Principal coordinate analyses based on microbiota composition showed a separation by the DBPC response (p = 0.039). For Bacteroides (p = 0.05) and Parabacteroides (p = 0.007), the frequency of amplicon sequence variants was lower, and that for Blautia (p = 0.009) and Streptococcus (p = 0.004) was higher in NCGS individuals at multiple regression analyses. No difference in the mycobiota composition was detected between the groups. In conclusion, almost half of the individuals with suspected gluten sensitivity reported symptoms with placebo; they showed lower mental health scores, increased risk for eating disorders, and a different gut microbiota composition.The aims of this observational “proof-of-concept” study were to analyze the clinical/psychological characteristics and gut microbiota/mycobiota composition of individuals with suspected non-celiac gluten/wheat sensitivity (NCGS/WS) according to responses to the double-blind-placebo-controlled (DBPC) crossover gluten challenge test. Fifty individuals with suspected NCGS/WS were subjected to the DBPC challenge test; anthropometric measurements, psychometric questionnaires, and fecal samples were collected. Twenty-seven (54%) participants were gluten responsive (NCGS), and 23 were placebo responsive, with an order effect. NCGS individuals displayed a significantly lower risk of eating disorders and a higher mental health score when compared to placebo-responsive participants, confirmed by multiple logistic regression analyses (OR = 0.87; 95% CI 0.76–0.98,  Non-celiac gluten/wheat sensitivity (NCGS/WS) is a clinical entity characterized by intestinal and extraintestinal symptoms induced by gluten ingestion in the absence of wheat allergy (WA) or celiac disease (CD) ,2. CompoThe pathogenesis of NCGS/WS is still uncertain; a higher intestinal expression of toll-like receptors suggesting a stronger implication of the innate immune mechanisms, the infiltration of immune cells in the gut mucosa, increased expression of antibodies, and dysregulated gene expression profiling and epigenetic mechanisms have been described ,6,7,8,9.The aim of the present observational “proof-of-concept” study was to analyze the clinical and psychological characteristics and the gut microbiota and mycobiota of individuals seeking medical help for suspected NCGS/WS according to their response to the DBPC gluten challenge test.The individuals seeking medical assistance for any form of gluten/wheat intolerance at the outpatient gastroenterology and/or allergology clinics of the “Città della Salute e della Scienza” Hospital of Torino, were evaluated for enrollment, starting from January 2018 .Inclusion criteria included the following: adults (age ≥ 18 years), and gluten/wheat sensitivity demonstrated by the following self-reported conditions (i) intestinal and extraintestinal symptoms associated within hours or days after the ingestion of gluten-containing food, (ii) a clear benefit while they were on a gluten-free diet, (iii) relapse of symptoms with the ingestion of gluten-containing foods.H. pylori infection; lactose intolerance; history of gastrointestinal surgery; liver/pancreatic diseases; active or recent infectious diseases; known psychiatric diseases; any systemic diseases; pregnancy or breastfeeding; active use of antibiotics, probiotics, immunosuppressive drugs, non-steroidal anti-inflammatory drugs, or corticosteroids; CD in first-degree relatives; inability to give written informed consent.Exclusion criteria were as follows: presence of CD, WA, , and other gastrointestinal diseases; CD was diagnosed in the presence of CD compatible clinical symptoms, positive IgA tissue transglutaminase antibodies and/or endomysial antibodies, and villous atrophy of the duodenal mucosa according to the Marsh–Oberhuber criteria . WA was The first consecutive 60 individuals with possible NCGS/NCWS and without CD, WA, IBS, and the other exclusion criteria were enrolled for the present study.All the participants gave their written informed consent to participate. The local Ethics Committee for Human Research approved the study protocol.The primary outcome was evaluating the participants’ response to gluten by assessing their symptoms as measured by the modified version of the Gastrointestinal Symptom Rating Scale (GSRS), according to the Salerno Experts Criteria  after thSecondary outcomes were evaluating the clinical, psychological characteristics, and the gut microbiota and mycobiota composition of participants by their responses to the gluten challenge.Two registered, trained dieticians evaluated all patients at baseline, gave dietary recommendations, and checked diet compliance during the whole test. Participants were encouraged to immediately contact the dieticians by phone in case of any doubt or problem related to the diet.During the nutritional evaluation, anthropometrical parameters, nutritional status, and usual dietary patterns were evaluated. The same trained dieticians assessed the usual dietary intake before the experiment and checked every 2 weeks the adherence to the given dietary recommendations during the whole experiment  by collecting an accurate food history for each participant by a standardized interview.Weight was measured using a mechanical column scale  with the participants wearing light clothes and no shoes to the nearest 0.1 kg, height was measured to the nearest 0.1 cm with a stadiometer , and waist circumference was assessed using a plastic tape meter at the umbilicus level.Arterial blood pressure values were measured on the left arm, in a sitting position, after at least 10 min of rest, with a mercury sphygmomanometer with appropriate cuff sizes . Two measurements were taken with the arm supported at heart level, and the values reported were the means of the two.The first step included a period with a gluten-containing diet  for six weeks . The selThen, a six-week, gluten-free, low-FODMAPs diet was administered to all participants by the same dieticians, with detailed verbal and written explanations. During a 1 h session, the gluten-free diet was illustrated by the dietitians who provided oral and written recommendations, a list of allowed and unallowed foods and instructions on how to read the food labels. Patients were requested to avoid foods with a high content of FODMAPs, and a comprehensive list of foods with high and low FODMAPs content was supplied including fruits, vegetables, cereals, beverages, and condiments to be limited or consumed, respectively .The modified GSRS questionnaire was compiled during the last week of this second period. A decrease of at least 30% of the baseline score was considered a positive response. Diet adherence during the different phases of the study was checked by phone contact by the dieticians every 2 weeks. Adherence to the gluten-containing diet and to the gluten-free, low-FODMAPs diet, respectively, was considered adequate if at least 10 g/day gluten was consumed, and <10 mg/day gluten and <3 g/day FODMAPs were consumed based on the participant-reported diet .Out of the 60 enrolled participants, there were six dropouts because of unwillingness to continue the gluten-containing diet, two with acute conditions occurring during the first step and requiring drugs, and two who failed to show a symptomatic improvement with the gluten-free diet . TherefoDuring the following step, the participants continued the same diet  and the DBPC challenge test was performed: a one week challenge , followed by a one week washout, and then a further one week crossover challenge . Participants filled the above-described, modified GSRS questionnaire daily during the three weeks of the challenge. A variation of symptoms of at least 30% between gluten and placebo challenge discriminated a positive from a negative result .A pharmacist, with no clinical involvement in the study, prepared the randomization sequence, according to a computer-generated series. All participants and study team members were blinded throughout the study. The participants were given the capsules (gluten or placebo) in identical anonymous bottles, marked with the number corresponding to the order of consumption . Participants consumed for each of the two challenge weeks 10 capsules/day, ingested on no more than two occasions over the day, with a total daily amount of 5.6 g gluten, corresponding to the gluten content of 80 g pasta, in line with previous published protocols ,29. The Placebo capsules contained rice starch for a total daily amount of 5.6 g; it has been used by most protocols because of its low fermentable capacity due to its rapid absorption ,21,29. G–The Minnesota Leisure Time Physical Activity Questionnaire: the physical activity level was calculated as the product of the duration and frequency of each activity (in h/week), weighted by an estimate of the metabolic equivalent (MET) of the activity and summed for the activities performed .–The Hamilton rating scale for depression: mild depression was defined by scores ranging 8–17, moderate depression with scores ranging 18–24, and serious depression with scores >25 .–The Hamilton rating scale for anxiety: mild anxiety was defined by a score <17, mild-to-moderate by scores ranging 18–24, and moderate-to-severe by scores >25 .–ORTO-15 questionnaire: a 15 multiple-choice item tool investigating the obsessive attitude of the subjects in choosing, buying, preparing, and consuming food they consider to be healthy. A cutoff of 40 discriminates the presence or the absence of orthorexia .–Eating Attitude test (EAT) 26: an abbreviated 26-item version of the EAT-40 relate to attitudes, beliefs, and behaviors concerning food, weight, and body shape. A cutoff score of 20 or above denotes the presence of disturbed eating behavior .–Short-Form (SF)-12 health survey: to measure general health-related quality of life with different domains with scores ranging from 0 to 100  .Participants were administered the following questionnaires:Fecal samples were self-collected at the end of the first step, before the DBPC challenge test, by the participants, who were specifically instructed. All materials were provided in a convenient, refrigerated, specimen collection kit. Patients were provided with sterile containers to collect the feces . The fecal samples were self-collected at home and immediately transferred to the sterile sampling containers using a polypropylene spoon (3 spoons of about 10 g) and stored at 4 °C. The specimens were transported to the laboratory within 12 hours of collection at a refrigerated temperature. Containers were immediately stored at −80 °C for DNA extraction. No storage medium was used.Total DNAs from fecal samples were extracted using the RNeasy Power Microbiome KIT  following the manufacturer’s instructions. One microliter of RNase  was added to digest RNA in the DNA samples, with an incubation of 1 h at 37 °C. DNA was quantified using the QUBIT dsDNA Assay kit  and standardized at 5 ng/μL. Two microliters of each DNA was amplified for microbiota analysis by using primers and conditions for the amplification of the V3–V4 region of the 16S rRNA gene . The mycAfter sequencing, raw reads were analyzed by using the Quantitative Insights into Microbial Ecology (QIIME) 2 . PrimersThe data generated by sequencing were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and are available under the BioProject Accession Number PRJNA701870.Normally distributed variables were presented as mean ± standard deviation, while non-normally distributed variables were presented as median (range interquartile). The Kolmogorov–Smirnov test was used for the assessment of normality. Variables were compared by the t-Student test, chi-square test, or Mann–Whitney U test, as appropriate.QIIME2 diversity script was used to perform alpha and beta diversity analysis. A weighted UniFrac distance matrix was generated by QIIME2 and used both to build the principal coordinate analysis (PCoA) and to perform the permutational multivariate analysis of similarities (ANOSIM) by the “vegan” package in R environment. Box plots represented the interquartile range between the first and the third quartile, with the error bars showing the lowest and the highest value. The random forest model was performed by QIIME2 using the q2-sample-classifier plugin in order to identify variable importance by using the microbiota and mycobiota ASV tables. The receiver operating characteristic (ROC) curve and accuracy value (AUC) were calculated in order to verify the classification accuracy of a machine-learning model.Multiple logistic regression analyses were performed to evaluate the associations between the responses to the gluten challenge test (dependent variable) and the variables significantly different at univariate analyses, adjusted for age, gender, and education level. In case specific ASVs were not found in the gut microbiota of one of the two groups, standard errors and p-values were computed by a resampling method (the jackknife technique) .The adherence to the given dietary recommendations  was considered adequate for all the 50 enrolled participants. All the subjects took all the capsules, too. Median values of scores from the modified Gastrointestinal Symptom Rating Scale (GSRS) questionnaire were 12.0 during the gluten-containing diet and 4.0 during the gluten-free, low-FODMAPs diet.Most of participants were females, around 40 years old, with normal body mass index (BMI) and a high level of education . All disTwenty-seven out of fifty individuals (54%) had a symptomatic relapse during the gluten ingestion and were considered as having NCGS. Twenty-three out of fifty participants (46%) had a negative response and reported worsening of their symptoms after consuming the placebo. The median (interquartile range) of the scores of the reported symptoms after gluten and placebo in the two groups are reported in The most frequent gastrointestinal symptoms in NCGS were abdominal pain, bloating, and meteorism, while in individuals responsive to placebo they were bloating, abdominal distension, and meteorism. The extra-gastrointestinal symptoms were lesions of the oral cavity, dermatitis, and headache in gluten-responsive individuals, and headache, dermatitis, and pain in the limbs in placebo-responsive individuals .p = 0.048 with respect to the null hypothesis of 50%). Being exposed to gluten rather than placebo during the first week of the test was a predictive factor for receiving a final diagnosis of NCGS; in fact, among patients who were first assigned to gluten consumption, 17/25 (68%) received a diagnosis of NCGS, while among patients who were first assigned to placebo consumption, only 10/25 (40%) received a diagnosis of NCGS.Data suggested the presence of an order effect. In fact, overall, 32/50 patients (64%) reported worse symptoms during the first week compared to the third week , Bacteroides (10% and 16%), Dorea (9% and 11%), Collinsella (8% and 5%), Gemmiger (7% and 8%), Bifidobacterium (7% and 5%), R-Ruminococcus (7% and 6%), Faecalibacterium (6% and 9%), Akkermansia (6% and 7%), Eubacterium (5% and 4%), Eggerthella (4% and 2%), Alistipes (2% and 3%), Clostridium (2% and 1%), and L-Ruminococcus, Coprococcus, Romboutsia, and Methanobrevibacter (>1% in both groups) . A box plot at genus level showed a significant reduction in the frequency of Bacteroides, Dorea, and Parabacteroides and a significant increase of Blautia and Streptococcus in individuals with NCGS (Blautia was the predominant ASV accounting for differences in the gut microbiome between participants with positive and negative responses (AUC = 0.92). In a multiple logistic regression analysis, the associations between the response to the gluten challenge and the relative frequency of Blautia, Parabacteroides, and Streptococcus remained statistically significant  . Dialistis PCoA, , left ofith NCGS . By usinnificant .Saccharomyces (36% of the relative frequency and 38%), Pichia (20% and 19%), Cyberlindnera (6% and 3%), Meyerozyma (5% and 7%), Cladosporium (4% and 2%), Wickerhamomyces (3% and 1%), Geotrichum (3% and 2%), Candida (3% and 2%), Kurtzmaniella (2% and 0.2%), Galactomyces (2% and 1%), Malassezia (2% and 3%), Hanseniaspora (2% and 5%), Saitozyma (1% and 5%) and Kluyveromyces (1% and 0.05%)  . No sepad 0.05%) , left. Pryomyces . Kluyverobserved .Around half of the participants with a suspected intolerance to gluten/wheat reported worsening of their symptoms during the placebo consumption; those individuals showed an increased risk for eating disorders and a low mental health. Furthermore, an order effect was found, prompting a possible nocebo effect. Otherwise, the possibility that these subjects were intolerant to some other components of wheat and/or have a different form of hypersensitivity cannot be excluded.About half of our participants were gluten responsive, in line with previous studies reporting around one-third of positive responses to the DBPC gluten challenge ,20,21,22Our participants showed high scores for anxiety, depression, and orthorexia—the unhealthy obsession with healthy eating—in line with other studies ,15,17,20In patients with NCGS/WS, a systemic immune activation has been demonstrated; it was documented by increased serum levels of soluble CD14 and LPS-binding protein, as well as antibody reactivity to bacterial LPS and flagellin , enhanceActinobacillus (Gammaproteobacteria) and Finegoldia (Clostridia) and members of the Ruminococcaceae family were found in the duodenum and feces of NCGS/WS patients, without differences in α-diversity [Pseudomonas after four weeks of gluten-free diet was reported in these patients, and the authors hypothesized that some members of Pseudomonas can act as protective agents with low abundance prompting a higher sensitivity to dietary allergens [Roseburia and Faecalibacterium and a decrease in the Bacteroides, Blautia, Dorea, Coprococcus, and Collinsella genera, with an overall microbial profile with anti-inflammatory properties [Only a few studies have examined the gut microbiota of patients with NCGS/WS . Increasiversity . Furtherllergens . Higher llergens . Intrigullergens . Similaroperties . Bacteroides and Parabacteroides was in line with the beneficial changes observed in NCGS individuals only after the gluten-free diet [Bacteroides genus have been shown to promote beneficial anti-inflammatory and probiotic activities [Bacteroides has been correlated with the development of pathological conditions, such as CD and IBD [The evaluation of the microbiota of our participants was performed after 6 weeks of gluten-free, low-FODMAPs diet; therefore, we were not able to determine possible changes in its composition with respect to the gluten-containing diet, and our results are difficult to compare with other studies, where nutritional intakes have not been standardized. Nevertheless, a distinctive gut microbiota composition was evident between gluten-responsive and placebo-responsive individuals. In particular, the reduced relative abundance of ree diet , and theree diet . Literattivities ,56,57 or and IBD . Blautia in gluten-responsive participants, in line with what was reported in IBS patients [Blautia of utilizing the large gas amount produced in the intestine of these patients [Blautia, one of the major butyrate-producing ones in the human gut, is still unclear in patients with bowel diseases [We observed a significantly higher relative abundance of patients ,60, whicpatients . Indeed,diseases . Candida, Saccharomyces, Cladosporium, Penicillium, Pichia, Malassezia, and Aspergillus are the most abundant fungi in the gut [Saccharomyces, Candida, and Malassezia than reported in literature [Kluyveromyces and NCGS was almost threefold higher, though not statistically significant. Accordingly, Kluyveromyces was found to be one of the few fungi identified in pediatric patients with inflammatory bowel diseases [The human mycobiota is a neglected component of the microbiota; most species derive from diet, and a low gut abundance characterizes healthy individuals, while some fungi contribute to the aggravation of the inflammatory response in gastrointestinal diseases; in particular, fungal dysbiosis resulted in enhanced immune responses ,64,65. T the gut ,67. In oterature ,65,68; tterature ,68,69,70diseases .Around half of our participants reported symptoms during the consumption of the placebo. The differences in the reported symptoms between open (during the step 1 of the study) and blinded  gluten consumption were strongly in support of a nocebo effect in our individuals. The nocebo effect has been reported in up to 40% of patients with suspected NCGS in a metanalysis of 10 double-blind, placebo-controlled, gluten challenge trials, comprising 1312 adults . Among tOther possibilities should be considered. These individuals may be intolerant to other wheat components, such as amylase trypsin inhibitors (ATIs) or wheat germ agglutinin (WGA), as proposed by many authors ,2. ATIs The difference between the two groups suggested that gluten-sensitive and placebo-sensitive individuals were affected by two distinct conditions, with dysbiosis and a likely impaired immune response in the former, and a predominant nocebo effect in the latter. Recently, NCGS/WS has been considered as a multifactor-onset disorder, probably transient and preventable, related to quality and balance of the diet and not to the presence of gluten in itself . Caution in the interpretation of these findings is required owing to the low sample size. This was a proof-of-concept study and, therefore, no explicit estimations of the sample size were made at the time of study design; however, in a post hoc analysis, given the observed results and considering 0.05 as significance level, this study had a power of 82% for the detection of differences in the mental health score. Most of our participants were women (86%), but this finding is in line with the gender distribution reported in literature. We have evaluated one fecal sample only, when participants were on a gluten-free low-FODMAPs diet, and the human microbiota tends to be resilient, i.e., returns to its original state, after being subjected to a perturbation. Indeed, the careful standardization of the dietary habits of the participants for a long enough period (12 weeks) reasonably eliminated the potential interferences on the gut microbiota due to dietary differences between groups. The lack of a control group did not allow us to define the presence of dysbiosis in our participants. However, imposing six weeks of a gluten-free, low-FODMAPs diet to healthy controls, in order to avoid confounders in the comparison of microbiota compositions, would be unacceptable and ethically incorrect. Further strengths of the present study were the careful selection of participants, in order to exclude other gastrointestinal diseases or confounding conditions; the double-blind-placebo controlled test, by using indistinguishable capsules; the good level of participant compliance to the given dietary recommendations and test challenge; and the novelty of the mycobiota analysis, which has never been studied before in these individuals.In conclusion, around half of the individuals with suspected NCGS reported worsening of their symptoms during gluten consumption; they showed a reduced risk for eating disorders and a better mental health than placebo-responsive participants, prompting a possible nocebo effect in the latter. On the other hand, the gut microbiota composition of placebo-responsive individuals was different from that of gluten-responsive individuals. These results are worthy to be confirmed by further studies, testing in sequence different wheat components other than gluten.


Here we report on a pilot clinical study to evaluate the activity of an ATO-combined chemotherapy approach for the treatment of ARMS patients.Alveolar rhabdomyosarcoma (ARMS) is a subtype of rhabdomyosarcoma characterized by its aggressive behavior and poor prognosis, highlighting the need for novel treatment options. Arsenic trioxide (ATO) has been shown to specifically inhibit tumor growth and the metastasis of ARMS via an IV for 10 days combined with a chemotherapeutic regimen of vincristine, actinomycin, and cyclophosphamide (VAC regimen) on the third day, which was repeated every 21 days. A total of eight cycles of ATO-combined chemotherapy were applied throughout the entire scheme.We designed a therapeutic schedule of an ATO-combined chemotherapy, incorporating comprehensive management according to the Intergroup Rhabdomyosarcoma Study Group protocol. ATO was administered at 0.16 mg/kg per day over 8 h A total of three refractory/recurrent and one untreated ARMS patient, three male and one female, with a median age of 5.8 years , were enrolled in the present study. All patients were sensitive to combined chemotherapy with ATO and achieved partial or complete remission during treatment. Except for reversible gastrointestinal reaction and myelosuppression, no other adverse events were observed during the process of treatment.The combined chemotherapy of ATO and the VAC regimen exhibited beneficial activities against ARMS in pediatrics and was well tolerated, but prospective large-scale clinical trials are warranted to determine the long-term efficacy, optimal courses, and late toxicity in this population. Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in pediatrics, is a highly malignant mesenchymal tumor considered to originate from immature striated muscles. RMS can present as a heterogeneous array of differing histological, genetic, and clinical features, and the most recent World Health Organization classification scheme subdivides RMS into four subtypes: embryonal RMS, alveolar RMS (ARMS), pleomorphic RMS, and spindle cell/sclerosing RMS . ARMS acThe management of ARMS is facing a bottleneck, as chemotherapy still plays a critical role in the treatment of patients with ARMS. Presently, a combination of vincristine (VCR), actinomycin, and cyclophosphamide (VAC) remains the gold-standard protocol for ARMS therapy since few candidate drugs have shown superior durable objective response rates in clinical trials over the last few decades , 4. Recein vitro experiments have confirmed the anti-tumor effects of ATO in inhibiting tumor growth and the invasion of ARMS cell lines  signaling pathway is prevalent in ARMS tumors –7. Arsenll lines , 9. Furtding VCR . VCR is ding VCR . More sp3–2.0 μM . This su3–2.0 μM , 14, couThis was a case series pilot phase 2 clinical study. The eligible participants were ARMS patients under 18 years old without a major organ dysfunction. All patients received eight cycles of the ATO-combined chemotherapy incorporating comprehensive management according to the Intergroup Rhabdomyosarcoma Study Group protocol Figure 1Therapeutic response was evaluated at 4 weeks after the eight cycles of ATO-combined chemotherapy using the Response Evaluation Criteria in Solid Tumors, version 1.1. Adverse events were monitored and graded by the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0.In total, four ARMS patients, three male and one female, with a median age of 5.8 years , were enrolled in this study. The patient characteristics are summarized in via PET–CT, and the bone marrow examination suggested complete remission (CR). A total of eight courses of ATO treatment were performed, and the patient remained in CR for >7 months. By the end of a subsequent therapy without ATO, new tumor nodules in the brain were identified by PET–CT and magnetic resonance imaging (MRI), and the patient died after giving up treatment. The overall survival was 10.3 months from the beginning of ATO treatment.Patient 1 was a 5-year-old, male child who presented with a mass in the right inguinal and popliteal fossa and marked edema of the homolateral leg. The pathological diagnosis of ARMS with PAX-FOXO1 fusion-positive was defined using an excision biopsy of the right inguinal mass, while a large number of tumor cells were found in the bone marrow examination. A positron emission tomography–computed tomography (PET–CT) scan showed a large mass in the mediastinum and the right lower extremity, and distant metastases were found in the parietal pleura, pancreas, multiple bone lesions, celiac, and iliac lymph nodes. Chemotherapy was started with two courses of the VAC regimen, but this failed to alleviate oncothlipsis, with a <30% decrease in the sum of the diameters of the tumor lesions. Then, ATO was added in the third course of the VAC regimen. Notably, the edema disappeared soon after, and the superficial masses shrank by 83% within 3 weeks. Meanwhile, residual disease in the bone marrow converted negative. After four courses of the ATO-combined chemotherapy, tumor response was evaluated Patient 2 was diagnosed with ARMS when he was 2 years old with a single lesion in the left ala nasi. He had received 30 weeks of standard chemotherapy and midterm radiotherapy but exhibited a tumor recurrence in the left mandibular area. The patient once gave up further treatment after a radical operation. After 1 year, when he came back to the hospital, the general imaging examination showed the presence of a mandibular mass of 44 × 38 × 28 mm with multiple lymph node metastases in the bilateral carotid sheath Figure 3Patient 3 was a 12-year-old child with ARMS (89 × 6 × 60 mm) on the right forearm. ATO-combined chemotherapy was based on the results of core needle biopsy. As determined by MRI, the tumor volume was reduced by 98% after four courses of the novel regimen. Radical resection and proton radiotherapy were employed successively to eliminate the residual lesion, and four more courses of the ATO-combined chemotherapy were performed to consolidate its curative effect. Presently, the patient remains CR and sticks to the following consolidation therapy.Patient 4 was a 4-year-old girl who suffered from refractory and recurrent ARMS disease with distant metastases. Then, 2 years ago, she was diagnosed with a primary tumor on the left buttock, which was classed as stage 3 (T2bN0M0). She received first-line comprehensive therapy consisting of VAC and an ifosfamide-and-etoposide regimen but relapsed after radical operation and radiotherapy. Orienting for a possible targeted therapy, several gene amplifications were found by targeted NGS, including PIK3CA, MER proto-oncogene, tyrosine kinase, insulin-like growth factor 2, and GLI2. However, although a variety of second-line drugs, including vinorelbine, paclitaxel, nedaplatin, keytruda, pazopanib, anlotinib, temsirolimus, and everolimus, were tried successively, the primary tumor progressed, developing a distant metastasis in the process. At the time she was treated with the ATO-combined chemotherapy, large masses on the left buttock (66 × 46 × 29 mm) and the right back (33 × 26 × 18 mm), with metastasis to the orbicular mass 36 mm in diameter in the lung region, were observed. After eight cycles of ATO-combined chemotherapy, the tumor volumes of the primary lesion and metastases reduced by 64.7 and 59.2%, respectively. Due to the lack of other effective treatments, three more courses of ATO-combined chemotherapy were added, but the evaluation resulted in stable disease of the tumor. In the follow-up visit, the patient was transferred to another hospital by her own will and has now survived with the disease being stable.All patients exhibited grades II and III nausea and vomiting during treatment, but this was suitably controlled with antiemetic treatment and nutritional support. Myelosuppression occurred in every patient after each course of ATO-combined chemotherapy, and hematologic recovery was achieved at 2 to 3 weeks later. Grades III and IV neutropenia were the most common form of myelosuppression; two patients therein suffered from febrile neutropenia without focal infection. Chemotherapy-induced thrombocytopenia and anemia were mild with component blood transfusion. Except for these, no other adverse event was observed during the period of treatment.in vitro experiments, certain SMO inhibitors, such as cyclopamine, GDC-0449, LDE225, and HhA, have been preliminarily shown to exhibit a significant inhibitory effect on tumor growth and the metastasis of ARMS or transplanted tumor models (At present, the treatment of ARMS remains a difficult task in view of its unsatisfactory response to conventional treatment. Therapy is limited due to local tumor recurrence, development of metastases, and multidrug resistance. Patients with progressive or recurrent ARMS have a poorer prognosis, with a 5-year survival of <20% , 15. In r models . Howeverr models . Furtherr models , 19. Thuin vitro and in vivo by targeting overexpressed GLI1 (ATO is an old drug approved by the FDA for APL treatment and was later rediscovered as a Hh pathway inhibitor. The Hh pathway is an important signal transduction system for embryonic development, tissue regeneration, and homeostasis maintenance. The main signal axis of the canonical Hh pathway consists of the ligand protein SHH, the transmembrane receptor proteins PTCH and SMO, the suppressor of fused protein SUFU, and the nuclear transcription factor GLI. GLI regulates the expression of downstream genes involved in proliferation, angiogenesis, and stem cell self-renewal . Among tsed GLI1 . Boehme sed GLI1 . The antsed GLI1 . This insed GLI1 . Additiosed GLI1 . Hence, sed GLI1 Figure 5sed GLI1 . Therefosed GLI1 . As a coSince ATO has been used clinically for several decades, a variety of studies have demonstrated possible ATO-related toxicities. At pharmacological doses of ATO, expected toxicities of acute arsenic exposure include somatic symptoms and organ damage, which usually present in the gastrointestinal, hematological, cardiovascular, and nervous systems . When siOur study evaluated the activity and safety of ATO-combined chemotherapy with a VAC regimen in ARMS patients for the first time. As was shown in this pilot clinical trial, all of the four patients, including those with untreated and pretreated ARMS, were sensitive to ATO-combined chemotherapy and achieved partial remission or CR during treatment. It is noteworthy that patient 2 showed a continuous good response to the ATO-combined chemotherapy even though he exhibited repeated relapse. Although patient 1 died of relapse and patient 4 seemed to lose sensitivity to additional combined chemotherapy, these results do show that refractory patients are likely to benefit from ATO-combined chemotherapy with prolonged remission and survival times. Furthermore, ATO-combined chemotherapy with the VAC regimen seemed to cause no additional acute toxicity. From the details mentioned above, it is suggested that ATO, combined with the VAC regimen, exhibits beneficial activity against ARMS in pediatrics. There may be some limitations, such as small sample size, lack of a control group, and potential for selection bias in this study. Therefore, prospective large-scale clinical trials are required to determine the long-term efficacy, optimal course, and late toxicity of ARMS patients.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Sun Yat-Sen Memorial Hospital. Written informed consent to participate in this study and consent for publication of any potentially identifiable data or images were provided by the participants’ legal guardian/next of kinYL designed the study and reviewed the manuscript. XP performed the study and wrote the manuscript. XX and HL assisted in performing the experiments and in collecting the materials. CF, PW, CL, and WW supervised the study and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grant SYS-C-202007 from the Sun Yat-Sen Clinical Research Cultivating Program of Sun Yat-Sen Memorial Hospital.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.


SARS-CoV-2 is known to cause severe bilateral pneumonia and acute respiratory distress syndrome or COVID-19 in patients, which can be debilitating and even fatal. With no drugs or vaccines available yet, a wide range of treatment regimens used are being repurposed. The need of the hour is to analyze various currently available regimens and devise a treatment plan that is most effective for COVID-19. Here we describe the case of a 68-year-old man with hypertension and diabetes, exhibiting symptoms of cough and shortness of breath, who presented at the emergency department of our hospital. Chest computed tomography revealed bilateral ground glass opacities that were indicative of COVID-19, and a computed tomography score of 24 was indicative of severe pulmonary pneumonia. He tested positive for COVID-19. His treatment regimen included the use of convalescent plasma, oxygen therapy, steroids, high-dose antibiotics, broad-spectrum antiviral remdesivir, and anti–interleukin-6 monoclonal antibody (Tocilizumab) at various stages of the disease. Oxygen supplementation was required at the time of admission. The patient initially developed a cytokine release storm, and oxygen supplementation was initiated to manage his condition. Supportive care and multiple treatment regimens were used to successfully recover the patient’s health. With a rapid increase in number of confirmed cases worldwide, COVID-19 has become a major challenge to our health care system. With no available vaccines currently, the establishment of a combination of therapeutic drugs that effectively reduce disease progression is of utmost importance. COVID-19 is a severe acute respiratory infection that has spread worldwide since the first confirmed case in Wuhan  on December 31, 2019 . The golCurrently available treatment alternatives for hospitalized patients with COVID-19 with severe or critical disease presentation are limited to drugs that aid in the resolution of symptoms and provide supportive care. These include the use of convalescent plasma, oxygen therapy, steroids, and broad-spectrum, high-dose antivirals and antibiotics . Here we2) of 87% in ambient air. After the initial physical examination, the on-duty physician admitted the patient to the intensive care unit for the management of possible pulmonary pneumonia and suspected COVID-19, an RT–PCR test, and chest computed tomography (CT). Chest CT revealed bilateral multifocal patchy and confluent areas of ground glass opacity (GGO) in the lungs. Associated interlobular septal thickening with an erratic paving appearance, subpleural fibrosis, and prominent lower lobe bronchi were also noted bilaterally, with more marked effects on the right side. The CT severity score was 24, which was indicative of severe disease. On admission to the intensive care unit (day 1), the patient was initially administered a one-time intravenous dose each of convalescent plasma (450 mL) and paracetamol (1 g) and administered supplemental oxygen through a non-rebreathable mask at 4 L O2/min to maintain an SpO2 of >90%. He was also started on a regimen of intravenous methylprednisolone  and enoxaparin sodium . On Day 2, his laboratory findings indicated lymphopenia, neutrophilia, increased C reactive protein (CRP), and raised serum lactate dehydrogenase (LDH); hence, a 7-day course of two antibiotics, meropenem trihydrate  and moxifloxacin , was also added to the regimen. On days 3 and 4, the patient’s condition deteriorated considerably. He was semiconscious and developed ARDS. The flow velocity of oxygen had to be increased to 15 L O2/min, which was still insufficient to maintain an SpO2 of 90%-91%. Serum ferritin levels of the patient increased, with a further increase in lymphopenia, CRP, and LDH, and as the patient developed signs of cytokine release syndrome (CRS), tocilizumab (80 mg) was administered intravenously with the dose repeated after 12 hours. On day 5, the patient was started on continuous positive airway pressure ventilation where an SpO2 between 88%-92% was maintained, along with a 50% fraction of inspired oxygen (FiO2), positive end-expiratory pressure (PEEP) of 6 cmH2O, and pressure support ventilation (PSV) of 14 cmH2O. The patient was then maintained in a prone position for up to 12 hours to manage the respiratory distress. In addition to ventilator support, dexamethasone  was administered intravenously. On day 8, remdesivir (200 mg) was administered intravenously with a dose halved to 100 mg for the next 4 days. A one-time dose of 20% albumin infusion (50 mL) was also administered intravenously, and a regimen of intravenous linezolid  was added. On day 10, the patient’s condition began to improve and an SpO2 of 97% was achieved in the same ventilator setting. Serum ferritin, CRP, and LDH levels also reverted to baseline. On day 11, the patient displayed signs of recovery, having achieved an SpO2 of 97%, FiO2 reduced to 45%, PEEP of 6 cmH2O, and PSV of 14 cmH2O. On day 12, FiO2 was further reduced to 40%, and the PEEP and PSV were 6 cmH2O and 14 cmH2O, respectively. The patient was advised to sit upright for 1 hour after regular intervals. Since the patient displayed signs of improvement, he was gradually weaned off the ventilator and instead provided an oxygen mask, which helped maintain an SpO2 of 94%-96% at 2 L O2/min. On day 13, patient was prescribed oral doses of dexamethasone , moxifloxacin , and linezolid  for a 3-day course. Furthermore, a reduced intravenous dose of enoxaparin sodium  for the next 7 days was advised. The RT–PCR test for COVID-19 was repeated, which yielded a negative result, and the patient was discharged on day 14 of admission .On the evening of June 10, 2020, a 68-year-old Pakistani male physician with a history of hypertension and diabetes mellitus presented with fever, cough, and shortness of breath. Difficulty in breathing had started 3 days prior to reporting to the emergency department of our hospital and seemed to worsen with time. His vital parameters at the time of presentation included a temperature of 98.6°F, pulse rate of 85 beats per minute, blood pressure of 110/70 mmHg, and oxygen saturation (SpO2 (of 87%), and chest CT findings (CT severity score of 24), our patient was suspected with COVID-19 and was at the risk of developing severe disease. A positive RT–PCR finding for COVID-19 and later stages of disease progression during the course of his hospitalization confirmed this notion; however, timely recognition of risks and the provision of immediate, effective treatment most likely ensured his recovery and survival.This case study summarizes the clinical characteristics upon presentation, diagnosis, and treatments administered to a 68-year-old male physician with diabetes and hypertension who presented with fever, cough, and shortness of breath at our hospital. Considering his age, history , symptoms , low SpOIn a study describing the clinical characteristics, treatments, and outcomes of 138 confirmed COVID-19 cases, Wang et al  analyzedAs many of the factors were not indicative of a possibly favorable outcome, the patient was administered a number of therapeutic interventions including convalescent plasma, anticoagulants, antibiotics, corticosteroids, immunomodulatory drugs, and antivirals during the course of hospitalization.2 and had a better survival rate.Based on the hospital protocol at the time, one of the first interventions our patient received was intravenous convalescent plasma therapy (CPT). CPT is being used in different countries to provide passive immunization against SARS-CoV-2. It has been hypothesized that if CPT is administered in early stages of COVID-19, it reduces the overall viral load, improves disease prognosis, and increases the chances of survival among patients with COVID-19 . In a reThe pathogenesis of SARS-CoV-2 and its effect on host physiology is currently under intense investigation. In cases of severe COVID-19, the progression of viral infection manifests in the form of a massive inflammatory response termed as the cytokine storm or CRS, which initially affects the lungs, causing oxygen insufficiency and leading to ARDS, but can also spread to other organs such as the heart, kidneys, and liver, leading to multiple organ failure and eventually death ,17.The hallmarks of CRS include high serum levels of proinflammatory cytokines and chemokines, especially the IL-1 family and IL-6, which initiate the inflammatory cascade resulting in lung inflammation, fever, and fibrosis. These changes can be monitored through laboratory parameters including the lymphocyte count, which typically decreases , and an increase in serum levels of CRP, LDH, and ferritin . The preUse of glucocorticoid therapy for the management of CRS  has beenIn our case, the initial use of CPT and methylprednisolone was unable to curtail the cytokine storm and effectively prevent the onset of ARDS. Consequently, the patient’s condition deteriorated rapidly to the point where he was started on continuous positive airway pressure on day 5. The patient was then administered a regimen of combination therapy with 2 daily doses of dexamethasone and tocilizumab every 12 hours. Tocilizumab is an anti–IL-6 antibody preparation, which has been shown to be highly effective in improving disease severity by neutralizing IL-6, which plays a major role in inflammatory cascades and results in ARDS . In a reIn addition to the aforementioned immunomodulatory therapies, our patient received remdesivir on day 8. Remdesivir is a broad-spectrum antiviral agent that has previously been investigated for its inhibitory effect on viral replication in the Ebola virus, severe acute respiratory syndrome coronavirus, and Middle East respiratory syndrome coronavirus. The double-blind, randomized, placebo-controlled trial by Beigel et al  concludeFurthermore, it has been suggested that remdesivir may be more effective in combination with an immunomodulator such as dexamethasone or tocilizumab. A number of trials investigating the use of remdesivir in combination with tocilizumab are currently underway, and the final verdict regarding its efficacy is still pending ,30,32,33In our patient, however, the combination of remdesivir, tocilizumab, and dexamethasone, administered at a favorable time  resulted in timely resolution of the cytokine storm, resulting in an improvement in his ARDS symptoms and subsequent recovery. The underlying reason could be attributed to the effective recognition of risks and a timely administration of the available drugs.Despite being the focus of medical and scientific studies for more than a year, worldwide availability of a safe and effective COVID-19 vaccine still remains to be accomplished. Similarly, a number of antivirals and immunomodulators are being actively tested; however, an effective combination is still unavailable. Therefore, to effectively control COVID-19, there is a need to continuously explore and identify safe, effective combinations of the already available therapeutic agents. In order to ensure the safety and survival of patients, there is also a constant need to stay informed about the latest evidence. In our patient with CRS with normal liver and renal functions, combined treatment with tocilizumab, dexamethasone, and remdesivir helped reduce CRS and the subsequent requirement of mechanical ventilation. However, mixed evidence regarding the use of tocilizumab in combination with remdesivir suggests that caution should be exercised until more evidence is available for or against this combination for it to become a treatment regimen nationwide.


We observed oxidative stress triggering by the increase in malonaldehyde and nitrite levels in motor cortex. In the proteomic analysis, the motor cortex presented alterations in proteins associated with neural functioning, morphological organization, and neurodegenerative features. In addition, it was observed a decrease in the number of mature neurons. These findings, associated with previous evidences observed in spinal cord, cerebellum, and hippocampus under the same Pb administration protocol, corroborate with the motor deficits in the rats towards Pb. Thus, we conclude that the long-term administration to Pb in young Wistar rats triggers impairments at several organizational levels, such as biochemical and morphological, which resulted in poor motor performance.Lead (Pb) is a toxic metal with great neurotoxic potential. The aim of this study was to investigate the effects of a long-term Pb intoxication on the global proteomic profile, oxidative biochemistry and neuronal density in motor cortex of adult rats, and the possible outcomes related to motor functions. For this, Wistar rats received for 55 days a dose of 50 mg/Kg of Pb acetate by intragastric gavage. Then, the motor abilities were evaluated by open field and inclined plane tests. To investigate the possible oxidative biochemistry modulation, the levels of pro-oxidant parameters as lipid peroxidation and nitrites were evaluated. The global proteomic profile was evaluated by ultraefficiency liquid chromatography system coupled with mass spectrometry (UPLC/MS) followed by bioinformatic analysis. Moreover, it was evaluated the mature neuron density by anti-NeuN immunostaining. The statistical analysis was performed through Student's  Lead is one of the most abundant elements found on Earth, present as metallic form, lead salts, and organic lead –3. The IRecently, we investigated the effects of long-term exposure to lead from adolescence to adulthood on different regions of the Central Nervous System (CNS) –14. In tConsidering the brain regions responsible for movement control, the motor cortex is responsible for motor planning and programming, in addition to command both from the spinal cord and brainstem that modulate reflexes and gross movements , 16. ThuRattus norvegicus), with 40 days old, weighing between 150 and 160 g. The animals were randomly divided into two groups, control and treated, with different numbers of animal in each group for the different analyses. In the treated group, it was administered a daily dose of 50 mg/kg of lead acetate  for 55 consecutive days. The control group received distilled water  by intragastric gavage. During the intoxication period, water and food were given ad libitum, and the animals were weekly weighed to dose adjustment.This project was approved by the Ethics Committee from Federal University of Pará with protocol number 2237110716, following the NIH Guide for the Care and Use of Laboratory Animals. For this, we used 50 male Wistar rats  were anesthetized with a solution of ketamine hydrochloride  and xylazine hydrochloride  and then euthanized. The motor cortex was collected, frozen in liquid nitrogen, and stored at -80°C until further analyses. Then, the samples were thawed, resuspended in Tris-HCl buffer , and disaggregated sonically. The samples were separated into two aliquots: for nitrite and lipid peroxidation determinations. The detailed protocol for nitrite measurements was previously established by Green et al. [At the end of the behavioral tests, the animals from each group were randomly divided to the different analyses and then euthanized. Firstly, the animals  + 108 μL of 3% acetonitrile and 0.1% formic acid.All proteomic analyses were performed according to protocols previously described elsewhere , 25. Ninp < 0.05 for downregulated proteins and 1 − p > 0.95 for upregulated proteins. It was used the Rattus norvegicus proteome downloaded from Uniprot. After, the proteins identified were analyzed by a bioinformatic approach using Cytoscape 3.6.1 (Java®) with ClueGO plugin [The reading and identification of the peptides were performed on a nanoAcquity UPLC-Xevo QTof MS system , which were interpreted by Protein Lynx Global Server (PLGS) software applying the Monte-Carlo algorithm. After comparing the experimental groups, it was considered O plugin .n = 6/per group) were anesthetized with a solution of ketamine hydrochloride (90 mg/kg i.p.) and xylazine hydrochloride  and perfused through the left ventricle of the heart with solution heparinized 0.9% saline, followed by 4% paraformaldehyde. The samples were postfixed in Bouin solution for 6 hours and then processed and embedded in paraplast  [To evaluate morphological changes in the motor cortex, the animals  .μm of thickness. All histological analyses were performed in M1 area of motor cortex, specifically between layers 3 and 4 , through the coordinates: 2.5 mm lateral, 1.2 mm posterior, and 4.5 mm below from the pial surface  [After inclusion, the samples were sectioned by a microtome to obtain sections with 5 9.20 mm) .2 grid attached to the ocular and using objective lens of 40×, evaluating the density of anti-NeuN+ cells [The slides with sections were dewaxed and immersed in PBS for 3 min before antigen retrieval in citrate buffer at 70°C for 25 min. Afterward, the sections had endogenous peroxidase inhibited by immersing in methanol-hydrogen peroxide solution (3%). We used anti-NeuN antibody  for immunolabeling of mature neurons , 29, 30.N+ cells . The phot-test. In order to evaluate the body mass gain over the time, we applied the repeated measures two-way ANOVA test. The level of significance was set at p < 0.05.Results were tabulated and analyzed by GraphPad Prism 7.0 software , and the Shapiro Wilk normality test was performed. The hypotheses were tested by Student's p > 0.05; At the end of the experiment, all animals increased the body mass as expected, and no difference between groups was observed . It was observed a total of 34 proteins that were downregulated and 239, upregulated in Pb group, 112 proteins exclusively expressed in the control group, and 87 unique in the Pb group , regulation of neuron projection development (14.8%), axonogenesis (12.7%), regulation of serine/threonine kinase activity (9.1%), glycolytic process (3.9%), and others, including others as cerebral cortex development (3.6%) and negative regulation of calcium ion transmembrane transport (2.7%) .+ cells in primary motor area (M1) from motor cortex of rats that received lead when compared to control group , Superoxide Dismutase (P07632), Peroxiredoxin 2 (P35704) and 5 (Q9R063), and unique expression of Catalase (P04762) in the control group. In this way, these data suggest that the breakdown of oxidative biochemistry homeostasis may contribute to lead toxicological mechanism of damage, also related to the proteomic alterations.In this perspective, considering the systemic distribution of lead after intragastric administration, the motor cortex is susceptible to changes on oxidative biochemistry, by increasing MDA and nitrite levels. The increase on lipid peroxidation, visualized by higher MDA levels, is associated with the oxidation of polyunsaturated lipids present in cell and organelles membrane by reactive oxygen species (ROS)  that mayLooking to the proteomic approach, several proteins are indicative of a redox status imbalance, as the heat shock proteins (HSP), that have been present in several previous works from our group which investigated the effects of metals on CNS proteome , 43–46. Citrate Synthase (Q8VHF5), Cytochrome C Oxidase subunits , Pyruvate carboxylase (P52873), and Pyruvate dehydrogenase (P26284) that are involved in glucose metabolism and affect the cell energy balance, suggesting an impairment on cell energy metabolism.Associated to the discussion raised above, the energy metabolism process is intrinsically linked to both, oxidative stress triggering and further cell loss of function and death . We obseExcitatory amino acid transporter 1 (P24942) and 2 (P31596), besides the synaptic communication as shown by the up-regulation of Syntaxin 1B (P61265) and Syntaxin-binding protein 1 (P61765), Synasin 1 (P09951), and 2 (Q63537). In addition to that, the preservation of myelin sheath structure is important to the action potential, and it was observed an up-regulation of CD9 antigen (P40241), Myelin Basic Protein (P02688), and Myelin-Associated Glycoprotein (P07722); it was also observed the down-regulation of Myelin Proteolipid Protein (P60203). Considering these components, we suggest that neuroglial function might be compromised due to several issues in the background, as energy metabolism, oxidative stress, hence, synaptic communication and signal transduction.The failure on energy metabolism may impact directly the activity of neurons and glial cells due to its high demand , 51. In Synatogyrin-1 , that is, involved in short- and long-term synaptic plasticity [Synapsin-1 and 2  that are associated with neurotransmitter release [Regarding the synaptic activity, the proteomic approach revealed the modulation of asticity , 53. Mor release , and som release . In thisApolipoprotein E  showed the same status of regulation in hippocampus, while Protein S100B (P04631) was up-regulated in motor cortex and down-regulated in hippocampus, on the other hand, these proteins were not changed in cerebellum [CB1 cannabinoid receptor-interacting protein 1 (Q5M7A7) found up-regulated in this study was exclusively expressed in the control group in cerebellar global proteomic profile. These are very distinct proteins involved mainly in lipid transport (P02650) [Interestingly, it is worthy to point out that in previous proteomic data of hippocampus  after threbellum . However(P02650) , glial a(P02650) , and neu(P02650) . Althoug(P02650) –61, and (P02650)  suggesti2+, and due to that, several processes as energy metabolism, cell signaling, neurotransmitter release, and apoptosis may be affected [The literature has extensively described the mechanism of lead neurotoxicity related to the- interaction with Caaffected . When loaffected  and methaffected , 16, andaffected .Our behavioral tests support the evidence that the neurodegeneration and modulation of synaptic communication proteins observed in the motor cortex were reflected in ethological behavior alteration, with a reduced spontaneous exploration profile. This first evidence is supported by the results of the open field test, in which the decrease in the total distance, with changes in the peripheral distance. It is noteworthy that the central distance traveled was not modified by lead administration, which reflects that the motor impairment observed was not accompanied of anxiogenic-like behavior. In addition, our results showed that lead administration decreased fall angle on the inclined plane task. Such result leads to other evidence of cortical damage and motor repercussion, with negative reflexes on the index of hind limb strength.Considering all the changes observed in our study, we conclude that the long-term lead administration at a dose of 50 mg/kg/day causes poor motor performance associated with molecular and morphological impairments in motor cortex of adult Wistar rats. The lead administration triggered oxidative stress, modulated the global proteomic profile, including key proteins related to neurotransmission, cell metabolism, and signaling, leading to a motor function impairment.


Moreover, K+ has also been implicated in various defense mechanisms against abiotic stress. The first part of this review discusses the main negative consequences of the current climate, how they disturb the quality of grape berries at harvest and thus ultimately compromise the potential to obtain a great wine. In the second part, the essential electrical and osmotic functions of K+, which are intimately dependent on K+ transport systems, membrane energization, and cell K+ homeostasis, are presented. This knowledge will help to select crops that are better adapted to adverse environmental conditions. Grapevine is one of the most economically important fruit crops due to the high value of its fruit and its importance in winemaking. The current decrease in grape berry quality and production can be seen as the consequence of various abiotic constraints imposed by climate changes. Specifically, produced wines have become too sweet, with a stronger impression of alcohol and fewer aromatic qualities. Potassium is known to play a major role in grapevine growth, as well as grape composition and wine quality. Importantly, potassium ions (K In the cytosol, the K+ concentration is found in the 80–100 mM range [+ homeostasis is essential to keep the cell alive [+ is a highly mobile osmolyte and a major component of the cation/anion balance. K+ is also crucial for the control of the membrane polarization and the neutralization of organic acids and anionic groups [+ concentrations are required for optimal protein synthesis, activation of more than 70 enzymes, and photosynthesis [+ is involved in the plant responses to biotic and abiotic constraints inducing quite often a K+ loss from the cell [+ starvation is known to affect crop yield and the use of potassium fertilizers is a vital input in agriculture aiming to maintain optimal levels of nutrient.In plants, potassium , which is one of the most economically important fruit crops, climate change factors disturb the synthesis and degradation of primary and secondary metabolites directly, via the regulation of their biosynthetic pathways, or indirectly, via their effects on vine physiology and phenology [+ loading in the berry. This excess K+ during ripening leads to a decrease in grape acidity at harvest [However, nowadays, persistent climate changes and abrupt fluctuations in climatic conditions are imposing greater environmental constraints, with increasing temperatures, multiple droughts, and intense solar radiation. All these abiotic stressors represent serious threats to plants, impacting plant growth and development worldwide. Among other effects, abiotic stress disturbs Kchanisms . In graphenology ,19,20,21henology ,23. Thishenology ,24,25,26 harvest ,15,27,28 harvest . This di+ fluxes at the plasma membrane of grape berry cells in relation with climate changes. Because this control is strongly dependent on K+ transport systems and that the Shaker-like voltage-dependent K+ channels have been intensively studied, their involvement is particularly discussed. After a short description of grape berry development and its requirement for K+, the first part of this review discusses the detrimental climate constraints on grapes during their development and ripening which in turn disturb the production of wine. The second part focuses on the role of K+ in the initiation and the maintenance of berry loading during ripening, and its involvement in the defense mechanisms against abiotic stresses in relation to climate change.This review deals with the fine control of K+ and this cation also drives sugar accumulation via the phloem [+ channels are involved in the control of K+ and sugar translocation from source to sink via the phloem [+ between phloem cytosol and berry apoplast that allows the berry loading process [The development of grape berries consists of two successive growth periods separated by a lag phase. The first period comprises a rapid cell division phase followed by marked cell enlargement, and is characterized by the synthesis and vacuolar storage of the two main grape organic acids, tartaric acid and malic acid. At the onset of ripening, the berry starts to change color and soften, and the fruit undergoes major biochemical changes . From the phloem ,27,33,34e phloem , meaninge phloem . It is ie phloem ,36, usin process ,38.+ availibility is a prerequisite for a normal berry development. Grapevine is able to adapt to different availabilities of K+ via responses that mainly involve changes in root architecture and regulation of K+ transport systems. This plant also invests energy for the uptake of K+ and its distribution throughout the plant. Indeed, although the K+ concentrations of the soil solution are generally very low, highly variable, and usually in the range of 1 to 0.1 mM [+ to maintain high cytosolic concentrations at around 100 mM [+ availability using fertilizers to try to maintain an optimal K+ level. But, with regard to vines cultivated on sandy soils located on the sea front, the occurrence of additional salt stress disturbs K+ nutrition causing deficiency [+. It is widely known that potassium fertilization improves water relations and osmotic adjustment and the response of vine are often variety dependent [+ starvation reduces also the ability to use N and induces chlorosis at the tip of older leaves disturbing plant metabolism [For the whole grape maturing period, an adequate Ko 0.1 mM , grapevid 100 mM ,41. Vitificiency ,43. Droutabolism . 2 concentrations have led to increased global temperatures associated with episodes of poor water availability and drought. In addition, the incidence, intensity, and duration of extreme heat waves have increased [Due to the greenhouse effect, higher atmospheric COncreased ,46. Grapncreased . Among t+ during fruit ripening, and leads to wines whose ethanol content increases steadily over the vintages, which is associated with reduced acidity because over-accumulated K+ ion leads to an excessive neutralization of organic acids. This loss of acidity strongly affects wine quality since acidity normally allows the flavor and the aroma to develop during vinification. Upon high-temperature exposure, various damages that disturb the plant’s carbon balance and cellular homeostasis also occur, potentially leading to a severe delay in growth and development [+ homeostasis is crucial for maintaining the many physiological processes in which K+ is involved [In grapevine, heat stress causes permanent injuries that affect its phenology and grape yield. Heat stress also promotes vegetative growth, disturbing flower set, and the development of young berries . In addielopment . The maiinvolved ,5,50.In addition to an increase in atmospheric temperature, a warmer climate leads to more solar exposure for grape berries, which can be detrimental to grape quality. However, the classical viticultural practice of removing leaves in the fruit zone is known to control the source-sink balance and improves grape composition and the resulting wines ,52. When+ content, flavonoid compounds, and the organoleptic quality of the produced wines [The water requirements of plants increase in warmer climates since higher temperatures promote plant transpiration and contribute to soil dehydration, resulting in the occurrence of longer and more frequent water stresses. Water restriction has major effects, which are not always detrimental, on grape production and composition. The impact of water stress on grapevine depends on its intensity, duration, and occurrence within the development cycle . Indeed,ed wines .2 uptake and the downregulation of photosynthetic capacity and growth. It must be noted that the stomata are key players in the plant response to drought, and they close in order to reduce transpiration and avoid water loss. In grapevine, the different varieties exhibit various sensitivities to water deficit, meaning that some of them tend to close their stomata earlier than others [In contrast, even moderate water stress close to bloom time leads to poor fruit setting and the abortion of the entire clusters. Stress applied during the short initial berry cell division phase results in the significant reduction of berry size and quality . Indeed,n others ,62,63.Arabidopsis thaliana [+ concentration is observed leading to a lower Na+/K+ ratio, a better performance of cell water status and photosynthesis as compared to salinity alone [+ is added to watering, a better performance of the antioxydant enzymes and photosynthesis parameters are noted for this stress [Abundant documentation exists on the description of vine responses to abiotic stresses, which involves adaptive changes and/or deleterious effects to grape quality ,63,64,65thaliana  and othethaliana ,70,71. Ity alone . MoreoveA. thaliana, an extensive study on the impact of multi factorial stress, including heat and high light stress combination, shows that the expression of genes belonging to the ABA signaling are significantly regulated [+ and anion fluxes across cell membranes occur, mediated by different transport systems. Zandalinas et al. showed in their RNA-seq analyses that genes coding for targets of environmental stresses, including Shaker-like K+ channels and their molecular regulators, are significantly up- or down-regulated in combined stress conditions [In the plant model egulated . The phyegulated ,74 operaegulated . At the nditions . In grapnditions . Moreovenditions .+ is achieved by the precise adjustment of K+ fluxes that are controlled by K+ transport systems expressed in the different membranes. Shaker-like voltage-dependent K+ channels allow large passive fluxes at the cell plasma membrane and have been therefore intensively studied.To cope with solar radiation, warm temperature, and water stresses imposed by the current climate, plants need to sense nutrient levels in the surrounding organs and tissues and inside their cells. Then, they must accordingly adjust various transport steps leading to nutrient accumulation into the cells with respect to their homeostasis range. Maintenance of high cytosolic K+ channels are referred to as “plant Shakers” in reference to animal Shaker channels, which were named for a phenotype of jerky movements and shaking observed in fruit flies deficient in K+-channel activity [+ channels and metazoan Shaker channels are derived from distinct prokaryotic ancestors [+ channels, which are localized in the plasma membrane of expressing cells, play crucial roles in sustained K+ transport [In planta, the preferential assembly of these subunits as heterotetrameric structures increases channel functional diversity since the newly obtained channels acquire functional properties that differ from those of homotetrameric channels [HA that is rich in hydrophobic and acidic residues and involved in channel clustering [+-selective channels. In addition, a voltage sensor is found within the S1-S4 region with the S4 transmembrane segment containing important positive charges [Plant voltage-gated Kactivity . Althougncestors ,79. Planransport ,14,80. Fchannels ,82,83. Schannels ,85 is fochannels ,87), andustering . At the A. thaliana, as in grapevine. Phylogenetic analyses have shown that they belong to five subfamilies, although the number of members within each subgroup is not strictly conserved between the two species  that are activated by hyperpolarizing potentials and are closed when the driving force for K+ becomes outwardly directed. They can only mediate an inward K+ flux allowing the uptake of K+ into the cytosol. The members of group 1 and 2 differ in their cytosolic part as the KAT1-like channels (group 2) lack the ankyrin repeat domain which is present in AKT1-like channels (group 1). In A. thaliana and in grapevine, group 3 contains only one subunit. These subunits form a weakly inwardly rectifying potassium channel (Kweak) at the plasma membrane. Depending on their phosphorylation status, these channels named AKT2 in Arabidopsis and VvK3.1 in grapevine can exist in two different gating modes: a time-dependent voltage-activated mode mediating K+ influx (Kin) into the cell (mode 1), or a non-rectifying mode with a channel locked in the open state in its phosphorylated form (mode 2) across the entire physiological voltage range allowing influx and efflux dependent on the K+ electrochemical gradient [weak channels to mediate both, K+ efflux and K+ influx, and so they can function in both phloem K+ loading in source tissues and phloem K+ unloading in sinks [Group 4 contains the “silent” subunits , which includes SKOR and GORK in A. thaliana [out channels mediate only outward K+ fluxes from the cytosol to the apoplast. Finally, thaliana ,104 and thaliana . These cA. thaliana, the role of these channels has been investigated in planta. The obtained results indicate that the Kin channel AKT1 is involved in K+ uptake from the soil, preferentially in a heterotetramer structure together with the regulatory subunit AtKC1 [+ secretion in the xylem sap is driven by the Kout channel SKOR [weak channel that allows K+ influx and efflux, is involved in K+ (re)circulation, by controlling the phloem K+ loading/unloading, and in regulation of the cell membrane polarization [+ fluxes responsible for stomatal movements are mediated by the Kin channels KAT1 and KAT2 and the Kout channel GORK [In it AtKC1 ,105. K+ nel SKOR . Finallyrization ,37,97. Inel GORK ,106,107.+ levels in berries may have a negative impact on wine quality, the molecular determinants of K+ transport are currently under investigation. The grapevine VvK3.1 channel is a weakly rectifying channel (Kweak) that mediates both inward and outward K+ currents and is highly expressed in the phloem [+ efflux from the phloem cell cytosol to the berry apoplast. Switching to its non-rectifying mode, VvK3.1 drives the K+ efflux that allows K+ ions to move down their transmembrane concentration gradient . This ipoplast; , which e+ can also allow sucrose retrieval in energy-limited conditions [in channel VvK1.2, an AKT1-like channel (group 1) that mediates inwardly rectifying K+ flux, is expressed in the plasma membrane of perivascular and flesh cells. The activity of this channel is regulated by its interactions with specific VvCIPK/VvCBL pairs, and is strongly enhanced by protons at the membrane vicinity [+ into these cells to maintain the apoplastic K+ concentration at low levels . Thus, lular K+ ,110,111)lular K+ ,112.+ is stored in the vacuole of these cells by the transporter NHX1, a member of the NHX family [+ antiporter that mediates Na+/H+ and K+/H+-coupled exchange, with a higher affinity for K+ than Na+ [+ during ripening, driving the uptake of water that generates vacuolar expansion [It is important to note that once loaded into grape flesh cells by the VvK1.2 channel, KX family  . HAK5, the most studied transporter, has been shown to mediate K+ uptake in roots [Several other transport systems have been reported in grapevine, although they do not appear to be expressed in berries at the onset of ripening or throughout the grape ripening period. The current literature can therefore be briefly summarized in the following three points. (i) The Kepisodes . (ii) Thnctional . (iii) Tnctional . HAK-KUPin roots ,121. In in roots .+ homeostasis, plants control their K+ transport activity via a plethora of regulators that can precisely adjust K+ translocation at subcellular and cellular levels [+ channels at both the transcriptional and posttranslational levels. In particular, expression studies have revealed that transcript levels of the K+ channels AKT1, AKT2, KAT1, KAT2, SKOR, and GORK in A. thaliana and VvK1.1 and VvK1.2 in grapevine are sensitive to ABA [weak (AKT2) and Kout (GORK) channel transcripts are upregulated under heat and drought stresses [+ channels VvK1.1, VvK1.2, and VvK3.1 are upregulated upon drought stress [To maintain the Kr levels ,122. Stue to ABA ,123,124.stresses ,125,126.t stress ,32,33.As end effectors of environmental signals, channel proteins also undergo post-translational regulations, mainly governed by phosphorylation/dephosphorylation mechanisms, in response to stresses induced by climate change .A. thaliana CIPK/CBL/Shaker partners, since not all CIPKs interact with a given channel, and not all CBLs interact with a given CIPK [Xenopus laevis oocytes. This demonstrated that the regulatory actions of CIPK/CBL complexes are conserved in the plant kingdom.To date, the CIPK family is the only described family of kinases known to have an effect on the activity of grapevine Shaker channels. CIPKs are soluble protein kinases that are activated by interacting with CBL calcium sensors within network hubs that control the transport of most major ions . The binven CIPK ,130. CBLven CIPK ,87. The ven CIPK ,129, wasX. laevis oocytes. Among the 20 CIPKs and 8 CBLs within the grapevine genome, two couples have been selected and tested: VvCIPK03/VvCBL02 and VvCIPK04/VvCBL01 , of which the latter couple is more efficient at VvK1.2 activation [+ channels are preferentially activated by the CIPK/CBL couple, which is the most similar to that found for AKT1; the activation mechanism is likely phosphorylation as in Arabidopsis [The other inwardly rectifying channel of phylogenetic group 1, VvK1.2, is less closely related to AKT1 than VvK1.1 , and neetivation . Thus, gbidopsis .X. laevis oocytes. Surprisingly, the kinase catalytic domain of AtCIPK6 was not required for the activating effect, which rules out the activation by a phosphorylation/dephosphorylation mechanism. Instead, the C-terminal regulatory domain of AtCIPK6 could interact with the AtCBL4 protein, and AtCBL4 could drive the AKT2-CIPK complex to the plasma membrane via the myristoylation and palmitoylation sites in its polypeptide sequence. Similar to AKT2 , VvK3.1 is activated in X. laevis oocytes by VvCIPK03 (or VvCIPK02/05) and VvCBL04, the closest relatives of AtCIPK6 and AtCBL4 [The mechanism of activation by CIPKs is expected to be quite different for VvK3.1, a member of group 3 . The ortd AtCBL4 . This acType 2C protein phosphatases (PP2Cs) involved in abscisic acid signaling: ABA plays a prominent role in the initiation of berry ripening [+ channels [A. thaliana, clade A PP2Cs of the ABA signaling pathway directly regulate the activity of Shaker-like K+ channels [+ channels during the initiation and maturation of grape berries, or in response to abiotic stresses.ripening ,132 and ripening . During ripening . In the ripening . PP2Cs cchannels . In A. tchannels ,136 and channels . Twelve channels ). At thechannels ,138,139.channels , and thechannels . It is tSnF1-related protein kinase 2 (SnRK2) kinases: The OST1/SnRK2E/SnRK2.6 kinase, which is directly regulated by clade A PP2Cs [A. thaliana channel KAT1 [ A PP2Cs , interacnel KAT1  and phosnel KAT1 . Howevernel KAT1 , suggestnel KAT1 . Two-hybnel KAT1 , the granel KAT1 .Calcium-dependent protein kinases (CPK/CDPK): Besides CIPKs, other calcium-dependent kinases involved in ABA and calcium signaling are known to control the activity of Shaker K+ channels in Arabidopsis. These CDPKs (or CPKs) have different effects (activating or inhibiting) on channel activities expressed in X. laevis oocytes [ oocytes ,148. Add oocytes . CDPKs c oocytes . The gra oocytes , includi oocytes . Most of oocytes . Some of+, which is the most abundant cation in plant cells and is involved in multiple functions, needs to have its cytosolic homeostasis maintained in a concentration range compatible with biochemical processes necessary to cell life. This process is crucial to the development of plants and their resilience to hostile environments. Under stressful conditions, the plant adaptation response helps to protect K+ homeostasis via significant changes in the activity of K+ transport systems in stressed tissues and cells  would be needed to adjust the cytosolic K+ concentration in an energy-consuming process [Because nutrient homeostasis is crucial for maintaining the internal, physical, and chemical conditions of living systems, the underlying mechanisms involved in this control have been a topic of investigation in the past decade. In particular, Kbove and ,153,154) process .+ homeostasis. Specifically, plants respond to warmer climates by producing reactive oxygen species (ROS), which are key players in both developmental processes and stress responses [2, including singlet oxygen (1O2), hydrogen peroxide (H2O2), the superoxide anion (O2•−), and the hydroxyl radical (OH•) [Nevertheless, climate change can disturb this Kesponses . ROS areal (OH•) . Additioal (OH•) . Whereasal (OH•) .+ loss in stress-affected tissues [+ efflux from the cytosol of stress-affected cells has been confirmed for multiple stresses including anoxia, heavy metals, drought, and temperature extremes [+ efflux is activated by hydroxyl radical (OH•) [+ cytosolic concentration, consequently resulting in significant changes in metabolic reactions and physiological processes. In A. thaliana, this K+ efflux is mediated by the outwardly rectifying Shaker channel GORK (Kout) [+ homeostasis is crucial to maintain cell viability, and it is known that dramatic K+ loss from the cytosol activates the catabolic enzymes involved in programed cell death (PCD) [Like all fruits, grape berries are a source of antioxidants including anthocyanin and phenolic compounds, with numerous adequate metabolites that allow their synthesis and regeneration. These antioxidant compounds can process ROS to delay or avoid cell damage, and for signaling processes . At the  tissues . Initial tissues , this K+extremes ,163,164,al (OH•)  and leadK (Kout) ,100. UndK (Kout)  and phloK (Kout) ,112. Howth (PCD) ,165,166.+ sensing, voltage gating, phosphorylation, hormonal signaling, calcium, and ROS [+ efflux from the cytosol of stressed cells could be a switch that inhibits metabolic reactions in order to favor defense and adaptation mechanisms [In plants, the GORK channels are modulated by K and ROS ,167,168.chanisms ,12. The chanisms . One way+ is involved in integrated mechanisms necessary for grape development and maturation, it explains why it is so important for berry and wine quality. Starting at ripening, K+ is a major actor of the initiation and the maintenance of berry loading from the phloem owing to the fine tuning of K+ fluxes by K+ transport systems. This participates both to the control of membrane potential and the maintenance of the transmembrane K+ gradient at the phloem cells. During ripening, K+ as an osmolyte is involved in osmotic adjustment and osmoprotection of the flesh cells by the control of the vacuole turgor of mesocarp cells together with sugars. This also means that K+ ion accumulation, in the flesh cell vacuoles, surpasses purely nutritional requirements and participates in cell expansion mechanisms. At the whole plant level, K+ fluxes are involved in leaves in stomata opening and closure, and root K+ transport systems drive K+ uptake from the soil and control the emergence of root lateral primordium [+ to be present in high concentrations in all cell types, it is necessary for the grape growers to ensure that K+ availibilty is sufficient to allow appropriate berry growth and functioning. On the other hand, upon the current and ongoing climate change period, it is a true challenge since an excess of K+ in the fruit will neutralize the organic acids, thus increasing berry pH and a decline of wine quality. Considering the importance of berry composition at harvest as a prerequisite of the production of great wines, the grape community is active on this issue. Many research focalize on the improvement of our understanding of the underlying mechanisms controlling the vine and grape responses to climatic parameters that can appear individually or in combination. QTL analyses to identify key genes involved in the control of the characters studied [The negative impacts induced by climate change are among the major causes of crop yield decrease worldwide. Many researchers are working on this issue, investigating and testing various solutions to reach more crop resilience to challenging environment. In grapevine, temperature, high solar radiations, heat waves, and drought stresses are the climatic parameters that disturb the grape composition the most at harvest with a detrimental impact on wine quality. Since, Kimordium . Given t studied  and adeq studied ,171,172.