We enrolled pediatric subjects with developmental dyslexia and, as a control group, healthy age- and sex-matched subjects without developmental dyslexia. Thyroid function was evaluated in subjects with developmental dyslexia measuring serum concentrations of thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), and free thyroxine (fT4). Thyroid autoimmunity was evaluated in all subjects measuring antithyroid peroxidase (TPO-Ab) and antithyroglobulin (TG-Ab) antibodies. In subjects with developmental dyslexia, thyroid ultrasonography (US) was also performed. p < 0.001), while no significant difference was found in TG-Ab positivity (16% vs. 5.8%). Thyroid US performed in 49 subjects with developmental dyslexia revealed a thyroiditis pattern in 60%.  We enrolled 51 subjects with developmental dyslexia  and 34 controls . TPO-Ab positivity was significantly higher in subjects with developmental dyslexia compared to controls (60.8% vs. 2.9%,  We found an extremely high prevalence of thyroid autoimmunity in children with developmental dyslexia. Further studies are needed to confirm our observations, but our findings may change the approach to this disorder and eventually lead to a systematic determination of thyroid autoimmunity in children with developmental dyslexia. Developmental dyslexia is defined as a persistent reading difficulty not explained by sensorial or cognitive deficits, lack of motivation, or inadequate education , 2. Its An association between developmental dyslexia and autoimmune disorders has been postulated in previous studies. This supposition started from the observation of a higher prevalence of both immune disorders and learning disabilities in left-handed subjects, the so-called Geschwind-Behan-Galaburda hypothesis , 5. It wThe aim of our study was to evaluate the prevalence of thyroid autoimmunity in pediatric patients with developmental dyslexia.In the period June 2015–January 2017, we enrolled pediatric subjects with developmental dyslexia referred by patients' associations. Inclusion criteria were the presence of a certified diagnosis of developmental dyslexia, a normal intelligence according to Wechsler Intelligence Scale for Children  , 10, andAs a control group, healthy age- and sex-matched subjects without developmental dyslexia were enrolled. Control subjects were outpatients of the Surgery Unit of our hospital, who underwent a blood sample collection before a surgical intervention for the treatment of minor diseases . To be enrolled, they should have no history of chronic disease and receive no treatments.The study protocol was approved by the local Ethics Committee (CE 161/15), and written informed consent was collected from the parents or legal guardians of all subjects and patients themselves, where appropriate.μIU/mL for TSH, 0.2 pg/mL for fT3, 0.1 ng/dL for fT4, 28 IU/mL for TPO-Ab, and 15 IU/mL for TG-Ab assay. Reference values were 0.450–3.500 μIU/mL for TSH, 2.30–4.20 pg/mL for fT3, and 0.89–1.76 ng/dL for fT4. TPO-Ab and TG-Ab titers higher than 60 IU/mL were considered positive, according to our laboratory range.All the enrolled subjects underwent a blood sample collection in the morning after an overnight fast. Thyroid function was evaluated in subjects with developmental dyslexia measuring serum concentrations of thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), and free thyroxine (fT4). Thyroid autoimmunity was evaluated in all subjects measuring antithyroid peroxidase (TPO-Ab) and antithyroglobulin (TG-Ab) antibodies. Serum TSH, fT3, fT4, TPO-Ab, and TG-Ab levels were assessed in the Clinical Chemistry Laboratory of our University Hospital by direct chemiluminescent technology with acridinium ester as a label and paramagnetic particles as a solid phase . Sensitivity was 0.010 In subjects with developmental dyslexia, thyroid ultrasonography (US) was also performed by a single trained operator, who was not aware of the results of laboratory tests. Thyroid size and morphology were evaluated using a high-resolution 7-13 MHz linear transducer, with the subjects sitting and their necks slightly extended. The volume (milliliters) of each lobe of the thyroid gland was calculated using the following formula: 0.479 × depth × length × width. Thyroid volume was defined as enlarged according to the reference values for sex and body surface area . Thyroidp value of <0.05 was considered statistically significant. All analyses were performed using SPSS version 21.0 .Data were expressed as mean ± SD and range, for continuous variables, and as number and percentage (%), for categorical variables. Comparisons between the prevalence of positive antithyroid antibodies in subjects with developmental dyslexia and control subjects were performed using a chi-squared test or Fisher test, as appropriate. A We enrolled 51 subjects with developmental dyslexia (M : F = 39 : 12), aged 7.0-19.2 years (mean 12.4 ± 9 years) and 34 healthy control subjects (M : F = 24 : 10), aged 6.0-18.0 years (mean 10.8 ± 4 years). Two out of 51 subjects with developmental dyslexia refused their consent to the US examination.We collected data on the dominant hand in 46 of 51 subjects: 40 were right-handed (87%) and 6 left-handed (13%).μIU/mL (range 0.718–5.478 μIU/mL). Mean fT3 and fT4 were, respectively, 3.86 ± 0.56 pg/mL (range 1.30–5.20 pg/mL) and 1.25 ± 1.6 ng/dL (range 0.91–1.60 ng/dL). Subclinical hypothyroidism was found in 5 subjects (9.8%). None had overt hypothyroidism. TSH and thyroid hormones levels were similar in males and females.TSH, fT3, and fT4 levels were measured in subjects with developmental dyslexia. Mean TSH was 2.266 ± 1.003 p < 0.001). The prevalence of subjects with developmental dyslexia having at least one antibody positivity was significantly higher than that of controls (p < 0.001)  and TG-Ab positivity in 8 subjects with developmental dyslexia (16%). Overall, 32 subjects with developmental dyslexia (63%) had at least one positive antibody, and 7 subjects (14%) had both positive antibodies. The prevalence of positive antithyroid antibodies was compared to that found in age- and sex-matched healthy controls. TPO-Ab positivity was found in 1 control (2.9%) and TG-Ab positivity in 2 control subjects (5.8%). Overall, 2 control subjects (5.8%) had at least one positive antibody and 1 control subject (2.9%) had both positive antibodies. TPO-Ab positivity was significantly higher in subjects with developmental dyslexia than in controls (< 0.001) . The preThyroid US was performed in 49 subjects with developmental dyslexia: 29 of them (60%) presented a thyroiditis pattern. Thyroid nodules were found in 2 subjects (4%).Fifteen subjects (29%) with positive antithyroid antibodies had a negative ultrasound, whereas 14 subjects (27%) with a suggestive ultrasound did not show antibody positivity.The presence of thyroid autoimmunity (defined by antibody positivity and/or thyroiditis pattern at ultrasound) was observed in 46 subjects (90%). The prevalence of thyroid autoimmunity was not different according to gender or right/left handedness.In the present study, we found a striking elevated prevalence (over 90%) of thyroid autoimmunity, defined as the presence of positive antithyroid antibodies and/or US pattern suggestive of thyroiditis, in children with developmental dyslexia. Almost two-thirds of them had positive antithyroid antibodies, represented in 60.8% of cases by elevated TPO-Ab. This rate was significantly higher than that found in age- and sex-matched healthy subjects. The previously reported prevalence of TPO-Ab or TG-Ab positivity in healthy children ranges from 2.5% to 7% –19. TaubThe prevalence of thyroid autoimmunity became even higher including subjects with a thyroid US pattern suggestive of thyroiditis. In our series, 14 subjects presented a suggestive thyroid ultrasound pattern without circulating antithyroid antibodies. As stated in a precedent study , it is pElevated prevalence of immune disorders in subjects with developmental dyslexia was previously reported, even if thyroid autoimmunity was not specifically addressed. In the 80s-90s, Hugdahl et al.  studied Conversely, Gilger et al. found no association between developmental reading disability and immune disorders . But in particular, the presence of thyroid autoimmunity was assessed only by a survey and not by antibody testing .To explain our results, the Geschwind-Behan-Galaburda hypothesis regarding the presumed association between left-handedness, developmental dyslexia, and immunity is undoubtedly fascinating . HoweverMost of the enrolled subjects were euthyroid, with less than 10% of dyslexic subjects showing subclinical hypothyroidism. Nonetheless, the prevalence of subclinical hypothyroidism in our cohort was higher than that reported in pediatric age, being about 2%. This finding is consistent with the high prevalence of thyroid autoimmunity found in our cohort, and it is known that the presence of elevated antithyroid antibodies may be suggestive of the persistence of subclinical hypothyroidism over time or even of the progression to overt hypothyroidism .A limit of our study was that family history for thyroid disorders was not investigated. However, the possible influence of positive family history for thyroid diseases could hardly explain such a higher prevalence of thyroid autoimmunity in our children with developmental dyslexia, as it is known that only about 30% of children with Hashimoto's thyroiditis have at least one first-degree relative with thyroid diseases . At the A second limit is that US evaluation was performed only in subjects with developmental dyslexia and not in controls, so that we cannot exclude that some healthy controls would have displayed thyroid US alterations. However, the prevalence of thyroid autoimmunity merely assessed on the basis of positive antibodies is much higher in dyslexic subjects than in controls that it is improbable that the findings at US evaluation in controls would decrease the strength of our results.Furthermore, pubertal status was not evaluated in our patients, preventing the detection of differences in thyroid autoimmunity prevalence according to puberty.Moreover, only thyroid autoimmunity was evaluated, but the current results pave the way to the investigation of other autoimmune profiles in our cohort of dyslexic subjects, in particular antinuclear antigen, rheumatoid factor, and celiac antibodies.Finally, it would be informative to know the actual value of TPO-Ab, but it is a limit that could not be overcome, as the overflow values are presented just as >60 IU/mL according to the kit used in our lab.In conclusion, the detected association between thyroid autoimmunity and dyslexia may change the approach to children affected by developmental dyslexia. From a clinical point of view, asymptomatic children with circulating antibodies and characteristic ultrasound patterns are at greater risk of developing thyroid dysfunction. Although further studies are required to confirm our findings, they may eventually lead to a systematic determination of thyroid antibody profiling and thyroid function tests in dyslexic children. From a pathophysiologic point of view, this association may suggest a relationship between the pathogenesis of thyroid autoimmune disorders and developmental disorders, even if our findings do not claim to demonstrate a causative link between thyroid autoimmunity and developmental dyslexia, but just to describe an unexpected high association between them. In this regard, further studies are needed to explain this association and to evaluate in subjects with developmental dyslexia the prevalence of other widespread autoimmune diseases, such as type 1 diabetes mellitus and celiac disease, which are often in comorbidity with thyroid autoimmune disorders. It still remains a challenge to understand if autoimmunity is comorbidity or if it is implicated in the pathophysiology of developmental dyslexia.


Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). Metalloantibiotics showed improved antimicrobial activity against several clinical isolates, especially MRSA. Synergistic activity was evaluated in combination with ciprofloxacin and ampicillin by the disk diffusion and checkerboard methods. Synergistic and additive effects were shown against MRSA isolates. The mechanism of action was studied though enzymatic assays and atomic force microscopy (AFM) experiments. The results indicate a similar mechanism of action for FQs and metalloantibiotics. In summary, metalloantibiotics seem to be an effective alternative to pure FQs against MRSA. The results obtained in this work open the way to the screening of metalloantibiotics against other Gram-positive bacteria.Fluoroquinolones (FQs) are antibiotics commonly used in clinical practice, although nowadays they are becoming ineffective due to the emergence of several mechanisms of resistance in most bacteria. The complexation of FQs with divalent metal ions and phenanthroline (phen) is a possible approach to circumvent antimicrobial resistance, since it forms very stable complexes known as metalloantibiotics. This work is aimed at determining the antimicrobial activity of metalloantibiotics of Cu(II)FQphen against a panel of multidrug-resistant (MDR) clinical isolates and to clarify their mechanism of action. Minimum inhibitory concentrations (MICs) were determined against MDR isolates of  The cell envelope is the first natural bacterial barrier against antimicrobials, comprising two major components, the cell membrane and the peptidoglycan ,2. In geFluoroquinolones (FQs) are a family of antibiotics with a large spectrum of action, affecting Gram-negative and some Gram-positive bacteria, and being widely used in clinical practice . These aEscherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) were chosen and reference strains and multidrug-resistant (MDR) clinical isolates, including methicillin-resistant S. aureus (MRSA), were used. The compounds that revealed improved MIC values in comparison to pure FQs were also tested in combination with some antibiotics, through the disk diffusion and the checkerboard methods, to assess possible synergistic effects. To determine if they show a similar mechanism of action as FQS, the metalloantibiotics capacity to inhibit DNA gyrase and topoisomerase IV was evaluated through enzymatic inhibitory assays. Additionally, Atomic Force Microscopy (AFM) was used to evaluate the effect of FQs and metalloantibiotics on bacterial membrane features (shape and size).This work aims to evaluate the antimicrobial activity of five metalloantibiotics and their respective free FQs against clinical isolates of Gram-negative and Gram-positive bacteria, through the assessment of minimum inhibitory concentrations (MICs). Three bacterial species of clinical importance  (Escheri3)2.3H2O, FQs  and respective metalloantibiotics were prepared in 10 mmol dm−3 HEPES buffer , with the exception of the copper solution, prepared only in double deionized water. The solutions were filter-sterilized and stored in small aliquots, protected from light, at −80 °C. The stock solutions were then thawed as needed and used in the same day. The metalloantibiotic solutions used were prepared by mixing the three components  and phen) in stoichiometric proportions (1:1:1), as previously reported for other FQs metalloantibiotics [3)2.3H2O solution used to prepare the metalloantibiotics solutions was previously titrated in alkaline medium with EDTA and using murexide as indicator.Ciprofloxacin (cpx), enrofloxacin (erx), levofloxacin (lvx) and sparfloxacin (spx) , were purchased from Sigma-Aldrich. Moxifloxacin (mxfx) was a gift from Bayer. All other chemicals were from Merck. Stock solutions of phen, Cu(II)/phen (1:1), Cu, eight MDR E. coli  and 18 MRSA . All the strains and isolates were stored in glycerol at −80 °C and thawed at room temperature before being plated on non-selective agar. The antibiotic resistance profiles of the clinical isolates are compiled in Four bacterial reference strains from ATCC (LGC Standards) were used: two Gram-negatives, 5 colony-forming units (CFU) per mL [The MIC values were determined for all tested compounds by the broth microdilution method, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) . Briefly) per mL . Control) per mL , was detE. coli and MRSA were tested in combination with other antibiotics in two-drug combination studies. Selected compounds were combined with cpx (5 μg) or ampicillin—amp (10 μg) against E. coli and MRSA isolates. The combined effect of compounds with antibiotics was tested by the disk diffusion and the checkerboard methods.The compounds that revealed improved activity against some clinical isolates of MDR 600 = 0.1 (≈108 CFU/mL). The suspension was then inoculated on the surface of a MH agar plate (150 mm diameter). Each plate housed up to three paper disks. Control disks  and commercial antibiotic-impregnated disks  were loaded with the tested compound solution (15 µL of a 2048 μg/mL stock solution to reach 30 μg of each compound/per disk). The plates were then incubated at 37 °C, for 16 to 20 h. The diameter of the zones of growth inhibition observed around each disk was measured (in mm) [A suspension of each selected MDR isolate was prepared in MHB by picking overnight colonies, and its turbidity was adjusted to OD (in mm) . Each co5 CFU/mL [The compounds that exhibited potential synergism in the previous assay were also tested in two-drug combinations through the broth microdilution checkerboard method. MIC values were determined for each tested compound alone and in combination. The method was performed using a two-fold dilution series in 96-well plates and final bacterial concentration of ≈ 5 × 105 CFU/mL , as prev5 CFU/mL ,8. Two i5 CFU/mL .−3. Gyrase supercoiling inhibition assays were performed by incubating relaxed pBR322 plasmid (0.5 μL per assay) with 1 unit of gyrase (prepared in the dilution buffer supplied) and the assay buffer supplied, in the absence and presence of a concentration range of the solutions being tested. Topoisomerase IV relaxation inhibition assays were performed by incubation of supercoiled pBR322 plasmid (0.5 μL per assay) with 1.5 (E. coli) or 2 (S. aureus) units of topoisomerase IV (prepared in the dilution buffer supplied) and the assay buffer supplied, in the absence and presence of a concentration range of the compounds tested. All the reactions were carried out according to the manufacturer’s instructions . Three controls were used in the study: a negative control, consisting of a mixture of the plasmid, water, assay buffer and dilution buffer (in the absence of the enzyme), and two positive controls, comprising a mixture of the plasmid, water, assay buffer and water or HEPES buffer (in the presence of the enzyme). Cpx was used as a drug control in all experiments, due to its known enzymatic inhibitory activity. After each reaction, the plasmid (relaxed or supercoiled) was separated by agarose gel electrophoresis. All gels were stained with GreenSafe Premium (NZYTech), visualized and photographed under UV light. All determinations were performed in at least three independent experiments.The study was carried out with two metalloantibiotics, Cucpxphen and Cuspxphen, and six compound concentrations were evaluated: 0.5; 1.0; 5.0; 10.0, 50.0 and 100.0 µmol dm8 CFU mL−1, was reached. Control samples (without antibiotic treatment) and samples containing FQ or metalloantibiotic in a concentration below the MIC (0.002–0.008 µg mL−1) were prepared. The study was carried out with two FQs (cpx and spx) and their metalloantibiotics, Cucpxphen and Cuspxphen. E. coli ATCC 25922 and S. aureus ATCC 25923 strains were used. Drops of 20 µL of each bacterial suspension (control and treated) were loaded onto a glass plate, followed by drying for 30 min at 37 °C, then gently washed with isotonic solution, subsequentially dried for 30 min at 37 °C and washed again with ultra-pure water and dried for 1 h at 37 °C. Samples were observed under a TT-AFM instrument from AFM Workshop with a 50 µm scanner in vibrating mode. Probes were purchased from Applied Nanostructures, Inc. , and had approximately 300 kHz resonant frequency, probe diameter <10 nm . Multiple areas per sample were imaged and representative images were selected. For E. coli experiments, three independent experiments, each composed by three independent replicates for each control and treated samples, were performed. For S. aureus experiments, however, due to time constraints, only one experiment composed by three independent replicates was performed. Nevertheless, at least 10 individual cells were analyzed in terms of height and size (length and diameter) in both experiments. Gwyddion software was used to process the images [® software .Bacterial cultures were grown in Luria broth (LB) until an optical density of about 0.1, corresponding to approximately 10e images . Statist3)2.3H2O salt was tested against bacterial reference strains and MDR clinical isolates.The antibacterial activity of five free FQs  and the respective metalloantibiotics of CuFQphen, phen, Cu(II)/phen (1:1) and Cu(NO−1) [Metalloantibiotics ≈ 2 × MW FQs  ,35. Neve× MW FQs , the MICP. aeruginosa ATCC 27853, whose value  was lower compared to the range recommended in the guidelines (0.25–1 µg mL−1). This result may arise from the absence of cation content in the MHB medium used. The CLSI guidelines refer that the cation content of the broth used in the antimicrobial susceptibility testing may affect the MIC results. Moreover, the supplementation of the broth with cations, in cases where lower MIC values were observed in P. aeruginosa ATCC 27853, are suggested in the guidelines [−1 for cpx against this control strain [The MIC values determined for all compounds against reference strains are presented in idelines . Besidesl strain , which iE. coli ATCC 25922 by Feio et al. [3-fold higher) than those of FQs and metalloantibiotics. These outcomes were also previously stated by Feio et al. [Regarding metalloantibiotics, the MIC values were similar between each metalloantibiotic and the respective free FQ, which is in agreement with the data previously reported for o et al. . In turno et al.  and may o et al. ,37,38.Concerning MDR isolates, the MIC values obtained against Gram-negative bacteria were visibly different from those obtained against Gram-positive bacteria . The MIC−3). According to EUCAST guidelines, all the twelve MDR E. coli isolates tested were shown to be resistant to the tested FQs [E. coli isolates (−1) suggests a difference of 2-fold (128 μg mL−1 for cpx and 64 μg mL−1 for Cucpxphen), while the comparison in μmol dm−3 discloses a difference of 4-fold (386.3 μmol dm−3 for cpx and 93.0 μmol dm−3 for Cucpxphen). Besides the improved antibacterial activity of Cucpxphen compared to free cpx, the MIC value is still quite high.MIC values could be obtained for all compounds, within the range of concentrations tested, against Gram-negative MDR isolates , with thsted FQs . The MICisolates  were comisolates . These rP. aeruginosa isolates was comparable . Despite the lower MIC values of metalloantibiotics compared to pure FQs, the absolute value of the MICs can still be considered rather high. Therefore, some synergistic assays were also performed to evaluate the effect of the combination of these metalloantibiotics with other antibiotics.Interestingly, the results were more promising against E. coli and MRSA were tested in combination with some antibiotics (cpx and amp), in two-drug combinations. Cucpxphen and Cuspxphen were combined with cpx (5 μg) or amp (10 μg) against one E. coli isolate (HSJ Ec002) and two MRSA isolates (Sa1-SA3 and Sa3-SA3). The combination of phen, Cu(II)/phen (1:1) and Cu(NO3)2.3H2O salt with antibiotics was also evaluated.According to the results obtained from MIC experiments, the metalloantibiotics that revealed improved activity against most clinical isolates of MDR E. coli isolate HSJ Ec002, a slight zone of inhibition (8 mm) for Cuspxphen in combination with cpx was observed, while each one alone caused no zone of inhibition (0 mm)  . For allConcerning Gram-positive isolates, the diameter of zones of inhibition obtained are shown in E. coli isolate HSJ Ec002 and of Cucpxphen in combination with amp and cpx against S. aureus isolate Sa1-SA3.Regarding the MRSA isolate Sa3-SA3 , a growtThe checkerboard method allowed the calculation of the FIC index to further characterize the interactions among the different combinations . The comTherefore, the strategy based on the combination of these metalloantibiotics with different antibiotics should be further explored. Nevertheless, when combining different compounds, it is important to understand their mechanism of action. Thus, this study finishes with enzymatic inhibitory assays and Atomic Force Microscopy studies in order to assess the mechanism of action of the metalloantibiotics.E. coli and S. aureus. Two metalloantibiotics, Cucpxphen and Cuspxphen, and cpx (as a drug control due to its known inhibitory enzymatic activity) were tested. The enzymatic activity of cpx was always evaluated using two different concentrations (one able and one unable to inhibit the enzyme). The precise concentration (units—U) of enzyme required for each enzymatic inhibitory activity assay was previously determined , AFM is a technique that uses the physical interaction of a sharp probe with the surface of the sample to build a map of the height of the sample’s surface, and is widely used to study micro-organisms, especially bacterial cells. Despite being slower and less suitable for cell-counting studies when compared to common optical microscopy methods, AFM has the great advantage of allowing high resolution three-dimensional images of the bacterial cell with clearer details of cell morphology and sub-cellular features under ambient conditions . This teE. coli and S. aureus, respectively. Two FQs (cpx and spx) were chosen due to their different mechanism of permeation across the bacterial membrane: cpx translocation is strongly dependent on porins, while spx is expected to cross through the lipid bilayer [As membranes of Gram-negative and Gram-positive bacteria are largely distinct, we tested one model for each bacterial type:  bilayer . TherefoE. coli and S. aureus cells, respectively, and also depict an example of the plot of height and size measures analyzed in this study. For S. aureus, profile measurements show control cells with a height of about 500–700 nm and a diameter of about 1.0–1.5 µm (E. coli, AFM images of control cells show a height ranging from 100–300 nm and a length of about 3–5 µm (0–1.5 µm . For E. t 3–5 µm .E. coli cells showed a clear filamentation after treatment with all the compounds (E. coli cells. This result is consistent with other published studies on E. coli morphology after treatment with cpx at a sub-MIC concentration [E. coli cells independently of the compound used in the treatment. No statistically significant differences in cell length were found upon treatment with the free FQ or the metalloantibiotics (E. coli cells significantly (Analyzing the AFM images of treated bacterial cells  it is clompounds . This pantration ,45. Filantration . Filamenibiotics . Therefoficantly . In thisficantly B–E. ThisS. aureus cells produced slightly different results (E. coli (S. aureus size upon treatment with FQs or other DNA targeting drugs. It has only been shown that gatifloxacin caused morphologic alterations on MDR S. aureus cells [S. aureus cell size [S. aureus samples was hampered by the presence of artefacts and an unclean background, while it was not the case for the control samples (representative images The antibiotics’ action on  results . The siz(E. coli . In factus cells  and novoell size . OverallMIC values revealed improved antimicrobial activity of metalloantibiotics in comparison to pure FQs, especially against MRSA isolates. Additionally, the combination of these complexes with cpx and amp exhibited synergistic or additive effects against MRSA isolates. Metalloantibiotics showed a similar mechanism of action to FQs, acting on topoisomerase IV and DNA gyrase of bacteria. Moreover, in the case of Gram-positive bacteria these complexes showed preference to act on topoisomerase IV. AFM analyses showed that metalloantibiotics cause no damage on bacterial membranes, allowing us to infer a probable intracellular mechanism of action.As FQs are no longer used to fight infections caused by MRSA due to the bacterial resistance mechanisms developed against these drugs, the increased antimicrobial activity of metalloantibiotics against several MRSA clinical isolates may anticipate their potential use as alternatives to fight MDR Gram-positive bacteria.


The continuing use of nonsteroidal anti-inflammatory drugs (NSAIDs) usually increases the side effects such as peptic ulcer and acute gastric lesions in the gastrointestinal tract. Cuttlebone (CB), isolated from Sepiella maindroni de Rochebrune, was reported to have antioxidant activities, but its role in the treatment of indomethacin-induced gastric lesions has not yet been confirmed. In this research, we investigate the protective effect of cuttlebone on indomethacin-related ulcers in rats and possible mechanisms. Here, gastric ulcers were induced by oral administration of indomethacin, and then the rats were treated with omeprazole (4 mg/kg) or different doses  of cuttlebone. We evaluated lesion index, inflammation score, and a series of oxidant/antioxidant parameters. The data demonstrated that cuttlebone could protect against gastric ulcers induced by indomethacin in a dose-dependent manner (positive correlation). Also, these effects were associated with attenuating the expression of malonaldehyde (MDA) and increasing the levels of some protective ingredients like epidermal growth factor (EGF), prostaglandin E2 (PGE2), and superoxide dismutase (SOD). Thus, considering its ability to protect indomethacin-induced acute gastric mucosal lesions and the underlying mechanisms, CB might be a potential candidate for treating gastric damage caused by NSAIDs. Nonsteroidal anti-inflammatory drugs (NSAIDs) are proverbially used in rheumatic diseases, cardiovascular diseases, and the control of neoplasia development , 2. HoweCurrent treatment strategies for indomethacin-induced gastric mucosal lesions involve the use of proton pump inhibitors (PPIs), such as omeprazole, and antagonists of histamine receptor type 2 like ranitidine . Howeverβ (TGF-β) and vascular endothelial growth factor (VEGF) [4) [Cuttlebone (CB), the internal structure isolated from Sepiella maindroni de Rochebrune, is traditional Chinese medicine used in the treatment of stomach ulcers, gastric hyperacidity, and a variety of bleeding . It has r (VEGF) . Moreover (VEGF) . Also, cEGF) [4) . This ef2, MDA, and SOD. We aim to test the feasibility of CB with its antiulcerative and therapeutic properties and to determine whether the antiulcer mechanism of CB is linked with EGF, PGE2, MDA, and SOD.In the present study, we firstly established the model of indomethacin-induced gastric mucosal lesions in rats. Then, we observed the efficacy of CB with different doses in this model and measured the amount of EGF, PGECB was first tested and processed through quality standards. Water was added into the material and boiled 3 hours for extraction. Then the extract was filtered using filter paper. The filtrates were combined and concentrated into the paste with a certain proportion. Furthermore, the paste was dried in a shaded area, sieved, and mixed in the form of particles. Finally, the extract was kept in a package until use.2O) by gavage to establish the model of acute gastric mucosal injury as previously described [A total of 60 male Sprague Dawley (SD) rats (150 ± 15 g) were supplied by Shanghai SLAC Laboratory Animal Co., Ltd SCXK:2012-002). During the experiment, rats were provided with a specific pathogen-free state on a strict light cycle (lights on at 08:00 hours and off at 20:00 hours), temperature of 20–26, and humidity of 40–70%. After 24 h of food starvation, animals were treated with indomethacin , ulcer area = 1–3 mm2 (Level II), and ulcer area >3 mm2 (Level III), and then the ulcer indexes were calculated: ulcer index = 1 × (number of Level I ulcer) + 2 × (number of Level II ulcer) + 3 × (number of Level III ulcer).The stomachs were cut off, spread along the greater curvature, and then fixed on a board for the purpose of the megascopic gastric lesion analysis. After repeatedly rinsing with ice saline, the tissue was fixed on a board, and the area of visible erosive lesions was gauged with a vernier caliper. Then, the lesion index was calculated in line with the method of Andrade et al. , 22. ClaTo measure the levels of PGE2 and EGF (pg/ml) in blood serum, blood was obtained from arteria femoralis in rats and centrifuged (3000 rpm) for 10 min. After separation, the specimens were cryopreserved in vials at −20, followed by estimation using Rat-PGE2 and Rat-EGF enzyme-linked immunosorbent assay (ELISA) kits .The concentration of PGE2, MDA, and SOD inhibition rate was measured by Rat-PGE2 ELISA kits , thiobarbituric acid (TBA) method using MDA kit , and water-soluble tetrazolium (WST-1) method using SOD kit  and then divided by the protein concentration in tissue measured by bicinchoninic acid (BCA) protein quantification kit (purchased from Biyun Sky Institute of Biotechnology).The stomach tissues were excised from SD rats and washed with ice saline. Later, the samples were fixed with 10% neutral buffered formalin, routinely processed with dehydration, and embedded in paraffin wax. Then the gastric tissue was fixed with 10% formalin, dehydrated in increasing concentrations of ethanol, paraffin-embedded, sectioned, stained with hematoxylin-eosin (HE), and scored according to Rauws classification standard . BrieflyP < 0.05 was considered to be statistically significant.Statistical analysis was performed using one-way analysis of variance (ANOVA). All values were reported as the means ± standard deviation (SD). All statistical calculations were performed with Statistical Product and Service Solutions (SPSS) 13.0 software. To explore the curative function of CB on megascopic gastric lesions, we did macroscopic examination by ciphering lesion number and size on all rats in CB  groups, omeprazole group, and control group. In all rats, the lesion foci were formed by mucosal defects like punctate- or linear-shaped and scattered to all of the stomach surfaces. In addition, hyperemia in the stomachs of rats in the control group was more distinct than that of rats in all other medicine groups. The average lesion index decreased with increasing CB dose Figures . We can Next, the inflammation score of the gastric surface was computed to identify gastric inflammation for further demonstration of the curative effect of CB against indomethacin-induced gastric lesions. The average inflammation score decreased with increasing CB dose Figures . At the ∗P < 0.05) (∗P < 0.05) (∗∗P < 0.01) (∗∗P < 0.01)  . In the  < 0.05) . In the  < 0.01) . Similar < 0.01) .∗∗P < 0.01)  .∗P < 0.05) (∗∗P < 0.01) (∗P < 0.05)  . The con < 0.01) . In a lo < 0.05) .∗∗P < 0.01)  . Among a < 0.01) .∗∗P < 0.01) (∗∗P < 0.01)  . The act < 0.01) .In China, about 3.6% of adults were reported to take NSAIDs periodically . It has 2O2), and LPO, which will sabotage membrane lipids, DNA, cellular proteins, and organelles in a frank way [Indomethacin is one of the most common drugs to make an ulcerative model by producing gastric mucosal damage, the main mechanisms of which is overproduction of free radicals. By attaching close to ubiquinone and complex I of mitochondrial electron transport chain, indomethacin sums the formation of reactive oxygen species (ROS) through framing free radicals , 33. Furrank way .PGE2, whose synthesis is catalyzed by COX-1, is responsible for gastric mucosal protection. Previous research studies had confirmed that the exogenous administration of PGE2 protected against indomethacin-caused gastric mucosal damage , 37. EGFThe character of toxic oxygen radicals in the course of indomethacin-induced gastric damage has been confirmed . AccordiAlthough there are vital discoveries revealed by these studies, limitations still exist. First, we did not knock down the expression of PGE2, EGF, MDA, and SOD. Thus, saying that the antiulcer mechanism is directly interrelated with them may not be accurate. Second, to determine whether CB has severe side effects, we are supposed to treat the rats with CB periodically. Here, we only investigated the effects of CB on acute gastric mucosal lesions in a short time. The experiment should last for a time, and the results should include whether the rats show adverse effects.Our study indicated that CB can protect against indomethacin-induced gastric injuries at different doses and the antiulcerative effect shows a positive correlation with different dosages. Furthermore, our results suggest a possibility of a correlation between the antiulcer mechanism and increased secretion of EGF and PGE2, prevention of lipid peroxidation, and activation of radical scavenging enzymes. Thus, CB, a natural compound extracted from a kind of cuttlefish, may be a potential therapeutic candidate for preventing and treating indomethacin-induced acute gastric mucosal lesions.


Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by extraskeletal heterotopic ossification. It is well recognized that FOP can lead to a devastating condition of disability. However, the mortality rate of FOP patients in China and risk factors for mortality are still largely unclear.We conducted a retrospective research on a cohort of 65 cases of FOP patients in China from 2008 to 2018. We reviewed medical records of these FOP patients to retrieve information such as date of birth/death, gender, clinical features, genotypes and biochemical parameters and analyze the correlation of these parameters with the mortality.P < 0.05), and spearman correlation and logistic regression analysis indicated that serum osteocalcin and alkaline phosphatase levels were negatively correlated with the mortality. Furthermore, the receiver-operating characteristic curve analysis showed serum osteocalcin had the largest area under the curve of 0.855 among four biochemical parameters, and serum osteocalcin < 65.9 ng/ml displayed a good capacity to discriminate the non-survivors from survivors in FOP patients aged 18 years and younger at diagnosis.92.3% (60/65 cases) patients were classic FOP patients, 3.1% (2/65 cases) were FOP-plus and 4.6% (3/65 cases) were FOP variants. 9 cases of this cohort were dead during the ten-year period, and the overall mortality rate was 13.8%. c.617G > A mutation was confirmed in all non-survivors. In FOP patients≤18 years at diagnosis, non-survivors demonstrated significantly lower blood osteocalcin and alkaline phosphatase levels compared with survivors (Our findings showed that the mortality rate of FOP was 13.8% in China. Serum OC level was negatively correlated with the mortality in Chinese FOP patients ≤18 years at diagnosis. Fibrodysplasia ossificans progressiva (FOP) (Mendelian Inheritance in Man [MIM] #135100), also known as myositis ossificans progressiva, is a rare genetic disease characterized by heterotopic ossification (HO) in anatomical sites and congenital skeletal malformations  which evAnkylosis of affected joint, which is the most common complication, may result in progressive immobility and lifelong wheelchair-bound . SubmandIt is estimated that there are at least 650 cases of FOP patients in China, the most populous country in the world . HoweverFrom December 2008 to December 2018, 65 cases of individuals who were diagnosed as FOP in Tongji Hospital of Tongji University were recruited, consisting of 30 males and 35 females. The diagnosis of FOP was based on the two clinical features [The medical records were reviewed and the following data were collected in FOP patients: date of birth/death, birthplace, gender, personal history, family history, clinical manifestations, physical examinations, and blood biochemical tests. All blood tests were performed at the clinical laboratory of Tongji Hospital of Tongji University. Blood biochemical tests included serum parathyroid hormone level , osteocalcin , total serum calcium , alkaline phosphatase ALP 50–130 U/L), phosphorus , erythrocyte sedimentation rate , alanine aminotransferase , aspartate aminotransferase , creatinine , and blood uric nitrogen . According to the previous protocols [0 U/L, phP-value < 0.05 was considered significant.Statistical package for social sciences (SPSS) 19.0 was used for the statistical analysis. The Kolmogorov-Smirnov test was used to determine whether the parameters followed a normal distribution. All continuous variables were presented as mean ± standard deviation. Data were illustrated as medians (interquartile ranges) if not conforming to normal distribution. Comparisons of the means and proportions were performed with independent samples t-test and the chi-square test, respectively. Spearman correlations and logistic regression analysis were performed to explore the risk factors for the mortality of FOP patients. Receiver-operating characteristic (ROC) curves were constructed to estimate the cut-off, sensitivity and specificity of the risk factors which potentially predicted the early death of FOP patients. Tests were two-sided and a 65 cases of FOP individuals recruited in our study were all Han people from 25 provinces of China. 46.2% (30/65 cases) were male while 53.8% (35/65 cases) were female. According to criterion of classification, 92.3% (60/65 cases) patients were diagnosed as classic FOP patients based on classic defining features of FOP, 3.1% (2/65 cases) of individuals were FOP-plus since they had classic defining features of FOP and other atypical features such as cryptorchidism or childhood glaucoma, and 4.6% (3/65 cases) were determined as FOP variants for their mild malformations of great toes. ACVR1 gene analysis was performed in 50 patients and revealed that 48 cases were canonical c.617G > A mutation. Two of FOP variants did not present canonical c.617G > A mutation: one of FOP variants showed a variant mutation in ACVR1 at c.774G > C (p.R258S), while the rest one had a mutation in ACVR1 at c.1067G > A (p.G356D). (Table During the past 10-year period (Dec 2008 to Dec 2018), 9 patients with FOP  were dead while 56 patients  were survival. Hence, the total mortality rate was 13.8% (9/65 cases). The mortality rate in FOP patients aged 18 years and younger at diagnosis was 11.1% (5/45 cases), whereas it was 20% (4/20 cases) in patients aged over 18 years. All non-survivors were confirmed c.617G > A mutation, and were diagnosed as classic FOP for their congenital malformations of great toes and progressive HO  and FOP patients ≤18 years at diagnosis . However, in FOP patients aged over 18 years at diagnosis, no association between serum OC level and the mortality was found.Logistic regression analysis indicated that serum OC level was negatively associated with the mortality in all FOP patients and FOP patients aged 18 years and younger at diagnosis. Further adjustment for age and gender did not change the correlation between serum OC and the mortality in all FOP patients  of blood biochemical markers were shown in Fig. Previous researches of FOP usually focused on clinical features and genetic patterns of FOP patients , 13, 14.In our study, we found serum OC level was negatively associated with the mortality in FOP patients, and this correlation only exist in the FOP patients aged 18 years and younger at diagnosis. OC is a major non-collagenous protein in bone and serves as a biomarker of bone formation mostly. Serum level of OC is significantly correlated with the age . In prevCardiorespiratory failure from thoracic insufficiency syndrome was a severe complication and a common cause of death in FOP patients . It has Bone transformation markers including OC are highly dependent on gender, age and pubertal stage , 22. In Our research has several limitations. Firstly, 65 of FOP patients were recruited in our study, which only represent 10 % of all FOP patients in China . SecondlIn summary, our study described the mortality rate and related risk factors for the mortality of FOP patients in China for the first time. We found that the mortality rate of FOP was 13.8% and serum OC level was negatively correlated with the mortality in Chinese FOP patients ≤18 years at diagnosis. Our study could be valuable in predicting lethal outcome of FOP patients aged 18 years and younger at diagnosis, allowing interventions to be conducted most effectively to reduce the associated mortality.Additional file 1: Supplementary Table 1. Spearman correlations between parameters and mortality in FOP patients. Supplementary Table 2. Spearman correlations between parameters and mortality in FOP patients with age≤18 years. Supplementary Table 3. Spearman’s correlations between parameters and mortality in FOP patients with age>18 years.


Ostrinia furnacalis, ORs that respond to sex pheromones have been characterized, but general ORs for plant odorants, especially for repellents, have not been identified. Nonanal is a plant volatile of maize that could result in avoidance of the oviposition process for female adults in O. furnacalis. In this study, we identified a female-biased OR that responds to nonanal using a Xenopus oocyte expression system. In addition, we found that OfurOR27 was also sensitive to two other compounds, octanal and 1-octanol. Behavioral analysis showed that octanal and 1-octanol also caused female avoidance of oviposition. Our results indicated that OfurOR27 is an OR that is sensitive to repellents. Moreover, the two newly identified repellents may help to develop a chemical ecology approach for pest control in O. furnacalis.Attractants and repellents are considered to be an environment-friendly approach for pest management. Odorant receptors (ORs), which are located on the dendritic membranes of olfactory sensory neurons in insects, are essential genes for recognizing attractants and repellents. In the Asian corn borer,  Chemical ecology is now established as an approach for pest management. Pheromones have been identified in many insects and are great attractants to interfere with mating behaviors by trapping large numbers of male adults . HoweverInsects have evolved a complex olfactory system to detect various odorants to search for mating partners, locate host plants, identify oviposition sites, and evade toxicants and predators . AntennaDrosophila melanogaster , feeds on various plants including the economic crop maize, causing serious damage and resulting in about 10–30% yield loss of maize in China , 21 IRs (ionotropic receptors), 5 GRs (gustatory receptors), 2 sensory neuron membrane proteins (SNMPs), and 26 odorant degrading enzymes (ODEs)  . Among ts (ODEs) . In addis (ODEs) . GeneralO. furnacalis  in females of rnacalis . BehavioOstrinia furnacalis colonies were maintained at laboratory conditions in the Chinese Academy of Agricultural Sciences, Beijing, China. Insects were reared on an artificial diet at 28°C and kept at 15:9 (L/D) and 80% relative humidity. Male and female adults were fed with 10% sugar solution. Tissues including antennae, proboscis, thorax, legs, and sex glands were dissected from 3-day-old adults, immediately placed in liquid nitrogen, and then stored at −80°C until use.A total of 95 odorants from Sigma-Aldrich were used for this experiment  and wereTotal RNA from 50 male or female adults was isolated using TRIzol reagent  following the manufacturer’s instructions. The RNA was dissolved in RNase-free water, and the integrity was assessed by gel electrophoresis. The concentration and purity of RNA were determined on a NanoDrop ND-2000 spectrophotometer . The first-strand complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions.OfurOR27 (Acc. No. LC002721) was obtained from antennal transcriptome and was 1,206 bp in length, encoding for 402 amino acids   and further sequenced to confirm the sequence. The full-length gene sequence of OfurOR27 was then ligated into a pT7Ts expression vector using a pair of newly designed primers with ApaI and XhoI restriction sites (OfurOrco (OfurOR2) was stored at −80°C (LC002697.The full-length open reading frame (ORF) encoding no acids . The seq States) . PrimeSTon sites . The pT7at −80°C . The accSmaI and subsequently used for cRNA synthesis with an mMESSAGE mMACHINE T7 kit . Mature healthy Xenopus oocytes (stages V–VII) were incubated with 2 mg/ml collagenase I at pH 7.6 in wash buffer consisting of 96 mM NaCl, 2 mM KCl, 5 mM MgCl2, and 5 mM HEPES (pH 7.6) at room temperature for about 1 h until almost all of them were separated (OfurOR27 and OfurOrco cRNA (27.6 ng each) was microinjected into the oocytes. After an incubation for 2–4 days at 18°C in incubation medium , oocytes were connected to a two-electrode voltage clamp, and then, the currents were recorded. Currents induced by odorants were recorded using an OC-725C oocyte clamp  at a holding potential of −80 mV. Oocytes were exposed to 10–4 concentrations of different compounds in a random order for 15 s each, and the interval between exposures allowed the current to return to baseline. Data acquisition and analysis were carried out with Digidata 1440A and Pclamp 10.0 software . At the same time, dose-response data were analyzed using GraphPad Prism 7 . Statistical comparison of responses to different odors of OfurOR27 was assessed using GraphPad Prism 7, followed by least-significant difference (LSD) tests.The expression vector was linearized by restriction enzyme eparated . After bOfurOR27 in different tissues of adults, quantitative polymerase chain reaction (qPCR) was performed using cDNA obtained from antennae (A), proboscis (P), thorax (T), legs (L), and sex glands (SG). OfurActin was chosen as the reference gene. The primers are listed in OfurOR27 relative expression levels were analyzed using the relative 2–ΔCT quantitation method, where ΔCT = CT (OfurOR27) – CT (OfurActin). Statistical comparison of expression of OfurOR27 was assessed using one-way nested analysis of variance (ANOVA), followed by LSD tests.To reveal the expression level of Behavior analysis for oviposition was carried on for nonanal and other identified candidate odorants. A net cage (25 × 25 × 25 cm) was used with two pieces of plastic wrap (15 × 15 cm) hanging on opposite sides that contained odorants and solvent, respectively. Each odorant was diluted into 100 ng/μl with paraffin oil as the solvent. Fifty gravid females were put into the cage, and after 24 h, eggs laid on the two pieces of plastic wrap were collected and counted under a stereomicroscope . Three rSensilla trichoidea from 2-day-old female adults were used for the recordings. Individuals were fixed in a 1 ml plastic pipette tip and the settings for recording were the same as discribed in OfurOR27, OR gene repertoires from 14 species in six orders were collected including O. furnacalis   . The dosectively . Responsectively .OfurOR27 was predominantly expressed in the antennae of adults, with relative expression levels of more than 0.1, compared to those in proboscis, thorax, legs, and sex glands, with the relative expression levels of 0.00291, 0.00192, 0.00223, and 0.00255, respectively , which was an effective broad-spectrum mosquito repellent  has been strongly advocated in modern agriculture. The new approach of pest management using chemical ecology is an important part of IPM. Attractants such as pheromones have been applied controlling various pests due to a high degree of commercialization. For repellents, only a few compounds have been made as commercial products. The most famous odorant is epellent . Howeverepellent . For agrepellent . Recentlepellent . This reAlucOR46 might be related to locate the host plants of Apolygus lucorum  may be accessed from the DDBJ website: GW and BY designed the experiments. JY, YC, and YZ carried out the experiments. JY and BY analyzed the experimental results. JY and BY wrote the manuscript.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.


Objective: To investigate the relationship of overnutrition (obese and overweight) with severity of illness in children hospitalized with acute lower respiratory infections (ALRIs), frequency of viral coinfections and leptin levels.Methods: We studied 124 children <2 years old that were hospitalized for ALRI. Nutritional status was calculated by z-scores according to weight-for-age z-scores, length or height-for-age z-scores, and weight-for-height z-scores. Nasopharyngeal aspirates (NPAs) were obtained and viral respiratory pathogens were identified using reverse transcription polymerase chain reactions (RT-PCR). Respiratory syncytial virus (RSV) load was assessed using quantitative RT-PCR. NPA and plasma leptin level were measured. Clinical data and nutritional status were recorded, and patients were followed up until hospital discharge. Viral coinfection was defined as the presence of two or more viruses detected in the same respiratory sample. Severity of illness was determined by length of hospitalization and duration of oxygen therapy.Results: Children with overnutrition showed a greater frequency of viral coinfection than those with normal weight . A lower RSV load was found in obese (5.91 log10 copies/mL) and overweight children (6.49 log10 copies/mL) compared to normal weight children . In multivariate analysis, obese, and overweight infants <6 months old were associated with longer hospital stays  as well as a greater duration of oxygen therapy . Obese children <6 months showed higher plasma leptin level than normal weight children .Conclusions: In infants younger than 6 months, overnutrition condition was related to increased severity of infections and high plasma leptin level. Also, children with overnutrition showed a greater frequency of viral coinfection and low RSV viral load compared to normal weights children. These findings further contribute to the already existent evidence supporting the importance of overnutrition prevention in pediatric populations. Acute lower respiratory tract infections (ALRIs) are a common illness in children <5 years old, with significant morbidity and mortality in infants and young children under the age of two –3. RespiThere are many risk factors related to ALRIs. Some of these are relative to pathogens, such as viral load , while oOvernutrition causes an excessive accumulation of body fat. It is a highly prevalent condition and has been ranked as the foremost epidemic of the twenty-first century . ObesityIn addition to immune modulation of obesity, alterations in membrane lipid composition have been observed in erythrocyte from obese children. The membrane composition has also revealed an increase in cholesterol content as has the cholesterol to phospholipid molar ratio, both of which have been positively correlated with a decrease in membrane fluidity, body-mass index, and plasma cholesterol levels . CholestHence, the objective of this study was to estimate the relationship of overnutrition on severity of illness in infants (aged between 0 and 5 months) and children (aged between 6 and 24 months) hospitalized with ALRIs. Moreover, frequency of viral coinfection, RSV viral load and levels of leptin according to nutritional status were evaluated.A cross-sectional study was conducted for 2 consecutive epidemic periods (from May to August) in 2016 and 2017. Patients were selected based on following inclusion criteria: children <24 months old that were hospitalized for ALRI at two medical centers in Santiago, Chile: Urgencia Materno-Infantil at Clínica Dávila and Dr. Exequiel González Cortés Hospital. Patients were classified as having bronchiolitis, bronchitis or pneumonia. Diagnoses were made in patients with dyspnea, signs of lower respiratory tract infections  and/or a positive chest x-ray . The clinical and demographic characteristics of each patient were also recorded. The exclusion criteria were: children with nutritional risk and/or undernourished children, newborns <28 days old, patients with a diagnosis of recurrent wheezing, previous hospitalization for any cause, primary or secondary immunodeficiency, gestational age of <37 weeks, bronchopulmonary dysplasia, previous mechanical ventilation, congenital heart disease, respiratory infection during the previous 2 weeks, corticosteroid intake 72 h before sample was taken, and children with negative samples for respiratory viruses. Approximately 3 ml of nasopharyngeal aspirate (NPA) were collected from each patient, usually during the admission process (<3 h) and always within the first 24 h of hospitalization. Secretions from both nostrils were aspirated without flushing using a soft catheter placed in a collection trap with 3 ml of sterile saline solution and immediately transported on ice to the laboratory. Aliquots of 2 ml were stored at −80°C for viral analysis. Aliquots of 1 mL were centrifuged at 1,000 g for 15 min at room temperature and the supernatants obtained were stored at −80°C until determination of leptin level.Viral RNA and DNA were simultaneously extracted from 150 μL of the NPA samples using the Viral RNA isolation kit NucleoSpin  following the manufacturer's instructions, and stored at −80°C, until use. Viral infection was detected with real-time PCR kit ARGENE® , for RSV, human metapneumovirus (HMPV), parainfluenza virus 1–4 (HPIV), human coronavirus (HCoV), adenovirus (AdV), bocavirus (HBoV), influenza A (FluA), influenza B (FluB), and rhinovirus/enterovirus (HRV/HEV), following the manufacturer's instructions. Viral co-infection was defined as the presence of two or more viruses detected in the same respiratory sample.10 copies/mL.Forty-five patients that tested positive for RSV were selected using the ARGENE® real-time PCR kit. A new aliquot was used with 150 μL of NPA. Nucleic acids were extracted using the Viral RNA isolation kit NucleoSpin . The qRT-PCR was performed using the Takyon™ Rox Probe MasterMix dTTP blue kit , using primers with N1 (5′-GGAACAAGTTGTTGAGGTTTATGAATATGC3′) and N2 probes (CTTCTGCTGTCAAGTCTAGTACACTGTAGT-3′) . The conAbout 2 ml of blood were collected in sodium heparin collection tubes (BD Vacutainer) usually during the admission process. The sample was centrifuged at 1,000 g for 15 min at room temperature. The plasma obtained was divided into aliquots and stored at −80°C.Leptin concentration in plasma and NPA was measured using the Magnetic Luminex®assay (R&D), according to manufacturer protocol.2, Fisher's exact or Mann–Whitney tests. Spearman's test was used to estimate the association between viral load and severity. Multivariate Poisson regression models were used to determine the factors that predict severity of infection (length of stay and duration of oxygen therapy). Multicollinearity analyses among the independent variables were performed and none were observed. The cutoff point for multicollinearity was determined as variance inflation factor <5 and tolerance >0.2. We did not include birth weight in the multivariate regression model because it increases the multicollinearity in the model (Variance Inflation Factors from 2.32 to 6.47). The relative risk (RR) for increased length of stay and oxygen therapy was calculated by taking the estimated Poisson regression coefficient (β) for each variable and transforming it by eβ [exp*confidence interval] of each independent variable for the model. Statistical significance was set at p < 0.05 for all analyses, along with a 95% confidence interval (CI). All analyses were performed with Stata 14.1 software .Descriptive analyses, medians (ranges), and frequency distributions were used to summarize the demographic and baseline attributes of patients. Associations among the dependent and independent variables were assessed by either the χA total of 160 children were screened, and 124 patients were eligible for this study . The cliA total of 63 (50.8%) patients tested positive for a single respiratory virus, while another 61 patients (49.2%) tested positive for more than one respiratory virus. Of these, 50 (82.0%) tested positive for two viruses and 11 (18.0%) for three viruses. RSV was the most frequently detected virus in 88 cases (70.9%), followed by HRV/HEV in 33 (26.6%) and HBoV in 27 (21.7%). Most viruses were found in coinfection . No diffp = 0.013 and p = 0.004 respectively) (p = 0.839).Nutritional status and coinfection (≥2 viruses) were evaluated to assess the effect of overnutrition in viral coinfection. Obese and overweight infants showed a greater frequency of coinfection (obese 71% and overweight 68%) than those with normal weights (37%) (ctively) . No sign10 copies/mL (min: 3.22 log10 copies/mL and max: 7.86 log10 copies/mL) and 6.49 log10 copies/mL (min: 3.71 log10 copies/mL and max: 8.11 log10 copies/mL) for overweight infants, while those with normal weights had a median of 8.06 log10 copies/mL (min: 4.15 log10 copies/mL and max: 9.17 log10 copies/mL). The difference between obese and overweight infants compared to those with normal weights was significant  (p = 0.6395). No correlation was evidenced between RSV load and length of hospital stay  or days of oxygen therapy .The effect of obesity and overweight conditions on the RSV load was assessed in 46 infants  with available nasopharyngeal samples. The median of RSV loads in obese infants was 5.91 logctively) . No signOW:NW = 1.27; CI: 1.18–1.34) and duration of oxygen therapy compared with normal weight children . However, when we studied patients under and over 6 months separately, we found that both obesity and overweight conditions were associated with a greater length of stay  and duration of oxygen therapy compared with normal weight . These results show that obesity and overweight conditions are significantly associated with the severity of viral respiratory infections in very young children.children  (RROW:NW of stay  (RROW:NWl weight  (RROW:NWp = 0.862).Also, we analyzed whether assisted ventilation is related to nutritional status and our results suggest that assisted ventilation, as a proxy of severity, was not statistically associated with overnutrition .No significant differences were seen between leptin level and nutritional status in both plasma and NPA samples . On the Obesity and ALRIs constitute two important morbidity factors in the world, and its relationship began to be studied after the influenza H1N1 pandemic , 8. In aOAS1 gene. Despite not finding significant differences, we found a tendency to decrease the expression of the OAS1 gene in overweight and obese patients compared to normal weight children (Data not shown). Some studies have related leptin level with hepatitis virus infection. In the study by Caner et al. . Most of children <6 months (69%) were exclusive breastfeeding at the time of sampling. Savino et al. . We found that children <6 months showed higher plasma leptin level than older children, regardless of nutritional status (5.26 vs. 1.29 ng/μl; o et al.  observedo et al.  which cao et al. . Overweio et al. . AccordiHere, we have shown for the first time to our knowledge, that obesity and overweight conditions are associated with viral coinfections in children hospitalized for LRTIs. Although it is still unclear why this is so, there is some evidence that can start to shed light on this association. It has been reported that the erythrocyte membrane responds very early to modifications of plasma lipoproteins and suggest that in childhood obesity a modified transfer of cholesterol from plasma to erythrocyte membrane may take place . SeveralObese and overweight patients, in addition to exhibiting high levels of viral coinfections as well as more severe infections, also presented lower viral loads than infants with standard weights. Through a mathematical model, HRV, the fastest-growing virus, reduces the replication of the remaining viruses during a coinfection, while PIV, the slowest-growing virus is suppressed in the presence of other viruses . AccordiA possible limitation of our study is that we analyzed NPA samples using the RT-PCR method. It is unknown if this highly sensitive molecular technology detects only pathogens that cause LRTIs or if some of the viruses detected could be commensal pathogens . Some ofEffective interventions are required to reduce childhood obesity. Meanwhile, further research is needed to determine the role of leptin and other immunological markers in the severity of viral infection and to understand the pathophysiology of viral coinfection as it relates to nutritional status as well as to explore the epithelial-specific response to respiratory viruses in primary cells of lean and obese child.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Scientific Ethics Committee, Universidad Autónoma de Chile. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.The concept and design of the study was carried out by LF, GA-B, JI, DG-D, and GV. LF, GA-B, and GV obtained the clinical data. GA-B and MR-F conducted the experimental analysis. LF and FZ-R carried out the statistical analysis. LF, GA-B, and FZ-R wrote the manuscript. All of the authors contributed to the interpretation of the data and critically revised the manuscript, providing important intellectual content and approving the final report.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.


Some studies have found that soft drinks consumption was associated with the risk of fractures. We aimed to assess the association in the Chinese population; (2) Methods: Data from 17,383 adults aged 20 to 75 years old attending the China Health and Nutrition Survey (CHNS) between 2004 and 2011 were analyzed. Soft drinks consumption and fracture occurrence were self-reported. The cross-sectional and longitudinal associations between soft drink and fracture was assessed using multivariable mixed-effect logistic regression and Cox regression; (3) Results: After adjusting for sociodemographic and lifestyle factors and dietary patterns, compared with those who did not consume soft drinks, participants with daily consumption of soft drinks had an odds ratio (95%CI) of 2.72 (95%CI: 1.45–5.09) for fracture. During a mean 5-year follow-up, there were 569 incident fracture cases. Compared with non-consumers, those with daily soft drinks consumption had a hazard ratio (95%CI) of 4.69 (95%CI: 2.80–7.88) for incident fracture; (4) Conclusions: Soft drinks consumption is directly associated with the risk of fracture. Reducing soft drinks consumption should be considered as an important strategy for individual and population levels to maintain bone health. Musculoskeletal diseases such as fractures pose a serious global burden of disease. Based on the Global Burden of Disease study, musculoskeletal illness accounted for 6.8% of total disability-adjusted life-years worldwide in 2010 . Within Although the direct causes of fractures are accidental falls or hits, the fundamental causes are low bone density and excessive bone loss ,6. UnheaDuring the past three decades, China’s urbanization has transformed the traditional dietary pattern into a modern dietary pattern . AccompaMost of the existing studies on soft drinks consumption have focused on bone mineral density, (BMD) with few studies on fracture . SeveralSoft drinks consumption is positively associated with obesity risk. Obesity is a risk factor for fractures in specific bone sites and a prospective cohort study which involving 17 sites in 10 countries found that obesity is a risk factor for upper arm/shoulder and clavicle fractures ,24. HoweCHNS is an open prospective cohort study which was conducted in nine provinces in China . Ten wavData of fracture were self-reported in each wave in 2004, 2006, 2009 and 2011 by the question “Have you ever had a fracture?” along with age when the first fracture occurred [Soft drinks consumption was determined in 2004, 2006, 2009 and 2011 by the question “Last year, did you drink soft drinks or sugared fruit drinks?”. When the participants answer “yes” to this question, they were asked to provide more information of what types of soft drinks ((1) Chinese brand soft drinks; (2) Non-Chinese brand soft drinks; (3) Sugared fruit drinks) and the frequency of the consumption: (1) almost every day; (2) 3–4 times/week; (3) 1–2 times/week; (4) 1–2 time/month; (5) no more than once a month; (9) unknown. In order to distinguish the difference between “unknown” and other answers of questions in the original questionnaire, when the answer is “unknown”, the “unknown” coded as “9”. Chinese and non-Chinese brands include carbonated drinks, sugar-sweetened dairy drinks, etc. The soft drinks consumption frequency was categorized into five levels: non-consumers, <1 time/week, 1–2 times/week, 3–4 times/week, and almost daily. The participants were also asked about the amount of each type of soft drinks (liter) consumption each week. We calculated the total amount of soft drinks consumption and categorized into three categories: (1) non-consumers; (2) <1 L/week; (3) ≥1 L/week.Socio-demographic, lifestyle, physical measurements and chronic conditions data were collected at each wave. The highest level of education achieved was divided into three groups: low (illiterate or primary school), medium (junior middle school) and high (high middle school or higher). The residency was divided into three urbanization levels based on an urbanization index which is a composite of 12 components that included population and other socioeconomic characteristics ,26. The Hypertension was determined based on diastolic (above 90mmHg) and/or systolic (above 140 mmHg) blood pressure measures or having doctor-diagnosed hypertension. Diabetes was determined based on having doctor-diagnosed diabetes.To compare differences between groups for categorical variables, the chi-square test was used and ANOVA was used for continuous variables. Mixed effect logistic regression was used to assess the association between soft drinks consumption and the prevalence of fracture. A set of models was used: model 1 adjusted for age and sex; Model 2 further adjusted for intake of energy and fat, smoking, alcohol drinking, income, urbanization, education, physical activity, and BMI; model 3 further adjusted for two dietary patterns .All the analyses were performed using STATA 15.1 . Statistical significance was considered when In general, there was a negative correlation between soft drinks consumption and age in 2004 . Men werp for trend <0.001), respectively  across soft drinks consumption levels of none, <1 time/week, 1–2 times/week, 3–4 times/week and almost daily were 1.00, 1.16 (0.94–1.44), 1.32 (0.95–1.84), 1.70 (0.99–2.92), and 2.72 (1.45–5.09)  years. Compared with non-consumers of soft drinks, daily consumers had a hazard ratio of 4.69 (95%CI 2.80–7.88) for fracture after adjusting for sociodemographic and lifestyle factors including overall dietary patterns. However, the actual consumption of soft drinks did not show a statistically significant association with the fracture. Compared with non-consumers, those who consumed ≥1 L/week had an HR of 1.16 (95% CI 0.83–1.61) for fracture .In the sensitivity analyses, when we adjusted for dietary patterns without soft drinks, the above associations between soft drinks and fracture remainedA high consumption of soft drinks was associated with an increased risk of fracture in the current large prospective cohort study. Daily consumption of soft drinks was associated with a doubled risk of fracture independent of sociodemographic and lifestyle factors as well as overall dietary patterns.Despite the large number of studies on the association between soft drinks and non-communicable diseases, studies on soft drinks and fracture among adults are limited ,28,29. TOur findings have public health significance. The prevalence of soft drinks consumption in the 2004 survey of this study was 24%, which is lower than in the USA. However, there has been a rapid increase in soft drinks consumption in China. Data from the 2010–2012 national survey suggest that the prevalence of soft drinks consumption reached 50% . The conSeveral mechanisms may explain our findings on the direct association between consumption of soft drinks and fracture. The Chinese population may be at a high risk of fracture due to a low intake of calcium. Calcium intake was below 400 mg/d in all the soft drinks consumption groups. Soft drink contributes to the dietary intake of phosphorus. High phosphorus but low calcium diet may stimulate parathyroid hormone and cause bone resorption . A high Certain ingredients in soft drinks can affect bones. Sugar and sodium in soft drinks can increase the loss of calcium ,34. IncrAnother possible mechanism for the indirect effect of soft drinks on bone fractures is the mediating effect of obesity. It has been shown in many studies that the consumption of soft drinks increases the obesity risk . Fat affPhthalates may also play a role in the association between soft drinks consumption and fracture. Phthalates are widely used to make bottles for soft drinks. In animal studies, female rats treated with phthalates showed significant dose-dependent fetal skeletal malformations and bone homeostasis imbalances  . It can There are some limitations in the current study. Firstly, the fracture was based on self-report. The fracture date was based on the participants’ recollection of the age at which the fracture occurred, which may be affected by recall bias. However, a large cohort study has shown that this approach is a feasible alternative ,52. SecoIn summary, a high consumption of soft drinks is associated with fracture risk. Daily soft drinks consumption was associated with a doubled risk of fracture independently of sociodemographic factors and overall dietary patterns. This study highlights the important role of soft drinks in fracture risk among adults. To increase or maintain bone mass and reduce the risk of fracture, public health and clinical interventions should take into account reducing soft drinks consumption as an important strategy for the individual and population levels.


Increased anxiety was frequently reported during the 2020 global COVID-19 pandemic. An association between anxiety and increased body weight has been documented. Identifying associations between diet quality and anxiety may facilitate the development of preventive dietary policy, particularly relevant since obesity appears to increase the risk of adverse COVID-19 outcomes. In this study we aim to examine associations between changes in diet pattern and body weight and anxiety levels during the COVID-19 pandemic among Israeli respondents to an international online survey.Conducted between March 30–April 252,020, this was cross-sectional, international and online study. The questionnaire was developed and tested in Hebrew and translated into six other languages: English, Arabic, Spanish, French, Italian, and Russian. The survey was conducted on a Google Survey platform, the link to which was posted on several social media platforms. Adults aged 18 or older who saw and responded to the link on a social media site comprised the study population.Of the 3979 eligible respondents, 1895 indicated their current location as Israel. Most Israeli respondents completed the survey in Hebrew (83.2%) followed by Arabic (9.4%), though responses were recorded in all seven of the survey languages. The median age was 33 (IQ = 22) years, and 75.7% were female. Almost 60% indicated that their pre-pandemic diet was healthier than their current diet, and 25.2% indicated they had gained weight during the pandemic. The median Mediterranean diet score was 9 (IQ = 3). While the median General Anxiety Disorder (GAD-7) score was 5 (IQ = 8), only 37.3% of participants reported at least mild anxiety (a GAD-7 score of 5 or more), while 10.7% reported moderate anxiety or greater (a GAD-7 score of 10 or more). In a multivariate logistic regression model of at least mild anxiety, being male and completing the survey in Hebrew significantly reduced odds of at least mild anxiety, while a worsening of diet quality during the pandemic, weight gain, and isolation significantly increased odds of at least mild anxiety.During the COVID pandemic, changes in nutrition quality and habits were associated with greater anxiety. These findings suggest the need for routine and continuous surveillance of the nutritional and psychological consequences of outbreaks as part of healthcare preparedness efforts. Organizations responsible for community-based health services  should adopt specific interventions to improve case finding and support individuals at increased risk of anxiety and declining nutrition status within primary healthcare settings. These interventions should include the provision of appropriate diagnostic instruments, training of medical staff, feedback to physicians and nurses, and raising awareness among the relevant patient population and their caregivers. Primary care physicians should refer people with high anxiety or substantial weight gain during the pandemic to appropriate mental health and dietetic treatment, as needed.NCT04353934. The global COVID-19 pandemic has created health uncertainty, especially among groups at risk for adverse COVID-19 outcomes . COVID-1Social isolation and home quarantine can produce changes in eating behaviors, food intake, and physical activity patterns, thus influencing body weight and overall health and wellbeing , 8. An aThe Mediterranean diet (MedDiet), characterized by frequent intake of olive oil, fruits, vegetables, whole grains, legumes, fish and nuts, and infrequent intake of red and processed meats and added sugars, is associated with reduced all-cause and cause-specific mortality , 17. MorFew studies have evaluated changes in eating patterns and mental health symptoms in the general population during a pandemic , 21–24. In this study, we examined whether diet quality and weight changes are associated with anxiety during the first stage of the COVID-19 outbreak in Israel.The present cross-sectional survey was conducted online using a convenience sample. Anxiety was assessed using the General Anxiety Disorder (GAD-7) subscale. The survey assessed the extent to which the current participant diet is similar to the MedDiet ; respondent-reported change in diet quality and body weight; and demographic characteristics.The study was approved by the Institutional Ethics Board (Helsinki Committee) of Ariel University, Israel. Each participant provided informed consent prior to responding to the survey. Individuals who did not provide informed consent (indicated by clicking on the appropriate button) could not proceed with the survey.This international study was conducted online using a Google Survey platform. The survey was uploaded to the Ariel University Department of Nutrition Facebook page; and the r/Coronavirus community on reddit, which created a page for pandemic-associated research; and individual social media pages. Participants were encouraged to re-post the survey link on their own social media to expand exposure.The study population included all adult individuals who elected to complete the survey online and who provided informed consent by clicking on the appropriate button. Only the first response to the survey was included among individuals who responded more than once.All adult individuals (aged 18 or older) who responded to the survey were included in any of the study languages: Arabic, English, French, Hebrew, Italian, Spanish or Russian.Subjects who indicated that their age is younger than 18 years and those who did not provide informed consent were excluded. Only the index response of a given participant was included in the analysis.The survey was posted to public and personal social media pages. The survey could not be completed without first providing informed consent. Responses were automatically recorded in Google Survey.The survey was translated from its original Hebrew to the following languages: Arabic, English, French, Italian, Russian, and Spanish. Each translation was performed by a native speaker of the target language who was also fluent in Hebrew. Once translated, the translation was back-translated to Hebrew by an individual bilingual in both the target language and Hebrew, thus validating the translation.Data were anonymous, though respondents had the option of providing an e-mail address to which information about his/her MedDiet score was mailed. The following data were elicited in the survey: 1) demographic information: age; sex; country of residence; education; work setting; current occupational status; current quarantine/isolation; health status; 2) nutrition questionnaire  3) anxiety questionnaire (GAD-7).The Israeli Mediterranean diet screener (I-MEDAS) used in the current study was adapted from the original, 14-item questionnaire. The I-MEDAS predicted mortality in a prospective multi-ethnic, population-based cohort of Israeli adults. The I-MEDAS is scored such that a given question receives a value of 1 if the criterion was met, and 0 if it was not, so the total I-MEDAS score can range from 0 to 17 points .The 7-item Generalized Anxiety Disorder Scale (GAD-7) was used to measure anxiety. This questionnaire is validated for use in the general populationin each of the seven languages in which the present survey was conducted . The parThe sampling method is best described as a convenience sample with a snow-ball distribution as the survey was uploaded to both public and personal social media sites, and respondents were requested to re-post the survey on their own social media.p < 0.05.Data were downloaded from Google Survey to Excel (Microsoft USA) and analyzed on SPSS v25 Statistical Analysis Software (IBM Inc.). Distributions of continuous variables were assessed for normality using the Kolmogorov-Smirnov test. All continuous data had distributions significantly deviating from normal, so they are described as median (interquartile range). Nominal variables were presented as n (%). Associations between continuous variables were described by calculating the Spearman’s correlation coefficient. Continuous variables were compared by nominal variables using the Mann-Whitney U test or the Kruskal-Wallis test as appropriate. Associations between nominal variables were assessed using the chi-square test. A multivariate logistic regression model was developed to predict the cut-point for mild anxiety or greater (GAD-7 score ≥ 5) , 29 usinSample size was calculated with Raosoft® With 2144 anticipated responses, the total study was powered to provide a 95% confidence level of a population proportion and a confidence of 2%. A total of 1895 participants indicated Israel as their current location, providing 95% confidence level and a confidence limit of 2.1%.On April 25, 22:00 Israel time, the questionnaire was closed to responses, and the database was downloaded. Participant dispensation is presented in Fig. Characteristics of the study population are presented in Table p < 0.0001. A greater proportion of participants who reported a worsening of diet quality also reported weight gain compared to those who did not report a worsening of diet quality .Table No weight change was reported by 35.3% of respondents, and 27.5% did not know if weight change had occurred during the lockdown. Of 704 (37.2%) participants who did report weight change, most (67.8%) reported weight gain while the rest reported weight loss. Median weight change was 2 kg, whether individuals gained or lost weight.p = 0.002), and perceptible via mean values:8.92 ± 2.21 vs. 9.33 ± 2.37. Among people who gained weight, 84.5% indicated that their pre-outbreak diets were healthier than their current diets. By contrast, 15.5% of participants who reported no weight change or weight loss indicated that their pre-pandemic diets were healthier, p < 0.0001.Weight gain was significantly, inversely, associated with the change in diet quality. While the median MedDiet score was 9 (3) among participants who gained weight and among those who did not, the difference was significant by group (p = 0.72). Respondents who gained weight in the course of the week prior to the survey (during the pandemic lockdown) reported 30 (120) minutes of exercise/week, while those who did not gain weight reported 40 (118) minutes of exercise/week, p = 0.002.A 50% relative reduction in time devoted to exercise was observed during the pandemic, declining from a median of 60 min per week to 30 min per week, p < 0.0001. The amount of time reported spent exercising prior to the outbreak did not differ by weight change status, and was a median 60 (147) in both people who gained and those who did not gain weight (p = 0.62). After the outbreak, people whose diet worsened reported 30 (102.5) minutes/week of exercise, compared to 60 (147) minutes/week among those whose diets did not worsen, p < 0.0001.Similarly, time spent exercising prior to the pandemic did not differ by a subsequent change in diet quality and was 60 (147) minutes/week in people whose diet worsened as well as among those whose diet did not worsen during the outbreak  vs. 5 (2); the difference is significant (p < 0.0001), and the difference is apparent when examining mean values: 4.3 ± 1.2 prior to and 4.1 ± 1.3.Measures of anxiety and emotional stress are shown in Table p < 0.0001. Similarly, the GAD-7 score was significantly higher in individuals who reported weight gain during the pandemic vs. those who did not report weight gain: 5 (6) vs. 3 (5), p < 0.0001. Consistent with this, the GAD-7 score was significantly, positively associated with weight change, rho = 0.207, p < 0.0001. The GAD-7 score was also positively associated with the degree to which the respondent felt isolated: rho = 0.331, p < 0.0001. The amount of exercise reported by the respondent was weakly, inversely associated with the GAD-7: rho = − 0.051, p = 0.029.Among individuals reporting that their diet was healthier prior to the coronavirus pandemic, the median total GAD-7 score was 4 (5), compared to 2 (5) among those who reported no change or an improvement in diet quality since the start of the outbreak, The GAD-7 score was not significantly associated with age. Table Table The present study indicates that diet quality was inversely associated with anxiety during the 2020 COVID-19 pandemic: every one-point increase in the MedDiet score was associated with an almost 7% decrease in the odds of moderate to severe anxiety. More than 50% of study participants had a GAD-7 score consistent with at least mild anxiety. This rate is considerably higher than the prevalence of anxiety in the general population, which is estimated to be 1.9–5.1% .High levels of anxiety were reported in other countries during the first outbreak stages, using online cross-sectional surveys. A study among the Chinese population assessed anxiety using the GAD-7 questionnaires and reported on 35.1% with moderate anxiety . A surveIt has been shown that metabolic status (including obesity), age, and sex influence the clinical severity of COVID-19 . Poor diIsrael has an efficient primary care healthcare system, providing a broad basket of services , which cTherefore, steps should be taken to improve case-finding of people with higher anxiety among primary physicians and those with adverse eating behaviors among dietitians. This requires the provision of appropriate diagnostic instruments, training of medical staff, feedback to physicians and nurses, and raising awareness among the relevant patient population and their caregivers.Findings herein must be considered in the framework of study limitations. The data are cross-sectional. Associations between diet quality and anxiety are correlational only. Because the exposure  and outcome (anxiety score) were measured simultaneously, and, as such, causality cannot be inferred.In summary, this online survey conducted in a variety of widely-spoken languages in Israel demonstrates a high frequency of anxiety symptoms in Israel and an inverse association between diet quality and anxiety level. Poor diet quality was associated with weight gain, which could contribute to obesity. These results could inform the development of new preventive strategies aimed at bettering diet quality and eating behaviours and mental health problems during the COVID-19 pandemic. Further prospective studies are needed to confirm our findings in different populations with different degrees of COVID-19 pandemic severity.


Epinephelus awoara (♀) ×E. tukula (♂) was obtained by PCR amplification. The circular genome was 16,801 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region (D-loop region). The overall base composition was as follows: A: 28.46%, T: 27.27%, C: 27.27%, G: 16.49%. The new results may provide valuable data for the genetic and taxonomic research on artificial hybrid grouper.The complete mitochondrial genome of hybrid grouper from  Epinephelus awoara and E. tukula are both to the subfamily Epinephelinae, family Serranidae and orders Perciformes. E. awoara lives in rocky areas as well as on sandy-mud bottoms, mainly distributed in Northwest Pacific: Korea, Japan, China and the like. It is not only a marine fish of high economic value but also an ornamental fish in some countries because of its gorgeous body color. E. tukula is mainly found in deep reef channels and seamounts, mainly distributed in Indo-West Pacific: Red Sea and East Africa to southern Japan, Australia, Paracel Islands in the South China Sea, with strong disease resistance and fast growth rate. For the past few years, hybridization was commonly used in fish breeding as it allowed for a combination of advantageous traits from different species  × E. tukula (♂). This hybrid offspring has the advantages of low deformity rate and fast growth rate, and has potential economic value, but its genetic characteristics remain poorly understood  ×E. tukula (♂) was sequenced to provide useful information for the genetic and taxonomic research on artificial hybrid grouper.In this study, the sample was collected at Laizhou Mingbo Aquatic Co., Ltd., Shandong province, China (372506.7300 N 1200015.1100E). The sample was numbered QJ2 and stored in a −80 °C refrigerator with accession number 20190705QJ before sequencing. Total DNA was extracted with standard phenol-chloroform methods  methods in MEGA 7. The mitochondrial genome sequences were aligned using Seqman and subsequently edited and trimmed. Numbers on each node are bootstrap values of 1000 replicates. As shown in E. awoara♀ × E. tukula♂ had a closer relationship with E. awoara  than the other species, which demonstrated that the mitochondrial genome DNA of the hybrid was also maternal inherited. In addition, the hybrid had close genetic distance with Carangoides equula of Carangidae family, while they were belong to deferent families. This results deserved further study.The phylogenetic tree was reconstructed based on the complete mitochondrial genome nucleotide sequences of the hybrid of