Italy is definitely a high-risk country for multiple sclerosis (MS). Over the last 50 years, several epidemiological studies, including longitudinal surveys, have disclosed that MS incidence and prevalence in Italy mainland and Islands (Sardinia and Sicily) have progressively increased, picturing a semi-parabolic curve. Based on the comprehensive scrutiny of 58 papers, we conclude that the latitude risk gradient does not fit to the Italian map of MS. The genetic heterogeneity of the Italian ethnicities, that likely forms the basis of MS predisposition, does not account for the dramatic increase of MS incidence and prevalence observed in Italy over the last half century that, rather, seems better explained by the effect of environmental factors.The online version contains supplementary material available at 10.1007/s00415-021-10782-5. After removing duplicates, the titles and abstracts were screened independently by two investigators to evaluate whether they were eligible or potentially eligible literature or not. Only full-text papers were considered and meeting or conference abstracts were excluded. All the available literature, up to November 30, 2020, was screened by two independent investigators (MP and PG). If there were any differences between two investigators’ screening, differences would then be discussed together with a third investigator of the panel to meet an agreement. Papers were excluded if they met one of the followings: (i) hypothesis, case reports, review articles, letter to editor comments, case reports; (ii) studies available only in abstract form; (iii) unclear methodology; (iv) unclear statistics. After scrupulous selection, we collected and analyzed the data of 58 papers that were considered relevant for this analysis. Twenty-eight provided data from 35 prevalence or incidence time-points for Italy’s mainland, 13 for Sardinia and 17 for Sicily.The data on MS prevalence collected in Italy’s mainland over the last half century describe a semi-parabolic curve, with no plateau or slowing-down period even when these were reasonably expected Fig. . Indeed,Nevertheless, significant differences in MS prevalence are noticed not only between Regions, but also within the same Region and even within the same Province. In 2015, the estimate number of MS patients in Italy was 109,000 , with a Worth of interest are the results of the epidemiological studies done in the Republic of San Marino, a little independent State in the Northern Italy, 150 km South from Ferrara, whose population  is almost exclusively Caucasian . In this State, MS prevalence increased with the same trend: 51.6/100,000 in 1984 .Intriguingly, significant variations in MS incidence and prevalence were observed in neighbouring areas within the same Regions. In Tuscany, where the mean MS prevalence was settled at 208.7/100,000 on January 1, 2017 , a strikA number of incidence studies have also been performed in Italy mainland. However, longitudinal data are available only from a few studies. Methodologically, it has to be stressed that incidence studies based on small population cohorts and/or on short periods of observation  generally produce overestimated figures. These studies were not considered in this review. In other studies, the diagnostic criteria and/or the sources of cases were not adequately described. In general, the most accurate incidence rates available come from prospective studies based on the direct screening of the patients. Retrospective case-finding procedures, that include at least 2 years of post-observational period in order to cover the actual diagnostic gap, provide more useful incidence estimations. Even taking into consideration the limitations above described, a progressive increase of MS incidence occurred in Italy mainland as well as in the islands over the last half century. Indeed, since the ‘60ies to the ‘90ies very similar increase in MS incidence were scored in the Province of Padua  where MS incidence increased from 0.9/100,000/year to 5.5/100,000 , in the Several studies , 25–31 pHowever, MS prevalence throughout the island is far to be homogeneous with values ranging from 217 in the district of Olbia-Tempio to 425 in the districts of Ogliastra and Nuoro, located less than 100 km South  As for Sardinia, the surveys conducted in different Sicilian localities have disclosed a high-risk for MS in this island and pointed out that the latitude hypothesis does not fit to the epidemiological trends of the disease in this island Fig. . AlthougMS is an autoimmune disease, i.e., the results of a complex interaction of genetic and environmental factors. A common polygenic background is supposed to predispose to autoimmunity , but itsOn the other hand, many environmental factors have been suggested to play a role in either triggering MS or modulating the subsequent disease course, but results vary substantially between studies . In addi10 concentration associated with higher risk of MS relapses and brain magnetic resonance imaging (MRI) inflammatory activity [2.5 exposure [2.5 concentrations in both Provinces [The emerging role of air pollutants in the Padan Plain, that may partly explain the MS epidemiological trends described above, is worth of consideration. Indeed, increasing evidence strongly associates PM to MS risk in this geographic area that is one of the most polluted Region of Europe. Studies independently conducted in the Provinces of Padua and Pavia showed quite concordant results and indicated a strong association between MS and PM. In Lombardy Region, a fourfold PMactivity . In the exposure . More rerovinces . In addirovinces . Thus, a10, CO2, NO and SO2 were associated with MS prevalence and the risk of relapses [10, SO2, and NO2 concentrations. An association between PM10 exposure and risk of MS relapses was observed in Strasbourg (France) [2.5, CO, SO2, and Pb values in the upper quartiles of emissions were all significantly related to higher odds for pediatric-onset MS [An increasing amount of data supporting a role for PM in MS risk has risen from studies conducted in other Countries, at different latitude, and recently object of a meta-analysis . A pivotrelapses . A studyrelapses  found a (France) . Particuonset MS .Furthermore, a recent study has investigated the risk of MS in the industrialized area of Southwest Finland, having the city of Turku as Capital, compared to that the green region of North Karelia, located at the same latitude. A significant higher incidence (12.1 vs 6/100.000 year) and prevalence (275 vs 167/100.000 at the end of 2016) of MS was observed in the more polluted region of Southwest Finland . In TurkRegional differences in MS prevalence were also observed in Croatia in the ‘90ies, ranging from 194/100,000 in Gorski Kotar to 27/100,000 in the neighbour Istria . In thisAs a matter of fact, striking loco-regional differences in MS prevalence are observed in several European Country, both in the Mediterranean area and in the Scandinavian area Fig. .Fig. 3LoNevertheless, the idea that a single environmental factor may play a dominant role in MS in all the geographic areas and ethnic groups is questioned by the regional variation in the incidence rate observed in other European regions. For instance, in Scotland in the period 2010–2017  MS incidIncidence and prevalence of MS in Italy mainland and Islands (Sicily and Sardinia) have constantly increased all over the Country over the last 5 decades, with a clear latitude-independent trend. In Italy, the latitude-effect is also questioned by the significant differences observed between areas located at the same latitude or even adjacent, a finding shared with others European Regions.Although the genetic background may account for the basic predisposition to develop MS, that characterizes the different Italian populations, the epidemiological data collected in Italy over the last half century indicate that environmental factors play a major role in determining the risk of developing MS in the Caucasian people living in Italy.Supplementary file1 (DOCX 15 KB)Below is the link to the electronic supplementary material.


Although idea connections at verbal and conceptual levels have been explored by remote associates tests, the visual-spatial level is much less researched. This study investigated the visual-spatial ability via Chinese Radical Remote Associates Test (CRRAT), wherein respondents consider the positions of the stimulus and target Chinese radicals. Chinese Compound Remote Associates Test (CCRAT) questions also feature stimuli of a single Chinese character; therefore, it was adopted for comparison to distinguish the roles played by verbal and visual-spatial associations in a remote associative process. Thirty-six adults responded to CRRAT and CCRAT; their brain activities were analyzed. Upon excluding the influence of age, verbal comprehension, and working memory, it was found that the caudate, posterior cingulate cortex, postcentral gyrus, and medial frontal gyrus were activated when the respondents answered CCRAT, but only the caudate showed significant activation when they answered CRRAT. The Chinese radical remote association minus the Chinese compound remote association showed that the middle frontal gyrus, inferior parietal lobule, and precuneus demonstrated significant activation. Therefore, this study demonstrated differences in brain mechanisms between visual-spatial and verbal remote associations. Based on the associative theory of creativity, remote association refers to the ability of one to form new relations with seemingly irrelevant elements Mednick, . Those wIn addition, RAT has a short testing duration and objective scoring. Furthermore, its questions are relatively easy to compile, which facilitates the mass production of RAT questions to prevent respondents from knowing the test questions beforehand. Consequently, RAT has been widely used in creativity research on different dimensions  have three versions: the Chinese Radical Remote Associates Test (CRRAT)  consist of disparate test questions, for which respondents use different language knowledge and strategies during the question-answer process. When responding to CCRAT questions, research participants need to think of remotely associated concepts based on the stimuli in order to find out correct answers, because the usage frequency of the created words is set as “the least used”  and idea assessment (convergent thinking)  voluntarily participated in the study. Their ages fell between 20 and 30 years, with a mean of 24.1 ± 2.65 years. All of the participants were university students or had a university degree. The Edinburgh Handedness Inventory  and CCRAT unsolved (CU), respectively, and who had no <10 correct and 10 unsolved responses for CRRAT, which are denoted as CRRAT correct (RC) and CRRAT unsolved (RU), respectively. The brain images of 36 respondents were reserved for subsequent analysis, of which 13 were male and 23 were female, with a mean age of 23.88 ± 2.56 years.This study used CCRAT, CRRAT, and Wechsler Adult Intelligence Scale-IV (WAIS-IV) as research tools, as discussed below.The CCRAT employed in this study was compiled by Wu et al. . It consIn this study, CRRAT, compiled by Chang Y. L. et al. , was useChen et al.  translatThis study adopted an event-related functional magnetic resonance imaging paradigm. The gazing point “+” was presented the moment a stimulus appeared on the screen, which lasted 6 s  on average. Subsequently, a test question was presented on the screen for, at most, 20 s, which included three stimuli of Chinese characters. If the participants had answers to questions within the time limit, they could press the button and move on to question-answering screens, reporting their answer orally in 5 s. To determine whether the answers were correct, their answers were noted on the answer sheet. After finishing the check, the examiner presented the next question manually, asking the participants to continue answering questions. If the participants did not think of any answer within 20 s, the screen for the participants to report answers would appear automatically, but the examiner would not allow the participants to report their answers orally; the examiner would then manually move on to the next question and ask them to answer it. In summary, the stimuli onset asynchrony (SOA) was 25.3, 25.65, 26.1, 26.25, and 26.7, respectively. Each block consisted of 15 questions, which took up to 465 s. There were four intervals in total, with a 2-min break between each interval. The formal experiment took up to 37 min. The exact time relied on the actual question-answering situation.2, FOV = 224 × 224 mm2, slice number = 40, thickness = 3 mm, and spatial resolution = 3.5 × 3.5 × 3 mm3.Magnetic resonance imaging (MRI) scan was conducted using a Siemens high-field magnetic resonance scanner  equipped with a 64-channel head coil with a 90-degree phase difference and input/output (I/O) functions at the Research Center for Mind, Brain, and Learning of the National Chengchi University. Stimuli were presented with E-prime 3.0, and respondents read the stimuli that were projected on a projection screen via a mirror in front of them. The axial view was utilized to scan the whole brain with the anterior commissure-posterior commissure (AC-PC) line as the imaging reference line. Echo-planar imaging (EPI) was performed with the following parameters: TR = 2,000 ms, TE = 25 ms, FA = 90°, matrix size = 224 × 224 mm2; FOV, 256 × 256 mm2; slice number 192; thickness, 1 mm; and spatial resolution, 1 × 1 × 1 mm3.Subsequently, a high-resolution T1-weighted anatomical image scan, which was employed to digitize activated and functionally connected brain regions, was conducted. The pulse sequence of the anatomical image was 3D-MPRAGE, and the related parameters were as follows: TR, 2,530 ms; TE, 3.3 ms; FA, 7°; matrix size, 256 × 256 mmThe collected data were analyzed using SPM12 . Functional images were corrected for differences in slice acquisition time to the middle volume, and they were realigned to the first volume in the scanning session using affine transformations. The movement in any plane did not exceed 4 mm. Co-registered images were normalized to the standard Montreal Neurological Institute EPI template and resampled to a voxel size of 3 × 3 × 3 mm. The statistical analyses were performed on data that had been spatially smoothed using an 8-mm (FWHM) Gaussian kernel, with a high-pass filter (with a cutoff period of 128 s) at full width at half maximum to remove low-frequency artifacts.For the event-related analysis, functions corresponding to the onset of different event types were constructed and convolved with a canonical hemodynamic response function (HRF) and its temporal derivative. During the first-level single-subject analysis, different types of events  were defined, and voxel-wise parameter estimation was calculated for each regressor. In this study, “unsolved” implied that the participants did not submit any response during the trial. Canonical HRF was used to analyze stimuli as separate events, which were divided into solved and unsolved (2 s before pressing the button or the end of the response time). To increase the statistical sensitivity and remove motion-related artifacts, we also included six motion parameters as regressor/nuisance covariates of no interest in the first-level general linear model.t-tests, which allowed the study to parse its main effects on solving the two types of remote association (CCRA/CRRA). Meanwhile, the difference in correct events between the Chinese Compound and Radical Remote Associates problem solving was also analyzed by paired t-tests.The individual contrast images were then entered into a second-level analysis using a flexible factorial design. Parametric modulation was analyzed by paired p < 0.05, corrected for the family-wise error rate across the whole brain for multiple comparisons at the voxel level, with a cluster size ≥20 voxels. To visualize significant signal changes in the brain regions, time courses were extracted from the beta values for the peak voxel.All reported areas of activation were considered significant at the peak threshold of SD = 0.07) while CRRAT was 0.48 (SD = 0.08). In terms of verbal intelligence, the mean verbal comprehension was up to 122.22 ± 8.31, while that of working memory was up to 110 ± 14.08. The demographic variables  were not significantly correlated with Chinese compound remote association and Chinese radical remote association .The contrast between the CC and CU suggests that the caudate, posterior cingulate cortex, postcentral gyrus, and medial frontal gyri were all activated. Similarly, the contrast between the RC and RU showed that only the caudate was activated. The results are presented in This study revealed the difference between Chinese compound remote association and Chinese radical remote association. Chinese compound remote association minus Chinese radical remote association showed no significant activation in the brain regions. On the contrary, Chinese radical remote association minus Chinese compound remote association showed that the middle frontal gyrus, inferior parietal lobe, and precuneus were significantly activated, as shown in This research explored the differences in brain activities from the neural perspective to differentiate verbal and visual-spatial associations, distinguishing the brain mechanisms of different types of remote associations. These results are partially consistent with the hypothesis that Chinese compound remote association involves the activation of the brain regions of the caudate, posterior cingulate cortex, post-central gyrus, and medial frontal gyrus. The Chinese radical remote association led to a significant activation of the caudate alone. Chinese radical remote association minus Chinese compound remote association showed that the middle frontal gyrus, inferior parietal lobule, and precuneus were significantly activated. The results showed that the brain mechanism of Chinese radical remote association that involves visual-spatial abilities is different from that of Chinese compound remote association, which features verbal association. This is one of the first studies to present the differences in brain activity between verbal and visual-spatial remote associations.The contrast between CC and CU reflects how one forms a remote association based on Chinese characters. The research results showed significant activation of the caudate, posterior cingulate cortex, postcentral gyrus, and medial frontal gyrus, indicating that these regions may all involve verbal remote association. The caudate is the center of language control (Crinion et al., In terms of Chinese radical remote association, the contrast between RC and RU reveals how one forms remote associations based on Chinese radicals. The caudate was the only brain region that was significantly activated during the test. As mentioned above, the caudate involves verbal fluency and transformation (Hsieh et al., In particular, the caudate was significantly activated in both Chinese radical remote association and Chinese compound remote association. This finding shows that the caudate involves common components of verbal remote association. The caudate plays a vital role in monitoring and controlling language (Crinion et al., The RC minus CC revealed brain regions that involve visual-spatial association. The results showed that the middle frontal gyrus, inferior parietal lobe, and precuneus were significantly activated. These regions are associated with creative cognition (Ellamil et al., There are some limitations to the implementation of this study. First, this study assumed that CRRAT involves visual-spatial association based on the previous research finding that CRRAT is positively associated with visual divergent thinking Wu, , but it In conclusion, this study adopted fMRI to examine brain activity when verbal and visual-spatial associations are formed, and the corresponding brain regions are involved. The brain regions of the medial frontal gyrus, inferior parietal lobule, and precuneus are associated with different cognitive functions, such as ideation (Bendetowicz et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institution Review Board of the National Taiwan Normal University. The patients/participants provided their written informed consent to participate in this study.C-LW collected and analyzed the data, wrote the initial draft of the manuscript, and assisted in literature review and discussion. H-CC designed this study. All authors approved the final version of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Amygdalus pedunculata Pall  belongs to the Rosaceae family and is resistant to cold and drought. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and metabolomics were used to track the changes in bioactive metabolites during several stages of Amygdalus pedunculata Pall growth. A total of 827 different metabolites were detected, including 169 flavonoids, 68 organic acids, 35 terpenoids and 2 tannins. Flavonoid biosynthesis and flavone and flavonol biosynthesis were the main synthetic sources of flavonoids. Quercetin, isoquercitrin, and epicatechin as biomarkers related to growth and development were found. Quercetin connects the biosynthesis of flavonoids and the biosynthesis of flavones and flavonols. The contents of isoquercitrin and epicatechin increased uniformly during the whole growth process from the flowering stage to the fruit ripening stage, indicating that play key roles in the fruit growth and ripening stages of this plant. The tissue location and quantitative analysis of flavonoids in leaves at different stages were performed by confocal laser scanning microscopy. The flavonoids were mainly distributed in the palisade tissue and spongy tissue, indicating the need for protection of these sensitive tissues in particular. Through comprehensive and systematic analysis, the temporal distribution of flavonoids in the process of their leaves growth was determined. These results clarify the important role of flavonoids in the developmental process of Amygdalus pedunculata Pall. Rosaceae are dicotyledonous plants, and their species resources are very abundant. It is recorded that there are more than 3,300 species in 124 genera in this family, which are widely distributed all over the world, especially in the northern temperate zone. Rosaceae plants are important landscape plants, oil plants, perfume plants and so on. There are approximately 51 genera and more than 1,000 species from this family in China . Among tAmygdalus pedunculata Pall (A. pedunculata) is a deciduous shrub of the almond genus in the family Rosaceae and is native to arid regions in northwest China, Mongolia, and eastern Siberia. Due to its cold and drought-tolerant characteristics and deep root system, A. pedunculata is a candidate plant suitable for desert reclamation and can adapt to a variety of soil types and soil moisture conditions . Combined with chemometrics for non-targeted metabolomics analysis, the metabolic changes of several bioactive compounds during the growth A. pedunculata leaves were studied. In addition, confocal laser scanning microscopy (CLSM) was used to determine the tissue location and quantitative analysis of the flavonoids in leaves at different stages. Through these analyses, we clarified the changes in the important secondary metabolites of A. pedunculata leaves during the growth process.Recently, metabolomics has been widely used in the study of plants. For example, the effects of different temperature regimes on the metabolism of three species of eucalyptus were assessed , wheat rrevealed , the varredicted  and so oredicted . The metredicted . In thisMethanol, formic acid, water, and acetonitrile were all purchased from CNW Company . L-2-chlorophenylalanine was purchased from Shanghai Hengchuang Biological Technology Co., Ltd. . LysoPC17:0 was purchased from American Avanti Company (America). 2-Aminoethyl diphenylborinate was purchased from Acmec Biochemical Company . All chemicals and solvents were of analytical grade or chromatographic grade.A. pedunculata was planted at the base of the Shaanxi Desert Control Research Institute in Yulin, Shaanxi, China. The leaves of A. pedunculata were collected manually during the flowering period, fruit formation period, fruit hard core period and fruit ripening period. The sampling time was April 18, 2019 , May 16, 2019 , June 2 2019 , June 17 2019 , July 3 2019 , and July 19, 2019 . Well-grown leaves were chosen for collection. The leaves collected on each sampling date were collected from the same region of different plants, and the leaves were basically the same size and shape. Six duplicate samples were collected at each time point, immediately placed into dry ice, and sent back to the laboratory for storage at −80°C.Eighty milligrams of sample was weighed, and 20 μL of internal standard  and 1 mL of methanol:water (V:V = 7:3) were added. Two small steel balls were added, pre-cooled at −20°C for 2 min, added to a grinder , and ultrasonically extracted for 30 min. The mixture was allowed to stand for 20 min at −20°C and centrifuged for 10 min . A total of 300 μL of the supernatant was collected, dried and then reconstituted with 400 μL of methanol-water (V: V = 1:4). The mixture was then vortexed for 30 s, sonicated for 2 min, and centrifuged for 10 min . A total of 150 μL of the supernatant was filtered through a 0.22 μm organic phase syringe filter, transferred to an LC injection vial, and stored at −80°C until LC-MS analysis. To optimize the LC-MS system before sample analysis and to evaluate the stability of the mass spectrometer, quality control (QC) samples were prepared from equal volumes of extracts from all samples . Each QCAn ACQUITY UPLC from Waters Corporation was used in series with an AB Triple TOF 5600 high-resolution mass spectrometer from AB Sciex Corporation. Chromatographic separation was performed on an ACQUITYUPLC BEH C18 column  from Waters Corporation. The mobile phase consisted of eluent A  and eluent B . The column temperature was 45 °C, the flow rate was 0.4 mL/min, and the injection volume was 5 μL. The applied elution conditions were as follows: 0–2 min, 5% B; 2–4 min, 20% B; 4–9 min, 25% B; 9–17 min, 60% B; 17–19 min, 100% B; and 19.1–20.1 min, 5% B . The ins1), Lipidmaps2, METLIN database and self-built databases based on accurate masses, secondary fragments and isotopic distributions. After obtaining the qualitative data, the qualitatively obtained compounds were screened. Finally, the positive and negative ion data were combined.UNIFI 1.8.1. software was used to collect raw data. Prior to pattern recognition, the raw data were subjected to baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization using the metabolomics processing software Progenesis QI v2.3 . An Excel file was obtained with three-dimensional data sets, including m/z values, peak retention times (RTs) and peak intensities, and RT–m/z pairs were used as the identifier for each ion. The resulting matrix was further reduced by removing any peaks with missing values (ion intensity = 0) in more than 50% of the samples. The internal standard was used for data QC (reproducibility). Metabolites were then qualitatively analyzed using HMDB (The Human Metabolome Databasep < 0.05) and fold change (FC) analysis to compare the differential metabolites between the two groups.For statistical analysis, the data were normalized and then introduced into the R ropls package (RStudio 1.2.1335) for pattern recognition. Multivariate statistical analysis and univariate statistical analysis were carried out to visualize the metabolic alterations among experimental groups after mean centering (Ctr) and Pareto variance (Par) scaling, respectively. Principal component analysis (PCA) was used to observe the overall distribution between samples and the stability of the entire analysis process. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was then used to distinguish the overall differences in metabolic profiles between groups to find differential metabolites between groups. To prevent the model from overfitting, we used sevenfold cross validation and 200 response permutation testing (RPT) methods to examine the quality of the model. Afterward, the results used Tukey’s test , was placed in 0.1 mol/L phosphate buffer (with the addition of 1% NaCl (w/v), pH = 7). An appropriately shaped section was chosen and placed onto a microscope slide with a drop of buffer solution, and an image was taken as a control. Next, the leaf tissue was stained with 0.1% 2-aminoethyl diphenylborinate (w/v) in methanol (NA solution) for 5 min, phosphate buffer (pH = 7) was used to wash off the excess staining solution, and images were taken as the experimental group. Confocal images were collected by a Nikon A1 confocal microscope used in DU4 mode with a 40£/1.2 water immersion objective. A 488 nm argon ion laser was used to excite the dye molecules. Fluorescence images were collected with emission wavelengths of 500–560 and 570–630 nm. NIS-Elements software was used to adjust and process the image acquisition parameters. Triplicate experiments were performed, and each tissue section was photographed in triplicate.A. pedunculata metabolome in different periods, a non-targeted metabolomics method was adopted for AP1, AP3 and AP6, and 5,187 metabolites were identified, including benzenoids, lignans, neolignans and related compounds, lipids and lipid-like molecules, nucleosides, nucleotides, and analogs, organic acids and derivatives and so on  to fruit ripening stage . AP2  is the fruit formation stage. The AP3  and AP4  samples are both in the fruit hard core stage, and AP5  and AP6  samples are both in the fruit ripening stage. To explore the comprehensive changes in the nd so on .First, unsupervised PCA was used to reflect the overall variability in metabolite profiles between and within the sample groups and to observe the overall distribution trend between samples . The scaIn order to establish a more predictive model, we performed supervised OPLS-DA on the basis of PCA to enhance the difference between the experimental group and the control group. The scatter plot of OPLS-DA scores for all samples is shown in p-values from a two-tailed Student’s t-test on the normalized peak areas, where metabolites with VIP values greater than 1.0 and p-values less than 0.05 were considered differential metabolites  values were obtained. Differential metabolites were selected on the basis of the combination of a statistically significant threshold of VIP values obtained from the OPLS-DA model and abolites . A totalabolites . Compari3) website for metabolic pathway enrichment analysis. p-value) values for samples AP3 and AP6 in comparison with sample AP1, including three common primary metabolic pathways  and two common specialized metabolic pathways (flavonoid biosynthesis and flavone and flavonol biosynthesis). In the AP3/AP1 group, the metabolic pathways of flavonoid biosynthesis ranked first, and flavone and flavonol biosynthesis ranked eighth. In the AP6/AP1 group, the metabolic pathways of flavonoid biosynthesis still ranked first, and flavone and flavonol biosynthesis ranked fourth. These results showed that the pathways flavonoid biosynthesis and flavone and flavonol biosynthesis were significantly different from the flowering stage to the fruiting stage of the plant.To understand the differences in the metabolic network between the three groups of samples, the differential metabolites were submitted to the KEGG (Kyoto Encyclopedia of Genes and GenomesA. pedunculata leaves stained with NA (Naturstoffreagenz A) solution in different periods were observed by CLSM. At an excitation wavelength of 488 nm, flavonols and flavonoid glycosides emitted green and red fluorescence. The samples showed chloroplast autofluorescence before staining, and this interference was eliminated by subtracting the background signal. The laser confocal image of the leaf cross section shows that the fluorescence intensity was higher in the spongy tissue and palisade tissue than in other tissue of the leaves. Moreover, the fluorescence intensity of guard cells on both sides of the stomata and the nucleolar area of epidermal cells were relatively high. However, the fluorescence intensity in the cytoplasm of epidermal cells was relatively low, and the fluorescence intensity in the vascular bundle was lowest. The relative fluorescence intensity reached a peak in AP3 samples, and the relative fluorescence intensity was the smallest in the AP6 samples. The relative fluorescence intensity of the AP3 sample was 2.13 times that of AP6, the relative fluorescence intensity of the AP3 sample was 1.24 times that of AP1, and the relative fluorescence intensity of the AP6 sample was 0.58 times that of AP1  radicals, hydroxyl (OH⋅) radicals, hydrogen peroxide (H2O2) and superoxide anion radicals (O2–)  . HoweverSince secondary metabolites often play a very important role in the growth and development of plants, and they change significantly in different growth stages of plants, we focused on the changes of secondary metabolites during plant growth and development. A total of 827 differential metabolites were obtained from 5187 metabolites. Among these metabolites, a total of 274 specialized metabolites, including 169 flavonoids, 68 organic acids, 35 terpenoids and 2 tannins, were shown and discussed in Flavonoids are widely present in the leaves, fruits, roots, and skin of plants. These compounds play a variety of physiological roles in plant growth, development, and reproduction , and theA. pedunculata are rich in flavonoids, including flavonoid glycosides, flavones, biflavonoids and polyflavonoids, homoisoflavans, and isoflavans . The metabolic pathway of flavonoid and flavonol biosynthesis was only significant in the AP6/AP1 group. There were 11 metabolites related to these two pathways: naringenin, delphinidin, epicatechin, holy phenol, pinusin, quercetin, isoquercitrin, rutin, mountain kaempferol, asperin, and quercetin. These substances have an important influence on the synthesis of flavonoids, and they are synthesized by plants to serve many actions as visual attractors, feeding repellents, photoreceptors, oviposition stimulants, allelopathy, phytoalexins, antioxidants, and antimicrobials  can be well proven.Chloroplasts have long been reported to contain flavonoids and appear to be capable of flavonoid biosynthesis . In the  tissues . Since feat load . CombineA. pedunculata at different growth stages, especially the types and biosynthetic pathways of flavonoids. CLSM was used to analyze the tissue location of the flavonoids contained in A. pedunculata leaves at different times. It was quantitatively verified by fluorescence intensity that the fruit development stage had the highest flavonoid content in A. pedunculata leaves. We clarified the key role of flavonoids in the different growth and developmental stages of A. pedunculata leaves. This study provides a theoretical basis for future research on artificially increasing the content of flavonoids in A. pedunculata leaves. In addition, this study will provide a theoretical basis and scientific basis for the development of the social benefits of A. pedunculata as a medicinal material and the economic effects of edible medicinal plants, increasing comprehensive utilization value of A. pedunculata.This study provides a comprehensive evaluation of the temporal changes in bioactive metabolites in The original contributions presented in the study are included in the article/YS, CL, and YH conceived and designed the research. YH and LP searched for relevant literature. YH, LP, and TY performed the experiments. CL and YH analyzed the experimental data and wrote the manuscript. BC, WW, and LP assisted in analyzing the experimental data. YS and CL guided the manuscript preparation. All authors read and approved 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.


Bacillus subtilis is naturally endowed with several enzymes targeting complex vegetable polymers. In this work, a raw bacterial culture supernatant was used to assist in the extraction of bioactives using isothermal pressurization cycles. Besides a wild-type Bacillus subtilis strain, a new strain showing increased secretion of cellulases and xylanases, pivotal enzymes for the digestion of the plant cell wall, was also used. Results indicate that the recovery of compounds correlates with the amount of cellulolytic enzymes applied, demonstrating that the pretreatment with non-purified culture broth effectively promotes the release of bioactives from the vegetable matrix. Therefore, this approach is a valid and sustainable procedure for the recovery of bioactive compounds from food waste.The market for nutraceutical molecules is growing at an impressive pace in all Western countries. A convenient source of bioactive compounds is found in vegetable waste products, and their re-use for the recovery of healthy biomolecules would increase the sustainability of the food production system. However, safe, cheap, and sustainable technologies should be applied for the recovery of these beneficial molecules, avoiding the use of toxic organic solvents or expensive equipment. The soil bacterium  The agri-food sector generates massive amount of waste that not only causes the loss of market profit but, more importantly, represents a waste of precious environmental resources, such as land, water, fertilizers, chemicals, and energy expended in the production phase . MoreoveIn line with the circular economy concept, fruit and vegetable waste might be turned into a valuable resource as a natural and unlimited supply of biologically active compounds, including vitamins, polyphenols, dietary fibers, glucosinolates, essential oils, and organic acids, among the others, with an enormous economic potential as nutraceutical, pharmaceutical, cosmetic, and agro-pharmaceutical ingredients ,4. HowevAmong the new emerging techniques, Enzyme Assisted Extraction (EAE) of biomolecules represents one of the most environmentally friendly and safe methodologies which can be used either as a stand-alone technique, or as a pretreatment that increases the efficiency of a coupled extraction system ,7,8. EAEBacillus subtilis. B. subtilis is already heavily exploited for the industrial production of several biocommodities, such as degradative enzymes, heterologous proteins, bio insecticides, and antibiotics, and its products are considered as Generally Recognized as Safe (GRAS) [One of the most formidable enzyme-producers is the Gram-positive soil bacterium e (GRAS) ,10,11. Ie (GRAS) ,10,11. B. subtilis lives predominantly in the soil, and this type of habitat has evolutionarily shaped its genome, leading to the accumulation of a large array of genes associated with the ability to degrade complex carbohydrates from decaying vegetable biomass. According to the Carbohydrate-Active Enzymes database [B. subtilis spent growth medium represents an inexpensive, rich, and wide-range collection of GRAS enzymatic activities, ideally suited to break down the plant matrix and improve the release of bioactives.database , the bacdatabase . Moreovedatabase ,14. ThanB. subtilis culture supernatants, obtained from a wild-type (WT) strain and from a new B. subtilis strain, overproducing cellulases and xylanases (OS58). The sustainability of the process was maintained by carrying out the extraction process in an aqueous environment, avoiding the use of any polluting solvent. The substrate, cauliflower (Brassica oleracea L. conv. botrytis (L.) Alef. var. botrytis L.), rich in phytochemicals endowed with a large range of beneficial biological activities, derived from the quota of raw products that did not meet the qualitative standards for commercialization and were discarded by an industrial agro-food processing plant. The aim of this work was to evaluate the efficacy of a method for the sustainable extraction of bioactive compounds from vegetable waste based on a low-cost EAE pretreatment. The economic viability of the procedure relied on the use of crude B. subtilis strains were used. The WT strain corresponded to the commonly used lab strain JH642 [B. subtilis through genetic engineering of the WT strain. The cellulases and xylanases activities released in the growth medium by the two strains were determined after 24 h incubation. The optimization process led to a drastic enhancement in enzyme secretion. As shown in B. subtilis is known to secrete, might contribute to the final effect on the substrate [To evaluate the effectiveness of the low-cost enzymatic pretreatment, two in JH642  in whichubstrate .3 pieces. The fragmented material was incubated at 50 °C with culture supernatants from either the WT strain, OS58, or the sterile growth medium, which was used as a negative control for the treatment. Subsequently, each mash was subjected to 60 cycles of pressurization at room temperature (20°–23 °C) for the extraction of bioactive compounds. Part of the liquid flow, collected from the apparatus after each extraction, was dried and resuspended in 50% methanol for the analyses of the nutraceutical compounds.In order to verify whether the crude enzymatic mixture released by the bacterial strains represented an effective pretreatment for improving the extraction of bioactive compounds, cauliflowers discarded from the food supply chain were collected from an industrial processing plant during different seasonal periods. The biomass, originated from different regions across Italy, was treated according to a standardized protocol. The processing began with the grinding of thawed cauliflowers into 0.2–0.5 cmImportant differences in the content of bioactives were observed among the four replicates, which were performed on different stocks of cauliflowers; such differences are presumably linked to seasonal effects and the geographical origins of the vegetable material. For this reason, the correlation among replicates was estimated by including the variable “replicate” as a random effect in the statistical model .p < 0.1, weak significance); however, the recovery further increased  when the treatment was performed with the optimized strain OS58, which is a better enzyme producer , corroborating the hypothesis that the effect is indeed due to higher amount of enzymes released by the optimized strain.For the phenolic acids class of polyphenols, the recovery of chlorogenic acid was examined in detail in the three different extracts. As shown in p = 0.082). The reasons for these results are discussed below.For this class of compounds, the yields were below our expectations. Only within the OS58-treated group, a very modest increase in the recovery (1.3-fold) was observed , which, Bacillus subtilis strain (OS58), with optimized production of at least two enzymes relevant for EAE (cellulases and xylanases), was developed and applied as a pretreatment before cyclic pressurization extraction for the recovery of valuable secondary metabolites from cauliflower. The performance of OS58 was compared to that of a WT B. subtilis strain and a control containing the sterile bacterial growth medium.Enzymatic pretreatments have been shown to favorably impact the extraction of valuable compounds from different vegetable sources in a green and sustainable manner ,7,8,18. B. subtilis, avoiding expensive commercial enzymes, is a cost-effective strategy that guarantees the economic viability of the process. Moreover, being a soil microorganism, B. subtilis grows efficiently over a wide range of conditions and media and can even be fed with agro-industrial waste [Bacillus subtilis is ideally suited to industrial applications for the above-mentioned characteristics, and because it secretes many different enzymes directly in the medium, simplifying their recovery [The recovery of polyphenols, and in particular, of chlorogenic acid and catechins , was enhanced by the pretreatment procedure using the enzymatic mixture derived from the WT strain, and in all cases but one, it further improved with the enzymatic mixture derived from the optimized OS58 strain, demonstrating that the efficiency of the extraction was proportional to the enzymatic activity applied . It is wal waste . Bacillurecovery ,10,13,16recovery ,22. For The sustainability of the extraction procedure described here is further enhanced by the fact that the vegetable material used was a waste generated from a real agri-food supply chain because it simply did not meet the aesthetic quality standard requirements for large-scale retail channels. Cauliflowers from this source were collected over three different seasons , and originated from farms located in different geographical areas in Italy. It is known that the content of several secondary metabolites in fruits and vegetables is strongly dependent on several parameters, such as specific cultivar, seasonal harvesting time, cultivation site, endogenous circadian rhythms, and soil and pest-control strategies ,24,25,26Moreover, the solubilisation, and thus, the extraction efficiency, of some compounds occurs in polar organic solvents, which were not used in this work. For example, isothiocyanates, relevant anticancer compounds abundantly present in Brassicaceae , are usuIt is important to highlight that, as opposed to most of the literature data, this work was carried out on a pilot scale, using 5 kg of raw material for each sample and for each of the four experimental replicates. To our knowledge there are no previous reports on such large-scale extractions.Brassica oleracea L. conv. botrytis (L.) Alef. var. botrytis L.; the unpurified bacterial enzymes notably improved the retrieval of valuable phytochemicals from the matrix in a concentration-dependent manner. The applicability of the promising sustainable enzymatic pretreatment, coupled with solid-liquid extraction at high pressure, is currently being tested on raw vegetable materials with different structural characteristics and bioactives composition, to validate the applicability of raw enzymatic mixtures for nutraceuticals recovery.In conclusion, the OS58-based sustainable extraction procedure was demonstrated to be more effective than the WT strain-based extraction in the recovery of bioactives from Bacillus subtilis strains used in this study are a WT strain and its derivative, OS58. The WT derives from PB5249, a swrA+ spontaneous derivative [trpC2 pheA1 strain JH642 (GenBank accession no. CM000489.1) [trpC and pheA genes, obtained from the genomic DNA of the wild NCIB 3610 strain (GenBank accession no. CP020102.1). Amplifications of the trpC and pheA genes were performed using the primer pairs trpC_Up 5′-AGTGAAAACACTGGTTCTGCCG-3′ and trpC_Dw 5′-GATGGATTGCTTTACGCTGAGAAG-3′ followed by pheA_Up 5′-AACAGCCTTTGCCAATCGTGGG-3′ and pheA_Dw 5′-GTATACATGGATGCAGCCGCTCAG-3′, respectively. For the first transformation, the selection of the trpC+ prototroph occurred on minimal medium containing 1.5% agar, 1 mg/mL glucose and phenylalanine (50 µg/mL). Subsequently, pheA+ transformants were selected on minimal medium without amino acids.rivative  of the a00489.1) , which wOS58 was obtained from the WT strain by genetic engineering. A patent application is under preparation for this strain; for this reason, the details of the engineering design are undisclosed herein.B. subtilis WT and OS89 spores were revitalized on LB  1.5% agar plates and incubated overnight at 37 °C. Isolated colonies were inoculated in Antibiotic Medium 3 (Difco Laboratories) containing 5 mg/mL glucose and grown 16 h at 37 °C with shaking. In the same medium, a second pre-inoculum was set at optical density of 600 nm (OD600) 0.2 and grown for 6 h at 37 °C with shaking. The synthetic medium CMD  was inoculated at OD600 0.2 and grown for 24 h at 37 °C with 150 rpm orbital shaking. Bacterial growth was followed by OD600 readings. Culture supernatant was collected after 40′ centrifugation at room temperature at 2268× g. The spent medium (500 mL) was immediately used for the maceration of 5 kg-smashed cauliflowers. For cellulase and xylanase production, ®), following the protocol provided by the manufacturer.Cellulase  and endo-xylanase  activities were assayed in an aliquot of the culture supernatants after acidification, as previously described , using CBrassica oleracea L. conv. botrytis (L.) Alef. var. botrytis L.) were obtained, during spring, summer, and autumn 2021, from a food processing plant that collects vegetables from farms located in southern and northern Italy. The fresh material was weighted, aliquoted, and stored at −45 °C ± 1 °C  for over one month. For each treatment (including controls) and for each of the four experimental replicate, aliquots of 5 kg each (fresh weight before freezing) were thawed at room temperature for 2 days. After draining the residual water, each whole cauliflower, including flowers, leaves and stems, was cut into four parts and ground using an industrial mill ; the material was completely shattered into small pieces (0.5–1.0 cm in diameter) for subsequent processing. The set of extraction experiments  was independently carried out four times, on four different lots of cauliflowers.Fresh cauliflowers . For each of the four replicates, the material was incubated with 500 mL of sterile CMD instead of the spent medium, as a negative control.An isothermal cyclically pressurized extraction process (rapid solid–liquid dynamic extraction) was applied to pretreated cauliflowers using the Naviglio mechanical extractor . The principle is based on cyclic compression and decompression phases that exert a suction effect on extractable compounds present in the vegetable matrix . The solThe total content of polyphenols was measured using the Total Polyphenols Colorimetric Assay Kit  according to the manufacturer’s instructions. Absorbance was measured at 725 nm and results were expressed in gallic acid equivalents using a gallic acid standard curve.A 20-µL aliquot of the filtered sample was injected into the HPLC pump  equipped with a C18 column . For catechins and chlorogenic acid, HPLC analyses were performed with a 0.8 mL/min flow rate and setting the detector at 280 nm; the mobile phases consisted in 5% acetic acid (A) and pure methanol (B), and the chromatographic gradient conditions are summarized in Isothiocyanates were analyzed by HPLC after cyclocondensation, according to the protocol described by Tang et al. , with moThe variation in the amount of the compound extracted among the three experimental conditions, considered as response variable, was explored by using a series of linear mixed models (LMMS). Each model had a common fixed effect, represented by the experimental treatment adopted , and a common random effect (random intercept) represented by the different samples of organic matter . The estimated means, related confidence intervals, and planned comparison among treatments were computed using the emmeans and contrast functions of the emmeans package (version 1.7.2) . All modAll statistical analyses were performed in R  .


With the increased global temperature, the threat from climate change has already affected the livestock industry. Exposure to heat stress is a major factor responsible for impacts on the overall livestock production, which ultimately results in economic losses. With no exception, poultry is among the most vulnerable livestock to environmental stress. Hence, a comprehensive study is required to understand the molecular mechanisms and to improve the breeding program to overcome economic losses. Therefore, we investigated growth related phenotypes and performed transcriptome analysis to understand the heat stress response in chickens. Animal experiments were designed with two groups, which were kept at 21 and 33 °C for 2 weeks as the control and treatment groups. The transcriptome analysis used blood samples from each chicken. In this study, we identified a total of 245 differentially expressed genes (DEGs) with important roles in various biological processes, such as cell protection, energy conversion in the mitochondria, and protein quality control. The results indicate that the heat stress environment regulates genes and alter the metabolism to adjust for the heat environment in chickens. These findings could be useful to help understand the heat stress response in poultry.MYLK2, and BDKRB1 genes were identified as key genes in heat stress. The KEGG pathway analysis showed involvement in the ATP metabolic process, MAPK signaling pathway and calcium signaling pathway with related protein processing and synthesis. In conclusion, with induced heat stress, such changes in physiologic parameters alter the neuroendocrine system, and we observed that the heat stress environment regulates such Heat shock protein genes to protect the cells and proteins from an altered metabolism. These findings provide a more comprehensive understanding of the heat stress response in poultry.Chicken is important livestock that serves as a vital food source which remain largely affected by heat stress. Therefore, we performed the transcriptome analysis to help understand the mechanisms of heat stress response in chickens. In the animal experiments, we grouped them into a normal and severe at 21 and 33 °C, with identified physiologic parameters for 2-weeks. Subsequently, RNA-seq analysis was performed to identify DEGs with a false discovery rate < 0.05 and a fold change ≥ 1.5. In the physiological parameters, we observed average daily gain was declined, rectal temperature and respiration rate was increased in severe group. Among total 245 DEGs, 230 and 15 genes were upregulated and downregulated, respectively. In upregulated DEGs, HSPs,  Animal husbandry practice involves breeding, farming, and care of farm animals. Higher production in poultry farming mainly depends on nutrient intake, climatic condition, and surrounding humidity . In receIn poultry farms, the optimum temperature for growing chicken breeds varies from 18 to 22 °C. Induced heat stress, due to increased terrestrial temperature, is a visible threat directly affecting the poultry physiology and immune system . These pHSP70) gene is the most extensively studied member and widely used as a stress biomarker in the HSP family of genes [HPA1A, HPA1B, HSPA8, and HSPA5 have a critical role in reducing extracellular damage by altering gene expression and have an important role in immunity function and homeostasis [The heat shock protein (HSP) family and heat shock factors (HSF) are the typical genes that respond to heat stress, and their functions are reported in different studies . Studiesof genes ,17,18. Teostasis ,20,21. Heostasis . In preveostasis ,23.ERK5. Among the MAPK cascades, extracellular signal-regulated kinase 1 and 2, c-Jun N-terminal kinase, and p38 have been reported to correlate with the HSP genes in previous studies. Moreover, MAPKs, which activate due to the stress response with HSPA, regulate intracellular substrates through phosphorylation to execute their functions [The HSP gene signaling pathways involved in HSP induction includes mitogen-activated protein kinase (MAPK) genes . A stronunctions . With liunctions ,30.Therefore, a detailed analysis is required for further investigation to understand the detailed mechanisms underlying MAPK and the HSPA role in the heat stress mechanism. In this regard, this study was conducted to identify the responsible genes in heat stress and the genetic pathways through RNA sequencing analysis in the ROSS 308 chicken breed. This study will contribute to a better understanding of the transcriptomic mechanism of heat stress in chickens.The animal experiments were performed following ethical guidelines, approval number 2020-429, in the National Institute of Animal Science (NIAS). In the pre-experimental period (1 to 13 days of age), female broilers (Ross 308 breed) were housed in batter cages and fed a standard commercial diet. At 14 days of age, a total 100 of broilers were divided into two environmentally controlled chambers. Taking into consideration their average body weight, the broilers were kept in individual metabolic cages  and acclimated to the chambers for 3 days under a thermos neutral condition (21 °C/60%), as described in previous studies ,32. AfteFrom each blood sample, RNA was isolated using the TRIzol:Chloroform method following the manufacturer’s instructions. The extracted RNA was checked for its integrity and purity and measured with the Agilent 2100 Bioanalyzer and the RNA Nano 6000 Assay Kit . Samples with RIN (RNA integrity number) values > 8 were used for library construction. The library was constructed with random cDNA fragments and acquired adapter-fragments of the cDNA using the TruSeq Stranded Total RNA LT Sample Prep Kit , following the manufacturer’s instructions. The constructed library was used to perform sequencing on the Illumina HiSeq 2500 platform and paired-end reads were generated. Finally, the generated BCL image files were converted to FASTQ raw reads using the bcl2fastq Illumina package which were used for the post-analysis.https://www.bioinformatics.babraham.ac.uk/projects/fastqc), and the trimming of N base and <Q20 reads was done using Trimmomatic v0.39 for quality control when generating the raw reads using the paired-end option [2 + 1 values to check the correction. Then, using the calculated PC value, the after correction effect with the before correction was identified using the principal component analysis (PCA) plot. Finally, each corrected count file was used for the differential expression genes (DEGs) analysis.First, a quality check was performed for the raw reads using FastQC v0.11.5 , ggplot2 v3.3.3 , and pheatmap package v1.0.12  in R for visualization.We used the edgeR package v3.32.0 in R v4.0.3 to detect the DEGs between the two groups . The norThe Database for Annotation, Visualization and Integrated Discovery (DAVID) web tool was used for the enrichment analysis of the identified DEGs, and gene annotation was performed for the molecular functions, cellular components, biological processes, and pathways of the genes ,41. In Dp < 0.05. The physiological parameter of the animals was compared in the two groups by t-test, and DEGs were compared as previously described.All statistical analyses were performed with an adjusted p < 0.05). Here, the Severe group was observed to have a reduced average daily gain with an increased FCR, respiration rate, and rectal temperature shown in The chicken broilers were divided into the Normal and Severe groups for the normal and heat stress conditions, respectively, for a time period of 2 weeks, and daily monitoring was done for the following parameters: the average daily gain, feed conversion ratio (FCR), respiration rate, and rectal temperature. The results indicate a decline in the average daily gain in the Severe group when compared with the Normal group , PGLYRP2 (peptidoglycan recognition protein 2), PCDH8 (protocadherin 8), USH1G (USH1 protein network component sans), and LOC100859850, and downregulated, BPGM (bisphosphoglycerate mutase), KRT7 (keratin 7), PRR5L (Proline rich 5 like), KRT80 (keratin 80), and CEP63 . Additionally, the MYLK2 (myosin light chain kinase 2), FGFR1 (fibroblast growth factor receptor 1), and DHDH (dihydrodiol dehydrogenase) genes were expressed with an FC > 2.0, which include the HSP genes in the principal pathways. The results of the detected gene expressions with their FDR values are provided in A total of 371 DEGs was detected using edgeR with FDR < 0.05 and logThe identified 245 DEGs were used to perform GO analysis for functional annotation. The cutoff of the FDR < 0.05 was set to identify significant genes, and the DAVID server revealed that a total of seven biological processes and two cellular components were involved. The functional annotation found for various biological processes includes regulation of cell differentiation (GO:0045595), anatomical structure morphogenesis (GO:0009653), system development (GO:0048731), multicellular organism development (GO:0007275), anatomical structure development (GO:0048856), developmental process (GO:0032502), and multicellular organismal process (GO:0032501). Similarly, the functional annotation found for the cellular components was the plasma membrane (GO:0005886) and cell periphery (GO:0071944). The detailed annotation results are provided in Later, we performed KEGG pathway analysis to elucidate the molecular interactions and biological functions of the DEGs. The identified KEGG pathways, shown in To select the top 10 enriched pathways representing the gene network, we set the parameters, such as the FDR value for the correction using the hypergeometric test and Benjamini–Hochberg (FDR < 0.05 and FC ≥ 1.5). The upregulated and downregulated genes are represented as elliptical shapes, and the pathways are represented in different colors . The repThis study investigated the effect of heat stress on chicken broilers. The chickens were kept in the controlled humidity 68 ± 2.5% and 60.3 ± 2.0% for the Normal and Severe groups to affect the statistical analysis during post-processing of the data, kept the temperature as the one source of variance, and monitored the two main factors, which are the physiological and transcriptomic changes during heat stress ,15.During the analysis, we confirmed that environmental heat stress leads to a change in the physiological parameters as observed in our animal experiments using 17-day old ROSS 308 female chickens in the THI 1 and four conditions for 2 weeks. Overall, the results showed significant changes between the two groups in the average daily gain, and the FCR was found to gradually decrease, whereas the respiration rate and rectal temperature were increased in the Severe group at the higher temperature. Similarly, the increased environmental temperature caused an increased body temperature. To prevent heat emission, the chicken homeostasis decreased the feed intake and body weight in the Severe group compared to the Normal group. Especially, an increased respiration rate alters the physiological response. As typical behavior in a heat stress environment, a severe respiration rate results in the loss of heat, and homeostasis keeps the body temperature from rising ,12. AddiIn this study, we performed transcriptome analysis to confirm the regulation pattern of DEGs in a heat stress environment. We prepared a cDNA library and performed RNA sequencing for each blood sample; during the analysis, we detected 245 mRNAs transcripts with 230 upregulated and 15 downregulated mRNAs. GO and KEGG pathway analysis that elucidates the molecular interactions and biological functions of the DEGs showed nine GO terms and 32 enriched KEGG pathways. The results also show that the enriched genes were mainly involved in the adenosine tri-phosphate (ATP) metabolic process, MAPK signaling pathway, calcium signaling pathway, protein processing in the endoplasmic reticulum and focal adhesion. Especially, the MAPK signaling pathway, calcium signaling pathway, and focal adhesion are shown to share several genes, and the HSP genes were shown to be shared between the ATP metabolic process and protein processing in the endoplasmic reticulum pathway.The identified genes contribute to the metabolic process known to have a crucial role in generating ATP for cellular energy conversion and protein synthesis in the mitochondria. Heat stress disturbs this metabolic process and affects the skeletal muscles and influences protein synthesis by altering the mitochondrial morphology ,48,49. SBDKRB1) gene, which generally is minimally expressed during normal conditions, has an important role in the calcium signaling pathway, in the response to inflammation, and in tissue damage [MYLK2, FGFR1, and DHDH genes were upregulated as DEGs. These genes were enriched in the calcium signaling pathway, MAPK signaling pathway, and ATP metabolic process, and these genes are known to be involved in the development of tissues, skeletal muscles, and the nervous system, and are overexpressed in response to inflammation [BDKRB1 gene differentially expressed due to heat stress which led to tissue damage. Moreover, HSP genes were upregulated to protect the cells, which leads to reconstruction of gene expression in response to the changing environment. The existing literature supports our study results because these genes are mainly involved in changing the gene expression patterns involved in metabolic alterations, mRNA synthesis, cell-type differentiation, cellular transport, and the cytoskeleton; those similar pathway genes were upregulated in our study [BDKRB1 gene expression is upregulated in inflammation and tissue damage; the BDKRB1 gene in the absence of the Bradykinin receptor B2 gene was found to be constitutively expressed; therefore, further investigation is required to decode the role of these identified genes [Among the identified genes, the MAPK cascade is known as the central signaling pathway mainly responsible for regulating and responding to stress and for maintaining cell harmony by controlling essential cellular processes ,57,58,59e damage ,69,70. Fammation ,73,74,75ur study ,17,23. Ted genes ,68,70. IMYLK2 and BDKRB1 genes. However, considering just the temperature condition in animal experiments requires further investigation to understand the causal effects of heat emission on the internal body. The results of this study suggest the MAPK signaling pathway is enriched, and HSP genes are upregulated in respond to heat stress, and those alter the metabolic pathway, which maintains homeostasis via cellular energy conversion and protein control. Therefore, this study contributes to a better understanding of the physiological parameters, gene expression, and metabolic pathways affected during the heat stress response in poultry.Two factors were mainly responsible for heat stress-triggered economic losses due to less production. First, alterations in the physiological parameters led to decreased body weight, average daily gain, FCR, respiration rate, and rectal temperature in ROSS 308 female chickens. The second factor responsible was the changed expression pattern of genes in response to heat stress, and in our observations, the genes involved in the ATP metabolic process, MAPK signaling pathway, calcium signaling pathway, protein processing in the endoplasmic reticulum, and focal adhesion pathway were enriched, and the heat stress response was the upregulation of the HSPs,


The results revealed a 10–25% increase in root or tuber yield at PMC2 compared to that in the control. A substantial increase in Zn, Fe, and Mn concentrations in vegetable root/tubers was also observed. Organic matter content and microbial biomass were improved in the soil with PMC application leading to better soil health and better nutrient availability. These studies led us to conclude that the application of PMC not only enhances the vegetable yield but also biofortifies vegetables with micronutrients such as Zn, Fe, and Mn extending agricultural sustainability and eliminating micronutrient malnutrition in humans.Intensive agricultural practices lower soil fertility, particularly micronutrients which are rarely applied to soils as chemical fertilizers. Micronutrient deficiency in soils results in inferior product quality and micronutrient malnutrition in humans. Application of compost to soil may improve crop yields and quality by enhancing macro- and micronutrients availability, enhancing soil microbial population, and improving soil physicochemical properties. Poultry mortality compost (PMC) was prepared by decomposing dead poultry birds with poultry litter in an aerated bin through indigenous microbial populations. The prepared PMC was used as an amendment in three field experiments during 2017–18 and 2018–19 to investigate the effect on yield and nutritional quality of potato, carrot, and radish. In these field trials, two compost levels, i.e., 1250 kg ha The human population is growing at a startling rate, and it is a big challenge for scientists and policymakers to meet the food requirements . AgriculDifferent organic treatments such as farm manure, crop residues, poultry litter, composts, vermicompost, and biochar are applied to the soil to enhance soil health and crop yields , 4. CompProduction of organic waste from food, agriculture, orchards, and process residues is inevitable in our society, and to mitigate the socio-economic and environmental risks of organic waste, it is essential to manage this organic waste effectively . ConversMore than 22 mineral nutrients (both micro and macro) are essential for the human body for its routine work . MicronuIn the present study, we used these plants as test crops to observe the effect of compost application on the biofortification of micronutrients and the biological properties of soils. It was hypothesized that the addition of composted poultry mortality compost would serve as a rich source of micronutrients for vegetables and improve soil biological properties. The specific objective of this study was to explain the impact of poultry mortality compost on vegetable yield and the micronutrient concentration in edible parts of the plant, considering the micronutrient malnutrition in most of the developing countries.-1 ECe, 0.72% organic matter [-1 available P (using Olsen method),129 mg kg-1 NH4-acetate extractable K2O (using flamefotometer), 0.90 mg kg-1 Mn, 0.66 mg kg-1 Zn and 0.89 mg kg-1 Fe using atomic absorption spectrophotometer [Solanum tuberosum L.), carrot (Daucus carrota L.), and radish (Raphanus sativus L.) during the years 2017–18 and 2018–19 at the research farm of the Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Pakistan. Before these vegetable trials, the same fields had a wheat trial with the same treatments followed by maize without any further application of compost. Therefore, compost treatments in vegetable trials may have some residual effect of already applied compost to the soil. The PMC was prepared by composting dead poultry birds  with poultry litter collected from the same poultry farms as described by following the methods described by Sivakumar et al. [The climate of the experimental site is semi-arid with warm and humid summers, and dry cool winters. The experimental site is situated in the middle of latitude 31.41 and longitude 73.07. The weather data of all experiments have been shown in c matter , 30% satotometer . Three fr et al. . The chiThe PMC was prepared at the University of Veterinary and Animal Sciences (UVAS) campus in Patoki, Pakistan. In composting operations, dead poultry birds and poultry litter were mixed with a ratio of 3:1, respectively, based on the C:N ratio of the mixture and to keep the moisture between 40–60%, generally recommended for compost . Compost-1 (PMC1) and 1850 kg ha-1 (PMC2) along with the control where no PMC was applied to each experiment. The chemical fertilizers, i.e., urea, diammonium phosphate, and sulfate of potash were used at recommended doses of 114: 84: 62 kg nitrogen, phosphorus, and potassium (NPK) per hectare for potato, 100: 62: 62 kg NPK ha-1 for carrot, and 62: 50: 62 kg NPK ha-1 for radish. However, the amount of NPK present in compost was subtracted from the recommended dose in compost treatments. The whole amount of calculated PMC, urea, diammonium phosphate, and potassium sulfate was applied manually in the soil at sowing. While urea was applied in three equal splits—at the time of sowing, first irrigation, and second irrigation. Potato (cv Rako), carrot (cv Maharaja), and radishes  were manually planted on ridges in a plot size of 9 m × 12 m with a plant to plant distance of 20 cm and row to row distance of 50 cm for potato, while plant to plant and row to row distances for carrot and radish were 5 cm and 40 cm respectively. Irrigation was applied when needed, and data regarding agronomic and chemical parameters were recorded.Three field experiments  were laid out in a randomized complete block design (RCBD) with three replicates. The PMC was applied by mixing in the soil at the rate of 1250 kg haBefore harvesting, chlorophyll contents were determined with a chlorophyll meter (SPAD 502 P) at the vegetative growth stage of all experiments. After harvesting, total biomass and yield attributes were measured. The shoots and roots were separated, weighed, and then oven-dried at 80°C. Phosphorus (P) concentration was determined by spectrophotometer (Model No CE 7400), potassium (K) by flame photometer (Model No JENWAY PFP 7), and micronutrients, i.e., zinc (Zn), iron (Fe), and manganese (Mn) by using atomic absorption spectrophotometer (Model No Agilent 200 series AA) by wet digestion procedure using a mixture of nitric acid and perchloric acids with 2:1 ratio. SPAD values that indicate chlorophyll contents were measured by SPAD meter (SPAD 502 P) at the vegetative growth stage. Post-experiment soil analysis was carried out for organic matter , where tCollected data were analyzed statistically by using statistical software (Statistix 8.1). The least significance difference (LSD) test was applied at a 5% probability level to compare the treatment means .The economic analysis was done considering the tuber/root yield of all vegetables. Total permanent cost includes the cost of land rent, while seedbed preparation, seeds, fertilizers, compost, irrigation cost, pesticides, herbicides, and harvesting costs were considered as variable cost data. The cost of compost is not the cost of compost production, rather it is the cost on which compost is normally available in the market. In compost application treatment cost of saved fertilizers (NPK) has been subtracted from variable cost. The economic analysis is based on standard expenditure and yield output without including improvement in nutritional quality due to the unavailability of market nutritional quality-based produce prices.-1. The PMC2 application increased the tuber/root yields of potato, radish, and carrot by 15%, 43%, and 39%, respectively, compared with control (where no compost was applied). The increase in tuber/root yields in PMC1 remained statistically insignificant if compared with control and with PMC2 for some cases. Total biomass production in carrot and radish were significantly (P<0.05) enhanced in both PMC1 and PMC2 treatments as compared to control (The highest tuber/root and biomass yields were recorded at PMC2 where compost was applied at the rate of 1850 kg ha control .-1), carrot , and radish  were recorded as compared to control value in shoot and tubers of potato , carrot  and radish . The PMC1 treatment also increased the P concentrations in shoot and root of all vegetables, however, the difference was not significant in most cases, compared with the control. An almost similar trend was observed for K concentrations in shoots and roots. At PMC2 maximum K, concentrations were 2.90, 2.73 g 100 g-1 in potato shoot and tubers, 3.38, 3.28 g 100 g-1 in shoot and root of carrot, and 3.0, 3.28 g 100 g-1 in shoot and root of radish, respectively  significantly improved the soil organic matter content by 0.87% in the potato field and 0.85% in the carrot field, whereas in the radish field organic matter by 0.86% as compared to 0.71, 0.68 and 0.65% in the control of potato, carrot, and radish, respectively . A high The economic analyses of crops used in the present experiments are given in Vegetables contribute a major portion of the human food requirement, including essential mineral elements . MineralDead poultry manure compost not only increased the tuber/root yields of vegetables but also gave a substantial economic advantage concurring to the report by Akande et al. . The pouApplication of PMC to soil enhanced the P and K concentrations in potato plants  Table 2Table 2. Organic matter can be a sustainable source of nutrients to enhance soil fertility since the beginning of agriculture due to having a wide range of essential elements and its beneficial impact on soil physical and biological characteristics , 40. It 3) contamination of groundwater [3 in drinking water can cause methemoglobinemia (blue baby syndrome), cancer, respiratory illness in humans, and abortions in livestock. As the disease fear is a concern, during composting, the continuous high temperature solves pathogenic diseases. There is no such study that shows the possibility of pathogenic diseases spread through the application of compost. The poultry birds used in composting belong to the same flock, which is later used as human food. There are very less chances that a well-decomposed matter can affect human health via soil and plant, if it is not causing any effect to humans who are directly eating. Hence, aforementioned concerns can be minimized by using balanced and well managed compost.As noted above, poultry litter has a high nutritional value and is used as an organic fertilizer in several countries, thus recycling essential nutrients such as nitrogen, phosphorous, and potassium . The litundwater . High le-1) and mineral nutrients can considerably improve the yield and nutritional quality of vegetables. Additionally, the application of PMC also improves nutrient use efficiency, which not only reduces the use of chemical fertilizers but also has a positive impact on environmental pollution-related concerns. Considering the United Nations’ Sustainable Development Goals (SDGs), the use of PMC to agricultural lands is a good way forward to sustain soils for profitable agriculture and reduce the malnutrition issues in humans. Once the economic benefits of compost use are made clear to farmers, its social and cultural adoption will be relatively easy.Poultry mortality compost (PMC) is a potential option to be used as a supplement to synthetic fertilizers to improve the nutritional quality of vegetables. Improved vegetable yields with PMC application are associated with improvements in soil organic matter, supply of essential micronutrients, and soil microbial biomass. Based on this study, it can be concluded that the combined application of PMC (1850 kg ha


Homo sapiens. Here, we describe a novel genetic cause of an “involution” phenotype in four patients, who are characterized by quadrupedal locomotion, intellectual impairment, the absence of speech, small stature, and hirsutism, observed in a consanguineous Brazilian family. Using whole-genome sequencing analysis and homozygous genetic mapping, we identified genes bearing homozygous genetic variants and found a homozygous 36.2 kb deletion in the gene of glutamate receptor delta 2 (GRID2) in the patients, resulting in the lack of a coding region from the fifth to the seventh exons. The GRID2 gene is highly expressed in the cerebellum cortex from prenatal development to adulthood, specifically in Purkinje neurons. Deletion in this gene leads to the loss of the alpha chain in the extracellular amino-terminal protein domain (ATD), essential in protein folding and transport from the endoplasmic reticulum (ER) to the cell surface. Then, we studied the evolutionary trajectories of the GRID2 gene. There was no sign of strong selection of the highly conservative GRID2 gene in ancient hominids  or modern humans; however, according to in silico tests using the Mfold tool, the GRID2 gene possibly gained human-specific mutations that increased the stability of GRID2 mRNA.Bipedalism, speech, and intellect are the most prominent traits that emerged in the evolution of  VLDLR (low-density lipoprotein receptor) and RELN (reelin) genes, both involved in neuronal migration in the developing brain [CA8 (carbonic anhydrase VIII) gene, which participates in the modulation of intracellular calcium signaling in the cerebellum [WDR81 (WD repeat domain 81) gene, which is highly expressed in Purkinje and photoreceptor cells and is involved in endolysosomal trafficking [ATP8A2 (aminophospholipid transporter-like ATPase) gene, which is responsible for the transport of aminophospholipids via the lipid flipping process [TUBB2B (tubulin beta 2B class IIb) gene, which encodes a beta isoform of tubulin—an essential component of the microtubule cytoskeleton [A very rare case of cerebellar hypoplasia, quadrupedia or cerebellar ataxia, mental retardation, and disequilibrium syndrome (CAMRQ) or disequilibrium syndromes (DSs) is characterized by the lack of capacity for bipedal locomotion, ataxia, intellectual disability, and speech disturbance. A similar phenotype was described in several consanguineous families in Turkey ,2,3,4 anng brain ; the CA8rebellum ; the WDRfficking ; the ATPskeleton .GRID2 gene to bipedalism and speech development.In this study, we performed a comprehensive genetic analysis of a neurological case of quadrupedal locomotion with intellectual disability and the complete absence of speech, found in a Brazilian family . We descThis family case was previously described . BrieflyThe study protocol was approved by the Ethics Committee of the Vavilov Institute of General Genetics, the Russian Academy of Sciences, in accordance with the regulations and guidelines outlined in the Declaration of Helsinki. Blood samples of the members of the Brazilian family were collected with their informed consent.GRID2 gene 3 oligonucleotides, one direct sequence, and two reverse sequences, one corresponding to the deleted region (345 bp wild type fragment) and another to the flank region (432 bp mutant fragment), were designed . SNP profiling was performed using the Affymetrix Genome-Wide Human SNP Array 6.0 . Homozygosity analysis was performed using Homozygosity Mapper . We obtadesigned . Both prGRID2 gene was studied using a primate dataset for 23 representatives, and a Rodentia (1 representative) species as an outgroup. Coding sequences of the studied genes were obtained from the NCBI Reference Sequence Database [The molecular evolution of the protein-coding sequence of the Database , EnsemblDatabase , and theDatabase . Only spDatabase . EvolutiDatabase ,26. PostDatabase  was chosThe influence of the synonymous variants on mRNA thermodynamic stability was analyzed via the estimation of alterations in Gibbs free energy using Mfold software with default parameters ,29,30. GTo determine genetic loci linked to the pathogenic mutation in the Brazilian consanguineous family , we perfVLDLR, WDR81, CA8, ATP8A2, RELN, and TUBB2B). We checked all homozygous rare variants in these genes. We only found one homozygous variant, rs140526335 (MAF 0.0005), in the VLDLR gene. CADD prediction placed this variant among the 1% most deleterious substitutions in the human genome. However, using SIFT and PolyPhen programs, the variant was predicted as “tolerated” (0.142) and “benign” (0.083), respectively , causes glycine to change to alanine at an evolutionarily conserved position, the wild type variant is present in most placental animals and reptiles—except lizards, in which alanine is present. A minor variant is present in the human population with an MAF of 0.001, but no carrier with a homozygous state exists [Another nucleotide substitution in the e exists ,39. For e exists ,40, and e exists . AlthougGRID2 gene   . The del,272del) A,B. This,272del) C. Disrupr ataxia ,51,52,53r ataxia  . Thus, we assessed possible changes in selective pressure on the reconstructed phylogenetic tree of the primate dataset by consecutively assigning each crown branch as the foreground branch during CodeML analysis. The ω value across branches was investigated under the one-ratio model (M0), which assumed an equal ω value for all branches, and the free-ratio model (M1), which assumed an independent ω ratio for each branch. Even if the hypothesis of the uniformity of evolutionary rates (molecular clock) was rejected, the M0 and M1 models were not statistically different (GRID2 gene was investigated using branch-site-specific model A (MA0/MA1). The MA1 model was rejected, and the MA0 model was the best fit in all of the branches (Pan troglodytes and Pan paniscus) differs by 13 substitutions, 12 of which are synonymous   . This noHomo sapiens, Denisovan, and Pan troglodytes CDS using the Mfold tool. In terms of energy, a full-length Homo sapiens and Denisovan CDS is more efficient than Pan troglodytes CDS: dG −954.5 and −954.4 kcal/mol as opposed to −947 kcal/mol, respectively. As Mfold tools are more effective for use with short sequence fragments, we analyzed regions across two non-synonymous and thirteen synonymous variants over 25, 51, 75, 151, and 301 nucleotides (nt) of GRID2 CDS , Homininae (six substitutions), and Hominoidea (five substitutions)  . Gibbs fy levels . Moreovey levels . Thus, iGRID2 gene. Previously, the disruption of this gene was linked to progressive autosomal recessive ataxia. In all previously described cases, the common clinical signs, including coordination disorder and global development delay, are results of cerebellar atrophy [GRID2 defects, bipedal locomotion was possible with or without support, despite the gross motor development delay. Here, we focused on patients from a Brazilian family, first described by Garcias and Roth, who possess distinctive features which were not specified in other cases of GRID2 gene pathology (SCAR18): quadrupedal locomotion, the absence of speech, severe intellectual disability, seizures, and some appearance features . In some other previously reported cases of patients with GRID2 mutations, speech disorders in patients were characterized by dysarthria without limits in speaking, or consisting of simple sentences. Potentially, other genetic variants could contribute to the morphological or neuropathological features of the “involution” syndrome in the Brazilian family (CDS1 gene located in the same largest homozygous 4q22 region. The CDS1 is involved in phosphoinositide biosynthesis and lipid droplet biogenesis, and is expressed in the brain and other tissues [CDS1 is predicted to be functionally insignificant. We also found rare genetic variants in genes from genomic regions with relatively low homozygous scores (ARNT2 gene. The ARNT2 gene has a major expression in different brain regions, and was linked to autosomal recessive Webb–Dattani syndrome in one family [Our study expanded the genetic and evolutionary constituent of quadrupedal locomotion and severe intellectual disability in a Brazilian family. The exceedingly rare phenotype that is presented in this case is caused by deletion in the  atrophy ,53,59,60n family . For exa tissues ,40,41,61s scores . Most ine family . HoweverGRID2 gene pathology [GRID2 gene which encode half of the ATD adjacent to the LBD. Such deletion affects all of the predicted protein isoforms of GRID2, whereas in the majority of the previously reported cases [In the Brazilian patients, coordination disorder was accompanied by seizures, which have not been noted in any other described cases; moreover, there is no mention of hirsutism and stunting in previously described cases regarding athology ,53,59,60ed cases ,53,59,60ed cases , the obsed cases , which aGRID2 gene in the regulation of locomotion, speech ability, and intellect across primates. The GRID2 protein sequence is not altered in humans as compared to chimpanzees; moreover, there are no branches with statistically significant evidence of positive selection. However, synonymous selection in Homo sapiens CDS was predicted using the mutation-selection model. An additional test of mRNA stability predicted several significant alterations in Gibbs free energy levels for Homo-sapiens-specific synonymous substitutions in highly conserved positions. Two substitutions in the central part of the GRID2 CDS lead to an increased level of Gibbs free energy, while another two, localized in the second part of the CDS, decrease the level of Gibbs free energy. Moreover, additional analyses across Hominoidea taxa also showed a tendency toward a decrease in the level of Gibbs free energy. Changes in the spatial structure of RNA affect the further transport and translation of the protein; the formation of the optimal spatial structure of RNA contributes to better production, accuracy, and folding of the synthesized protein [GRID2 achieves a high level of expression in Purkinje cells, starting from embryonic development and remaining at a high level throughout an individual’s life, and is almost absent in other parts of the brain (GRID2 gene is conserved among mammals (GRID2 is expressed in the cortex neurons of layer five, which is involved in the formation of motor functions (Anthropological analysis attributes bipedalism to Australopithecus ,64. Thus protein ,66. Intehe brain . Additio mammals . A low lunctions . As showGRID2 gene, and studied the evolutionary value of this alteration. The results of this study indicate a high degree of evolutionary conservation of the protein-coding sequence of the GRID2 gene. The disruption of these regions leads to the loss of bipedal locomotion and affects ancient, evolutionary biological functions in the brain that are responsible for locomotion, cognitive function, and speech. Importantly, other genetic events, such as synonymous substitution, which influences mRNA spatial structure and stability, could have an additional or modifying effect on disease manifestation.In this study, we described a very rare clinical syndrome with quadrupedal locomotion, speech, and learning disabilities caused by a mutation in the highly conserved


Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune astrocytopathic disease affecting central nervous system (CNS). CSF pressure in these patients is usually normal.A 30-year-old woman was admitted with complaints of headache and lower limbs paresis. Lumbar puncture (LP) and magnetic resonance imaging were performed for the patient. Opening pressure was 42 cm H2O in the first LP. According to the clinical evidences, imaging, and the patient's positive aquaporin-4 antibody, the diagnosis of NMOSD was established.High intracranial pressure headache; however rare, may be the first sign of the onset of the acute exacerbation phase of NMOSD. Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune astrocytopathic disease affecting central nervous system (CNS). The disease has a variety of manifestations, both clinically and radiologically. On the one hand, none of the clinical symptoms are pathognomonic for NMOSD; and on the other hand, it includes a wide range of clinical symptoms , 2. NMOSIn NMOSD, the cerebrospinal fluid (CSF) profile can also cover a wide range . There iA 30-year-old female patient presented to another center in September 2019 with primary manifestations of upper limb muscle spasms and was diagnosed as multiple sclerosis (MS). In November 2019, she was admitted in our center with complaints of headache and lower limbs paresis. The patient's headache was positional aggravated by lying down. Opthalmoscopy examination revealed bilateral papilledema. Paresis was progressive and within 4 days, had increased from force level 5 from 5, to 1 from 5, and also she had a sensory level belowT4, which all representing acute myelitis. Brain MRI revealed periventricular lesions, periependymal enhancement and linear enhancement of corpus callosum . CervicaBrain MR venography (MRV) was normal. Lumbar puncture (LP) was performed for her. Opening pressure (OP) was 42 cm H2O in the first LP, so we began tablet acetazolamide 250mg twice daily and tablet lasix 20mg daily for the patient. CSF profile was white blood cell (WBC) count 99 cells , glucose 45, protein 55, and red blood cell (RBC) count 10. The patient's CSF samples were sent for rule out of tuberculosis, brucellosis, and Cryptococcus, which all of them were negative and also all other vascular, autoimmune diseases, and malignancies were considered and ruled out. The second patient's LP also had OP 27 cm H2O. Due to severe presentation of disease, we also began the plasmapheresis and corticosteroid simultaneously  or secondary to medications like corticosteroids . TherefoIn conclusion, according to this case, high ICP headache, although it is rare, may be the first sign of the onset of the acute exacerbation phase of NMO. Keeping this in mind can help in more quickly diagnosing the disease, and to initiate treatment faster to prevent further complications.