Whether this HLA allele plays a part in susceptibility to pain, has not, as far as we are aware, been previously investigated. This study utilizes information on DQB1*03:02 alleles as part of the EIMS, GEMS, and IMSE studies in Sweden. It also uses register data for 3,877 MS patients, and 4,548 matched comparators without MS, to assess whether the DQB1*03:02 allele is associated with prescribed pain medication use, and whether associations with this genotype differ depending on MS status. Our results showed no association between the DQB1*03:02 genotype and pain medication in MS patients, with an adjusted odds ratio (OR) of 1.02 (95% CI 0.85–1.24). In contrast, there was a statistically significant association of low magnitude in individuals without MS [adjusted OR 1.18 (95% CI 1.03–1.35)], which provides support for HLA influence on susceptibility to pain in the general population. Additionally, the effect of zygosity was evident for the non-MS cohort, but not among MS patients, suggesting the DQB1*03:02 allele effect is modified by the presence of MS.Murine models have demonstrated that the major histocompatibility complex (MHC) is associated with pain-like behavior in peripheral nerve injury, however, the same association has not been shown when considering injury to the central nervous system (CNS), which more closely mimics the damage to the CNS experienced by MS patients. Previous research has indicated the DQB1 Individuals with MS experience a wide variety of symptoms, one of which is pain. Recently, we reported that MS patients are affected by pain, in particular neuropathic pain, to a greater extent than the general population through considering prescribed pain medications as a proxy for a pain diagnosis . The neuI allele . MoreoveI allele , 5. The elopment –8. In hurniation . The DQBrniation . Pain isrniation  which ar*03:02 had an increased risk of pain and if such an effect would be different in MS patients and those without MS. Both general pain and neuropathic pain can be identified using prescriptions of pain medication as a proxy, which can identify pain with a reasonable degree of accuracy, , , 13 GEMSRegisters include information on inpatient and outpatient diagnoses through the National Patient Register (NPR), and prescription dispensations from the Prescribed Drugs Register (PDR). The PDR only contains information on prescriptions, there is no information on over the counter (OTC) purchases. Additionally, demographic information and data on socioeconomic circumstances are available using the Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA) maintained by Statistics Sweden. Datasets were merged using the unique personal identification number issued to all Swedish residents at birth or immigration.p < 0.0001) were removed from analysis. After quality control, 94,607 SNPs were included in the dataset. Individuals with > 2% failed genotype calls, with increased heterozygosity (> mean + 2 standard deviations), related individuals (increased identity by descent), or individuals where the recorded sex differed from genotype were removed from analysis. Population outliers identified using the SmartPCA program were removed. A principal component analysis (PCA) was conducted using Eigensoft  were determined using a previously described Illumina custom array, the MS Replication Chip  . SNPs wiigensoft , 17 and , and highest educational attainment .Logistic regression, with record of prescribed medication for pain  as the dependent variable was used. Possession of the DQB1n = 166) and depression (n = 575), respectively, were conducted to ensure results did not significantly alter whether or not these patient groups were included. For the main analysis, these patients were retained because it is possible the treatment was given for a dual use. Only prescribed pain medication was included in the outcome, as it is not possible to identify OTC purchases using the registers.Given that pregabalin and gabapentin are commonly prescribed for the treatment of epilepsy, and amitriptyline and nortriptyline are often used in the treatment of depression, two sensitivity analyses which excluded patients with a diagnosis of epilepsy . Analysis was also stratified by matching factors to assess the possibility of effect modification.SAS version 9 and Stata version 13 were used for analysis.*03:02 (2% compared with 0.8% of the MS cohort). The proportion within each cohort with one allele was comparable for MS and non-MS, with 23 and 24%, respectively, possessing at least one allele.The distribution of characteristics including the matching factors and highest educational attainment were fairly evenly spread between the two cohorts, with a slightly higher percentage of the non-MS cohort achieving further/higher education relative to the MS cohort . After adjustment, the magnitude of the difference between those with and without at least one DQB1*03:02 allele was larger for women than for men. The sensitivity analyses which excluded firstly those diagnosed with depression, and subsequently individuals diagnosed with epilepsy, showed no significant differences to the main results.Overall, the increased risk of pain medication use for those with one or more DQB1*03:02 genotype appeared to be associated with a greater risk of pain medication use among the non-MS cohort, but not among MS patients. In particular, the presence of the DQB1*03:02 genotype increased the strength of the association with pain medication use relative to those without the genotype among non-MS women, with an odds ratio of 1.21 (1.03–1.41). A zygosity effect was evident in the non-MS cohort, with a particularly high magnitude evident for women without MS, jumping from an OR of 1.16 (0.99–1.36) for those heterozygous for the allele relative to those with no DQB1*03:02 gene, up to 2.26 (1.25–4.06) for those homozygous for the allele. Such an association was not present for the MS cohort, with no statistically significant associations with pain medication by number of DQB1*03:02 alleles . In addition, MS pain likely stems from lesions in the CNS, and our data suggest that there is no major genetic influence on this MS sub-phenotype, and may mainly be dependent on stochastic factors determining the location of MS lesions.When considering a possible association between risk of pain and DQB1*03:02 genotype on pain medication use for women relative to men amongst the non-MS cohort, suggesting sex acts as an effect modifier. It has been previously documented that women are more likely to experience or report pain than men  meant reduced power to detect associations, which may be why results for this pain phenotype were not statistically significant.Alongside considering pain in a more general sense, this study also investigated specifically neuropathic pain in relation to the presence or absence of the DQB1Interestingly, the findings of this study mirrors several features previously described in experimental models of pain. The susceptibility to develop neuropathic pain-like behavior among inbred and congenic rat strains subjected to a standardized peripheral nerve injury has been shown to be linked to the MHC , 7. ThisThis study had several strengths. The definition of exposure to MS was robust and required attendance at an MS clinic, improving specificity. The ability to identify pain through prescribed pain medications allowed an objective approach to measurement of the outcome rather than, for example, questionnaires being used . The useSome potential limitations to the study should also be considered. MS patients were recruited during their visits to the neurologist, when blood samples used for genetic profiling were collected. Non-MS study recruits had to attend their local health center to deposit their blood samples, which may result in bias to the health profiles of the non-MS group. Individuals with frequent visits to health centers due to health problems may be more likely to participate, due to frequent contact with health services. Conversely, those with mobility problems or poorer health may be less inclined to visit the health center to provide their sample, so bias could be acting in both directions, making it difficult to estimate how results might be affected. The sampling of MS patients is not based on probability sampling, but is rather convenience sampling whereby patients agree to participate and are then recruited into the study during their visits with an MS specialist. This could result in selection bias, and impact on the generalizability of the results, because self-selecting samples are not reflective of the population , 31. In *03:02 genotype and pain in patients with MS, however, the risk of pain medication was increased, albeit modestly, for those with one or more DQB1*03:02 alleles among the non-MS cohort. Furthermore, this finding was strengthened by an association with zygosity among the non-MS cohort, particularly for women. The same pattern was seen with neuropathic pain, however smaller numbers reduced power and makes conclusions difficult due to a lack of significance. Although the HLA complex also includes non-immune genes, a growing body of evidence suggest that immune functions are involved in the development of pain and the findings presented here further strengthen this notion.Overall, our results indicated no association between possessing the DQB1jan.hillert@ki.se).The data that support the findings of this study are available from the Swedish National Board of Health and Welfare , Karolinska Institute (MS register), and Statistics Sweden . The register data supporting these findings can be obtained by applying to the relevant register holders as listed above. The data for EIMS, GEMS, and IMSE can be obtained by applying to the data holders. Enquiries can be sent to Jan Hillert (The studies involving human participants were reviewed and approved by Regional ethics committee of Stockholm. The patients/participants provided their written informed consent to participate in this study.SBu conducted the data analysis and wrote the initial draft of the manuscript. KS and PS worked with SBu on the drafting of the first draft, and were involved in interpretation of the data and provided scientific input. IK was involved in the data application, interpretation of results and provided comments on the manuscript. JH was involved in the data application, interpretation of results and provided comments on the manuscript. HL was involved in the interpretation of results, and provided comments on the manuscript. LA was involved in the data application, interpretation of results and provided comments on the manuscript. TO was involved in the data application, interpretation of results and provided comments on the manuscript. FP was involved in the data application, interpretation of results and provided comments on the manuscript. SM was involved in the data application, interpretation of results and provided comments on the manuscript. SBa was involved in the data application, interpretation of results and provided comments on the manuscript. All authors contributed to the article and approved the submitted version.FP has received unrestricted academic research grants from Biogen, Genzyme, Novartis, and received research grants from Genzyme, Merck KGaA, and Novartis, and fees for serving as Chair of DMC in clinical trials with Parexel. IK has received funding from AstraZeneca, and a speaker honorary from MercSernono. JH has received funding from Biogen, Novartis, Merck-Serono, Bayer-Schering, and Teva and has received speaker honoraria from Sanofi-Aventis. LA has received speaker honoraria from Teva. TO has received funding from AstraZeneca, and speaker and advisory board honoraria from Biogen, Novartis, and Genzyme. SM has received funding from AstraZeneca, and funding for MS related research from F. Hoffman-La Roche AG, and Novartis International AG. The remaining 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.


Background: The aim of this cross-sectional study was to assess the dementia literacy of community-dwelling adults in four cities  of the Greater Bay Area of China, and to determine their mass media preferences for receiving dementia information. Methods: The survey was completed by 787 community-dwelling adults. Dementia literacy was indirectly measured using two validated scales—the 30-item Alzheimer’s Disease Knowledge Scale and the 20-item Dementia Attitude Scale (DAS). Participants were also asked to indicate whether they wanted to receive dementia information via digital or traditional media. Chi-square tests, logistic regressions, and MANOVA analyses were conducted. Results: Unemployed or retired people had poor attitudes towards dementia and lower levels of knowledge about dementia. Single, cohabiting, or divorced people in Hong Kong and Macau had lower DAS scores than married people. Young people and those with a secondary education preferred to get their dementia information from social media. People with a tertiary education and employed people enjoyed searching government or hospital websites for information. Middle-aged, unemployed, or retired people tended to learn about dementia from television or radio. Conclusion: It is worth educating the public about dementia and developing strategies consistent with their preferences for types of mass media. Dementia is a growing health concern in many countries, including China. In 2016, there were 46.8 million people in the world living with dementia, about 25% of them in China [Dementia literacy refers to knowledge of dementia and attitudes towards persons with dementia . With inMass media play an important role in health promotions. There is evidence that the mass media promote healthy behaviors (such as safe sex and the prevention of skin cancer) through descriptive norms . NonetheThis is a multi-city, cross-sectional study with the samples representing their respective ratio of the population in the selected cities/regions. The target was to recruit 200 subjects in each city. A convenience sample was used in this study. The study population consisted of adults aged 18 or above, who were cognitively intact , able to read Chinese, and had lived in their respective city for at least 1 year. Those who were hospitalized or living in a residential care home were excluded. In this study, we only included respondents aged 18–64 years, in the analysis. University students in different cities were trained to be data collectors. The number of data collectors varied in each city—5 students each in Macau and Guangzhou, 10 in Hong Kong, and 27 in Zhuhai. Before they embarked on the process of collecting data, all of data collectors underwent a 2-day training session held by the project team. The collectors approached as many people as they could in community centers or public places , screened them for eligibility to participate in this study, obtained their written consent to participate, and provided assistance to those who needed help in completing the questionnaires. Data collection was conducted from July 2017 to May 2018, in the four cities.The outcome measure was dementia literacy. Since there was no single validated instrument for measuring dementia literacy, we measured the variable indirectly by mapping the two most relevant concepts—knowledge about dementia and attitudes towards dementia, as suggested by Low and Anstey .Knowledge about dementia was measured using the Alzheimer’s Disease Knowledge Scale (ADKS). The ADKS contains 30 true/false questions that assess knowledge related to AD assessment, diagnosis, caregiving, life impact, prevalence, prevention, risk factors, symptoms, treatment, and management . A compoAttitudes towards dementia were measured using the Dementia Attitudes Scale (DAS), which is a 20-item instrument. Each item is rated on a 7-point Likert scale ranging from 1 (strongly disagree) to 7 (strongly agree), and the total score of the scale could be from 20 to 140. The higher the total score, the more positive was the attitude toward dementia. The result of the confirmatory factor analysis indicated that DAS is a model with a reasonably good fit. The Cronbach’s alpha of this scale was 0.83 [Both ADKS and DAS contributed to dementia literacy and were considered dependent variables in the study.Another outcome measure was the channels for receiving dementia information. The participants were asked to indicate their preferred types of mass media for receiving relevant information about dementia, and they could choose more than one type. A total of 12 options were given. The options were then categorized into four types—social media, websites, paper-based materials, and television/radio. Social media included WhatsApp, Facebook, QQ, Instagram, Baidu, WeChat, and blogs, while websites referred to websites from government units, hospitals, health-related organizations, and non-governmental organizations. Paper-based materials referred to posters, newspapers, magazines, and books.Written consent was obtained from the eligible subjects before the completion of the questionnaires. Ethical approval to conduct the study was obtained from the Human Subject Ethics Subcommittee of Sun-Yat Sen University, the Hong Kong Polytechnic University (HSEARS20170511002), Kiang Wu Hospital College of Nursing of Macau, and Zhuyi Medical University of Zhuhai.Descriptive statistics were used to summarize the sample characteristics. Considering that both ADKS and DAS contribute to dementia literacy, we put two continuous variables into the multivariate analysis of variance (MANOVA) model as the dependent variables. Two-way MANOVA was used because two or more independent variables could be tested simultaneously with two dependent variables. Demographics  and location  were the independent variables. Interaction terms between each demographic factor and location were also put into the model to assess the effect of demographics on dementia literacy, in different cities.p > 0.05. We then assessed the Mahalanobis distance of the two dependent variables (ADKS and DAS). Since the Mahalanobis distance of this data was 13.44 , multivariate normality was assumed. According to Levene’s test (p > 0.05), the criterion group variances were homogenous. We assessed the maximum likelihood criteria (Wilk’s lambda), and effect size was presented as partial eta (η2). When the MANOVA detected significant statistical differences between the IVs and the DVs, we proceeded to conduct a two-way ANOVA for the dependent variables and a post-hoc test, to assess the effect of categorical independent variables on the dependent variable.MANOVA was used after validating the normality and homogeneity assumptions. The existence of multivariate outliers was assessed by a test of normality, and outliers were eliminated, when detected. The normality of the data was also assessed using the Shapiro–Wilk test; normality was assumed at p < 0.05.Four logistic regression models, using a Wald stepwise backward elimination method with the probability of a stepwise entry alpha of 0.05 and removal alpha of 0.10, were conducted to identify the associated factors  in favor of a particular type of mass media, for receiving information on dementia. All statistical analyses were performed using the Statistical Package for the Social Sciences . The level of significance was set at A total of 787 community-dwelling adults (aged 64 or below) were included in the analysis, with 242 from Hong Kong (HK), 184 from Guangzhou (GZ), 177 from Macau (MA), and 184 from Zhuhai (ZH). p < 0.05, and partial η2 = 0.008. This indicated that students, the employed, and the unemployed had significantly different levels of dementia literacy. Looking at the individual components of dementia literacy (ADKS and DAS), employment status had a significant effect on DAS  = 4.130, p < 0.05, and partial η2 = 0.011), but not on ADKS  = 2.510,  = 0.010 . In addi see the . This imp < 0.05, and partial η2 = 0.014  = 3.494,  see the . Figure p < 0.05) preferred to get dementia-related information from these traditional media  are a useful tool for changing attitudes. Discussions about attitudes can be made in the chatrooms of social media sites.Our finding that married people in Hong Kong and Macau had more positive attitudes towards dementia than their single counterparts, contributed to our understanding of the relationship between marital status and dementia literacy. Most previous studies had found that married people had more knowledge about dementia than unmarried people ,18. HoweAs noted, Hong Kong and Macau residents and middle-aged people prefer paper-based materials for dementia education. Materials should be developed by taking into consideration the literacy levels of the readers. The use of pictures or photographs in educational materials is recommended. Comic books have been found to be effective at improving knowledge about medication adherence, the storage of drugs, and health literacy, among Chinese people . FotonovUsing social media to deliver public education in dementia was a strategy favored by the participants in the current study, particularly those from the young generation. Such a proposal was worth supporting, as social media are already embedded in daily life and would be a novel medium for disseminating public health information . NonetheImproving dementia literacy requires the use of multiple channels and techniques . TraditiDementia literacy is a multi-faceted concept, and its definition has been inconsistent in many previous studies . In the This study had several limitations. First, the use of a cross-sectional design limited the extent to which causal inferences of the observed associations could be established. The findings should be further examined in longitudinal studies to establish the temporal validity of any associations that were found. Second, some of the important factors, such as personal experience in dementia care or the impact of Chinese culture on dementia, were not measured. This precluded us from examining their independent and potential interaction effects on dementia literacy and preferences in the types of media for receiving dementia information. This could also partly explain the limited explanatory power of the associated factors for the outcome variables. Finally, the current sample was obtained using convenience sampling, which might not have produced representative samples of the population in the four cities. This limits the generalizability of the current findings to the larger population of the four cities.Since unemployed and retired people in the Greater Bay Area of China have inadequate dementia literacy, it is worth conducting public education about dementia, targeting these groups of citizens. Strategies targeted at specific groups of citizens in each city could be formulated according to their preferences in different types of mass media. These recommendations are in response to the World Health Organization’s Global Calls for Action, to develop and implement campaigns to raise public awareness about dementia and foster dementia-inclusive societies by 2025 .


The aims of the present work are to estimate the nutritional value and to evaluate and compare the levels of macroelements , microelements , heavy metals , and their ratios in extruded complete foods for adult dogs, their compatibility with nutritional guidelines, as well as food profile similarity. Basic composition was determined according to Association of Official Analytical Chemists (AOAC). Analyses for elements were performed using an atomic absorption spectrometer. All the evaluated dry dog foods met the minimum recommended levels for protein and fat. Eighteen tested dog foods (60%) did not meet at least one recommendation of nutritional guidelines. Four dog foods exceeded the legal limit of Fe and five foods exceeded the legal limit of Zn; in one of them, Zn level was almost twice higher. Dog foods with insect protein exceeded the legal limit for Mn content. Eight dog foods had an inappropriate Ca:P ratio. Heavy metals were below detection limit in all analyzed dog foods. The results seem to show the need for regular feed analyses of the elemental composition in raw materials before introducing supplementation and for the monitoring of the mineral composition of finished pet food. Companion animals’ population is increasing worldwide. According to the American Pet Products Association (APPA) , in 2018Other than the major nutrient requirements, companion animals require certain amounts of elements that must be provided in their food for a healthy, balanced diet. Calcium, phosphorus, and magnesium are associated with the development and maintenance of bones and teeth, as well as being necessary for the proper functioning of the muscular and nervous system of dogs . PotassiWhile major macroelements and trace elements in pet foods are regulated by the US NRC , the AAFIt is worth noting that there is a substantial scientific interest in the topic of grain-free trend in pet food sector ,23. For Most domestic dogs in industrialized countries are fed extruded dog food . The infThe aims of the present work are to estimate the nutritional value and to evaluate and compare mineral profiles, heavy metal concentrations, mineral ratios, and profile similarity in randomly selected extruded maintenance commercial complete food for adult dogs with a particular emphasis on the division into cereal and cereal-free foods, and also to establish if the mineral levels meet the recommended requirements.All tested dog foods met the recommended minimum levels for crude protein and fat according to FEDIAF , which aOne out of thirty analyzed dog foods did not meet the minimum recommended level (MRL) for Ca A. SixteeAll dog foods contained higher amounts of trace elements than the MRL established by the nutritional guidelines . Two outSignificant differences were found between foods with a presence or absence of cereals in the composition. Significantly more Fe, Zn, Mn, and Cu were found in cereal-free foods . Heavy mn = 4), Zn (n = 4) and Cu (n = 1). On the other hand, in several products (n = 9) the manufacturer did not declare the addition of Fe and Mn on the label, consequently 100% of the results obtained in present study were obtained through raw materials. Five dog foods have declared the content of these four trace elements under the heading “analytical constituents”  and food no. 9 (cr = 0.55). In relation to other foods, there was either a lack of similarity (18×) or dissimilarity (9×). The next two foods with a significant dissimilarity of mineral profiles compared to the other feeds are food nos. 11 and 12. Just like food no. 8 mentioned above, these foods have potatoes in their composition and at the same time belong to the group of cereal-free foods. Food no. 12 has a particularly different profile as it has 13 negative similarity coefficients, both in relation to foods with and without cereals, and 12 dissimilarity coefficients.In order to generalize the differentiation of ash composition of the tested foods, a comparison was made of the multivariate mineral composition profiles . Out of Similar observations come from the PCA analysis, but it should be stressed that its first two components only accounted for 47.85% of the total variance. Nevertheless, five groups of food were distinguished, as shown by the ellipses in The nutritional guidelines have only established MRL and MNL for the Ca:P ratio, which is shown as the gray area in Although calculations are preliminary and more research is needed to establish appropriate mineral ratios, in the tested dog foods some incompatibilities with the determined ratios of individual mineral components were found . Most deIn Europe, the most commonly used recommendations for the chemical composition of pet foods and their recommended minimum levels and nutritional or legal maximum limits come from the FEDIAF . MinimumDiptera and Coleoptera. Insects are considered as a sustainable protein source for future pet foods. Bosch et al. [Hermetia illucens, Musca domestica, and Tenebrio molitor, was high and the undigested insect fractions were at least partly fermented by the dog’s fecal microbiota. Therefore, insect protein could be potentially included in pet food as health-promoting ingredients. In our study, dog foods with Hermetia illucens larvae as the main animal protein source had an average protein level of 22.36 g/100 g DM.Of key importance is the source of protein, with its quality being decisive for the nutritive value of dog food . An inteh et al.  concludeCommercial dog foods contain lipids from plant oils (mainly sunflower), and/or animals fat , or often a mixture of both. Which fat sources a manufacturer will include in a dog food depends on several factors, such as essential fatty acid (EFA) content, effect on palatability, susceptibility to oxidation, and market price . In the n = 7), sweet potato (n = 5), and peas (n = 5) and had less NFE (average 6.71 g/100 g DM) than foods with cereals (average 7.45 g/100 g DM). Subsequently, the mean of crude ash content of all tested dog foods (7.13 g/100 g DM) was close to the results of a study of 20 commercial dry foods for adult dogs by Pereira et al. [Carbohydrates are a group of compounds and include digestible sugars (monosaccharides and disaccharides), starch (polysaccharides), and non-digestible fibers (oligo and polysaccharides). Digestible carbohydrates are a direct source of glucose, an important source of energy for the body, and the main source for some tissues like the brain and red blood cells. Dietary fiber is physiologically important for pets. Certain fermentable fibers, defined as prebiotic fibers, promote the development of a beneficial gut microflora, which can help reduce the colonization of harmful bacteria . Dependia et al.   can be life-threatening because of the risk of cardiac arrhythmias. Therefore, excessive K supplementation should be avoided, especially in dogs with heart disease and chronic kidney disease. Appropriately formulated, potassium-reduced diets are an effective alternative to treat hyperkalemia . In our The situation was different with Na, where all analyzed foods had from about 2.5 to 8 times the amount specified in the MRL A. DespitIt is worth noting that the mandatory declaration of analytical constituents is related to the type of feed and/or the target animal species . For comWhile Cu plays an important role in a variety of biological processes, including mitochondrial respiration, antioxidant defense, neurotransmitter synthesis, connective tissue formation, pigmentation, and Fe metabolism , when coIron is necessary for the synthesis of oxygen-transporting proteins, particularly hemoglobin and myoglobin, and for the formation of heme enzymes and other Fe-containing enzymes involved in electron transfer and oxidation-reductions ,49. DefiHermetia illucens, i.e., the species whose larvae were used in the production of the tested dog food (see 2+ content 433.0 ± 14 mg/kg biomass of the Hermetia illucens) points to an important role of this metal in its ontogeny [Hermetia illucens demonstrated that extrusion can contribute to increase in organic matter digestibility [There is a fairly wide acceptable range for recommended Mn levels . Howeverfood see , it was ontogeny . Therefoontogeny ,62,63. Sontogeny . Howevertibility . Hence, Zinc deficiency is well-known in dog nutrition and, among other things, may reduce Zn serum concentration, affect animals’ growth, cause skin lesions, behavioral problems, and compromise the immune function ,67. In pHowever, the level of an element in the feed is not the only factor as the bioavailability and metabolism of minerals are complex. Many factors influence the absorption and use of a mineral by the animal’s body. Intestinal absorption of trace elements such as Zn or Cu can be noticeably reduced if the diet has relatively high dietary fiber or phytate content ,53. As wThe quality of dog food depends not only on the amount of individual minerals consumed but also in their proportions. The dietary pattern approach should consider the role of all nutrients because ratios of nutrients in a complete diet, are more informative than each single nutrient’s contribution . Since nDog food should maintain Ca within the recommended minimum and maximum values, as well as in the appropriate ratio to P. Nutritional guideline established the Ca:P MRL ratio at the level of 1:1 and the MNL at 2:1 . InvertiRecommended ratios between other elements are not determined by any of the nutritional guidelines for dogs. Therefore, the remaining ratios have been calculated on the basis of the MRL of individual elements. It is worth noting that calculations based on the MRL of individual elements are preliminary suggestions.The Ca:Mg ratio gained a lot of attention in recent years. Inappropriate ratio increases the risk of metabolic, inflammatory, and cardiovascular disorders. Adequate Mg levels are essential for the absorption and metabolism of calcium and vitamin D . It has The appropriate K level is necessary to sensitize tissues to the effects of thyroid hormones. In adult dogs, too low K level in the diet leads to decrease blood pressure and cardiac output . The K eSodium deficiency can lead to increased heart rate, dehydration, increased thirst, ataxia, and lethargy . The accReduced iron bioavailability may cause anemia, manganese deficiency can cause slow or impaired growth and soft-tissue calcification , and zinEven in trace amounts, heavy metals can cause serious problems for all organisms, and their toxicity is related to its accumulation in tissues and depends on the frequency of exposure, the amount absorbed, and the absorption channel . This suThe research material consisted of thirty complete dry extruded maintenance diets for adult dog in packages of 0.8, 1, and 2 kg. Dog foods were selected because of the presence or absence of cereals in the composition and also based on the availability in major pet stores and commercial suppliers in the city of Szczecin . Key nutritional information provided on the label was recorded such as macronutrient content  alongside the country of origin and batch number. Although not exhaustive, the selected samples provide a snapshot of European dry dog foods from different market segments.n = 17, cereal foods, n = 13). The composition of the main ingredients of analyzed dog foods is shown in Dog foods were assigned one of two groups depending on whether cereal was a named ingredient on the label . Crude ash was measured by burning in a muffle furnace at 580 °C for 8 h. NFE content of each diet was calculated on the basis of the assessed chemical composition. The results are expressed in g per 100 g DM.DM, crude protein, crude fiber, ether extract, and crude ash were determined according to AOAC . ChemicaOn the basis of identified chemical composition, ME  of the foods was calculated, according to the equation provided by the NRC , using A2SO4) and perchloric acid (HClO4). Analyses for Ca, K, Na, and Mg were performed using an atomic absorption spectrometer . For the determination of Ca, K, and Mg the following wavelengths has been set: K: 766.5 nm; Ca: 422.6 nm; Mg: 285.2 nm. The calculation of the content of each element was started with a calibration curve, taking into account the mass of the tested portion and the dilutions used. After mineralization of the food in a solution of sulfuric (VI) acid and H2O2, phosphorus (P) analysis were performed by colorimetric method using ammonium molybdate at 470 nm [2O5 to the total phosphorus content was calculated according to a chemical equivalent (0.436). The reliability of the method used was confirmed by comparative studies, among others calibration curve, using the pattern series method. The results of Ca, P, K, Mg, and Na analyses were expressed in g per 100 g of DM.The material for macroelelement  concentration analyses was subjected to digestion in concentrated sulfuric acid (Ht 470 nm . The lev3) and perchloric acid (HClO4) mixture. Analyses were performed using an atomic absorption spectrometer . For the determination of Fe, Mn, Zn, and Cu the following wavelengths has been set: Fe: 248.3 nm; Mn: 279.5 nm; Zn: 213.8 nm; Cu: 324.8 nm. The calculation of the content of each element was started with a calibration curve, taking into account the mass of the tested portion and the dilutions used. The results of Fe, Zn, Mn, and Cu analyses were expressed in mg per 100 g of DM.Material for microelements  and heavy metal  concentration analyses was subjected to digestion in a nitric acid (HNO0.75 for dogs with moderate activity (1–3 h/day).The levels of all minerals were compared with recommendations from FEDIAF  considerKey nutritional information provided on the label was recorded, including, where available, the concentration of minerals added to each food . The declared levels of compounds of trace elements on the dog food labels (wherever available) were converted into the levels of actual elements . The shaThe nutritional guidelines have only established MRL and MNL for the Ca:P ratio. Recommended ratios between other elements are not outlined by nutritional guidelines. For this preliminary exploration of elements ratios the remaining ratios have been calculated on the basis of the MRL of individual elements. Ratios Ca:Mg, K:Mg, Na:K, K:Ca, Fe:Mn, Mn:Zn, and P:Zn have been calculated. However, these ratios are for illustrative purposes, supporting the concept that mineral levels are relative.p = 0.05. According to the test results the means was divided into homogeneous groups. The means denoted by different letters differ statistically at α = 0.05 . In order to compare the mineral profile of the dog foods, we determined their elemental content . The percentage of a given element in the profile is expressed by an arithmetic mean converted into units on a 9-point scale. For profile comparison, Cohen’s profile similarity coefficient cr was used which was calculated from the formula [iA, iB—unitarized values of traits included in the compared profiles A and B; n—number of traits in the profile; m—midpoint of the ranking scale.One factor analysis of variance (ANOVA) for all analyzed features and a principal component analysis (PCA) were carried out using the STATISTICA 13.0 software  . The sig formula :rC=∑i=1ncr were to boundary values (1/−1), the stronger the evaluated similarity/dissimilarity was. Inter-profile analysis was conducted using MS Office 2017.This coefficient value was measured in the range from −1.0 to 1.0 and its interpretation was dependent on the values: x ≥ +0.75 (high similarity); +0.75 > x > +0.30 (moderate similarity); +0.30 ≥ x ≥ −0.30 (no similarity); −0.30 > x > −0.75 (moderate dissimilarity); x ≤ −0.75 (high dissimilarity). The closer the values of All the evaluated dry dog foods met the minimum FEDIAF recommended levels for protein and fat;Comparative analysis of the mineral profile of foods showed differences between foods depending on the composition, which allowed to distinguish specific groups of foods—among the tested foods significant differences in the presence or absence of cereals in the composition were found, with higher P, K, Na, Fe, Zn, Mn, and Cu levels found in cereal-free foods;For a few individual products, mineral content often either far exceeded or did not meet the nutritional requirements, of which in two dog foods, the limit for two trace elements exceeded simultaneously and the concentration of exceeded element was supplemented with nutritional additives, which possibly led to their excess;Dog foods without the declared supplementation with nutritional additives met the minimum recommended levels of trace elements, and in two foods they even exceeded the legal limit;Diets with an insect source of animal protein had relatively high Mn content, which was supplied from raw materials, not nutritional additives; this may indicate the need for a more detailed analysis of this relatively new source of protein in dogs’ diets in order to avoid excess Mn in the finished product and thus its toxicity;Most of the incompatibilities concerned inappropriate mineral Ca:P ratio content (five dog foods below the minimum recommended levels and three above maximum), which may constitute a risk to the overall well-being and health of pets when they are exclusively fed with these products in the long term;Heavy metals  levels were below detection limit in all analyzed dog foods.The results of this study suggest that there is considerable non-compliance with current EU guidelines for pet food. Eighteen out of thirty tested dry dog foods (60%) differed from at least one recommendation established by the nutritional guidelines. The obtained results are as follows:Therefore, the results argue for the need to conduct more frequent, regular, more precise, and restrictive feed analyses to know the content of the elements of the raw materials before planning supplementation as well as to monitor the mineral content of finished pet food. The information provided on the dog food label should be more comprehensive and include detailed information concerning mineral content.


To establish a classifier for accurately predicting the overall survival of gallbladder cancer (GBC) patients by analyzing pre-treatment CT images using machine learning technology.This retrospective study included 141 patients with pathologically confirmed GBC. After obtaining the pre-treatment CT images, manual segmentation of the tumor lesion was performed and LIFEx package was used to extract the tumor signature. Next, LASSO and Random Forest methods were used to optimize and model. Finally, the clinical information was combined to accurately predict the survival outcomes of GBC patients.P = 0.012). After inclusion of clinical factors, we used multivariate COX to classify patients with GBC. The AUC values in the test set and validation set for 3 years reached 0.79 and 0.73, respectively.Fifteen CT features were selected through LASSO and random forest. On the basis of relative importance GLZLM-HGZE, GLCM-homogeneity and NGLDM-coarseness were included in the final model. The hazard ratio of the CT-based model was 1.462(95% CI: 1.014–2.107). According to the median of risk score, all patients were divided into high and low risk groups, and survival analysis showed that high-risk groups had a poor survival outcome (GBC survival outcomes could be predicted by radiomics based on LASSO and Random Forest. Gallbladder cancer (GBC) is the fifth most common tumor of the digestive system and accounts for 95% of malignant tumors in the biliary system . The lacLesions can be directly observed by radiological images in clinic and lesion information not visible to the naked eye can be provided by radiomics. In recent years, radiomics has been developed to focus on the extraction and mining massive medical imaging data. It is hypothesized that these selected imaging features reflect specific tumor phenotypes , 9. BecaGBC survival prediction model is of great significance for patients’ prognosis assessment, treatment mode selection, surgical patient selection, postoperative adjuvant treatment plan determination, high-risk recurrence patient identification, follow-up frequency formulation, and rational use of medical resources. In this study, we assessed a number of CT-based radiomics parameters to predict patient’s overall survival (OS). Patient cohort with a total of 141 patients was used to analyze image data, extract features, and perform model tests. All the selected parameters were evaluated for their predictive power and stability. Finally, we combined clinical information for a cost-effective prediction.The flow of data analysis and processing is shown in All procedures involving human participants comply with ethical standards bodies and/or national research councils. Ethics Committee of Sichuan University approved this retrospective study. Written informed consent (written informed consent for patients under 16 years of age must be signed by a parent or guardian) is required before radiological examination for all patients.CT scanning was performed using 64-MDCT Scanner  or 128-MDCT scanner  before going through any treatment.All CT examinations were performed under the following conditions: 120 kVp; 199 mAs; 12.9 ctdIVOL (mGy); 460.7 DLP (mGy*cm); pitch, 0.75–1.0; rotation time, 0.5–0.75 s; collimation, 0.625 mm; section thickness, 2.0 or 5.0 mm.The ROI area was sketched by two experienced radiologists.Due to the limited recognition ability of ordinary CT for GBC and the boundary of cancer is usually fuzzy, we followed the following principle when making segmentation: 1) delineate solid lesions with high density of GBC and avoid low-density areas such as bile, 2) delineate the definite tumor part when it is difficult to recognize the blurring around the lesion, and 3) excluded samples with disagreement among radiologists.We used the image feature extraction software LIFEx to obtain the texture signatures of CT images . Based oFirst, all the collected samples were randomly divided into test set and validation set according to a ratio of 7:3. We used the sample function of R software to make randomization, and conducted a hypothesis test on the age of the randomized patients between the two groups . After 1P = 0.012).First, we randomly divided the patients into a training group and a test group, with a split ratio of 7:3. Then, LASSO method was used to make simulation in the training group for up to 100 times and 15 signatures were selected. The results are shown in In order to achieve better performance, we analyzed a variety of clinical indicators of patients, including age, gender, and tumor stage. Through multivariate COX analysis, we screened out the prognostic indicators affecting the survival of patients with GBC, including surgery or not, liver metastasis and lymph node metastasis grade  in real clinical work. Secondly, limited by the size of the sample, there are not enough enhanced CT data and MRI data, thus, this study was not compared with a variety of advanced scanning techniques. Third, our research was limited by its retrospective data. These findings might have better clinical implications, if confirmed in prospective studies. Forth, the patients included in the study were all from a single center, which may result in the lack of sufficient extensibility of the classifier. Considering the differences of medical institutions in obtaining original images and the differences in manual segmentation of lesions, we cannot guarantee that this machine learning classifier performs well on external data sources. But all of the research methods and analysis used in the study come from open-source data packets, which mean that the analysis process needs to be repeated on other data.We found associations between established CT imaging parameters and overall survival. Radiomics-based non-invasive technology represented promising ability in predicting the overall survival of gallbladder carcinoma, although more extensive testing are necessary to perfect this technology in real clinical use.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Sichuan University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.ZL, XM, and JJ are responsible for conceiving and designing the subject. ZL, GZ, XJ and HZ conduct data analysis and article writing. YZ and HZ are responsible for data processing. ZL and GZ make the same contribution to this paper. 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.


To analyze the potential active ingredients and related crucial targets of the ShengDiHuang Decoction (SDHD) formula in the treatment of dysfunctional uterine bleeding (DUB) by using network pharmacology and verification experiment.  In this study, we determined the potential active ingredients from the traditional SDHD formula and their targets with the network pharmacology method. The network of “compound-disease-target” was constructed by the software of Cytoscape. Software of DAVID was used to enrich pathways for these 87 targets of SDHD. Further, the therapeutic effect of SDHD on DUB was verified by observing the morphological changes of the uterus and ovaries and determining the expression of ERS2 and progesterone in the plasma. Rheum and 5 compounds of Rehmannia were selected, and 87 potential targets were screened by network pharmacology. Furthermore, 7 main active ingredients were acquired by the ADME process. In addition, enrichment analysis of drug-target networks indicated that SDHD may play a role in overall coordination through “multicomponent and multitarget” in different organ patterns by regulating multiple pathways directly or indirectly. Finally, in the verification experiment of SDHD on DUB, it was found that SDHD can effectively repair the uterus and ovaries and also have an upregulation effect on the target ESR2 and increase the content of progesterone.  52 compounds of  Overall, this study revealed potential mechanisms of multitarget and multicomponent about SDHD in the treatment of DUB and provided a scientific foundation for further studying the mechanism. For women aged 40 years or more, before menopause, the treatment includes cyclic progestin therapy, oral low-dose contraceptives, levonorgestrel intrauterine device, or cyclic hormone therapy [Dysfunctional uterine bleeding (DUB) is defined as a state of abnormal uterine bleeding , which i therapy . AlthougRehmannia and Rheum (in the weight ratio of 30 : 1), is derived from the recipients of “Qianjinfang” of Sun Simiao in the Tang Dynasty [Rehmannia has the functions of improving hematopoiesis, promoting vascular endothelial cell proliferation, and improving immunity [Rehmannia, as the principle herb, has the effect of cooling blood and hemostasis and tonifying the yin and the kidney, and Rheum as adjuvant herbs assist the effects of Rehmannia to promote blood circulation to remove blood stasis [Traditional Chinese Medicine (TCM) was recognized as a popular complementary and alternative medicine in Western countries , and it  Dynasty . It is m Dynasty . Modern immunity , 12. Rehd stasis . AlthougNetwork pharmacology is a promising strategy for systemic analysis based on interaction networks of diseases-genes-targets-drugs , 15, whiRehmannia was purchased from Baoji Hanfang National Herbal Pieces Co., Ltd. Rheum was purchased from Lanzhou Xukang Pharmaceutical Co., Ltd. Co., Ltd. Gongxuening Capsules were provided by Yunnan Baiyao Group Co., Ltd., hydrocortisone came from Tianjin Jinyao Pharmaceutical Co., Ltd., hydroxycarbamide was from Beijing Jialin Pharmaceutical Co., Ltd., mifepristone was obtained from Zhejiang Xianyi Pharmaceutical Co., Ltd., and misoprostol was from China Resources Zizhu Pharmaceutical Co., Ltd. The ERS2 kit was purchased from Wuhan Huamei Biological Engineering Co., Ltd. The progesterone kit was supplied from the Nanjing Institute of Bioengineering.http://lsp.nwu.edu.cn/tcmsp.php), BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm), and TCM-MESH (http://mesh.tcm.microbioinformatics.org/). Compounds were selected with DL > 0.18 in TCMSP. According to the collected compounds, the potential targets of the ingredients were searched in the database of TCMSP, BATMAN-TCM, and TCM-MESH. Other than these, we also performed the validated targets which were obtained from the HIT database (http://lifecenter.sgst.cn/hit) and the predicted targets which were acquired from the ChemMapper database (http://lilab.ecust.edu.cn/chemmapper/).All compounds of the two Chinese medicinal herbs in SDHD were collected from the TCMSP (http://www.drugbank.ca/) and OMIM (http://www.omim.org/) with dysfunctional uterine bleeding as keywords, as well as obtained from DisGeNET (http://www.disgenet.org/web/DisGeNET/menu/home) using uterine hemorrhage as keywords.Disease-targets were collected from the Drugbank  and then merged and intersected by mapping the Venny (http://bioinfogp.cnb.csic.es/tools/venny/index.html). After that, 52 ingredients of Rheum, 5 ingredients of Rehmannia, and 87 related targets were collected and used as an input to the software Cytoscape (version3.2.1) to construct the predicted-target (PT) network.All the gene names of OB is one of the most important pharmacokinetic parameters in the ADME process . High OBFour topological properties, “degree,” “betweenness centrality,” “closeness centrality,” and “average shortest path length” were analyzed in Cytoscape (version3.2.1) and then the degree values of which are more than two folds of the median degree of all nodes in the network were filtered, which are considered to be the important targets .http://david.abcc.ncifcrf.gov/home.jsp, version 6.8) and p < 0.05 was selected, and then the pathway data obtained from the Kyoto Encyclopedia of Genes and Genomes  were selected.To clarify the pathways which are involved in putative SDHD targets, KEGG and Go enrichment analysis was performed using the Database Visualization and Integrated Discovery software32  rats (230 ± 20 g) were used and supplied by the Chengdu Dashuo Animal Experiment Center. Rats were raised under standard temperature, humidity, and light conditions and fed standard rodent diet and water. The study was approved by the Ethical Committee of the Shaanxi University of Chinese Medicine.Rats were caged with female : male in the ratio of 2 : 1. The next day, the female rats were examined for vaginal suppository and vaginal smear to find whether there is sperm in the vaginal suppository and vaginal smear as the first day of pregnancy. From the first day of pregnancy, all groups were oral administered with hydroxycarbamide at a dose of 450 mg/kg at 8:00 am and injected 3.7 mg/kg of hydrocortisone every day, except for the control group. The treatment was continued for 7 days to make a model of functional dysfunctional uterine bleeding. On the 8th day of pregnancy, mifepristone and misoprostol were administered orally at 8:00 am and at 18:00, respectively. At the same time, a sterile cotton ball was placed in the vagina of each pregnant rat. The next morning, the cotton ball of the rat's vagina was collected, and blood was seen on the cotton ball, indicating that the model was successful. After successful modeling, twenty one successful model rats were randomly divided into the model group, the positive group, and the treatment group with seven rats in each group. The positive group and the treatment group were administered with 0.156 g/kg Gongxuening and 0.0646 g/kg SDHD on the second day for 8 consecutive days, respectively. After the last administration, the rats were fasted with free access to drinking water. On the next day, the rats were anesthetized and blood was taken from the abdominal aorta. Afterwards, the uterus and ovary tissues were taken and fixed in a 10% formalin solution for 24 hours and then sequentially dehydrated with an ethanol gradient, embedded in paraffin, and sliced, and then hematoxylin-eosin (HE) staining was done. The morphological and quantity changes of the uterine glands in the uterus and follicles in ovaries were observed under a microscope. In addition, the determination of ERS2 and progesterone in the serum was performed.Rheum and 8 of Rehmannia. 1085 proteins were predicted for Rheum ingredients, and 319 proteins of Rehmannia ingredients were fished. 415 related targets for dysfunctional uterine bleeding were collected.A total of 65 constituent compounds of the 2 herbs contained in SDHD were collected, including 57 of Rheum, and 5 compounds of Rehmannia were screened. The chemical names, structures, and molecular formulas of all compounds are shown in Supplementary As shown in Rheum meet the requirements of OB ≥ 30%, DL ≥ 0.18, and Caco-2 > −0.40, including aloe emodin (anthraquinone), (−)-catechin (phenols), beta-sitosterol (triterpene), daucosterol (triterpene), eupatin (flavonoid), rhein (anthraquinone), and toralactone (phenols). Although no ingredients in Rehmannia meet the requirements, catapol as the highest content compound in Rehmannia can be considered as the main active ingredient [Through the screening of main active ingredients by the ADME process, we found that seven components in gredient . The valPredicted SDHD target-known therapeutic targets of the dysfunctional uterine bleeding network were constructed using the links between targets of SDHD and known therapeutic targets of disease. As previously reported , if the in vitro [α, plays an important role in female reproductive activity. Related studies have shown that deregulation of estrogen expression may lead to changes in endometrial function [β, is a nuclear hormone receptor and binds estrogens with an affinity similar to that of ESR1 and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner. It has been well determined that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor-α can promote the proliferation of cancer tissues, while ER-β can protect against the mitogenic effect of estrogen in breast tissue. The expression of ER-α and ER-β may greatly influence the development, treatment, and prognosis of breast cancer [β have shown that deficiency of ER-β leads to hyperproliferation and loss of differentiation of the uterus [PTGS2 expresses cyclooxygenase (COX-2), and it was reported that cyclooxygenase is the main rate-limiting enzyme in the synthesis of prostaglandins and thromboxane from arachidonic acid, COX-1 is a structural enzyme, and COX-2 is an induced enzyme . Symptomin vitro . Under nin vitro . Currentin vitro . Therefofunction , 29. Thefunction . Abnormafunction . ESR2, et cancer . Previoue uterus , ventrale uterus , and cole uterus . Thence,e uterus , 37. Rele uterus . Angiogee uterus . Factor e uterus . It is re uterus . At the e uterus . So, it p value, and the FDR. As shown in p value less than 0.05 were possible pathways for SDHD treatment of DUB. The results indicated that these proteins are closely related to the immune system, endocrine system, and cancer, such as complement and coagulation cascades, pathways in cancer, estrogen signaling pathway, bladder cancer, proteoglycans in cancer, and hepatitis B. GO analysis involved three main terms, including biological processes (BP), cellular components (CC), and molecular functions (MF). For GO-BP, 89 pathways were obtained (Supplementary p value 0.01, and these pathways are shown in p value less than 0.05 are considered more likely to be involved in the treatment of DUB. As shown in Supplementary p value of less than 0.05, which is shown in In order to understand the mechanism of SDHD on treating DUB and to identify signaling pathways associated with SDHD protein targets, DAVID software was used to enrich pathways for these 87 targets of SDHD. A total of 30 pathways of KEGG were obtained, and the results of all pathways are shown in Supplementary Rehmannia, participates in the activation of thrombus, which significantly prolongs the closure time of whole blood and the occlusion time of platelet embolism, and plays an important role in hemostasis [Rehmannia, has antiplatelet aggregation effect, thereby inhibiting blood coagulation [Rheum, may play a key role in the regulation of pathways in cancer. In the estrogen signaling pathway, estrogen, as a main steroid sex hormone, can regulate a plethora of physiological processes in mammals, including the growth, development, and function of the female reproductive system, bone integrity, cellular homeostasis, and behavior. There is also substantial evidence for an effect of estrogen on the cardiovascular system [α and ER-β in coronary arteries of premenopausal and postmenopausal women. It was found that increased expression of ER-β is associated with advanced atherosclerosis and calcification regardless of age or hormone status [α deletion, the cardioprotective role of estrogen in ischemia/reperfusion injury is lost [α [The pathway of complement and coagulation cascades is a proteolytic cascade in blood plasma and a mediator of innate immunity, a nonspecific defense mechanism against pathogens. There are three pathways of complement activation: the classical pathway, the lectin pathway, and the alternative pathway. All these pathways generate a crucial enzymatic activity, which in turn produces the effector molecules of complement. The main consequences of complement activation are the opsonization of pathogens, the recruitment of inflammatory and immunocompetent cells, and the direct killing of pathogens. Blood coagulation is another series of proenzyme-to-serine protease conversion, culminating the formation of thrombin, the enzyme responsible for the conversion of the soluble fibrinogen to the insoluble fibrin clot. The coagulation system has evolved to limit blood loss at sites of vascular injury, and its biological effects are usually mediated directly by enzymes. In the case of vascular injury, thrombin is produced very rapidly and relatively in large quantities at the site of injury. Therefore, effective inhibitors are needed to ensure that excessive coagulation does not occur . It was mostasis . Acteosigulation . The abogulation . Therefor system . A studye status . Further is lost . Thus, t lost [α .The structure of rat ovary was observed by HE staining. In The morphology changes of uterus are shown in β leads to hyperproliferation and loss of differentiation of the uterus [ESR2, the nuclear hormone receptor, has well determined that estrogen is involved in the pathophysiology of breast cancer. Deficiency of ER-e uterus , ventrale uterus , and cole uterus . It was Progesterone, a female sex hormone produced by the corpus luteum in early pregnancy, induces secretory changes in the uterine lining . It was Rehmannia is less studied, there may be a lack in data collection, and at the same time, the mechanism of action of some targets is still unclear due to less experimental verification. Therefore, we will use more systematic methods in subsequent experiments.At present, we screened SDHD for DUB targets through network pharmacology and combined with experiments, it was found that SDHD further affects the uterine and ovarian damage caused by DUB by improving the reduction of ESR2 and regulates the changes of endometrial tissue by increasing progesterone level. This method and results provided the basis for further mechanism research on SDHD for DUB treatment and also supplied a novel strategy for exploring the molecular mechanisms of traditional medicines. However, since the compound-targets of In this study, the protein targets of DUB and active components of SDHD were collected and then the complex network between drugs and diseases were revealed by using network pharmacology. We found that the pharmacological activities of SDHD on DUB might be associated with the regulation of five main targets that include PTGS2, ESR1, PGR, ESR2, and F10. Also, SDHD has an upregulation effect on target ESR2 and increases the content of progesterone that we have confirmed through experimental verification. However, further experimental validations of these prediction results are required in our future studies.


Gonodactylaceus randalli, is the first stomatopod species that has been identified in a biofouling community. In this study, we sequenced and analyzed the complete mitochondrial genome sequence of this species for the first time. Its mitochondrial genome is 15,907 bp in length and comprises 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a non-coding A + T-rich region. The overall base composition in the heavy strand is as follows: A: 37.3%, T: 31.3%, G: 11.9%, and C: 19.4%, with a G + C content of 31.3%. The phylogenetic analysis revealed that G. randalli belonged to the families Protosquillidae, Gonodactylidae, and Takuidae, in the same clade, within the superfamily Gonodactyloidea. This is the first record of the complete mitochondrial genome sequence of the genus Gonodactylaceus.Biofouling has long been known as a major route for the invasion by non-indigenous species. The mantis shrimp,  Gonodactylaceus randalli is the first stomatopod species that has been identified in a biofouling community  are known to negatively affect biodiversity and ecosystems, human and wildlife health, and the economy , on 11 February 2019. A voucher specimen was preserved in 80% ethyl alcohol and was deposited at the Research Institute of EcoScience, Ewha Womans University, Seoul, South Korea (Registration number: EWNHMAR770). Total genomic DNA was extracted from the leg muscle tissue using the MGIEasy DNA Library Prep Set . Whole genome sequencing was performed with the MGISEQ-2000 platform . MITObim . In the case of COX1, the start codon is ACG, as often observed in the malacostracan mitochondrial DNA . Based on the mitogenome sequences previously deposited in GenBank and using a lysiosquilloid species as an outgroup, the analysis revealed that G. randalli belonged to the genera Chorisquilla (Protosquillidae), Gonodactylus (Gonodactylidae), and Taku (Takuidae), in the same clade, within the superfamily Gonodactyloidea. Notably, Gonodactylaceus randalli and Gonodactylus chiragra, two species belonging to the same family, are more closely related to protosquillid and takuid species, respectively, than to each other. Further taxonomic studies of this superfamily are required (To explore the phylogenetic position of required .Gonodactylaceus. Along with our results, further mitogenomic analyses of undetermined taxa within stomatopods would improve our understanding of their taxonomic relationship and phylogeny.This is the first record of the complete mitogenome sequence of a stomatopod that is member of a biofouling community. Furthermore, it is the first record of a complete mitogenome sequence in the genus


Background: Breast cancer is the leading cause of cancer death in women worldwide with more than 1. 7 million new cases annually. Major advances have been made in the therapeutic management of this condition in many countries. However, neuropsychiatric disorders in patients with breast cancer constitute a significant concern due to their negative impact on patient's life and on the success of therapy itself.Methodology: In this study we aimed to evaluate psychological disorders in a population of 212 Moroccan women treated for breast cancer within the Mohammed VI Center for the Treatment of Cancers of Casablanca. A questionnaire was designed to this end on the basis of different validated scales, including the Hospital Anxiety Depression Scale (HADS), the Pittsburgh Sleep Quality Index (PSQI), and the Body Image Scale (BIS).Results: Data analysis has shown that 69.3% of participants had significant sleep disorders; 87% suffer from an anxiety-depressive syndrome (ADS), and 83.0% had significant body image dissatisfaction. A positive correlation was shown between ADS and both patients' national health insurance coverage and patients or husbands' education. Analysis further revealed that anxiety and depression were negatively correlated with different types of treatment. Similarly, both BIS and PSQI scores were positively associated with anxiety and depression disorders.Conclusion: The present investigation highlights the need to generalize and strengthen the psychological approach of patients treated for breast cancer in Morocco. We anticipate that such a strategy will alleviate suffering and promote therapy success in these patients and will diminish or prevent conjugal and familial impacts of the illness. Breast cancer is considered to be one of the most common public health issues in the world. It's the leading cause of mortality due to cancer in women in the world, with more than 1.7 million new cases and ~521,900 deaths each year , 4. AccoIn this setting, depression disorders consist of a combination of major depressive disorders, persistent depressive disorders—also known as dysthymic disorders—and a mixed form of both depression and anxiety disorders . In addiThe same Moroccan team working at the Moroccan National Institute of Oncology at Rabat, the capital, suggested many interventions that should be done in order to protect patients living with breast cancer against psychological distress, among them, social family support and both emotional and financial facilities .However, a study published by Haddou Rahou and his collaborators highlighted a lack of studies on the quality of life of women with breast cancer in different Muslim and Arab countries, including Morocco. Hence the need to conduct a comprehensive investigation .The aim of this study was to evaluate both anxiety and depression disorders in a population of Moroccan breast cancer patients within the region of Casablanca, the largest Moroccan city, and to identify the anxio-depressive impacts of some socio-demographic parameters and some clinical variables including types of treatment, sleep disturbance, and body image dissatisfaction.The present descriptive and cross-sectional analysis was carried out between February 2018 and May 2019 in the Mohammed VI Center for the Treatment of Cancers, Ibn Rochd University Hospital of Casablanca, Morocco. Casablanca is the largest city in Morocco and the economic capital of the country. During this study, 212 breast cancer patients were randomly chosen among patients coming for follow-up at the Oncology Department. The inclusion criteria were the following: women with confirmed diagnosis of breast cancer, those agreeing to participate in this study with written consent , patients with no previous follow-up of any psychiatric and mental disorders, and the ability to understand and speak the Moroccan dialect. The latter was to ensure efficient and easy communication during the completion of the questionnaire.Before proceeding with data collection using the Hospital Anxiety and Depression Scale (HADS), the Pittsburgh Sleep Quality Index (PSQI), and the Body Image Scale (BIS), two translators translated the questionnaire from English into dialectal Arabic, and then another team made up of two other translators performed a back translation of the questionnaire. The two versions of the questionnaire were studied by a team of experts  to produce a pre-final version. Finally, a pilot study was carried out on 30 cancer patients to test comprehension of the items. The reliability test calculation (Cronbach α) indicates good validity for the three scales .In this study we used a questionnaire that provided participants' socio-demographic data and the Hospital Anxiety and Depression Scale (HADS). The HADS, developed by Zidmond and Snaith , assesseThe Pittsburgh Sleep Quality Index (PSQI) used in the questionnaire has 19 items grouped into seven components, including: (i) subjective sleep quality, (ii) sleep latency, (iii) duration of sleep, (iv) habitual sleep efficiency, (v) sleep disturbances, (vii) sleep medication use, and (vii) daytime dysfunction due to lack of sleep. Each component score ranges from 0 to 3, where 3 refers to a severe sleep disturbance. The summation of the PSQI scores yields a total score ranging from 0 to 21. A total score >8 indicates a patient with a significant sleep disorder and poor sleep quality. The Cronbach α for the PSQI in this study is 0.67; this value is strong and approaches one of the original validations .In the current study, body image dissatisfaction regarding breast cancer patients was assessed using the body image scale (BIS) . This scThe descriptive analysis consisted of calculating absolute and relative frequencies for qualitative variables, in addition to positioning and dispersion parameters for quantitative variables .t-test, the Mann-Whitney test, ANOVA, and the Friedman test.The normal distribution of the variables was examined with the Kolmogorov-Smirnov test. In bivariate analysis, the continuous variable comparison involved the Student p < 0.05. The statistical analysis was carried out using SPSS software version 19.0.The significance level was set at n = 212), with a mean age of 49.66 (SD = 12.50). About half of the participants (49.53%) and their husbands (56.60%) were unschooled and exhibited a low educational level, and about 52% were of low socioeconomic status. As for insurance coverage, the vast majority of our study population (93.4%) had RAMED, which is a national health insurance program intended to allow low-income Moroccan citizens access to health facilities  received surgery and/or chemotherapy or hormone therapy. In all, 32.55% of patients received only surgery and chemotherapy. A minority of our participants (5.66%) had surgery, radiotherapy, chemotherapy, and hormone therapy together .As for the assessment of sleep quality, the total score of the Pittsburgh Sleep Quality Index (PSQI-T) revealed that more than two thirds of our breast cancer patients (69.34%) had significant sleep disorders and exhibited poor sleep quality .The monitoring of neuropsychiatric parameters revealed that our population sample exhibited severe psychiatric comorbidities. In fact, the HADS and the BIS data analysis showed that 86.67% of our breast cancer patients suffered from an anxiety-depressive syndrome (ADS). Results further showed that almost 83% our population suffered from not being attractive and then expressed significant body image dissatisfaction (BIS-D) .In this study, three components of the HADS scale were used: HADS-A, for anxiety; HADS-D, for depression; and HADS-T, for anxio-depressive syndrome. The three components allowed the assessment of the presence or absence of these neuropsychiatric disorders, which usually occur in women with breast cancer .p > 0.05). However, a strong correlation was found between educational levels and the onset of both anxiety and depression in our sample (p = 0.0001). This correlation was also seen between the husband's education levels and both depression and ADS (p = 0.05).Throughout the present study, we noticed that our general population showed suspected or well-proven anxiety and/or depression. The suspected anxiety or depression is assessed from a HADS-A or HADS-D score from 8 to 10. Anxiety or depressive disorders are proven by HADS-A or HADS-D scores ranging from 11 to 21. Data also indicate the presence of anxio-depressive syndrome (ADS) for the whole of our breast cancer population  . Accordip > 0.05), whereas our results showed a noticeable association between national health insurance coverage (RAMED) and both anxiety disorder (p = 0.034) and ADS (p = 0.014)  .p = 0.0008), depression (p = 0.0005) and ADS (p = 0.0002) , and body image dissatisfaction (BIS-D score). In light of this, different types of treatments received by our patients, including surgery, chemotherapy, radiotherapy, hormone therapy, and immunotherapy, had a negative effect on the mental health of our study population, which exhibited a strong association with anxiety disease ( 0.0002) .p = 0.0007) and ADS (p = 0.0009)  and both anxiety disease ( 0.0009) .p = 0.001), depression (p = 0.004), and ADS (p = 0.0001).Our results show also that the psychological disturbances revealed in this study are strongly related to the side effects of the surgery seen on patients' bodies. Thus, the assessment of body image dissatisfaction (BIS-D) exhibits solid association with anxiety . The results show a significant improvement of sleep quality as well as in relief of anxiety and fatigue .Overall, breast cancer is known to be an alarming disease that causes women both physical and psychological suffering. In this study we have demonstrated that our population exhibited severe psychiatric comorbidities, especially anxiety and depression. Besides low socioeconomic status and low educational level, we found that most types of treatments, sleep disorders, and body image dissatisfaction encountered by women with breast cancer exacerbate both anxiety and depression levels inducing in patients a worse psychological health status.From this study, it seems that the pathological triad of anxio-depressive syndrome, sleep disorders, and body image dissatisfaction forms a kind of vicious circle and that each of them potentiates the effects of the others. When these three comorbidities are associated in the same breast cancer patients, the patients' survival rates decrease.Given the results of this study, we recommend that health care providers and decision-makers in the field of health strengthen psychological care teams for breast cancer patients and consider establishing mobile psychological health care units in order to visit patients in their homes, especially those living in isolated areas or with low socioeconomic status.This study has some limitations. On the one hand, our sample may not be representative of the general population of Moroccan women with breast cancer, due to the recruitment of a limited sample in a single counter hospital. On the other hand, our work is conducted only at the university hospital center, which welcomes patients coming from both urban and rural areas of the Casablanca-Settat region, the country's economic capital. For future studies, we plan to initiate a multicentric study which would include other hospital universities and also the private sector.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Mohammed VI Center for the Treatment of Cancers, Ibn Rochd University Hospital Center Casablanca, Morocco approved the study presented here. The patients/participants provided their written informed consent to participate in this study.OE, AA, and AE: conception and design of the study. MD, AA, and MG: data collection. NE, AA, OE, and ES: acquisition and data analysis. AB: recruitment of patients. OE, MM, AJ, and AE: interpretation of data. OE and AA drafting of the work. MM, AB, AJ, and AE: revising the manuscript critically and final approval of the manuscript. All authors: approved the final version of the manuscript to be submitted.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.


In amyotrophic lateral sclerosis (ALS), magnetic resonance imaging (MRI) allows investigation at the microstructural level, employing techniques able to reveal white matter changes. In the current study, a diffusion tensor imaging (DTI) analysis, with a collection of apparent diffusion coefficient (ADC) and fractional anisotropy (FA) indexes, was performed in ALS patients to correlate geno- and phenotype features with MRI data, to investigate an in-vivo correlation of different neuropathological patterns. All patients who underwent the MR-DTI analysis were retrospectively recruited. MRI scan was collected within three months from diagnosis. FA and ADC values were collected in corpus callosum (CC), corona radiata (CR), cerebral peduncle (CR), cerebellar peduncle (CbP) and corticospinal tract at posterior limb of internal capsule (CST). DTI analysis performed in the whole ALS cohort revealed significant FA reduction and ADC increase in all selected regions, as widespread changes. Moreover, we observed a higher value of FA in rCR in bulbar patients. A positive correlation between ALS Functional Rating Scale-Revised and FA in rCP was evident. In consideration of the non-invasiveness, the reliability and the easy reproducibility of the method, we believe that brain MRI with DTI analyses may represent a valid tool usable as a diagnostic marker in ALS. The complex of motor neuron diseases (MNDs) includes a heterogeneous group of conditions, in terms of clinical, genetic and biological aspects. Amyotrophic lateral sclerosis (ALS) is the most common MND, with the involvement of the cortical and spinal motor neurons (MN) . The cauThe use of neuroimaging in ALS, including magnetic resonance imaging (MRI) and positron emission tomography (PET), has recently grown rapidly, becoming two well-established technical tools in MNDs to define in vivo markers of underlying pathological alterations . Recent The damage of axons and myeline of WM fiber tracts is associated with a decrement of FA values but to an increase in ADC values accordingly.In the literature, several cross-sectional DTI studies have consistently shown a reduction in FA in the CST in ALS, with a concomitant increase in the ADC values in the same region and in frontal and parietal WM ,14,15,16Similarly, a few studies have employed DTI measures as disease biomarkers: in 2019, Du et al. observed significant direct correlations between the ALS Functional Rating Scale-Revised (ALSFRS-R) scores and FA in bilateral CST, as well as between disease duration and local mean diffusivity in right CST ; in addiIn the current study, a diffusion analysis, with a collection of ADC and FA indexes, was performed in ALS patients, aimed at investigating the invivo correlation of different neuropathological patterns and, in turn, to correlate geno- and phenotype features, and disease severity as well, with MRI data. In detail, we tried—in a single study—to link the DTI findings as FA and ADC, collected at several levels , both with clinical and genetic data. In addition, all patients underwent a complete neuropsychological evaluation and genetic analysis.All patients who underwent the MR-DTI analysis from January 2015 to September 2020 at the Tertiary ALS Center at Maggiore della Carità Hospital, Novara, Italy, were retrospectively recruited. Patients’ charts were reviewed retrospectively for all patients included in the study. The study included patients who met El Escorial Criteria for ALS , and diaDetails of demographics, clinical data including ALSFRS-R  for diseThis study was conducted retrospectively and the patients underwent the MRI scan in our center. As such, patients were exempt from signing consent and our study did not require the approval of the Ethical Committee because we subjected all patients to neuroimaging evaluation as routine in a multidisciplinary approach. No risks were expected for the subjects for the retrospective study design and the results of the study did not have an impact on the diagnosis, prognosis or management of study participants.Patients underwent a complete battery of neuropsychological tests in order to evaluate the executive and visuospatial functions, behavior, language and memory. The neuropsychological battery tested global cognition with the Mini Mental State Examination (MMSE), logic and deductive skills with Raven’s Progressive Colored Matrices, executive functions with Cognitive Estimates Test, Frontal Assessment Battery (FAB) and Clock Drawing Test, memory with the Digit Span test and the Short Story Test, attention with the Trail Making A-B Test and Attentive Matrices and language with verbal fluency and comprehension.Neurobehavioral dysfunctions were determined using the Neuropsychiatric Inventory (NPI). Patients were divided, based on the battery score and Strong criteria published in 2017, into normal cognition, ALS-cognitive dysfunctions (ALS-ci), ALS-behavioral dysfunctions (ALS-bi), ALS-cognitive and behavioral dysfunctions (ALS-ci/bi) and ALS-frontotemporal dementia (ALS-FTD).All were screened for the main genes causing or associated with amyotrophic lateral sclerosis . All genes were sequenced with the TruSightTM one Sequencing Panel (Illumina) on a Miseq (Illumina) instrument. C9Orf72 GGGGCC repeat was analyzed also for repeat expansions associated with ALS .2); TE and TR were 83 and 6466 ms. The 3.0 T DTI study protocol consisted of 52 slices, 92 × 90 matrix, slice thickness 2.5 mm, pixel size 2.5 × 2.5 mm, the directional resolution was 32 with two b factors (1000 and 0 s/mm2); TE and TR were 84 and 3000 ms. The number of scans acquired at each scanner was almost the same between the patients and the healthy group, matched for number of scans at the different scanners, age and sex.All MRI scans were performed within three months of diagnosis. MRI scans were obtained with two scanners, a Philips Ingenia 1.5 Tesla and a Philips Ingenia 3.0 T . The 1.5 T DTI study protocol comprehended 50 slices, 112 × 110 matrix, with a slice thickness of 2.6 mm, pixel size 2 × 2 mm, the directional resolution was 32 with two b factors . The more the tissue is loose and free from structural constraints for water molecule movements, the more the diffusion is “free”. On the contrary, the more the movement is restricted by ultrastructural obstacles, the more the diffusion is “restricted”. Thus, MR imaging measures and reflects the “in vivo probe” of what happens to a tissue. In detail, ADC and FA reflect the interstitial extracellular space width indirectly, by measuring the water molecule movements, and in the brain tissue, this is correlated with axonal density, which is afflicted by ALS pathology: ADC increase and FA lowering reflect in fact a neuronal loss .The analysis of the DTI data was performed using the software Philips IntelliSpace Portal , using the “MR diffusion” application analyzer. After reconstructing the FA directional map on the basis of the DTI imaging, we proceeded to the evaluation of the corticospinal tract using the “MR FiberTrack” application. Images were evaluated by applying the FA directional map as overlay and the 3D FLAIR sequence as anatomical underlay. The corticospinal tract was reconstructed with a multi-ROI manual tracking, which allowed us to calculate the FA and ADC values in different portions of the motor pathway. FA and ADC values were collected in corpus callosum (CC), right and left corona radiata (rCR) and (lCR), right and left cerebral peduncle (rCP) and (lCR), right and left cerebellar peduncle (rCbP) and (lCbP) and right and left corticospinal tract at posterior limb of internal capsule (rCST) and (lCST).For the reliability of fiber tracking measurements, two raters experienced in fiber tracking independently performed measurements at each level. Reliability was evaluated using the intraclass correlation coefficients with a score of 0.98 (average measures), and the spatial agreement of tract shape was evaluated with Cohen’s kappa (κ), which found that it was 0.82 (both considered as excellent) .t-test was used to examine the between-group  difference in every DTI measurement. We performed comparison analyses in ALS patients based on disease phenotype, cognition, genetic data and progression rate. Univariate analyses were completed using either a chi-squared test  or independent samples t-test (for continuous variables). A comparative analysis between groups was performed using one-way ANOVA for continuous variables and Pearson’s chi-square tests with Bonferroni correction for categorical variables. Survival was calculated from diagnosis to death/tracheostomy or censoring date. The Pearson and Spearman’s rank correlation were used to correlate FA and ADC with clinical features. For each result, a p-value of < 0.05 was used to demonstrate a statistically significant difference.All statistical procedures were carried out with SPSS Version 25.0 . The two-sample Forty-one ALS patients underwent MRI with DTI and were included in the study. The demographic, clinical and disease progression features are summarized in p-value < 0.05), with diffuse lower values of the FA in patients and, in turn, higher levels of the ADC values. In detail, the FA map revealed the lowest values in the ALS population in the bilateral CST, in the bilateral CR and in the right CbP, while the highest ADC values were detected in the CC and in the bilateral CP  and a trend for ADC in the left CST . No significant correlations were found with sex. Considering clinical parameters, the ALSFRS-R at baseline was directly correlated with the FA values of the right CbP ; in addition, a correlation also between ALSFRS-R and right CP was evident as a trend . In terms of FVC% progression over the disease, downward relations were observed between delta FVC% and the FA values in the right CR and right CbP , meaning that a faster decrement in FVC% could be associated with lower FA values in these two regions at baseline. No significant correlations were observed between the FA and the ADC values and the ALSFRS-R subscores. Similarly, no significant correlations were found between diagnostic delay and diffusivity alterations.By correlating diffusivity measures in the selected regions and patients’ demographic variables, we observed a direct correlation between age at diagnosis and ADC in the left CP and in the right CR   .Disease phenotype. Of 41 ALS patients, 32 had spinal onset and 9 had bulbar onset. The mean ALSFRS-R score at baseline was significantly higher in the bulbar group compared to the spinal group ( = 0.05) , left. Ip-value = 0.04 for FA and 0.02 for ADC) (The median of ALSFRS-R rate of progression in our cohort was 0.79/month (SE: 1.80). Consequently, we divided our cohort based on the rate of disease progression in patients above and patients under the cut-off of 0.79/month. for ADC) , right.Gathering patients both for cognitive profiles  and gene mutations , we did not observe significant differences in the FA and ADC values between subgroups (data not shown).In the present study, we evaluated the white matter microarchitecture alterations, measured by the fractional anisotropy and the apparent coefficient diffusion, in a cohort of ALS patients, and the relationships between the diffusivity changes and the geno/phenotypic features of patients.Overall, we found widespread changes in DTI values in our cohort, with a significant reduction in the FA values measured in CC, bilateral CR, CP, CbP and CST, in ALS patients compared to the normative values acquired within our Department of Radiology. The greatest evidence exhibited a reduction in the FA values in the bilateral CR, extended to the CC, and the bilateral CP, more evident in the right side. In turn, higher values of the ADC were observed in the CC and the bilateral CP. FA reduction in the CR has been reported in previous DTI studies and suggested as an area allowing junctions with the frontal one ,30. In tALSFRS-R is a functional rating scale commonly used in evaluating patients with ALS. The scale measures the activities of daily living and global function and usually shows a relatively linear decline with disease progression, correlating with physiological measures of progress and with survival . In thisThe absence of significant correlations between ALSFRS-R subscores and the FA and the ADC values, in our opinion, does not have great clinical significance in consideration that all patients were at the disease onset and, consequently, the values of ALSFRS-R were most elevated and similar between items.In our cohort, we did not find any statistically significant differences between the FA and ADC values in sporadic patients and c9Orf72 mutation carriers. Clinically, our C9Orf72 group is relatively younger than the sporadic one; in addition, the c9Orf72 group shows a higher percentage of bulbar patients and higher incidence of cognitive impairment than the sporadic group. These findings are in line with the literature . SimilarThe main strength of our study is related to an accurate clinical characterization of the patients, both at baseline and during the follow-up, where patients were quarterly evaluated for the entire disease duration. Limitations of this study are, firstly, related to small sample size, particularly the number of familial patients in the absence of other mutations apart from C9Orf72 patients, making a strong DTI differentiation between sporadic and familial patients impossible. Secondly, the absence of a specific scale for UMN involvement may have led to underestimation of the correlation between DTI values and clinical features. In consideration of the non-invasiveness, the reliability and the easy reproducibility of the method, we believe that brain MRI with DTI analyses may represent a valid tool usable as a diagnostic biomarker in motoneuron diseases. However, to achieve this goal, further investigations must be carried out. Future directions should include a larger group of familial patients and longitudinal data in order to enhance the clinical and prognostic significance of our findings.