P  <  0.05). OLFM4 and C4BPB may be conducive to identifying patients with UC. Further verification studies could be conducted.Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease with an increasing incidence and prevalence worldwide. The diagnosis for UC mainly relies on clinical symptoms and laboratory examinations. As some previous studies have revealed that there is an association between gene expression signature and disease severity, we thereby aim to assess whether genes can help to diagnose UC and predict its correlation with immune regulation. A total of ten eligible microarrays  were included in this study, specifically with six microarrays  in the training group and four microarrays  in the testing group. After the data processing, we found 87 differently expressed genes. Furthermore, a total of six machine learning methods, including support vector machine, least absolute shrinkage and selection operator, random forest, gradient boosting machine, principal component analysis, and neural network were adopted to identify potentially useful genes. The synthetic minority oversampling (SMOTE) was used to adjust the imbalanced sample size for two groups (if any). Consequently, six genes were selected for model establishment. According to the receiver operating characteristic, two genes of OLFM4 and C4BPB were finally identified. The average values of area under curve for these two genes are higher than 0.8, either in the original datasets or SMOTE-adjusted datasets. Besides, these two genes also significantly correlated to six immune cells, namely Macrophages M1, Macrophages M2, Mast cells activated, Mast cells resting, Monocytes, and NK cells activated ( The clinical manifestation of UC mainly includes bloody diarrhea, frequent bowel movement, abdominal discomfort, pain, weight loss, fever, and fatigue2. Unfortunately, UC is deemed as an incurable disease despite plenty of therapeutic options available depending on the disease severity. UC patients are often suffering from alternating conditions of clinical relapse and remission that severely deteriorate their quality of life3. The incidence of UC is previously more prevalent in high-income countries of Europe and North America has shifted towards industrialized countries such as Asia. As a result, it has become a global refractory disease with worldwide shifting epidemiological characteristics. Previous studies have found that immune dysfunction contributes to the progression of UC4. Specifically, in B cells, UC patients showed an increasing percentage of CD23 B naive cells than the normal individuals, while intestinal CD11b + B Cells relieve colitis by secreting immunoglobulin A6.Ulcerative colitis (UC), one type of inflammatory bowel disease , is characterized as inflammation and ulceration in the rectum and colon, which may eventually affect the whole colon if left untreated7. There are some standard methods, such as Mayo score, Ulcerative Colitis Disease Activity Index (UCDAI), etc., which were widely used to identify the remission or active stage of UC8. Additionally, several other factors, including IL-6, TNF-α, and hs-CRP, were also helpful for the diagnosis of UC9. Besides, some scholars have compared the transcriptomic data of rectum biopsy in UC patients and healthy subjects, and high heterogeneity in the gene expression was observed in the UC group10. Particularly, genes with positive correlations were enriched among biological processes, including inflammatory response, neutrophil chemotaxis, and immune regulation11. Compared to other immune diseases , few studies have analyzed the diagnosis or transcriptome differences between UC patients and healthy individuals12. Moreover, although previous studies had reported the role of genes in the diagnosis of UC, the results were not satisfactory due to the database with only two microarrays or the area under the curve (AUC) were unstable14. Thus, it is necessary to develop a predictive model for UC diagnosis with stable AUC based on multiple microarrays.Generally, the diagnosis criteria of different UC stages are mainly based on clinical symptoms and the endoscope and biochemical examinations15. Among various ML methods, Support Vector Machine (SVM) has advantages in the diagnosis research through feature classification of disease and iconography transcriptomic datasets. It has been tested in the studies of various diseases, including cancer, schizophrenia, and postpartum hemorrhage20. Regarding UC, the characteristics of high stability and prediction accuracy of the SVM method have also been proven in previous reports22. Another method of ML, the Least Absolute Shrinkage and Selection Operator (LASSO) analysis, is commonly used in the biomarker identification of various carcinoma diseases25. Previous studies reported that various MLs could be used for the diagnosis prediction of UC, such as PCA analysis26 for metabolomics, the GBM analysis27 for microbiota, RF analysis28 for gene diagnosis, and NN analysis29 for immune-related signature. However, no studies identified the useful genes for UC diagnosis prediction based on the comparison among different ML techniques30. It is highly recommended to combine or compare different methods to increase the accuracy of classification and prediction for the diagnosis research33.Machine learning (ML), based on a series of complex algorithms process, is recently commonly used to identify biomarkers and to predict a wide range of diseases. For example, Random Forest (one type of ML) was used to forecast Crohn’s disease and UC with higher prediction accuracy (more than 90%), even exceeding the traditional prediction modelTherefore, according to the gene expression omnibus (GEO) database, we aim to select multiple microarrays  and to identify the potential useful genes in terms of UC diagnosis through comparing the results from multiple MLs. If applicable, we will further explore the relationship between selected genes and immune cells.http://www.ncbi.nlm.nih.gov/geo/) is a publicly accessible functional genomics database. We initially searched the keyword of “Ulcerative Colitis”, and then screened the data based on the following criteria: (1) inclusion criteria (i) diagnosed as UC in humans; (ii) derived from colon tissue with transcriptome; (iii) included the healthy control (derived from the UC microarrays with no diseases). (2) exclusion criteria (i) suspected carcinoma or other diseases; (ii) included pharmacological intervention(s) for the treatment of UC patients. The sva R package (version 3.36) was used to bias control and to minimize the batch effect among the included various microarrays. After that, we randomly grouped the microarrays as the training set and testing set according to the classic statistical ratio of 6:4. The training data was used to develop the predictive model, while the testing data was used to verify the results of the model.Gene Expression Omnibus  for quantile normalization. This process was composed of (1) transferred the primary dataset into a fixed data type of "matrix"; and (2) the function of "normalize. quantiles" was used for quantile normalization. Secondly, we screened the differently expressed genes (DGEs) in both UC patients and healthy subjects of the training set. Then, the functional analysis of DGEs was conducted through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Disease Ontology (DO) enrichment analysis, and Gene Set Enrichment Analysis (GSEA). Moreover, six machine-learning algorithms, namely LASSO, SVM, NN, GBM, RF, and PCA, were used to establish the models. Regarding the testing group, we divided two subgroups for verification, including one individualized set and all datasets of the testing group. Finally, a correlation analysis between identified genes and immune cells was performed.Based on the training and testing groups, we further analyzed the data. Firstly, we used R package https://www.r-project.org/) and the basement of RStudio (version 1.4.1717). For continuous variables, the independent Student’s t-test was adopted if the variables met Gaussian distribution, if not, the Wilcoxon test was used. For categorical variables, the chi-square test was used, and the Wilcoxon test was used for signed-rank variables. The Pearson or Spearman coefficients were adopted in the correlation analysis. A two-sided p value < 0.05 was considered as significant criteria.Statistical analysis was conducted with R software  was used to identify the DEGs. The raw data was processed as log2 transformation after the quantile normalization. The p value was adjusted to control the false discovery rate (FDR) based on the method of Benjamini and Hochberg. The DEGs were filtered with the criteria of the absolute value of fold change > 1 (|logFC| > 1) and FDR<0.05 (Student’s t test).The clusterProfiler, DOSE, and enrichplot package of R with version 3.16.1, 3.14.0, and 1.8.1 respectively. The GO consist of molecular function (MF), biological process (BP), and cellular components (CC).Functional enrichment analysis was conducted to compare the DEGs between the UC group and the healthy subjects, specifically including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Disease Ontology (DO) enrichment analysis, and Gene Set Enrichment Analysis (GSEA) based on glmnet (version 4.1) package. To avoid overfitting, the SVM algorithm was also used to adjust the premium genes with e1071 R packages (version 1.7–6) and the core of “svmRadial”. Specifically, the R pakage e1071 was applied to the SVM with the function of "rfe" with "sizes" from 2 to 40 with step size of 3, "rfeControl" with "functions" of "caretFuncs" and "cv", and the "methods" was "svmRadial". The SVM code in RStudion was setting as the followed: " rfe ), sizes = c ), rfeControl = rfeControl , methods = "svmRadial")". The R package randomForest (version 4.6–14) was adopted for the RF algorithm with 100 trees. And the PCA algorithm was performed by psych package (version 2.2.3). The variable importance in projection (VIP) values of PCA was used to estimate the importance of genes. The neuralnet (version 1.44.2) was used for the NN algorithm with 3 hidden. The h2o (version 3.36.0.3) was adopted for the GBM algorithm with 100 trees. The overlapping genes that existed in these two algorithm groups were included and the expression levels of candidate genes were further validated in the testing group. Furthermore, the SMOTE with R package DMwR (version 0.4.1) was used to expand the sample size when an imbalanced sample size appeared between the two groups. To estimate the prediction value for UC diagnosis, we used the pROC package (Version 1.17.0.1) in R. AUC of ROC was calculated to judge the accuracy of the predictive model. The greater value of AUC presents the higher accuracy of the predictive model. Additionally, the error rate was added for the assessment of accuracy among various MLs, and the lowest value of error rate could be indicated a better classification capacity.We adopted six machine-learning algorithms  for the identification of significant predictive genes in DEGs. LASSO was performed to screen the candidate genes with binomial deviance and 10-fold cross-validation for the discrimination of UC patients and healthy subjects with https://cibersortx.stanford.edu/), a bioinformatics algorithm, was conducted for correlation analysis. The putative abundance of immune cells was estimated using a reference set with 22 types of immune cell subtypes with 1,000 permutations. Correlation analysis and visualization of these 22 types of immune cells were performed using the corrplot R package (version 0.84). Using the vioplot R package (version 0.3.5), violin plots visualized the differences of immune cells between the UC group and the healthy control cohort. The Spearman's rank correlation analysis in R software was performed. The ggplot2 R package (version 3.3.5) was adopted to visualize infiltrating associations between various immune cells.To quantify the relative proportions of immune cells from the gene expression profiles, CIBERSORT , including GSE48634, GSE6731, GSE114527, GSE13367, GSE36807, GSE3629, GSE53306, GSE87473, GSE74265, and GSE96665 were included for subsequent analysis. Based on the random ratio of 6:4, we developed the training set with 6 microarrays , including GSE48634, GSE6731, GSE114527, GSE13367, GSE36807, and GSE3629, and further established the predictive model. Meanwhile, we developed the testing set with four microarrays , including GSE53306, GSE87473, GSE74265, and GSE96665, and further tested the results of the model. In addition, as the microarray GSE87473 included the largest sample size in the testing set, we also selected it as a separate group for the model testing. With the datasets, we further processed the following analysis just shown in the workflow  were used for classification, we adopted the calculated error rate for model evaluation. The SVM presented the lowest value among the four MLs Table . MoreoveWe adopted the ROC curve and AUC values to assess the diagnosis value of the model. When we set the 6 genes (as mentioned above) into the ROC curve Appendix , the resTo analyze the relationship between ten microarrays and 22 immune cells, we demonstrated the relative percentage of immune cells among 526 samples of ten microarrays Fig. A. Then, Spearman analysis  were 0.804, 0.897, and 0.895 in the training group, testing group, and individual GSE87473 testing group, respectively. In previous studies, OLFM4, as a cancer stemness gene induced by IL-22, was highly distributed in primary sclerosing cholangitis-associated ulcerative colitis35. It was also overexpressed in the active IBD36. In this study, we added a new result in terms of diagnosis values of OLFM4 for UC patients.In this study, we included ten microarrays with 526 samples for data analysis, and further selected a total of 87 DEGs. Based on the six MLs, we successfully established the predictive models and identified two useful genes in the diagnosis of UC, namely OLFM4 and C4BPB. The AUC values of C4BPB (average 0.873) were 0.812, 0.899, and 0.907 in the training group, testing group, and individual GSE87473 testing group, respectively. Compared to the previous studies, C4BPB presented the diagnosis value in Crohn's disease5. Regulatory T cells were also a key factor that exacerbated UC through immune imbalance37. Compared to Crohn's disease, the colonic mucosa samples in UC patients showed the expansion of IL17A+ CD161+ effector memory T cells and IL17A+ T-regulatory cells, expansion of HLA-DR+CD56+ granulocytes, and reductions in type 3 innate lymphoid cells38. The regulation of T cells for UC patients based on Bcl-6 and IL-21 could help to avoid the occurrence and development of IBD39. In a previous survey that included a global immune cell landscape of UC patients’ tissue, the results identified the increasing number of neutrophils, T CD4 memory-activated cells, active dendritic cells, and M0 macrophages, and decreasing number of T CD8, Tregs, B memory, and M2 macrophages40.Regarding the correlations analysis, these two genes  presented significant associations with 6 types of immune cells, including Macrophages M1, Macrophages M2, Mast cells activated, Mast cells resting, Monocytes, and NK cells activated. Some scholars have found that the UC patients presented an increasing percentage of CD23 B naive cells, compared to the normal individuals41. Although Epstein–Barr virus infection might trigger several immune dysfunctions, such as natural killer/T cell lymphoma arising, hemophagocytic lymphohistiocytosis, and malignancies, few studies focused on UC previously43. Furthermore, Epstein Barr Virus might be useful in the development of vaccines and immune cell therapy for EBV-Associated diseases, especially for several immune-related diseases44. The prognosis of Epstein–Barr virus infection in UC was less paid attention to45. In this study, the KEGG pathways results indicated that more studies of the Epstein–Barr virus infection pathway in UC could be conducted46. Particularly, IBD patients have a higher risk to develop autoimmune and inflammatory diseases, such as rheumatoid arthritis47.Through the functional enrichment analysis, we found three pathways, including Chemokine signaling pathway, Epstein–Barr virus infection pathways, and Rheumatoid arthritis pathways, might be closely related to the progress of UC. The previous study had emphasized the potential role of the Chemokine signaling pathway in the up-regulation of UC patients and further proposed the CXCL8-CXCR137 (a type of chemokine) in the treatment of UC48, particularly in the NN method which includes many layers, such as an input layer, an output layer, and hidden layers (count fluctuating)50. Among them, the characteristics of the hidden layers are hard to identify51. Thirdly, we have limited information about the clinical features, such as the patient's age30, ethnicity and race53 and stage of UC. Generally, some detailed information impacts the algorithm bias. Thus, further subgroup analysis could be included to identify more useful results in future research.Actually, there are many limitations to using machine learning in a clinical setting. As MLs include multiple factors, especially in statistics, clinical practice, and bioinformatics. To improve the study design and to facilitate the explanation of results from ML analysis, it is recommended to include a variety of experts of authors/researchers in a study. Individuals with rich clinical experience and MLs technique background are also conducive to playing an important role in clinical MLs studies. In this study, there are some limitations. Firstly, insufficient verification is a common type of limitation in bioinformatics studies. Although we designed testing groups to assess the stability of the predictive model based on AUC values, and included ten microarrays to increase the sample size in this study, more research works, either in clinical trials or animal experiments, should be conducted to obtain solid verifications for these predictive results. Secondly, the machine-learning model itself includes some limitations, such as the black box phenomenonIn this study, we found two useful genes of OLFM4 and C4BPB which may help to identify UC patients. Further verification studies could be conducted.Supplementary Information.


The highest prevalence of the surveyed characteristics in both groups was for overweight/obesity and daily coffee consumption, followed by psychochemical drug intake, migraine, and peripheral vasospasm. Our data show that overweight/obesity, migraine, asthma, and smoking are major risk factors for conversion from OHT to OAG in this Spanish and Portuguese population.Open-angle glaucoma (OAG), the most prevalent clinical type of glaucoma, is still the main cause of irreversible blindness worldwide. OAG is a neurodegenerative illness for which the most important risk factor is elevated intraocular pressure (IOP). Many questions remain unanswered about OAG, such as whether nutritional or toxic habits, other personal characteristics, and/or systemic diseases influence the course of glaucoma. As such, in this study, we performed a multicenter analytical, observational, case–control study of 412 participants of both sexes, aged 40–80 years, that were classified as having ocular hypertension (OHT) or OAG. Our primary endpoint was to investigate the relationship between specific lifestyle habits; anthropometric and endocrine–metabolic, cardiovascular, and respiratory events; and commonly used psychochemicals, with the presence of OHT or OAG in an ophthalmologic population from Spain and Portugal. Demographic, epidemiological, and ocular/systemic clinical data were recorded from all participants. Data were analyzed using the R Statistics v4.1.2 and RStudio v2021.09.1 programs. The mean age was 62 ± 15 years, with 67–80 years old comprising the largest subgroup sample of participants in both study groups. The central corneal thickness (ultrasound pachymetry)-adjusted IOP (Goldman tonometry) in each eye was 20.46 ± 2.35 and 20.1 ± 2.73 mmHg for the OHT individuals, and 15.8 ± 3.83 and 16.94 ± 3.86 mmHg for the OAG patients, with significant differences between groups (both  Ocular hypertension (OHT) is the only known modifiable risk factor of glaucoma development. Intraocular pressure (IOP)-lowering therapy reduces the risk of glaucoma. Several risk factors for glaucoma conversion, mainly a higher level of IOP and age, have been established by the Ocular Hypertension Treatment Study (OHTS) and the European Glaucoma Prevention Study (EGPS). However, substantial variability exists in the measurement of the currently known risk factors, especially if the assessment is performed from a longitudinal perspective. Additionally, many factors are responsible for overdiagnosis ,2,3,4. TA GS is defined as a person who has one or more clinical features and/or risk factors that increase the possibility of developing glaucomatous optic nerve degeneration (OND) and visual deficiency ,6,7. TheOpen-angle glaucoma (OAG) is the most prevalent type of glaucoma worldwide, and its diagnosis depends on the demonstration of clinical signs such as IOP elevation and optic nerve damage, which can be documented using structural imaging techniques and functional tests ,11,12,13Recognized non-IOP risk factors for OAG mainly include older age, ethnicity, thinner corneas, and the degree of glaucoma severity ,11,13,14Increased knowledge of the pathological mechanisms and risk factors of OAG is urgently needed. Identifying key non-ocular factors that may influence the progression from OHT to OAG is critical for designing specific interventions to more appropriately managing patients at risk of glaucomatous OND. Because only few studies have been conducted on this topic in Spain and Portugal, a singular geographical area where family and public culture and lifestyle behaviors differ from other European countries , we deciWe conducted a multicenter, analytical, observational, case–control study of 635 participants, initially recruited from the ophthalmological departments of the collaborative hospitals from Spain and Portugal, who agreed to participate in the study and signed an informed consent form. We adhered the principles of the Declaration of Helsinki  and the Ethics Committee standards of the study centers (2021). The clinical research requirements to maintain the privacy of all data obtained from our study volunteers were met.n = 198) and (2) patients diagnosed with OAG . All patients diagnosed with OAG and some participants of the OHTG were treated with hypotensive eye drops, laser, or glaucoma surgery, depending on the individual needs and glaucoma stage. The major causes of the reduction in sample size from the baseline-selected participants to the end of study were the following: volunteer withdrawal, clinical findings that strongly recommended excluding the participant, personal reasons, or exceptional complications that prevented finishing the study course.Ophthalmologists from the glaucoma sections at the main collaborative university hospitals  performed a systematized ocular examination of 635 initial volunteers of both sexes and aged 40 to 80 years, presenting to glaucoma clinics, through a nonrandom consecutive sampling method. Suitable study participants were chosen by ensuring their appropriate status according to the inclusion and exclusion criteria, as listed in 2) (25 to 29.9 indicates overweight and >30 indicates obesity); coffee or tea consumption in the form of hot beverages, recorded as the average number of cups consumed per day or per week or lack of coffee or tea intake; smoking and/or alcohol habits, which was recorded as alcoholic beverages consumed per day or per week and/or how many cigarettes were smoked daily or weekly, with lack of alcohol or tobacco use also recorded; systemic disorders, which included questions about thyroid status (hyperthyroidism or hypothyroidism) and cardiovascular  and respiratory , and sleep apnea) conditions as well as the use of psychochemical drugs . Data were recorded as DEMO and QUEST in a Microsoft Excel spreadsheet that was specifically reviewed by the survey supervisors.We conducted interviews with each participant to determine the social and demographic factors associated with specific characteristics and lifestyle domains, systemic comorbidities, and commonly prescribed psychochemical medications. All surveyed volunteers answered questions regarding the following: height and weight, which were used to determine the body mass index (BMI) by dividing the weight (kg) by the height , morphological measurements , and optical coherence tomography (OCT) cirrus spectral-domain OCT , as well as functional ) performance, using the 24-2 Swedish interactive threshold algorithm  for evaluation. We considered the best-corrected visual acuity (BCVA) obtained by the logarithm of the minimum angle of resolution (LogMAR) for each eye. The IOP was measured three consecutive times during the ophthalmological visit within the time-period between 09:00 a.m. and 12:00 p.m. Only the study professionals were responsible for the IOP measurements by means of the Goldman tonometer. The mean IOP in the normal adult population is 15–16 mmHg. Normal IOP is defined as two standard deviations above the normal, i.e., 21 mmHg; any IOP above this level was considered being elevated. We report IOP values as the mean ± SD in millimeters of mercury for three determinations for each participant.® iPac®; Reichert/Ametek, Munich, Germany). The pachymeter was calibrated at the beginning of each session following the manufacturer’s recommendations. After instillation of anesthetic eye drops, on an undilated pupil, we performed the ultrasonic pachymetry using a probe tip of approximately 1 mm in diameter. The tip was perpendicularly placed on the plane of the central cornea. Three independent measurements were conducted in a random sequence of 3 min from each other. Normal CCT (ultrasonic pachymetry) was estimated at 533 μm. CCT values are reported as the mean ± SD μm of three determinations for each participant.Central corneal thickness (CCT) was measured using hand-held ultrasonic pachymetry -adjusted IOP (Goldman tonometry) for the study participants was calculated according to the Los Angeles Latino Eye Study  for a CCt-test for independent samples  or the Mann–Whitney U test . Comparison of more than two means was carried out through analysis of variance  or the Kruskal–Wallis test . Akaike information criterion (AIC) was used to estimate the prediction error  for a given dataset by providing a method for optimizing model election [Statistical analyses were conducted by two independent data scientists using R Statistics v4.1.2 and RStudio v2021.09.1, a set of integrated tools designed to help with R . R packages readxl, dplyr, ggplot2, ggcorrplot, and DemoTools were used for data loading and wrangling, exploratory analysis, and data plotting. R packages caret, MASS, Epi, questionr, and car were used for data modeling and model evaluation. Pearson chi square test was used for comparing two categorical variables, and Shapiro–Wilk test to check the distribution of quantitative variables. Comparison of two means was carried out using Student’s election ,24. BothThe final sample included 412 participants, who we clinically classified as 198 OHT individuals (OHTG) (48.06%) and 214 OAG individuals (OAGG) (51.94%). The distribution of data from each participating hospital was as follows: University Hospital Dr. Peset of Valencia (20%); University General Hospital of Valencia (18%); Clinic Hospital of Barcelona (17%); University Hospital “Morales Meseguer” of Murcia (11%); Punta de Europa Hospital of Algeciras (11%); Clinsborges of Porto, Portugal (11%); University Jerez Hospital of Jerez de la Frontera (6%); and San Juan de Dios Hospital of Sevilla (6%).The mean age of the study participants was 62.0 ± 15.0 years. The percentages in the different age subgroups among the participants are shown in p < 0.05) of the OHTG, respectively, versus 51.47% and 48.53% of the OAGG, respectively.Men and women constituted 39.39% and 60.61%  and left eye (LE) of each participant.p = 0.005; LE: p = 0.001. The mean CCT-adjusted IOP in the OHTG was 20.46 ± 2.35 mmHg (RE) and 20.1 ± 2.73 mmHg (LE), whereas in the OAGG, the mean IOP was 15.8 ± 3.83 mmHg (RE) and 16.94 ± 3.86 mmHg (LE). When comparing each eye from both groups, we found significant differences in IOP between the OHTG and OAGG . OCT parameters  and VF mean deviation for each eye were significantly different between the study groups (p < 0.001).Our data show that mean BC LogMAR VA was 0.00 (RE) and 0.10 (LE) in the OHTG versus 0.20 (RE) and 0.20 (LE) in the OAGG. Moreover, the CCT was significantly higher in the OHTG  than in the OAGG . Based on comparison between each eye from both groups, RE: We categorically considered the relevance of the clinical history to the OHT or OAG diagnosis in this cohort. Age, sex, anthropometric characteristics (BMI), thyroid state, cardiovascular and respiratory situation, smoking and drinking habits, coffee or tea intake, and the use of psychochemical drugs of all participants were determined to help manage the early diagnostic classifications and therapeutic decisions for OHT or OAG individuals. The highest prevalence of the studied characteristics in both groups was for overweight/obesity and daily coffee consumption. Additionally, psychochemical drug intake, migraine, and peripheral vasospasm were outstanding characteristics in this OHT and OAG population, as highlighted in p = 0.01), IOP , smoking (p < 0.001), height (p = 0.01), and COPD (p < 0.001).First, we performed univariate analysis to determine significant differences between the study groups in some of the variables , such asSecond, we analyzed the correlation between the studied variables to determine the power of the linear relationship between two variables as well as to record their association in relation to the primary study endpoint, as shown in p = 0.03), asthma (p = 0.003), COPD (p = 0.03), and migraine (p = 0.001).Third, we analyzed the diagnostic variable using logistic regression , asthma (p = 0.002), and migraine (p = 0.001). In addition, a fourth variable had a significant influence on the diagnosis: weight . These data may be interpreted as COPD being caused by cigarette smoking, which evidently is the main cause of the disease, and is thought to account for around 9 in every 10 cases worldwide. Thus, we decided to remove the variable COPD from the third analytical model, being now, and according to age and gender, the most significant characteristics in our study population: smoking (p = 0.041), weight (p = 0.009), BMI (p = 0.001); asthma (p = 0.002), COPD (p = 0.044), and migraine (p = 0.002). Overall, these data are reflected in Finally, we consider the possible influence of the variables age and gender on the diagnosis of HTO/OAG. Therefore, a third analytical model was created by adding these two variables to the ones pertaining to our previous model, as listed in We explored some classical and emergent risk factors in HTO or OAG men and women volunteers pertaining to a Spanish and Portuguese ophthalmological population aged 40 to 80 years, revealing the importance of identifying people more predisposed to progression from OHT into OAG, as well as to early undertaking precision measures for avoiding OND and visual impairment-blindness ,12,13,25In this concern, four comorbidities significantly influenced the diagnosis of OHT vs. OAG, being these: overweight/obesity, migraine, asthma, and smoking.OAG is a chronic optic neuropathy following the damage and death of the RGCs in the framework of IOP elevation, and it remains the leading cause of irreversible blindness worldwide ,26,27. HIndividuals with OHT constitute an important group to manage because they are frequently undiagnosed. Additionally, GSs are at high risk of OND with respect to the global population ,6,7,28. In this study, most participants were in the age range of 67–80 years in the OHTG and OAGG . SeveralExclusive reliance on clinical glaucoma tests may decrease the likelihood of identifying OHT subjects at higher risk of progression to glaucoma. Moreover, total reliance on factual data regarding glaucoma hallmarks may negatively influence the decision to treat individuals. Several authors ,27,28,40Overweight and obesity are conditions in which excessive fat storage poses a serious risk to health and wellbeing [ellbeing ,27,42,43ellbeing ,16,25,37ellbeing  demonstrellbeing . Jung etellbeing , in a naellbeing  describeCoffee and tea are the hot drinks most consumed worldwide, with consumption significantly increasing from 2012 to 2021 [ to 2021 . The med to 2021 . Tellone to 2021  reported to 2021 . Habitua to 2021 ,14,15,28 to 2021  first co to 2021 , single- to 2021  found th to 2021 . In our Psychochemicals are widely prescribed for the treatment of a variety of mental illnesses . Drugs of the new generation are currently the first line treatment for affected patients, among which includes the selective serotonin reuptake inhibitors (SSRIs) and serotonin noradrenaline reuptake inhibitors (SNRIs), among others [g others . These dg others . Psychocg others ,14,15,28g others . Howeverg others , in a syAlcohol and tobacco are considered lifestyle factors and sociocultural habits as well as the most addictive legal drugs with the highest popularity of consumption in the general population, with both having an important impact on morbidity and mortality worldwide [p = 0.01) and smoking (p < 0.001), similarly to the findings by Zanon-Moreno et al. [orldwide . Polydruorldwide . Severalorldwide ,61. We forldwide  found a orldwide  analyzedo et al. .Comorbidities are common in glaucoma [thyroid gland is located on the anterior cervical area and produces thyroid hormone, which plays a pivotal role in controlling general metabolism, immune system, mood state and cognitive functions, and nearly every system in the body [glaucoma ,45,46,47the body . Dysfuncthe body ,66. The the body , thyroidthe body . In our Migraine is a devastating condition that ranks among the most disabling disorders worldwide and has been considered an important OAG risk factor, with studies yielding controversial conclusions [Peripheral vasospasm is vascular dysregulation  of the microvasculature that is manifests as symptoms of cold hands and feet [Because of this, we also formulated some questions to obtain a global view of the trends related to vascular disorders in this population cohort. clusions ,70. A meclusions  suggesteclusions  found anclusions ,16,28,37and feet  and is aand feet ,75. We iAsthma is one of the most frequent respiratory illnesses globally, affecting both sexes of all ages [COPD is a multifactorial disease that includes emphysema and chronic bronchitis, associated with pulmonary symptoms  and a variety of extrapulmonary manifestations. Smoking is the major risk factor for COPD. Short-acting bronchodilator inhalers are the first line therapy for the disease [Sleep disordered breathing includes a wide spectrum of anomalies, including sleep apnea, with a common feature of hypoventilation–hypoxia, playing an important role in the pathogenesis of cardiovascular, neurologic, and metabolic disorders [Regarding the respiratory disorders that we analyzed in our study population , our data are described below. all ages . Hallmarall ages ,78. COPD disease . Occasio disease . Moreove disease  reportedisorders . Cesareoisorders  reportedOverall, our data show that overweight/obesity, migraine, asthma and smoking, are major risk factors for the progression from OHT to OAG in our study population.The limitations of the study are as follows: (1) Participants may have underreported their characteristics, lifestyle, and illnesses, either intentionally or due to recall bias. (2) Volunteers may have underreported their own tobacco and alcohol use habits. Moreover, binge drinkers  may have provided inaccurate answers our questions. (3) Although we could not accurately categorize systemic disorder for all cases, we considered the most relevant issues for each comorbidity in the study endpoints. (4) We did not include the medications for each systemic disease surveyed or for glaucoma. (5) The study produced a large amount of information, and the statistical processing created large volumes of data. Due to this, we focused on the main study objectives, and some of the information and data have been omitted from the final data in processing. Briefly, we took some actions to mitigate the above limitations, such as by confirming the patient data collection through an extensive review of their clinical charts and by speaking with the accompanying persons and/or family members. Moreover, we discussed any discrepancy in the selection of suitable participants, data screening, and results. We are aware of that any of these described potential limitations could have affected the study findings and interpretations. However, to ensure coherence in the obtained information, data scrubbing and normalization were independently performed by two researchers. All data reported herein will be deposited in a cross-disciplinary public repository. These actions represent a logical and useful way of improving our data power.Holistic approaches to glaucoma must be taken by selecting the proper strategies that permit ophthalmologists to address the whole person rather than only evaluating the ocular status. The patient account of their medical history is a fundamental step in any medical act. In the era of precision medicine, glaucoma specialists must properly evaluate the risk factors related to lifestyle, non-ocular-specific personal characteristics, and systemic comorbidities. The data from our study consistently indicate that individuals with OHT and OAG suffering from overweight/obesity, migraine, or asthma, and the smokers are at higher risk of progression from OHT to OAG and glaucoma. Through extensive control and/or modification of the above disorders, the development or progression of glaucoma can be positively altered in the affected persons.In this Spanish and Portuguese cohort, we identified some non-ocular-specific factors to help assess the risk of OAG and to prevent or delay glaucoma OND and blindness, namely overweight/obesity, migraine, asthma, and smoking. Based on the results of this study, we strongly recommend careful and considered care for the people suffering from these disorders to prevent subsequent visual disability and loss of quality of life.


There was a significant systematic increase in BMD in both genders at various skeletal sites, grouped by BSA quartile. The osteoporosis rates of the lumbar spine (97.9%), femoral neck (92.4%) and total hip (87.1%) in the female Q1 group were significantly higher than those in the Q4 group (P < 0.001), which were 80.0%, 57.9% and 36.9%, respectively, in the Q4 group; the osteoporosis rates of the lumbar spine, femoral neck, and total hip were 53.9%, 59.4%, and 36.3% in the male Q1 group, and 15.2%, 21.9%, and 7.03% in the Q4 group, which were significantly lower than those in the Q1 group (P < 0.001). In age-adjusted Cox regression models, the risk of fracture in the remaining three groups  for weight, BMI, and BSA for both genders, compared with the highest quartile (Q1 by descending quartile stratification) were significantly higher. In models adjusted for age and BMD, only men in the BSA Q3  and BSA Q4 groups  had significantly higher fracture risks. In models adjusted for age, height, weight, BMI, and BSA, low BMD was the greatest fracture risks for both sexes. Our results showed that BSA was closely related to BMD, prevalence of osteoporosis, and fracture risk, and that a decline in BSA may be a new potential risk factor for osteoporotic fractures in Chinese men.Clinical vertebral fractures and femoral neck fractures are severe osteoporotic fractures that increase morbidity and mortality. Anthropometric variables are associated with an increased risk of osteoporotic fractures, but it is not clear whether body surface area (BSA) has an effect on clinically severe osteoporotic fractures. The study included total of 3,694 cases of clinical vertebral fractures and femoral neck fractures  and 3,694 controls without fractures who were matched with the cases by sex and age. There was a significant positive correlation between BSA and bone mineral density (BMD) in female and male fracture patients (females:  Osteoporosis is a systemic bone disease that is characterized by a decrease in bone mass, a deterioration of the microstructure of bone tissue, and a decrease in bone strength, leading to an increase in bone fragility and susceptibility to fractures . ClinicaIt is well known that low bone mineral density (BMD) is an important risk factor for osteoporotic fractures , 25, butThe study was conducted from March 2011 to October 2021 at the Second Xiangya Hospital of Central South University, Changsha, China. Patients diagnosed with osteoporotic fractures by imaging were considered potential subjects for the case group. The inclusion criteria for severe osteoporotic fractures were patients who came to the hospital with symptoms of vertebral fractures or femoral neck fractures, patients who reported low-injury fractures that occurred from falling from a standing height or less, or occurred without falling. A vertebral body fracture was confirmed by a radiologist based on a lateral vertebral radiograph and a femoral neck fracture was confirmed a by radiologist based on a proximal femoral radiograph, using semi-quantitative methods . PatientThe data of 3,694 patients assigned to the control group were obtained from a reference population of a BMD database, which was established by us before the study , 36. A 1The lumbar spine (L1–L4), femoral neck, and total hip BMDs were measured by fan-beam dual-energy X-ray (DXA) absorptiometry . If the lumbar vertebrae of patients with vertebral body fractures were filled with postoperative artificial bone cement or contained installed metal brackets, these lumbar vertebrae were excluded from the analysis. The right hip was measured if the patient had a left femoral neck fracture or had a hip replacement. If patients had bilateral femoral neck or hip fractures, the hip measurements were discarded and these patients were excluded from the study. BMD was measured twice in 33 subjects. The root-mean-square coefficients of precision  were 0.86%, 1.17%, and 0.88% for the lumbar spine, femoral neck, and total hip, respectively. The long-term (> 17 years) CV of routine quality control phantom measured daily by DXA bone densitometer was < 0.45%. Using our own BMD reference database for women and men , 36, we 0.7271 × W0.3980; and its estimation formula for females was BSA = 84.4673 × H0.6997 × W0.4176; where BSA was expressed in cm2, height (H) in cm, and body weight (W) in kg. According to the BMI classification criteria for overweight and obesity in Chinese adults , the rest of the indicators  basically met the normal distribution standard . The indicators that did not meet the standard of normal distribution were expressed by median and range. If there was a significant difference between groups, test for two independent samples was used. Indicators meeting the normal distribution criteria were expressed as mean and standard deviation (SD) and one-way analysis of variance (ANOVA). The relationship between BSA and BMD at various skeletal sites was analyzed using Pearson’s correlation. The patients in the case group were divided into quartiles according to their BSA, and the differences in mean BMD, prevalence of osteoporosis, and fracture risks were compared among these four subgroups. The relationships of different variables with the risk of osteoporotic fracture were analyzed by multivariate Cox regression models, which produced multivariate hazard ratios (HRs) for fractures and their 95% confidence intervals (CIs). The multivariate analysis included adjustments for age, height, weight, BMI, and BSA or BMD. The differences in the prevalence of osteoporosis and osteopenia between genders and across different groups of fracture patients were compared using the chi-square test. A P < 0.05 was considered statistically significant.All analyses were performed using SPSS V23.0 for Windows . Kolmogorov-Smirnov test (K-S test) was used to explore normal distribution of the data. The K-S test results showed that the age, height and weight of the subjects of both genders and the age at menopause (AM) and years since menopause (YSM) of women did not meet the normal distribution criteria  were higher for females than they were for males . The correlation of BSA with Hip-BMD was higher than the correlation of BSA with LS-BMD and FN-BMD. P = 0.019 to < 0.001) than the reference group (Q1); only the female Q4 group with the smallest height (height ≤ 147.9 cm) had a significantly higher HR1 ; HR1 also was significantly higher in the Q3 and Q4 groups of males. In the models adjusted for age and BMD, the increases in HR2 for each quantile of weight, BMI, and BSA of females were no longer statistically significant, but there was a significant increase in HR2 for the height Q4 group of females . The HR2 was also significantly higher in the BSA Q3  and BSA Q4  groups of males. In the models adjusted for age, height, weight, BMI, and BSA, the HR2 in the female each quantile (Q2 to Q4) varied from 2.30 to 4.42 as BMD decreased; in the male each quantile, the same measures varied from 3.48 to 8.74   and total hip (61.9%), whereas the rates in male patients were very low , and about 2.87 times (89.6/31.2) and 3.14 times (61.9/19.7) higher in women than in men, respectively, yielding significant differences between the sexes. This suggested that severe osteoporotic fractures occur in only a small proportion of females and a large proportion of males who did not have osteoporosis. Our results were similar to those of previous research that found the osteoporotic rate of female fracture patients was significantly higher than that of males . The resd obese) , the obed obese) , 42, whi2 in group Q3 and BSA ≤ 1.5892 m2 in group Q4 (the results were not shown), so we determined when BSA ≤ 1.6895 m2, the risk of severe osteoporotic fracture in men was significantly increased by about 41–55%.We found BSA was strongly associated with BMD, prevalence of osteoporosis, and fracture risk in male and female cases. BSA was significantly and positively associated with lumbar spine, femoral neck, and total hip BMD in cases of both genders Figure 1Our study also showed when BMD was stratified by descending quartiles , and two models that adjusted for age (HR1) or adjusted for age, height, weight, BMI, and BSA (HR2) Table 3,This study has some limitations. First, it was not a multi-center study, and its results may only be representative of the population in and around Changsha City. Because of China’s vast territory, differences between the north and the south and between the east and the west are large, so more extensive multi-center studies are needed. Second, this study did not have a follow-up survey, and its results could not necessarily reflect causality. The third limitation is that the measure of height in patients with vertebral fractures, especially those with multiple vertebral compression fractures, may be unduly low, thereby affecting the accuracy of the BSA and BMI calculations. The BSA was not a direct measurement, but was estimated using a correlation formula based on the subject’s height and weight , and theOur study suggested that among patients with clinically severe osteoporotic fractures, the prevalence of osteoporosis in women was approximately three times that in men, and there was a significant difference between the two genders. Obesity rates among women and men with fractures were approximately 14% and 8%, respectively, of those in North American countries. In both genders, BSA was significantly positively associated with BMD in fracture patients, and the prevalence of osteoporosis decreased with increasing BSA. In models adjusted for age and anthropometric measures , decreased BMD or osteoporosis was the greatest risk factor for fracture risk in both genders, and increased fracture risk varied with sex and BMD at different skeletal sites. In age-adjusted models, fracture risk increased non-linearly and linearly in women and men, respectively, with decreasing BSA levels. In models adjusted for age and BMD, decreased BSA remained a risk factor for increased fracture risk in men. Therefore, it suggested that lower BSA may be a new potential fracture risk factor independent of BMD in men, and its importance should be considered when assessing clinical fracture risk.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Z-FS and X-YW designed the study and wrote the manuscript. X-YW, H-LL, YS, L-HT, and L-QY conducted data collection and data analysis. R-CD, HZ, Y-QP, and Z-JX acquired data from chart review and performed the analysis. Z-FS, X-YW, H-LL, YS, L-HT, L-QY, R-CD, HZ, Y-QP, and Z-JX reviewed and revised the manuscript. All the authors contributed to the article and approved the submitted version of it.This work was supported in part by grants from the National Natural Science Foundation of China (81500685), the Ministry of Health of the People’s Republic of China (200446850) and the Clinical Big Data Project of Central South University ([2013]15-86), China.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.


Triticum turgidum using bioinformatics tools and to investigate the expression patterns of some members in response to salt stress. Our results show that there are 80 TtAQP genes in T. turgidum, which are classified into four main groups based on phylogenetic analysis. Several duplications were observed between the members of the TtAQP gene family, and high diversity in response to post-translational modifications was observed between TtAQP family members. The expression pattern of TtAQP genes disclosed that these genes are primarily upregulated in response to salt stress. Additionally, the qPCR data revealed that TtAQPs are more induced in delayed responses to salinity stress. Overall, our findings illustrate that TtAQP members are diverse in terms of their structure, regulatory systems, and expression levels.During the response of plants to water stresses, aquaporin (AQP) plays a prominent role in membrane water transport based on the received upstream signals. Due to the importance of the AQP gene family, studies have been conducted that investigate the function and regulatory system of these genes. However, many of their molecular aspects are still unknown. This study aims to carry out a genome-wide investigation of the AQP gene family in  AQP cperoxide .AQP genes differs depending on the organs and tissues, as well as plant growth regulators and abiotic stress such as drought, heat, and cold [In recent years, the function of AQPs has been investigated in relation to plant responses to biotic and abiotic stresses ,9,10,11.and cold ,14. Betwand cold . The dowand cold ,17,18. Aand cold ,19.T. turgidum L. ssp. durum (Desf.), is a tetraploid wheat (2n = 4x = 28). T. turgidum is best adapted to areas with a relatively dry climate with warm days and cool nights during the growing season (corresponding to a Mediterranean and temperate climate) [T. turgidum. In the current study, we aimed to identify and characterize AQPs in T. turgidum (TtAQPs), and the expression levels of several TtAQPs were evaluated at different salinity concentrations. As the first report on this topic, new perspectives related to the structure, process of evolution, and function of members of this gene family are presented.Today, cereals are considered major crops, and wheat is considered a strategic crop among cereals across the globe. Wheat genotypes exist in three ploidy levels, which include diploid, tetraploid, and hexaploid . Durum wclimate) . As mentAQP genes in T. turgidum L. ssp. durum (Desf.) (TtAQPs) [Arabidopsis (AtAQPs) and rice (OsAQPs) were applied as the query sequences against the complete protein sequence of T. turgidum using the BLASTP tool of the EnsemblPlants database (Accessed on 24 November 2022) [To identify the (TtAQPs) , the proer 2022) . In the er 2022) . Then, ser 2022) .T. turgidum, a phylogenetic tree was constructed with the complete protein sequences of the TtAQP gene family members along with their orthologues from Arabidopsis, rice, barley, corn, and sorghum plants. AQP genes were identified in Arabidopsis , barley (Hordeum vulgare), corn (Zea mays), and rice (Oryza sativa japonica) using the EnsemblPlants database, as was T. turgidum. The sequence of AQP proteins was aligned using the ClustalW program using an online tool, Clustal Omega [To study the relationships of TtAQP proteins in al Omega . Then, tal Omega . The iTOal Omega  was usedal Omega  based onTtAQP genes were identifiedbased on similarity, more than 80% between pairs of TtAQP genes [TtAQP genes was estimated using the synonymous mutation rate of substitution λ per synonymous site per year, as T = (Ks/2λ (λ = 6.5 × 10−9)) × 10−6 [The duplication events between QP genes . In addiQP genes . Then, t) × 10−6 .Phosphorylation is one of the most important types of post-translational modification. The sites of phosphorylation are serine, tyrosine, and threonine residues on proteins. The potential phosphorylation regions of TtAQP proteins were predicted through the NetPhos 3-1 site, with a potential value higher than 0.80 .The three-dimensional structure of TtAQP proteins was predicted using the Phyre2 database . The valTtAQP genes was investigated as the promoter region. The sequence of the promoter region was analyzed to recognize the putative cis-regulatory elements using the PlantCARE database [To identify the regulatory regions in the promoter region, a region 1500 nucleotides upstream of the database . FinallyT. aestivum using the String database . Additionally, gene ontology (GO) enrichment analysis of proteins presented in the network was performed. To identify the significant GO terms, the FDR  was adjusted to <0.01.In the present study, we constructed an interaction network of TtAQP proteins based on their homologs in T. turgidum L. ssp. durum (Desf.) cultivar Yavaros were sterilized with 3% sodium hypochlorite for 10 min. Then, they were cultivated in pots containing sterilized perlite and peat moss (2:1). Five seeds were planted in each pot, and after germination, three of the best seedlings were left in each pot. The cultivation conditions were kept with a photoperiod of 16 h of light and 8 h of darkness and a temperature of 25 °C. Forty-day-old seedlings were subjected to salt treatment via irrigation. In the current study, two salt concentrations, 150 and 250 mM of NaCl, were applied twice at 24 h intervals. Some pots were also considered as controls . After applying salt stress, the leaves of seedlings were sampled at different exposure periods of 6, 24, and 72 h. The collected samples were immediately placed in liquid nitrogen, and then, were transferred to a −80 °C freezer for other analyses.Seeds of TtAQP genes, including TtAQP18, TtAQP29, TtAQP34, TtAQP79TtAQP58, and TtAQP42, were selected based on the phylogenetic result. The primers of the selected TtAQP genes were designed based on the coding region (CDS) using Primer3 online software [Actin7 was used as a reference gene  Master Mix  based on the manufacturer’s protocol and ABI Step One. The cycling patterns of qPCR were 95 °C for 10 min, then 35 cycles at 95 °C for 15 s, and 60 °C for 60 s. The relative expression of each gene was calculated using the delta delta Ct method [An RNX plus kit  was used for RNA extraction based on the manufacturer’s instructions. The quality and quantity of the extracted RNA were checked using a NanoPhotometer . Complementary DNA (cDNA) was synthesized by reverse transcriptase  based on the manufacturer’s protocols. The expression levels of t method . The expTtAQP genes were identified in the genome of T. turgidum. Detailed information on the 80 TtAQPs is provided in A. thaliana, H. vulgare, O. sativa, and Z. mays based on their physicochemical properties  and 392 (TtAQP27) amino acids, and their pI ranged between 4.65 (TtAQP42) and 11.05 (TtAQP09). The exon number of the deduced proteins varied from 1 (most members of the PIP2 subfamily) to 6 and their MWs ranged from 16.7 (TtAQP42) to 41.5 (TtAQP27) kDa. In addition, the GRAVY value of TtAQPs varied between −0.46 and 0.94, and 96% of the deduced proteins were predicted as stable proteins , 35 Arabidopsis AQPs (AtAQPs), 39 rice AQPs (OsAQPs), and 44 maize AQPs (ZmAQPs), were used to design a phylogenetic tree. According to the phylogenetic tree, all AQPs were classified into four main groups . In terms of the gene distribution on chromosomes, Chr6B and Chr7B had the highest distribution and frequency, with eight TtAQP genes on each. After these two chromosomes, Chr2A, Chr2B, Chr5A, Chr6A, and Chr7A were more abundant, with seven TtAQP genes on each. In addition, Chr1A and Chr1B, each with two genes, included the lowest number of genes. Finally, a TtAQP gene was located in unknown chromosomal positions. These results determined that TtAQPs are not uniformly distributed on T. turgidum chromosomes, and probably during the evolution and polyploidy processes, these genes increased randomly. Additionally, the investigation of the duplication process between TtAQP genes showed that many duplications occurred between members in the TtAQP family during evolution , and the highest expression levels were observed after 72h of salt stress. Overall, the expression patterns of the six selected TtAQP genes in durum wheat showed that TtAQPs are mostly involved in the group with late responses to salinity stress.The expression pattern of selected genes showed that t stress . Most ofTtAQP genes was screened to identify the putative cis-regulatory elements. The results disclose that important key regions involved in response to biotic and abiotic stresses, as well as phytohormones, are located in the upstream region of TtAQP genes (TtAQP genes (TtAQP genes. In addition, MRE elements, which are present in the MYB binding site and play a role in response to light, had the lowest frequency in the promoter site of TtAQP genes. Following that, the WUN-motif, which is an element involved in the response to abiotic stresses, and AuxRR-core, which is involved in the response to auxin, had the lowest frequency in the promoter site of TtAQP genes. In general, the identified regulatory elements can be classified into four groups, including hormone-responsive, stress, light, and growth elements. The highest of regulatory elements upstream of TtAQPs was predicted in the field of stress and hormone-related functions. The presence of these important regulatory elements in the promoter sites indicates that TtAQP genes are involved in the response of plants to stress conditions. Overall, TtAQP genes showed high potential to participate in various cellular processes, so it is recommended that focus be placed on this gene family in molecular works related to durum wheat breeding.The promoter region of QP genes . MoreoveQP genes . Then, CT. aestivum using the STRING database (The interaction network of TtAQPs was constructed based on their orthologues in database a. Accorddatabase a. Moreovdatabase b. The ceT. turgidum, so far. In the present study, 80 putative TtAQP genes were identified and characterized in the genome of T. turgidum, as in the first report. Based on previous studies, the number of AQP family members has been variable. For example, 32 AQPs were identified in Physic nut [Arabidopsis [Brassica oleracea [AQPs was higher in T. turgidum; there is a hypothesis that this gene family is more affected by polyploidy and duplication events under the evolution process [T. turgidum underwent an allopolyploid event, which led to extension of the homolog genes in the wheat genome. Moreover, according to the Ka/Ks index, the duplicated members of TtAQPs have been under negative selection; as a result, the functional diversity among TtAQPs has decreased. The exon/intron number was varied between TtAQP subfamilies, suggesting that each subfamily has had a different evolutionary process and each AQP subfamily was created before the derivation of monocotyledonous and dicotyledonous plants [The role of AQPs in regulating cell homeostasis by controlling the flow of water and some ions in the cell membrane is well known . The funysic nut , 35 in Abidopsis , 41 in pbidopsis , 26 in bbidopsis , 45 in cbidopsis , 71 in cbidopsis , 67 in Boleracea , and 51 oleracea . The num process ,51. Addis plants . Furthers plants ,54. It sPost-translational modifications tightly regulate the activity of aquaporin . In the TtAQP genes showed diverse expression patterns in response to salinity. In 150 mM NaCl conditions, candidate genes from the PIP2 subfamily showed downregulation, unlike the other selected genes from the PIP1, TIP, and SIP subfamilies. However, with increasing salinity concentration and duration of treatment, all TtAQPs were induced, and they showed the highest expression levels. Our results disclose that TtAQPs are a part of durum wheat responses to salt stress. The TIP and PIP subfamilies, which are more studied than others [PIP genes in durum wheat, TdPIP1;1 and TdPIP2;1, were identified as being involved in the response to abiotic stresses [PIP1;1 and TdPIP2;1 could improve drought and salt tolerance in durum wheat [TtAQPs are involved in the group of durum wheat with late responses to salinity stress.The candidate n others ,17. Addistresses . In addium wheat . We specTtAQP genes from the genome of T. turgidum were characterized. The results disclosed that subfamilies of TtAQP, including PIP1, PIP2, TIP, NIP, and SIP, have high genetic distances relative to each other. Furthermore, it was found that segmental duplications have played a major role in the extension of the TtAQP family. In addition, we predicted that PIP2 subfamily members have more potential to be influenced by phosphorylation modification and they are probably involved in signaling pathways related to kinases. According to their expression profiles, we conclude that TtAQPs are associated with late responses to salt stress. Overall, we conclude that TtAQPs are diverse proteins, based on their structure, regulatory systems, and expression. The results of this research can be used in further studies related to salt tolerance in durum wheat.In the present study, 80 putative


Histoplasma capsulatum is a geographically specific dimorphic fungus that can cause a spectrum of diseases. While most cases are asymptomatic pulmonary infections, in severe cases, particularly in immunocompromised patients, disseminated disease can occur. Histoplasmosis in California is limited to only a few case reports. In this article, we describe a rare case of disseminated histoplasmosis in a non-endemic region presenting with diagnostically challenging symptomatology, including altered mental status, status epilepticus, septic shock, and bilateral adrenal masses. Histoplasma capsulatum is a geographically specific dimorphic fungus that causes histoplasmosis (HP). In America, this fungus is most prevalent in the Eastern United States, specifically in the Ohio and Mississippi River Valleys.1 Rarely have cases and outbreaks been reported in non-endemic areas. Although California is highly endemic for another dimorphic fungus, Coccidioides, which causes coccidioidomycosis, HP is rarely reported. Three cases were reported in San Diego in 1966,2 2 cats in Central California were diagnosed with HP in 2004,3 and 1 case of disseminated HP in an AIDS patient was reported in southern California in 2019.45 Similarly, in 2008, another outbreak was reported after a group traveled to El Salvador to renovate a church.6 In 2013, another outbreak was associated with the renovation of an old house in Quebec, Canada.7 In this article, we aim to highlight the importance of retaining HP in the differential diagnosis while working up patients with similar presentations even in non-endemic states as we describe another case of HP in a patient from California whose only known risk factor was occupational exposure from years prior and military service in Germany when he was 18 years and in Texas when he was 21 years old. This case report highlights the importance of obtaining a thorough social history, including previous residence, travel, and occupational exposures.Nonetheless, there has been an increasing concern of occupational exposures to HP. In 2001, a large outbreak of HP occurred after a group of students traveled to the non-endemic region of Mexico where a hotel under construction was determined to be the common risk factor for the outbreak.Histoplasma capsulatum in a patient presenting with altered mental status and bilateral adrenal masses in a non-endemic region.To present a rare case of a Case report and literature search.A 58-year-old Caucasian male with a medical history of hypertension, remote history of coccidioidomycosis, and tobacco use presented with acute-onset altered mental status and 70-pound unintentional weight loss over the previous 6 months. The patient was found to be in status epilepticus refractory to anti-epileptic drugs secondary to traumatic brain injury (TBI) from multiple ground-level falls, and magnetic resonance imaging (MRI) of the brain demonstrated bifrontal hemorrhagic contusions (larger on the left), scattered subarachnoid hemorrhages without demonstrable mass effect or midline shift, and few scattered tiny hypointense areas on gradient echo . Subsequ3 8 mmol/L, and a corrected anion gap of 22 mmol/L, acute renal failure with a creatinine of 14.8 mg/dL, and hyperkalemia of 6.6 mmol/L  without contrast of the abdomen and pelvis, which revealed hepatomegaly with a craniocaudal length of 21 cm, splenomegaly with a length of 18.2 cm, bilateral adrenal masses measuring 4 cm each, and enlarged pericaval lymph nodes with a length of 2.3 cm × 1.7 cm × 4 cm  and 4. A9/L. Splenomegaly was seen on CT scan, raising the specter of possible hemophagocytic lymphohistiocytosis (HLH) in the setting of potential malignancy or possible infection.Despite empiric antibiotics throughout the hospital course, the patient continued to maintain low-grade fevers. Initial laboratory values noted to have an elevated ferritin 1186 µg/L, and the patient remained transfusion dependent due to pancytopenias with hemoglobin 7.9 g/dL and platelets 66 × 102O, white blood cell (WBC) count of 4 cells/µL, red blood cell (RBC) count of 55 cells/µL, glucose level of 55 mg/dL, and protein level of 66 mg/dL.All cultures, including fungal cultures, from cerebrospinal fluid (CSF), blood, and urine had no growth. Laboratory results were negative for HIV, hepatitis B and C, syphilis, and QuantiFERON-TB Gold. Given the patient’s history of coccidioidomycosis infection living in an endemic area, serology was ordered and showed immunodiffusion IgM weakly reactive and immunodiffusion IgG non-reactive, with complement fixation <1:2. A lumbar puncture was performed, which ruled out meningitis with an opening pressure of 19 cm HFurther collateral information was obtained from the patient’s family who reported that the patient had a productive cough and a 2-week history of dyspnea, suffered multiple ground-level falls, and had poor oral intake. The patient was working as an independent handyman and school maintenance worker. Much of his job entailed working with plumbing in attics, basements, underneath sinks, and sewer lines. He denied exploring caves or being involved with birds/bats. His travel history included military service: he was stationed in Germany when he was 18 years old and in Texas when he was 21 years old.Due to suspicion of possible malignancy with pancytopenia, significant weight loss, and hepatosplenomegaly, a CT-guided bone marrow biopsy was performed. Although it was suboptimal due to a lack of adequate core, the biopsy revealed a normocellular sample with unremarkable trilineage hematopoiesis; no evidence of hemophagocytosis, lymphoid aggregates, or metastatic malignancy; and no evidence of fungal elements by Grocott’s methenamine silver (GMS) stain.Subsequently, a left adrenal biopsy was performed and stained with hematoxylin and eosin, demonstrating small, round, shadowlike microorganisms that turned dark black when stained with GMS—consistent with HP . SerologThe patient was started on liposomal amphotericin B therapy for disseminated HP with improvement of mental and functional status, participating with physical therapy and was transferred to the Veterans Affairs hospital in the region to complete his treatment course.Histoplasma spores from soils contaminated by bird and bat droppings.8 These spores settle in the alveolar spaces, and once in the human body, the fungus transforms into its yeast form.9 The immune system responds with alveolar macrophage phagocytosis of the yeast. It is within these WBCs that the fungus begins to replicate, as these macrophages travel throughout the human body via the reticuloendothelial system.9 This mechanism for fungal dissemination allows for infection of various organ systems, the most common of which is the adrenal glands.11 This progressive dissemination occurs when the immune system is unable to formulate a sufficient T-cell-mediated response to clear the yeast from the human body Patients are often unaware of contracting the fungus, given that the majority of cases are asymptomatic.8 Nonetheless, symptomatic HP has 3 forms of presentation: acute primary HP, chronic cavitary HP, and progressive disseminated HP.12 Disseminated HP may present years after the initial infection, and it may be the primary presentation if the initial infection was asymptomatic.13 Although rare, the risk for disseminated HP is more probable in immunocompromised patients.9HP infection typically results from inhalation of 9/L, ferritin 1186 µg/L, and detected soluble IL-2 receptor α (CD25) 23 518 pg/mL, thus confirming the diagnosis of secondary HLH.Based on the HLH-2004 criteria, to make the diagnosis of HLH requires 5 out of 8 of the following: fever, splenomegaly, peripheral cytopenias in at least 2 lines, high triglycerides or low fibrinogen, ferritin levels over 500 µg/L, high-soluble interleukin (IL)-2 receptor (CD25), and no natural killer cell activity. Our patient met 5 of these criteria with fevers, splenomegaly seen on CT, cytopenias with hemoglobin 7.9 g/dL and platelets 66 × 10In the current literature, there has been approximately 26 cases of secondary HLH in the setting of disseminated HP, and within most of these cases, the patients were noted to be immunocompromised. HLH is caused by abnormal activation of macrophages and lymphocytes, which results in inflammation, tissue damage, and is life threatening. HLH is typically seen in infants; however, it can be diagnosed in adults as old as 70 years of age, with a higher predisposition seen in males, such as our patient. Successful treatment of HLH is often thwarted due to a delay in diagnosis. These delays can be caused by varied clinical presentations and overall rarity of the disease in the concomitant setting of another disease.The diagnosis of HP in non-endemic regions is often delayed and difficult to make. This case report highlights the importance of obtaining a thorough social history, including previous residence, travel, and occupational exposures. In addition, findings such as bilateral adrenal lesions in the adult patient should raise suspicion to broaden differential diagnosis to include atypical infectious etiology. The patient’s current residence in a geographical location highly endemic to coccidioidomycosis, which is caused by another dimorphic fungus, complicated the HP diagnosis due to similarities between the 2 fungal infections. Furthermore, the patients’ symptoms of altered mental status and associated weight loss are common to disseminated coccidioidal infection, which is endemic to the California San Joaquin Valley where our patient was born and resided in for a majority of his life. He worked as handyman and school maintenance worker, often immersed in confined humid spaces: attics, sinks, water pipes, and sewer lines. Additional differential diagnoses also included Whipple’s disease due to extensive and prolonged exposure to sewage and weight loss; however, there was no diarrhea or joint involvement.The clinicians reasoned that the sudden 70-pound weight loss was caused by poor oral intake which also led to hypotension and episodes of hypoglycemia with subsequent ground-level falls. Resultant TBI caused structural abnormalities seen on MRI of the brain . TBI sup15 Most cases of HP diagnosed in California are attributed to fungal exposure outside of the state, often through previous residence or travel, similar to this case.16 Both are contracted through the inhalation of fungal spores.17 The gold standard workup for diagnosis of disseminated HP to adrenal glands is ultrasound-guided fine needle aspiration and cytology (USG-FNAC). This usually follows a discovered localized suspicious focal area, best revealed by CT. Disseminated HP to the adrenals may be found as enlargement of the adrenals with central hypodensity, suggesting gland destruction along with enhancement of the internal septations and peripheral rim.13 Microscopic examination of the aspirate may reveal caseous granuloma and fungal elements, including hyaline granules and intracellular or extracellular single buds with narrow bases.18 The growth of Histoplasma is confirmatory. For our patient, abdominal and pelvic CT revealed hepatosplenomegaly, enlarged pericaval lymph nodes, and bilateral adrenal masses. USG-FNAC of the left adrenal demonstrated small, round, shadowlike microorganisms.On further investigation, it was found that his military service led him to travel to Germany for 2 years when he was 18 years old and then to Fort Hood in Texas for another year after that. HP has been described in both geographical regions.19 Management requires up to 12 months of pharmacological treatment with monitoring of adrenal function. Our patient was initiated on liposomal amphotericin B, given his severe presentation, and showed improvement with plans of transitioning to itraconazole in the near future.Infectious Disease Society of America (IDSA) recommends treating moderate to severe cases of HP with liposomal amphotericin B with the transition to itraconazole. In mild cases, treatment can be achieved with itraconazole alone.Diagnosing HP in individuals in non-endemic regions is challenging and mostly delayed. The exposure history of the endemic area is usually found after the diagnosis is already made by other means such as histopathology, serology, or cultures. Clinicians must obtain a thorough social history when reactivation of dormant infections is suspected.


Nutcracker syndrome (NCS) is a rare pediatric disease caused by left kidney vein compression. Besides the “Triade’s symptoms”, including hematuria, proteinuria, and flank pain, a wide spectrum of clinical manifestations has been reported. As the significant hemodynamic changes secondary to the dilatation of the left renal vein, serious consequences such as renal vein thrombosis and severe anemia might occur in these children. NCS diagnosis includes a variety of invasive and non-invasive imaging tools, but cutoff values need to be further validated. A conservative treatment represents the most common therapeutic approach for these patients, but operative options are available in selected cases. To complicate matters, a standard diagnostic and treatment algorithm is currently lacking and scientific pediatric evidence in this field is still poor and limited. In this perspective, early recognition of NCS is crucial but challenging for pediatricians. Therefore, a better knowledge of the disease is recommended. Starting from two different clinical presentations of NCS, we aimed to provide a comprehensive overview of the disease in children. Nutcracker syndrome (NCS) represents a rare disease in childhood ,2 defineWhile NCS has been largely characterized in adults ,4, pediaIndeed, the wide variability of NCS in clinical presentation in children (ranging from hematuria and proteinuria to hypertension and nephrolithiasis) has made difficult a comprehensive overview in this field .In an attempt to fill this gap, we aimed to provide a pediatric landscape of diagnostic work-up and treatment of NCS in children through the initial presentation of two different clinical cases.2 (10th percentile), and blood pressure levels were normal. Serum creatinine and albumin levels were within the normal range . At our observation, the presence of this proteinuria degree at 24 h urine collection and at UPr/UCr on spot urine collected during the day was confirmed. Evaluating the urine dipstick and UPr/UCr on urine sample collected in supine position, the absence of proteinuria with UPr/Cr 0.17 mg/mg was found. These data were confirmed 4 months later.A 10-year-old girl came to our attention for occasional finding of proteinuria at urine dipstick. She presented with a three-month history of isolated proteinuria between 350 and 400 mg/24 h  and with a spot urine protein/creatinine ratio (UPr/UCr) between 0.7 and 0.9 mg/mg . Symptoms as edema, gross hematuria, urinary tract infections, rashes, fatigue, joint pain, and drug use  were denied. Clinical examination was unremarkable. Her weight was 31 kg (10–25th percentile), height 145 cm (50–75th percentile), Body Mass Index (BMI) 14.8 kg/mAs orthostatic proteinuria might be attributable to NCS, the patient underwent kidney ultrasound with Doppler sonography . At rena2 (25th percentile), and normal blood pressure levels. Physical examination was regular. Serum creatinine and complement proteins C3 and C4 were within the normal range. In the past, he sometimes complained of discomfort in both flanks . His dipstick showed Hb+++ with 10 red blood cells  for high power field at urine microscopy. Proteinuria was absent both at 24 h urine collection (109 mg/24 h) and at UPr/Cr on spot urine (UPr/UCr 0.18 mg/mg). During the following 6 months, persistent micro-hematuria at urine dipstick (Hb++/+++) without proteinuria was detected. Therefore, a renal ultrasound with Doppler sonography was performed. Renal ultrasound was normal. At Doppler ultrasound, a reduction in the caliber of the left renal vein at the level of aorto-mesenteric compass (about 2 mm) with caliber of the upstream segment of about 7.5 mm was observed. In comparison, the caliber of the right renal vein was 6 mm accomplishing the diagnosis of NCS. Conservative management was chosen as the patient had minimal symptoms. He was scheduled for follow up visits every three to six months to evaluate blood pressure, complete blood count, and kidney function tests.A 13-year-old boy presented with 5 days of isolated macrohematuria during Epstein–Barr virus infection. His medical history was not contributive. A previous urine dipstick (at 10 years of age) was negative. At our observation, his anthropometric evaluation was as follows: weight 43.3 kg (10–25th percentile), height 160 cm (50th percentile), BMI 16.9 kg/mClinical presentation of NCS in children might widely vary as the hemodynamic consequences of anatomic changes . In factAlthough NCS might occur in patients of any age, a peak has been found in lean or underweight children aged 10–14 years, with a slight female predominance [From a clinical point of view, two different NCS subtypes such as typical  presentation and atypical (or urologic) presentation have been described ,2. TypicDue to the entrapment of LRV, orthostatic proteinuria (with or without microscopic hematuria) represents one of the most common symptoms in children with NCS with an estimated prevalence up to 75% ,16. In aAs the lack of robust evidence in this field, the mechanism of orthostatic proteinuria is still not fully elucidated. Changes of renal hemodynamic and elevated norepinephrine and angiotensin II levels have been supposed as potential pathogenic factors .Hematuria (both macro- and microhematuria) represents another frequent clinical finding of NCS with an estimated prevalence up to 76% . It is dAtypical left flank pain occurs in one-third of pediatric patients with NCS and is usually explained as referred visceral pain secondary to LRV dilation ,14. PartAn atypical diffuse abdominal pain encountered in 10% of the pediatric cases as a consequence of pelvic venous congestion ,11,14,15Hypertension does not represent a classic sign of NCS and only a few reports have described NCS accompanied by hypertension in children. In case of secondary hypertension not linkable to other factors and in presence of a history of hypertension unresponsive to antihypertensive drugs, NCS should be considered as a potential cause. The underlying mechanism is complex and could be explained by an increased plasma renin activity and aldosterone levels in the peripheral blood despite the absence of renal artery stenosis or a renin-producing tumor .Among uncommon NCS features, varicocele affects a small part of patients and usually occurs on the left side . Its devMoreover, chronic fatigue syndrome has been occasionally associated with NCS with high LRV-inferior vena cava pressure gradients . SymptomThe angle between the aorta and SMA is commonly between 38° and 65° ,2, and sThe clinical variability often might overlap with other clinical entities , and the lack of standard diagnostic criteria further complicates the diagnostic work-up of NCS . Of noteAlthough a dilatation of LRV without clinical manifestations is frequently found in radiological images ,21, the When a thorough history and physical examination suggest NCS , urine aIn this framework, extensive diagnostic procedures are required to confirm the diagnosis and the initial noninvasive work-up includes a Doppler ultrasound, computed tomography (CT) , or a magnetic resonance imaging (MRI) using angiography sequences ,27,28. TFirst-line diagnostic options include abdominal ultrasound and doppler studies with a documented sensitivity and specificity of 82.3% and 89–100%, respectively [For Doppler US, the application of ratio criterion can be challenging ,33. In fIn selected and rare cases, invasive assessment might require a measurement of the pressure gradient between the LRV and inferior vena cava (IVC) by catheterization . In the In addition to non-invasive tests  ,22,39, pAs the lack of diagnostic consensus on Pediatric NCS, its management is still controversial. Commonly, the best approach to be used for each case should be determined by the severity of symptoms, the age of the patient, and the stage of the syndrome ,2. In paIn this age group, during a wait and see monitoring of 24 months, instead of 6 months as for adults, a spontaneous and complete remission of symptoms has been demonstrated in 75% of cases ,40. SeveIn case of persistent orthostatic proteinuria, a treatment with angiotensin converting enzyme inhibitors (ACE-i) has been proposed ,43 with Remarkably, rare and selected cases presented with severe abdominal or (left) flank pain, recurrent gross hematuria, renal dysfunction, left varicocele, anemia, and persistent symptoms (after 24 months of conservative treatment) might require a more complex and invasive management such as surgical and endovascular techniques ,44. LRV Despite possible complications such as thrombosis, restenosis, retroperitoneal hematoma, and paralytic ileus, this technique has been associated with a long-term resolution of symptoms that exceed the aforementioned related risks ,39,42,44Left renal autotransplantation might represent an additional invasive option for LRV pressure levels normalization  but furtOther possible techniques for NCS operative treatment include SMA transposition, nephropexy, nephrectomy, renocaval bypass, left gonadal vein transposition, or laparoscopic procedures  ,29,44. AIn view of the current data from the literature ,2,25, a As the delicate phase of childhood, other treatment options should be considered in selected cases after a careful risk-benefit evaluation to make the best therapeutic choice.Owing to its limited knowledge in children, NCS represents a less-defined and often underdiagnosed disease with a large spectrum of potential symptoms. In this framework, both our cases confirmed and corroborated the wide clinical phenotypical variability  that might result in an easily missed diagnosis. NCS represents an uncommon cause of hematuria and proteinuria in children, but both features are the most common presentations of this syndrome. Therefore, a high index of suspicion and awareness of its clinical variability are required for pediatric nephrologists to avoid misdiagnosis or late diagnosis.To complicate matters, the available data from the literature in this field arise from small pediatric case series, further preventing a large and validated knowledge of the disease. More, the lack of standard clinical criteria for NCS undermines the correct timing of the diagnosis.Given the potential serious complications including renal vein thrombosis and severe anemia secondary to hematuria, increasing knowledge of NCS is crucial for its early identification. Although a conservative approach has been recognized as a first-line option, surgery might be a helpful approach in unsuccessful cases and persisting and/or severe symptoms, but several aspects limit its use in clinical practice.Taken together, NCS diagnosis and treatment represent a great challenge for pediatricians. Therefore, a deeper understanding of the disease is needed to achieve an accurate and in time diagnosis and to improve the overall NCS management in children.


Monascus purpureus (Went NRRL 1992). The response surface methodology was used to optimize total pigment and fungal biomass production. The influence of potato powder waste concentration, fermentation period, and peptone concentration on total pigment and biomass production was investigated using the Box-Behnken design method with 3-factors and 3-levels. The optimal production parameters were potato powder waste concentration of 7.81%, fermentation period of 12.82 days, and peptone concentration of 2.87%, which produced a maximum total pigment of 29.86 AU/ml that include, respectively, a maximum biomass weight of 0.126 g/ml and the yield of pigment of 236.98 AU/g biomass. The pigments produced were used as coloring agents for ice lolly. This study has revealed that the ice lolly preparations supplemented with these pigments received high acceptability. Finally, we recommend using wastewater and potato powder wastes for pigment and biomass production, which could reduce the cost of the pigment production process on an industrial scale in the future.During potato chips manufacturing, large amounts of wastewater and potato powder wastes are produced. The wastewater obtained at washing after cutting the peeled potatoes into slices was analyzed, and a large quantity of organic compounds and minerals such as starch (1.69%), protein (1.5%), total carbohydrate (4.94%), reducing sugar (0.01%), ash (0.14%), crude fat (0.11%), Ca (28 mg/L), Mg (245 mg/L), Fe (45.5 mg/L), and Zn (6.5 mg/L) were recorded; these wastes could be considered as valuable by-products if used as a fermentation medium to increase the value of the subsequent products and to exceed the cost of reprocessing. In this study, we used wastewater and potato powder wastes as a growth medium for pigment and biomass production by  Horticulture production has significantly increased to 23.7 million tons of food per day during the last 50 years. Some portion of biomass from agriculture products generates waste, which may not be used as food  and soaking was obtained from a potato chips factory at Assuit Governorate, Egypt.Second, potato chips waste powder produced after settling wastewater and then dried under sunlight was packed in polyethylene pages at 4°C until use.The contents of crude protein, crude fat, ash, reducing sugar, and carbohydrate as a percentage (W/V) were determined using standard methods of the Association of Official Analytical Chemists AOAC, .The mineral contents of potato chips manufacturing wastewater were determined by Perkin-Elmer Atomic Absorption Spectrophotometer 2,380 for calcium, iron, zinc, and magnesium. The determination was carried out in the Central Laboratory, Faculty of Agriculture, Assiut University, as described in AOAC .M. purpureus Went NRRL 1992 obtained from Microbiological Resources Center (MIRCEN), Ain Shams University Cairo, Egypt. It was maintained on a Yeast Extract–Peptone–Dextrose (YEPD) agar medium  at 4°C and subcultured periodically every 3 weeks. This strain was tested for its ability to produce citrinin as mycotoxin and found to be nonproducing  agar slope culture, 10 ml of sterile distilled water was added and the spores were scraped under strict aseptic conditions. The spores suspension obtained was used as inoculum (~7 × 105 spores per ml) as described by Babitha et al.  provided by Design Expert Software (2011) with a standard tool known as Box-Behnken design (BBD)  at 400, 470, and 500 nm for yellow, orange, and red pigments, respectively  to a 50-ml Erlenmeyer flask and 10 ml of ethanol 95% was added. The suspension was allowed to stand at 30°C for 12 h , which was adjusted to pH 3.0 with 2 N HCl as shown in C for 30 min to precipitate yellow pigment. The acetone layer containing red pigment was collected, filtered, and concentrated further, and then taken in 20 ml of water. The yellow crystals, which settled at the bottom of the flask, were dissolved in 20 ml of water and kept in a glass vial.In addition, the yellow pigment can be separated from the ethyl acetate extract by evaporating the extract in vacuo at 50°C. The semidry residue was re-dissolved in a small amount of water by adjusting the pH to 7.0 with 2 N NaOH. Then 50 ml of cold acetone was added and kept at 4C, and then taken in 20 ml of water separated water-soluble red, orange, and yellow pigments were used separately to prepare red, orange, and yellow ice lolly.The orange pigment, which eluted from the separation funnel, was collected, filtered, and concentrated further by evaporated in vacuo at 50The traditional methods manufactured ice lolly, and red, orange, and yellow pigments were added to the ingredients and then flavored with strawberry, orange, and banana, respectively. For ice lollies manufacturing, mix was standardized to sugar (20 %), stabilizer (0.5%), citric acid (0.3%), color (0.05%), flavor (0.45%), and water. Selecting ingredients, figuring the mix, blending, pasteurizing (68°C/30 min), transferring into molds, freezing (−18°C), and packing/storing until subjected to sensory evaluation  according to the method described by Reitmeier and Nonnecke . AttribuThe wastewater used for experimental work was collected from the potato chips industry. The wastewater was obtained after the cutting (slicing) and soaking process, so this water contains a low microbial charge. The results obtained from the chemical analysis of the wastewater are presented in The results show that the wastewater of cutting (slicing) and soaking from the potato chips manufacturing process contains a high amount of calcium and magnesium minerals and organic compounds of crude protein, starch, and carbohydrate. It also has a low content of crude fat, ash, reducing sugar, and zinc and iron minerals. As a result, these wastes could be considered valuable by-products if used as a fermentation medium to increase the value of the subsequent products and exceed the cost of reprocessing.This study results are rather different from the study described by Gautam et al.  who repoM. purpureus Went NRRL were listed in The results of BBD experimental design include 17 runs of 3 independent variables  for optimizing pigment production and biomass (dependent variables) by Second-order quadratic model equation (Equation 1) (coded units) was used to express total pigment and biomass production as the following:Pigment is the total pigment concentration, YBiomass is the biomass weight g/ml, (A) potato wastes %, (B) fermentation period days, and (C) peptone concentration %.Y2 = 0.987), indicating that the predicted and actual values were in reasonable agreement. The correlation coefficient (R2) is a tool to check the “goodness of fit” between the experimental and the predicted results  and low p-value (< 0.05). From p < 0.05) for total pigment production. In addition, the value of the quadratic potato wastes concentration, incubation period, peptone concentration, and the interaction between potato wastes concentration and peptone concentration also showed a significant effect on the production of total pigment. Non-significant lack of fit means that the model is adequate to describe the observed data.According to ANOVA , the modR2, R2adj, and Adeq precision were 0.972, 0.937, and 21.59, respectively. The normal probability plot indicated the quality of the adequacy of the current model where all residual points were on a straight line . The effect of potato wastes concentration and fermentation period was significant. Also, the interactions between potato wastes concentration and fermentation period and potato wastes concentration and peptone concentration were significant. The quadric effect of potato wastes concentration and fermentation period was significant.Based on ANOVA, which is listed in Monascus growth and pigment production  concentrations. Raman et al. . The incubation period in this study was shorter than that of which reported by Dikshit and Tallapragada .As indicated in M. purpureus NRRL 1992 pigments for coloring prepared food products has proved to be excellent. The pigment is distributed evenly in the food product giving a pleasing appearance.The average scores for taste, color, texture, odor, and overall acceptability were between 8.8 and 9.9 scored as “like extremely” for all tested food products samples as described by Wang and Zhao . The resM. purpureus pigments were used  was employed as a colorant in the manufacturing of food items (Kesari) and had a very nice color and look. They also investigated the use of MFR for coloring flavored milk, which revealed that pigment dispersed uniformly, resulting in an attractive hue and acceptable look with higher acceptance.Vidyalakshmi et al.  reportedM. purpureus Went NRRL 1992. The BoxBehnken (BBD) design was influential in modeling total pigment production as a function of changes in process variables, potato waste concentration, fermentation period, and concentration of peptones. The optimum conditions were potato wastes concentration of 7.81%, fermentation period of 12.82 days, and peptone concentration of 2.87%, which produce total pigments of 29.86 AU/ml. The produced pigments were applied as coloring agents for ice lolly . Applications of these natural colorants in the manufacturing of these products were judged to be extremely acceptable based on sensory assessment.The utilization of potato waste powder is beneficial in manufacturing pigment from The original contributions presented in the study are included in the article/HA-R and MA: investigation, draft writing, and software and methodology. SG and AH: writing and reviewing. SA, MH and DM: reviewing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.