera venom. Confirmed and unconfirmed allergies to other drugs were also evaluated.Skin tests were performed at least 2 weeks after the last DHR, using, at prick level: paclitaxel in concentration of 6 mg/ml, docetaxel 10 mg/ml, carboplatin 10 mg/ml, oxaliplatin 5 mg/ml, rituximab 10 mg/ml, adalimumab 50 mg/ml, and brentuximab 5 mg/ml. If the prick was negative, dilutions of 1:1,000, 1:100, 1:10, or 1:1 were used for intradermal tests, as described elsewhere , 27. A wThe desensitization protocols used were those proposed by the Brigham and Women's Hospital group, consisting of bags, at different dilutions, administered in 4, 8, 12, or 16 steps with 2- to 2.5-fold dose increments, along with increasing infusion rate every 15 min, except in the final step, where it was maintained until the full dose was reached .Based on the symptoms during the IDHRs, premedication was administered 30 min before desensitization, including cetirizine  and ranitidine (50 mg intravenously). Additional premedication included aspirin 300 mg (for flushing) and montelukast 10 mg (for bronchospasm/chest tightness). COX-1 inhibitors, acetaminophen, fluids, and opioids were used to prevent chills, rigors, fever, and pain, while benzodiazepines  were administered in case of anxiety. Patients were asked to withhold β-adrenergic blocking medications for 24 h before desensitization . A serum tryptase concentration > 11.4 μg/L was considered elevated, as were the values during the reaction which exceeded the product obtained by the formula  1.2 + 2 , 29. IL-Descriptive statistics of categorical variables were expressed through absolute and relative counts using frequency tables and graphs. Quantitative variables were described using measures of central tendency (mean and median) and dispersion [standard deviation (SD) and interquartile range (IQR) and/or range]. We used the IBM SPSS Statistics program, version 27 .Of the 116 patients evaluated for DHR to chemotherapeutic and biological drugs, 12 (10.3%) met criteria for CPh: 3 (25%) had reactions to paclitaxel, 3 (25%) to docetaxel, 2 (16.7%) to rituximab, 1 (8.3%) to adalimumab, 1 (8.3%) to brentuximab, 1 (8.3%) to carboplatin, and 1 (8.3%) to oxaliplatin.Most (91.7%) of the included patients were women, and the mean age at diagnosis was 41 (SD 13.6) years. Ten (83.3%) patients were diagnosed with cancer , and two had autoimmune diseases (Crohn's disease and multiple sclerosis). Atopy was present in 80% of the patients.The median number of drug exposures at the time of the NIDHR was 2.1 (range 0-16), and the median interval between drug exposure and reaction was 90.6 h (range 8-288 h). Patients reacting to taxanes had the least historical exposure to these drugs (83.3% had a reaction on their first exposure), while the carboplatin-allergic patient had been the most exposed (16 previous exposures prior to DHR). n = 7; 58.3%), CRR  and EPh .At the time of conversion, the mean exposure before developing the IDHR was 3.8 (range 1-17) and the phenotypes expressed during the IDHR were PhI  2 (57.1%) or G3 (28.6%); the remaining 14.3% had a G1 reaction. Skin tests were positive in 6 (85.7%) patients. Reactions were mainly (57.1%) triggered by biological drugs , followed by platins  and taxanes  .On the other hand, patients who developed CRRs had an average of 1.3 exposures to the drug at the time of the IDHR, all corresponding to taxanes (50% to paclitaxel and 50% to docetaxel), with G2 reactions and negative STs.Finally, one patient with an EPh developed a G2 reaction during the second exposure to paclitaxel, with a negative skin test. Clinical characteristics are summarized in Regardless of phenotype, none of the patients had positive results on the delayed reading of the skin tests.Altogether, 51 desensitizations were done, and all (100%) of the treatments were completed. There were 10 (19.6%) BTRs: 7 (70%) of which were immediate and 3 (30%) non-immediate.Regarding the phenotype expressed during the immediate BTRs, 5 (85.7%) were PhI, and 1 (14.3%) Eph; all were of the same phenotype as the IDHRs. On the other hand, non-immediate BTRs came from CRR in two patients who developed warmth and back pain some hours after desensitization. One carboplatin-allergic patient , developed a non-immediate BTR with fever and back pain a few hours after desensitization was completed; this patient developed an immediate PhI reaction during desensitization . There wSerum levels of tryptase and IL-6 were quantified in 5 out of 7 (71.4%) BTRs during desensitization. Two patients presented significant elevations of tryptase, including patient 11, allergic to oxaliplatin, who was identified by applying the formula ; both elevations corresponded to PhI reactions. In addition to the increase in tryptase, a slight elevation of IL-6 was demonstrated during an immediate BTR; this patient had a non-immediate BTR suggestive of CRR several hours after finishing desensitization .The phenotype-based approach of precision medicine allows us to predict how patients will respond to treatment . We descThe characteristic change in the CPh makes it unique. Previous publications by Gómez et al.  and TorrIn these patients, NIDHRs often developed on the first exposure to the drug and were characterized by the appearance of non-severe symptoms including maculopapular exanthema, erythema, and flushing. Several days after exposure, some patients also developed symptoms suggestive of cytokine release such as back pain, myalgias, arthralgias, chest tightness, and fever.We showed that after developing an NIDHR, at least one subsequent exposure was needed before changing the phenotype to develop an IDHR. Most patients developed PhI reactions, especially those treated with platins , 25, 30,On the other hand, almost all taxane-treated patients developed CRR during the first exposures. Patients who converted from an NIDHR to PhI reaction had mostly presented with symptoms such as myalgia, arthralgia, malaise, and fever, while those who developed CRR had presented maculopapular exanthema, chest tightness or pain. These findings suggest that during NIDHRs, different cytokines on which the symptoms depend could be released. In the first group, the release of IFN-γ would predominate, while in the second group excessive amounts of other cytokines such as IL-6, TNF-α and IL-10 would be produced .Regarding desensitization, the overall efficacy we observed, at 80.4%, was better than in previous publications, where it ranged from 74%  to 76.8%In addition, during BTRs we did not observe new changes in the phenotypes. Moreover, compared to Silver et al. , in our Regarding biomarkers, skin tests were very useful for identifying PhI reactions, even in patients who reacted after few exposures to the drugs. On the other hand, elevation of serum tryptase and IL-6 were related to the phenotypes involved in the reaction, supporting the idea that they are useful in the endophenotyping of the reactions , 11. In in vivo and in vitro tests  could inform the decision on which patients would benefit from a challenge or desensitization (Previous observations about CPh suggest that the proper allergology workup after the NIDHRs using tization  in ordertization . Althougtization .Finally, although our objective was not to clarify the mechanisms by which CPh could develop, our observations allow us to hypothesize. A logical explanation involves both innate and acquired immunity. During the NIDHR, the cytokines released would be responsible for the symptoms and for the polarization of the reaction, triggering a Th1 response with the subsequent appearance of a CRR in successive exposures, or on the contrary the cytokines could produce a Th2 response, responsible for the activation of mast cells as an IgE-mediated response. There are connection pathways between mast cells and macrophages that could explain mixed phenotypes .This descriptive study has several limitations. Firstly, our cohort is not representative of all patients with CPh because not all those who developed NIDHR were rechallenged, and some patients who finally changed their phenotype were not sent to evaluation by the allergist. Secondly, the size of the sample and the heterogeneity of the patients and drugs involved limit the performance of a robust statistical analysis and the extrapolation of the data. Thirdly, intravenous chemotherapeutic and biological agents are highly sensitizing drugs and the IDHR experienced by these patients could potentially be unrelated to the previous NIDHR. Finally, we did not use experimental methods that would allow us to verify our hypothesis, so we recommend carrying out basic studies that make it possible to elucidate the mechanisms that could be involved in this potential CPh as described in our study.Summarizing, CPh denotes a set of reactions characterized by an initial NIDHR and subsequent IDHRs after repeated exposures. These reactions, which had initially been described after exposure to taxanes, present a similar pattern after administration of other drugs, including platins and MAb.The symptoms presented during the NIDHR may indicate the need for an early allergological evaluation to assess the risk of future IDHRs. Rapid drug desensitization could prevent conversion or, if this has already occurred, prevent the recurrence of serious or life-threatening immediate reactions.The clinical characteristics of reactions, skin test results, and biomarkers can help predict responses to rapid drug desensitization, guiding clinicians on how to optimize therapy delivery while maintaining patient safety.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Ethics Committee for Research with Medicines of the Alicante Department of Health. The patients/participants provided their written informed consent to participate in this study.TWJ-R, IL-C, RM-A, VS-G, PG-D, and JF-S contributed to conception and design of the study. FM, RM-A, and NM-B coordinated the data collection. FM analyzed the biomarkers results. RM-A, AB-SJ, SC-B, and NM-B organized the database and analyzed the results. TWJ-R wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Uveal melanoma (UM) is the most common primary intraocular tumor in adults and usually has a very poor prognosis. Histologically, UMs have been classified in epithelioid cell type, spindle cell type, and mixed cell type. Balloon cells are large pale cells that contain small, hyperchromatic, central nuclei with vesiculated, clear, and lipid-rich cytoplasm. A balloon cell morphology is infrequently observed in naevi and even less frequently in malignant melanomas of the skin, conjunctiva, ciliary body and choroid. In this regard, UMs that exhibit balloon cell features are generally those previously treated with proton beam irradiation and then enucleated, rather than those that directly underwent primary surgery. To the best of our knowledge, very few cases of primary UM showing extensive balloon cell morphology have been reported in scientific literature to date. We herein present an unusual case of primary UM with diffuse balloon cell changes in a 69-year-old woman. BRAF kinase, GNAQ, GNA11, SF3B1, EIF1AX, and BAP1 are the most frequently mutated genes in UM [Uveal melanoma (UM) arises from melanocytes within the choroid, the ciliary body, and the iris of the eye, and, despite being generally considered a rare neoplasm, is the most common primary intraocular tumor in adults . One of es in UM .The anatomical location most frequently affected by UM is the choroid, then the ciliary bodies, and lastly, the iris. Iris melanomas (IMs) generally have a better prognosis than the others, possibly because IMs are usually visible from the outside of the eye and thus receive an early diagnosis . Most UMGenomic DNA was isolated from 8 FFPE sections 5 microns thick. After an initial step with deparaffinization solution  used to eliminate paraffin from the sample, the DNA was extracted with a High Pure FFPET DNA isolation kit  following the manufacturer’s instructions. DNA concentration was detected with Quantus Fluorometer utilizing QuantiFluor dsDNA kit . A total of 300 ng of DNA were used for quantitative real-time PCR. A mutational analysis of NRAS and BRAF genes was carried out with EasyPgX kits  according to the manufacturer’s recommendations. The evaluation of the mutational status of NRAS on exon 2 , exon 3 , and exon 4  was performed using 8 specific oligos. The mutational status on codon 600  of BRAF was analyzed using 4 specific oligo mixes. A total of 25 ng of genomic DNA was used as a template for each reaction. Real-time PCR was performed using AriaDx instrument .DNA was extracted from the FFPE sample using a Gene Read DNA FFPE kit (Qiagen), and its yield was assessed by a dsDNA HS Assay kit on a Qubit 3.0 Fluorometer . A total of 10 ng input DNA was used for next-generation sequencing (NGS) analysis. The library was generated with an Ion Ampliseq library kit plus and the Ion Ampliseq cancer HotSpot Panel v2 primer pool . An Ion Chef System was then used for automated template preparation and loading the sequencing chip, and sequencing was carried out on an Ion GeneStudio S5 Plus System . Ion Reporter v5.18.2.0  was employed for single nucleotide variant annotations.−6 mm2/s.  of the orbit showed a lentiform-shaped mass in the posterior chamber of the eye. The lesion demonstrated intermediate signal intensity on both T1- and T2-weighted images, restricted diffusion on diffusion-weighted imaging (DWI), and pronounced enhancement after intravenous administration of gadolinium-based contrast agent. The apparent diffusion coefficient (ADC) value of the lesion, measured on the ADC map derived from DW images, was 840 × 106 mm2/s. . These fNRAS, BRAF, and GNAQ genes, whereas we identified, according to the FATHMM score, the pathogenic GNA11 p.Gln209Leu mutation and two neutral variants on APC and TP53 genes  balloon cell morphology occurred in a UM primarily treated by enucleation of the eye; accordingly, we believe that, in our case, balloon cell morphology was an intrinsic feature of the tumor and not a degenerative cell reaction. Histopathologic differential diagnosis of balloon cell UM mainly includes ocular metastasis from clear cell neoplasms  and balloon cell choroidal nevus. In the present case, the former has been excluded based on the absence of other primary tumors on total body CT, but it could also be excluded by the immunohistochemical positivity for melanocytic markers, such as SOX10, Melan-A, S-100, and HBM-45, whereas the latter has not been taken into account due to the presence of overtly malignant histopathologic features.Functional MRI techniques, such as DWI with quantitative measurement of ADC, could be a useful diagnostic tool , whereas


Objective: This study aimed to detect serum vitamin D (VitD) levels in patients with primary restless legs syndrome (RLS). The further objective was to analyze the relationship of VitD levels with the severity of RLS symptoms, sleep, anxiety, and depression.Methods: The serum 25-hydroxyvitamin D [25(OH)D] levels of 57 patients with primary RLS and the healthy physical examinees in our hospital during the same period were detected. The International Restless Legs Syndrome Study Group (IRLSSG) rating scale for measuring RLS severity and Pittsburgh Sleep Quality Index (PSQI) Scale, 24-item Hamilton Depression Rating Scale (HAMD24), and 14-item Hamilton Anxiety Scale (HAMA14) were used to assess the severity of symptoms, sleep, and emotional state of patients with RLS. Based on VitD level and IRLSSG score, they were grouped for analysis.Results: The serum 25(OH)D level was significantly lower in patients with RLS than in healthy controls, and the incidence of insufficient serum VitD levels was significantly higher in patients with RLS than in healthy people (both P < 0.05). The serum VitD level was significantly lower in (extremely) severe patients with RLS than in mild to moderate patients with RLS (P < 0.05). The IRLSSG scale score and HAMD24 score were significantly higher in patients with RLS with insufficient serum VitD levels than those with normal serum VitD levels (both P < 0.05). Correlation analysis of IRLSSG scale score with serum VitD level and each scale score in patients with RLS showed that IRLSSG scale score was negatively correlated with VitD level, but positively correlated with PSQI, HAMA14, and HAMD24 scores. The results of correlation analysis between serum VitD levels and each scale score in patients with RLS indicated that serum VitD levels were negatively correlated with IRLSSG scale scores, PSQI scores, and HAMD24 scores.Conclusion: The serum VitD level is generally lower in patients with RLS than in healthy people, and lower serum VitD level is associated with more severe symptoms of RLS, worse quality of sleep, and worse depression. Restless legs syndrome (RLS) is one of the most common sleep-related sensorimotor disorders of the central nervous system, which is mainly manifested as indescribable limb discomfort, such as “prickly or stinging sensation,” “creeping sensation,” and “burning sensation” in the resting state, especially before going to bed or during quiet rest. It can occur on one side or both sides of the limbs, specifically in the lower limbs. The above limb discomfort symptoms are usually unbearable, forcing patients to move or massage their limbs to relieve the discomfort symptoms . The preAt present, relevant studies mainly focus on brain iron deficiency, neurotransmitter regulation disorders including dopaminergic system, and genetic factors. Brain iron deficiency is considered to be the key pathogenesis, which is jointly affected by environmental factors and genetic inheritance . VitaminRecent studies have reported –8 the prA total of 57 primary patients with RLS that were treated in the Department of Neurology, the First Hospital of Hebei Medical University from October 2019 to October 2020 were enrolled in this study. Another 57 age- and gender-matched healthy people who underwent physical examinations during the same period were selected as the control group.Patients that conformed to the diagnostic criteria of RLS. The consensus by the International Restless Legs Syndrome Study Group (IRLSSG) in 2014 proposed new diagnostic criteria of RLS/Willis-Ekbom disease (WES) .(1) RLS secondary to chronic kidney disease, renal failure, iron deficiency anemia, pregnancy, peripheral neuropathy, Parkinson's disease, and Parkinson's syndrome, drug-induced factors, including taking anti-depressants or sedatives and sleeping pills, familial and genetic factors; (2) other sleep disorders such as rapid eye movement sleep behavior disorder, sleep apnea syndrome, etc.; (3) metabolic syndrome, osteoporosis, cerebrovascular disease, respiratory diseases, malignant tumors, thyroid diseases, various mental diseases, liver and kidney diseases, and other diseases that affected serum VitD levels; (4) patients taking calcium, calcidiol capsules, and other drugs that affected serum VitD levels or drugs that alleviated RLS; (5) pregnant and lactating women. This study was approved by the Ethics Committee of First Hospital of Hebei Medical University, and written informed consent was obtained from the enrolled patients.The information such as the name, gender, age, chief complaint, present medical history, and previous underlying diseases of the patients and healthy controls was collected.24), and 14-item Hamilton Anxiety Scale (HAMA14).Professional physicians inquired about RLS symptoms, severity, sleep quality, anxiety, and depression of patients with RLS, and conducted corresponding scale assessments, including the IRLSSG rating scale for measuring RLS severity, Pittsburgh Sleep Quality Index (PSQI) scale, 24-item Hamilton Depression Rating Scale D] levels.According to the serum 25(OH)D level of patients with primary RLS, they were divided into normal VitD level [25(OH)D ≥ 20 ng/ml] group and insufficient VitD level [25(OH)D <20 ng/ml] group. According to the severity of limb discomfort , 11, patSD, and the independent sample t-test was used for the comparison between groups. The measurement data of non-normal distribution were expressed as the median and interquartile range, and the Mann-Whitney U-test was used for comparison between groups. The measurement data were expressed by the number of cases (percentage) method, and the comparison between groups was performed by χ2-tests. Pearson correlation analysis was used for analysis between serum VitD level and IRLSSG scale score. P < 0.05 indicated that the difference was statistically significant.The statistical software SPSS 25  was used for statistical analysis. The measurement data conformed to normal distribution were expressed as the mean ± P > 0.05). The serum VitD level was lower in patients with RLS than in the healthy controls, and the incidence of insufficient serum VitD level was significantly higher in primary patients with RLS than in healthy people (both P < 0.05)  and 38 women (66.67%). The ratio of men to women patients with RLS was 1:2. The age ranged from 15 to 78 years, with a mean age of (57.56 ± 13) years. A total of 57 cases were enrolled in the healthy control group, including 17 men (29.82%) and 40 women (70.18%), aged 18–79 years, with a mean age of (57.68 ± 12.9) years. No significant difference was found in age and gender between the two groups (.05) see .P > 0.05). The serum VitD level of (extremely) was significantly lower in severe patients with RLS than in mild to moderate patients with RLS (P < 0.05)  had mild to moderate RLS, including 12 men and 24 women aged 31–74 years, with a mean age of (56.81 ± 12.33) years. There were 21 (36.84%) (extremely) severe patients with RLS, including 7 men and 14 women aged 15–78 years, with a mean age of (58.86 ± 14.28) years. No significant difference was found in gender and age between the two groups (.05) see .P > 0.05). The IRLSSG scale score and HAMD24 score of patients with RLS were significantly higher in the insufficient serum VitD level group than in the normal serum VitD level group (both P < 0.05)  were in the normal VitD level group, including 5 men and 6 women aged 38–69 years, with a mean age of (59.36 ± 9.1) years; 46 patients (80.7%) were in the VitD level group, including 14 men and 32 women aged 15–78 years, with a mean age of (57.13 ± 13.81) years. There was no statistical difference in gender and age between the two groups (.05) see .r = −0.395, P = 0.002), but positively correlated with PSQI score, HAMA14 score, and HAMD24 score   see .24 score   see .P < 0.05); patients with RLS with VitD deficiency had more frequent symptoms of limb discomfort. Almeneessier et al. . This study also found that patients with primary RLS had low serum VitD levels, and the incidence was higher in patients with insufficient VitD levels than in healthy people.Vitamin D participates in the synthesis and release of neurotransmitters, and plays an anti-inflammatory, anti-oxidant, and neuroprotective role, exerting an important effect on regulating the occurrence and development of sleep, anxiety, and depression , 13. Vitr et al.  studied S showed  that ther = −0.47, P = 0.01). In this study, the ratio of men to women patients with RLS was 1:2. However, there was no difference in VitD levels between male and female patients, which might be related to the small sample size in this study.Restless leg syndrome occurs at any age, but the prevalence of RLS is higher in women than in men; and the incidence of RLS in women is approximately twice that of the general population , 17, whiP = 0.002). This preliminary study revealed that drug supplementation with VitD could ameliorate the severity of RLS symptoms. Tutuncu et al. (P < 0.001), the impact on sleep (P < 0.001), the assessment of symptoms (P = 0.002), and the assessment of the impact of disease on life (P < 0.001). There was a trend of improvement in the two sub-items of seizure frequency (P = 0.11) and mood (P = 0.051). Nonetheless, several opposite conclusions were also drawn from this study. Wali et al. (Serum VitD levels were lower in patients with (extremely) severe RLS than in those with mild to moderate RLS, which were negatively correlated with IRLSSG scores. The results of this study suggested that more severe symptoms of limb discomfort in patients with RLS were associated with lower serum VitD levels. Therefore, it is controversial whether correcting insufficient serum VitD levels can improve the symptoms of limb discomfort in patients with RLS. Wali et al.  recommenu et al.  conductei et al.  conducteP < 0.05). Turk et al. (P < 0.05). The probability of depression in patients with RLS was 2.5–5 times higher than that in patients without RLS, and the probability of depression and the severity of insomnia both increased with the worsened severity of RLS symptoms (Patients with RLS often experience various discomforts such as limb pain, swelling, burning, creeping sensation, which can lead to difficulty falling asleep and sleep interruption. Patients with RLS may suffer daytime sleepiness, fatigue, inattention, and decreased interest, depression, irritability, and other anxiety and depression emotions, affecting the quality of sleep and quality of life of patients , 23–25. k et al.  reportedk et al. . Yilmaz k et al.  found thsymptoms . This stIn conclusion, VitD deficiency is more common in patients with RLS. The lower the serum VitD level in patients with RLS, the more severe the symptoms of limb discomfort in patients with RLS, the worse the quality of sleep, and the worse the depression. Clinicians should pay attention to the detection of serum VitD levels and the evaluation of anxiety and depression during the diagnosis and treatment of RLS. However, whether VitD deficiency will cause the occurrence of RLS and the aggravation of symptoms as well as their causal relationship remains to be further studied. Conventional drug supplemented with VitD as an intervention for RLS is still controversial, and a large number of long-term prospective cohort studies and clinical trials are needed to confirm the findings. However, dietary supplementation with VitD, regular exposure to sunlight, and outdoor exercise is recommended for patients with RLS. Given that this is a clinical retrospective study with a small sample size, multi-center, large-sample clinical studies are needed in the future to further improve placebo-controlled study and explore the changes in RLS symptoms, mood, and sleep quality before and after supplementation with VitD. In our next study, we will aim to analyze the correlation between vitD level and RLS symptoms by age.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the First Hospital of Hebei Medical University. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The symbiosis in trypanosomatids is a mutualistic relationship characterized by extensive metabolic exchanges between the bacterium and the protozoan. The symbiotic bacterium can complete host essential metabolic pathways, such as those for heme, amino acid, and vitamin production. Experimental assays indicate that the symbiont acquires phospholipids from the host trypanosomatid, especially phosphatidylcholine, which is often present in bacteria that have a close association with eukaryotic cells. In this work, an in-silico study was performed to find genes involved in the glycerophospholipid (GPL) production of Symbiont Harboring Trypanosomatids (SHTs) and their respective bacteria, also extending the search for trypanosomatids that naturally do not have symbionts. Results showed that most genes for GPL synthesis are only present in the SHT. The bacterium has an exclusive sequence related to phosphatidylglycerol production and contains genes for phosphatidic acid production, which may enhance SHT phosphatidic acid production. Phylogenetic data did not indicate gene transfers from the bacterium to the SHT nucleus, proposing that enzymes participating in GPL route have eukaryotic characteristics. Taken together, our data indicate that, differently from other metabolic pathways described so far, the symbiont contributes little to the production of GPLs and acquires most of these molecules from the SHT. Angomonas, Kentomonas, and Strigomonas genera (strigomonads) evolved through a mutualistic relationship with an intracellular bacterium referred to as Trypanosomatid Proteobacterial Endosymbiont (TPE), belonging to the Candidatus Kinetoplastibacterium genus -orthophosphate. However, the PC amounts in the free symbiont are markedly reduced when compared to those measured in the intracellular bacterium, thus indicating that this prokaryote may obtain most of its PC from the host trypanosomatid [In fact, the endosymbiosis in trypanosomatids has been characterized by two-way metabolic changes. In this sense, the TPE enhances the mitochondrion’s oxidative phosphorylation and induces an increase in energy production by making the metabolic link between glycosomes and the mitochondrion. The result of the interaction between these three cell structures is the increased production but also the higher consumption of ATP. There are indications that this integrated metabolism benefits the bacterium, which is able to use part of the generated metabolites to build its own carbon skeleton ,22. The osomatid .Trypanosoma and Leishmania genera, PC and phosphatidylethanolamine (PE) are present in higher amounts, followed by phosphatidylinositol (PI), while phosphatidylserine (PS), phosphatidylglycerol (PG), and cardiolipin (CL) are present in minor quantities [Angomonas deanei, biochemical analysis showed that the major phospholipids are PC and PE, however PI and CL are present in similar quantities that correspond to those found in RTs [The glycerophospholipids (GPL) are amphipathic molecules composed by hydrophilic head groups linked via phosphate to glycerol-bound fatty acid or fatty alcohol chains. GPLs are present in both eukaryotic and prokaryotic cells and have structural and metabolic functions. They play essential roles as membrane constituents, regulating protein structure and function as well as the formation of specialized membrane domains ,25. Furtantities ,32,33,34d in RTs .A. deanei presents CL as the major phospholipid, followed by equal amounts of PC and PE and minor quantities of PI [A. deanei, genome analyses and phylogenetic data showed that sequences for PI biosynthesis were absent in the symbiont, thus indicating that the bacterium depends on the host to obtain PI [The symbiotic bacterium of es of PI . PC is ues of PI ,39,40,41es of PI ,43. In Abtain PI .In this work, we performed in-silico identification and phylogenetic analysis of the genes involved in glycerophospholipid synthesis considering strigomonad SHTs and their respective symbionts as well as other members of the family that naturally do not contain the symbiotic bacterium. The symbiont but not the host trypanosomatid has the gene that converts phosphatidylglycerol phosphate (PGP) to PG. Furthermore, the bacterium contains genes that may enhance the host production of phosphatidic acid (PA), which is essential for the construction of glycerophospholipids. Such sequences are also present in SHTs and have eukaryotic origin. However, most genes of the metabolic pathways using dihydroxyacetone phosphate (DHAP) and glycerol-3-phosphate (G3P) to produce PS, PE, PG, and CL are complete in the host trypanosomatid genome but absent in the symbiont. Here, our genomic data indicate that the symbiont seems to depend on the host cell for obtaining most glycerophospholipids that constitute its membranes.Bordetella species. Sequences for lysophosphatidate acyltransferase and PA citidyltransferase , but a few of them have more ambiguous scenarios.Bodo saltans, plus a few other eukaryotes  of 70. The few other eukaryotes clustering with the trypanosomatids with high BSV (100) are from diverse parts of the tree of eukaryotes: Vitrella brassicaformis (Alveolata), Ectocarpus silicosus , and Sphaeroforma antarctica .G3P dehydrogenase (1.1.1.8 and 1.1.5.3) was found in many trypanosomatids as well as The sister group of these eukaryotes in the tree, with BSV of 86, is composed of one unclassified Proteobacteria and one unclassified Deltaproteobacteria. All other nearby clades in the tree present very low statistical support and therefore do not allow for any confident definition of a more exact placement of this trypanosomatid enzyme in the tree. The only other eukaryotes present in the 1.1.1.8 tree are a group of three green algae; they are relatively near the trypanosomatid clade, but with very low BSV; therefore, their placement is not conclusive.Trypanosoma, Bodo, and Perkinsela; and a smaller one, comprised of mostly Leishmania but also containing one T. theileri and a few Leptomonas, Strigomonas, and Angomonas sequences. Since the two clades are located in two different subtrees separated by a long branch, it is possible that the two clades are either different families performing the same function (G3P oxidase) or different but related functions, and hence, the sequence similarity allows for the selection and alignment of both groups. Closer inspection of RPSBLAST search results (against the Conserved Domain Database of NCBI) representative couples, one eukaryotic and one bacterial, from each of these two clades, suggests these are indeed two separate families of 2-hydroxyacid phosphoglycerate dehydrogenases since the domains identified have the same functions but different sequences (data not shown).The tree for G3P oxidase  presentsA. deanei and S. culicis sequences as well as one Leptomonas pyrrhocoris sequence) as an outgroup to all other Kinetoplastida sequences, including B. saltans and Perkinsela sp.It is interesting to notice that the large clade contains two groups of Strigomonadinae sequences, one in their expected placement near the Leishmaniinae and another (composed of just Pseudocohnilembus and Oxytricha) and one dinoflagellate (Symbiodinium), with BSV of 58. More distantly, and with insignificant statistical support, there is a small group of Apicomplexa, plants, and Stramenopiles intermingled with Bacteria, mostly Deltaproteobacteria and Acidobacteria. The fourth and final clade containing Eukaryota is in the smaller subtree, and it does not cluster close to the trypanosomatids in that subtree.As seen for the G3P dehydrogenase, the G3P oxidase tree is mainly composed of enzymes from bacteria , being the most represented groups, but there are more eukaryotic groups present. In the large clade, the Eukaryota are near each other although with very low BSV, which does not allow for confident placement. The sister group of the Kinetoplastida clade in this subtree is comprised of two Ciliates  or G3P oxidase (1.1.3.21)) to produce G3P, followed by addiction of acyl chains to G3P molecule by G3P acyltransferase to produce PA , which uses DHAP for G3P synthesis, indicating the relevance of G3P to the bacterium and corroborating data on the ability of SHTs to ferment. Such protozoans prioritize carbohydrate consumption in glucose-enriched media, producing large amounts of G3P when oxidative phosphorylation is inhibited by KCN . In the Symbiodinium endosymbiont has the ability to influence the composition of PA fatty acids found in lipid bodies and other host structures [PA production also seems to be possible in symbiotic SHT bacteria since the gene of a G3P acyltransferase (2.3.1.51), which converts Lyso-PA to PA, is present. As stated earlier, in corals, the ructures . It is nructures ,64.T. cruzi and Plasmodium falciparum [DAG is used to produce PC and PE by the Kennedy pathway through a condensation reaction with the radical, choline, or ethanolamine, respectively. An assumption is that this pathway, present in the ER, uses exogenous Lyso-PE or Lyso-PC so that the trypanosomatid could obtain ethanolamine or choline from the external environment. Genes for enzymes involved in PE and PC production showed high similarities between protozoa species, such as lciparum ,66. GeneA. deanei. It is possible that this single enzyme is catalyzing the three methylations, thus producing PC, as described in L. major, which contains the LmjPEM2 gene [The Greenberg pathway is also present in trypanosomatids; in this case, three successive PE methylations generate PC, the major GPL of eukaryotic cell membranes. Although it is known that in eukaryotes, more than one PE N-methyltransferase (2.1.1.17) participates in this route, we have identified only one gene for this enzyme in EM2 gene . In striIn addition to DAG, PA is also a starting point for GPL production. The first step is the conversion of PA to CDP-DAG by the enzyme PA cytidylyltransferase (2.7.7.41). From there, CDP-DAG can be used to produce PS by the enzyme CDP-DAG-serine phosphatidyltransferase (2.7.8.8), whose gene has been identified in the trypanosomatids and symbionts analyzed in this work. The presence of this sequence suggests that the PS production pathway occurs in the protozoan ER. In addition, we have also identified the enzymes PS synthase 2 (2.7.8.29) and PS decarboxylase (4.1.1.65) genes, whose activities result in the conversion of PS to PE and vice versa, thus regulating the cellular levels of such GPLs. Interestingly, the enzyme PS decarboxylase (4.1.1.65) is located in mitochondria, whereas enzymes CDP-DAG-serine phosphatidyltransferase (2.7.8.8) and PS synthase 2 (2.7.8.29) are located in the ER . It has Another important point to discuss concerns the possible influence of the symbiont on CL production, a GPL that confers high permeability selectivity in membranes. In eukaryotes, CL is present in great amounts in the mitochondrial inner membrane, and its biosynthesis is the result of a biochemical reaction promoted by phosphatidyltransferase, also called as cardiolipin synthase (CLS), a phosphatidyltransferase that condenses PG with the Lyso-PA grouping of a CDP-DAG molecule . In prokBodo saltans, and few other eukaryotic species. The observation that eukaryotes present in the G3P dehydrogenase tree are clustering near each other and, without any sign of artifacts, such as long-branch attraction, suggests that they inherited the enzyme from a common ancestor, while all other eukaryotes lost this particular orthologue. It is not clear why so few and so distantly related groups would retain this version of the enzyme. Another interesting point was the phylogenetic analysis for enzymes G3P oxidase (1.1.3.21) and PA cytidylyltransferase (2.7.7.41) since they grouped trypanosomatids in two clades that are located in two different subtrees separated by a long branch. It is possible that the two clades are either different families performing the same enzyme function or distinct but related functions. Phylogenetic analyses showed that enzymes involved in GPL synthesis positioned symbionts of trypanosomatids as a basal group in the Alcaligenaceae family. Sequences for CDP-DAG and PG production showed a common ancestor for TPEs and bacteria of the Taylorella genus, which is in accordance to previous evolutionary data using genomic and phylogenomic analysis [Phylogenetic analyses revealed that most of the trypanosomatid enzymes involved in phospholipid synthesis analyzed here presented no sign of horizontal gene transfer, but a few of them have more ambiguous scenarios. The enzyme G3P dehydrogenase (1.1.1.8) was found in trypanosomatids, in se 2.7.7.1 since tEndosymbionts of trypanosomatids have a very small but highly functional genome . PreviouA. deanei that gene expression can be modulated by RNA interference and that the CRISPRCas9 gene deletion system is active in this protozoan [In-silico data generated here reinforce the idea previously obtained through biochemical analysis that the bacterium GPLs are mainly obtained from the host protozoan. Such results are relevant since they suggest that this phenomenon occurs in different species of strigomonads. More recently, it was shown in rotozoan ,75. ThusA. deanei and its symbiont, named Ca. K. crithidii, presented the target genes, we first searched the sequences by EC number in L. major in the TriTrypDB database [Escherichia coli in NCBI’s non-redundant (nr) protein database. Then, we searched the A. deanei and Ca. K. crithidii genomes for these sequences. and each sequence identified was used to create one dataset.For genomic analysis, protein sequences involved in GPL synthesis were identified by Enzyme Commission (EC) number , collectdatabase  and Esch−10.The strigomonad SHT and RT datasets, identified by EC number, are the results of BLASTP searches against the NCBI’s nr database, collecting up to five hundred (for less conserved sequences) to a thousand sequences (for more conserved ones) that passed the maximum threshold E-value of 1 × 10For TPEs, 18 GPL enzyme sequence datasets were created using Betaproteobacteria as reference: six were found in at least two TPEs and were used for phylogenetic analysis, whereas the other 12 sequences could not be used for this purpose . For tryFor each bacterial dataset, multiple sequence alignments (MSA) were performed with Mafft v.7.313 , and evohttps://inkscape.org/, accessed on 14 October 2021).For the eukaryotic datasets, protein alignments were performed using MUSCLE v. 3.8.31 , and phy


Mage = 12.30, SD = 1.74) completed a battery of self-report questionnaires regarding relative deprivation, prosocial tendencies, perceived social support, in-group identity, and demographic variables. The results indicated that relative deprivation was negatively correlated with migrant children's prosocial tendencies and this connection was partially mediated by perceived social support. Moderated mediation analysis further indicated that in-group identity moderated the effect of perceived social support on prosocial tendencies, with a high level of in-group identity strengthening the positive association between perceived social support and prosocial tendencies. Parents, educators, and other members of society concerned about migrant children's psychosocial adaptation should provide adequate social support resources and help them foster positive in-group identity to migrant populations to mitigate the adverse effects of relative deprivation and promote their prosocial tendencies.As a particularly vulnerable group, children from rural areas in China whose families migrate to urban areas often encounter social exclusion, prejudice, and discrimination as they adjust to city life. Hence, migrant children may experience a sense of relative deprivation when they feel they are treated unjustly when compared to their urban counterparts. Although previous research has demonstrated that relative deprivation is a risk factor for prosocial tendencies, this association has not yet been examined in the population of migrant children in China. Further, few studies have revealed the mediating and moderating mechanisms between relative deprivation and prosocial tendencies. Therefore, this study constructed an integrated model examining the possible mediating role of perceived social support and moderating role of in-group identity on the association between relative deprivation and prosocial tendencies. A large sample of 1,630 Chinese rural-to-urban migrant children (845 girls;  There were 246 (15.1%), 899 (55.1%), and 485 (29.8%) participants, whose length of residence in the city was <3 years, between 3 and 8 years, and over 8 years, respectively. In terms of the educational background of migrant children's parents, 357 (21.9%) reported that their fathers had primary school education or below, 758 (46.5%) reported junior high school education, 383(23.5%) reported senior high school education, and 132 (8.1%) reported college education or above. In addition, 574 (35.2%) of their mothers had primary school education or below, 673 (41.3%) had junior high school education, 275 (16.9%) had senior high school education, and 108 (6.6%) had college education or above. Regarding the family economic statuses, 284 (17.4%) of the participants had an average monthly household income <2,000 yuan, 731 (44.9%) between 2,000 and 5,000 yuan, and 615 (37.7%) had more than 5,000 yuan.In the present study, Chinese rural-to-urban migrant children were sampled from three coastal cities in southeast China: Fuzhou, Xiamen, and Quanzhou. From each city, we adopted the convenience sampling method to select target schools, namely, one primary school and one junior high school. In the target schools, we used a cluster random sampling method to choose 4th−6th graders from primary school and 7th−9th graders from junior high school. The eligibility criteria for migrant children included the following: (1) there must be no This study was approved by the Ethics Committee for Psychological Research at the corresponding author's institution. All participants, as well as their parents and teachers, provided informed consent before the survey. Participants were asked to complete all of the items regarding relative deprivation, prosocial tendencies, perceived social support, and in-group identity and were informed that their participation would be voluntary, and responses would remain confidential.Participants completed a brief demographic form that included background information on their age, gender, education level, length of residence in the city, parental education, and monthly household income.How do you think of your family economic status when compared with your urban counterparts?” and “How satisfied are you with this situation?”), measuring five aspects of migrant children's current situation . The 7-point Likert scale items of the cognitive dimension range from 1 (very good) to 7 (very bad), and the items of the emotional dimension range from 1 (very satisfied) to 7 (extremely unsatisfied); higher scores indicate higher levels of relative deprivation. This scale has been used in previous studies with good reliability and validity . In the present study, Cronbach's α for the scale was 0.92.The Relative Deprivation Scale for Migrant Children . This instrument has 26 items and uses a 5-point Likert scale ranging from 1  to 5 (describes me very much); higher scores indicate higher levels of prosocial tendencies. In the current study, Cronbach's α for the scale was 0.94. Structural validity  was in line with the standards of psychometrics.The Chinese version of the Prosocial Tendencies Measure  and instrumental support . Participants answered 18 items on a 5-point Likert scale ranging from 1 (nobody) to 5 (many people). The higher the scores, the more social support migrant children might receive in those cities. In the present study, Cronbach's α for the scale was 0.96. Structural validity  was in line with the standards of psychometrics.The Chinese version of the Perceived Social Support Scale ”] and cognitive identity . Each item was rated on a 6-point Likert scale ranging from 1 (strongly disagree) to 6 (strongly agree). The average score was taken as the index of in-group identity, with higher scores indicating a stronger identification with their inner group . In the current study, Cronbach's α for the scale was 0.90. Structural validity  was in line with the standards of psychometrics.Migrant children's group identities were measured using the Chinese version of the In-group Identification Scale Phinney, . The scaDescriptive statistics and correlation analyses were first conducted using the Statistical Package for the Social Sciences (SPSS) version 22.0. Then, we employed the SPSS macro PROCESS, developed by Hayes , to examThe potential common method bias effect was examined by using Harman's single factor test for all of the research items , perceived social support , and in-group identity . Perceived social support was positively correlated with prosocial tendencies  and in-group identity . In-group identity was also positively correlated with prosocial tendencies . These results were consistent with our expectations and supported hypothesis H1.The results of the descriptive statistics and correlation analyses are presented in B = −0.29, p < 0.001), and perceived social support positively predicted prosocial tendencies . The effect of relative deprivation on prosocial tendencies was also significant , which suggests that perceived social support partially mediated the link between relative deprivation and prosocial tendencies ; the bootstrap 95% confidence interval for the mediating effect of perceived social support did not contain 0. The mediation effect accounted for 62.5% of the total effect. Thus, hypothesis H2 was supported.As shown in Table 2Summary of mediation results.B = 0.07, p < 0.001; see M –SD, M, and M + SD. As shown in simple slope = 0.20, t = 7.27, p < 0.001). However, for participants with lower levels of in-group identity, perceived social support had no positive predictive effect on prosocial tendencies , which indicates that the predictive effect of perceived social support on prosocial tendencies decreased with the decline of migrant children's in-group identities. Thus, hypothesis H3 was supported.Table 3Moderated mediation analysis results with in-group identity as a moderator.We constructed a moderated mediation model to analyze the psychological mechanisms underlying the relationship between relative deprivation and prosocial tendencies in migrant children. The results showed that perceived social support acted as a mediator, and in-group identity acted as a moderator on the relationship between relative deprivation and prosocial tendencies. Furthermore, the mediating effect of perceived social support was moderated by in-group identity.Consistent with previous studies  and/or dimensions of prosocial tendencies, gaining a more comprehensive picture of the correlations of prosocial tendencies in migrant children. Finally, to avoid ethical risks, this study only uses self-reporting methods to identify migrant children. Further, the study does not distinguish between different types of migrant children, such as those who follow their father, those who follow their mother, and those who follow their parents. Future research should combine subjective reporting with objective criteria and distinguish different types of migrant children, to further validate the findings of this study.The focus of most previous studies on relative deprivation has been its effects on undesirable or destructive psychological and behavioral outcomes, neglecting the potential effects of relative deprivation on positive psychosocial outcomes. Further, previous studies have focused less on a vulnerable under-researched group, namely, rural-to-urban migrant children in China. Hence, we constructed an integrated model to fill in the gaps by testing the mediating role of perceived social support and moderating role of in-group identity on the association between relative deprivation and prosocial tendencies in Chinese migrant children. Relative deprivation was significantly negatively correlated with prosocial tendencies, and this connection can be partially mediated by perceived social support. Moreover, in-group identity moderated the effect of perceived social support on prosocial tendencies, with a high level of in-group identity strengthening the positive association between perceived social support and prosocial tendencies. This study provides a new direction for the scientific training of migrant children's prosocial tendencies. Parents, educators, and others who are concerned about migrant children's psychosocial adaptation should provide adequate social support resources and help them foster positive in-group identity to migrant populations to mitigate the adverse effects of relative deprivation and promote their prosocial tendencies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee for Psychological Research at the corresponding author's institution. The participants and their legal guardian provided their written informed consent to participate in this study.MX conceived and designed the study, performed the survey, and authored and reviewed drafts of the paper. LX analyzed the data, prepared figures and tables, and wrote it into the article. YY conceived and designed the study. All authors were involved in developing, editing, reviewing, and providing feedback for this manuscript and have given approval of the final version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GFAP gene. Recurrence in siblings from healthy parents provides a confirmation to the transmission of variants through germinal mosaicism. With the use of DNA isolated from peripheral blood, next-generation sequencing (NGS) of GFAP locus was performed with deep coverage (≥500×) in 11 probands and their parents (trios) with probands heterozygous for apparently de novo GFAP mutations. Indeed, one parent had somatic mosaicism, estimated in the range of 8.9%–16%, for the mutant allele transmitted to the affected sibling. Parental germline mosaicism deserves attention, as it is critical in assessing the risk of recurrence in families with Alexander disease.Alexander disease is a leukodystrophy caused by heterozygous mutations of  It is emponents . To dateosaicism   technology can facilitate the detection of low-level mosaicism, undetectable by conventional Sanger sequencing , deep coescribed , using DGFAP gene containing variants under analysis, namely, p.E75K, p.L76P, p.R79C/H , and p.R239C . The parameters for the variant calling were configured in the custom mode recommended for detecting mosaicisms .The genomic region included in the experimental design and sequenced is at chr17:4,2,982,001–42,997,000 (GRCh37/hg19), contains regulatory regions and UTRs, all nine exons, and flanking regions , is divided into 59 amplimers of 264 bases on average and has a final coverage of 95%, with 752 bases missing, mostly intronic. Our attention was focused on two amplimers of ®) and oligos suitable for the mutation site (p.R79C) on both strand , followed by capillary electrophoresis on automated DNA sequencer (ABI PRISM® 3130XL), according to a protocol already reported  following the manufacturer’s instructions. The plasmid DNA isolated from 51 clones was directly sequenced using the Sanger protocol, as already reported .de novo occurrence, all representing single instances of sibs having a mutation. In particular, the coverage of the amplicons containing the mutant codons is reported for each indicated patient’s parents (mother or father). All parents could be evaluated for possible mosaicism at the mutation site of corresponding patients, except the father of proband #147 (ID#147F), who had to be removed from the set because the coverage of the region, affected by the mutation present in his son, did not reach the minimum threshold established by the experimental design (500×).GFAP mutation already detected in her son, the c.236G>A (p.R79H). The ratios between mutant and total allele reads amount to 12.18%, which can be regarded as the extension of the mosaicism in blood cells for this mother. However, when the 12.18% value was compared with ratios obtained previously with known dilutions of the mutant allele of a heterozygous control, ID#180A (the patient of family #180) carrying the mutations GFAP c.227T>C (p.L76P), an estimate of the extension of mosaicism of 16% was achieved (The mother of proband #110 (ID#110M) revealed a mosaicism of achieved , a figurThe presence of a small proportion of mutant allele was confirmed through the single-nucleotide primer extension approach, evaluating the amount of the wild-type and mutant alleles obtained from ID#110 families by using both the F- and R-primers  and thenGFAP mutation c.236G>A (p.R79H). Overall, 51 colonies were obtained, 46 of which showed the presence of a wild-type G allele at the mutation site after Sanger sequencing. This allowed us to estimate a ratio of clones carrying mutant and wild-type inserts of 10% .Based on the different approaches applied and the comparably similar estimates obtained , it could therefore be concluded that ID#110M mother shows a rather low degree of mosaicism, although not irrelevant for A retrospective reanalysis, carried out on the Sanger sequences initially obtained from this mother’s DNA, showed a very small second/mutant peak allele underneath the reference allele, which however was not attributed to the mutant allele but rather to the background noise (data not shown).GFAP mutation that was detected in buccal DNA , but not in blood DNA, clearly arising in the embryo rather than in the parental germline  mutation, a genetic defect already shown in association with AxD and recurring in unrelated patients , and indeed genetic counseling and possibly prenatal testing during a second pregnancy are already recommended for families at risk of AxD recurrence, the discovery that the patient’s mother ID#110M is actually a mosaic has represented a confirmation for parents of their actual transmission hazard. Indeed, the discovery of germline mosaicism exposes this family to a much higher but unquantifiable risk of AxD recurrence: though the possibility that 100% of oocyte cells carrying this variant cannot be excluded, the real risk of recurrence in this family is presumably lower than the 50% risk typical of germline carriers (Finding a carriers .Although the search for parental somatic, and therefore germline, mosaicisms should make use, whenever possible, of tissues relevant to the disease, NGS with deep coverage at target sites of even less appropriate DNA sources may represent a fruitful approach to this type of investigation. Obviously, the test becomes informative only if a somatic mutation is found, while for subjects with negative results, mosaicism cannot be definitely excluded .The tools provided by NGS, and in particular the possibility of an in-depth analysis, allow for the detection of possible mosaicisms, thus providing safer and more informative genetic counseling, an opportunity that should be considered for many rare diseases, including AxD.