Little is known about skipping breakfast and breakfast patterns (BP) and their evaluation according to sociodemographic, clinical, lifestyle, cardiometabolic and nutritional data in children and adolescents with congenital heart disease (CHD). This cross-sectional study with 232 children and adolescents with CHD identified the prevalence and patterns of the breakfast, described these according to sociodemographic, clinical and lifestyle characteristics, and assessed their association with cardiometabolic and nutritional markers. Breakfast patterns were identified by principal components, and bivariate and linear regression analysis were applied. Breakfast consumption was observed in 73% of participants. Four BP were identified: pattern 1 “milk, ultra-processed bread, and chocolate milk”, pattern 2 “margarine and processed bread”, pattern 3 “cold meats/sausages, cheeses and butter/cream” and pattern 4 “fruits/fruit juices, breakfast cereals, yogurts, and homemade cakes/pies and sweet snacks”. Family history for obesity and acyanotic CHD were associated with breakfast skipping. Younger participants and greater maternal education were associated with greater adherence to pattern 1 and pattern 4. Older participants and longer post-operative time showed greater adherence to pattern 3. No association between skipping breakfast or BP and cardiometabolic and nutritional markers was observed. Nonetheless, the findings reinforce the need for nutritional guidance for healthy breakfast, aiming to reduce the consumption of ultra-processed foods and to prioritize fresh and minimally processed foods.  Children and adolescents with congenital heart disease (CHD) are more likely to develop overweight or obesity and changes in cardiometabolic markers, such as increased blood pressure . This isFeeding behavior during childhood plays an important role in preventing excess body weight and changes in metabolic markers related to developing acquired cardiovascular diseases in adulthood . EvidencBesides having breakfast, the composition of this meal has important implications for cardiometabolic health , consideIn children and adolescents with CHD, as far as we know, there has been only one study carried out in Brazil, which identified six DPs of the global diet and observed that an unhealthy DP  was associated with a higher risk of central adiposity, a healthy DP  was associated with a decreased risk of central adiposity, and a low dairy DP  was inversely associated with carotid intima-media thickness .In this context, the primary objectives of this study were to identify the prevalence of skipping and breakfast consumption, to describe the breakfast patterns, and to evaluate the sociodemographic, clinical and lifestyle characteristics according to the skipping and breakfast consumption and breakfast patterns of children and adolescents with CHD. Second, we aimed to investigate the association of skipping breakfast and breakfast patterns with cardiometabolic and nutritional markers in this population.A cross-sectional study was carried out with children and adolescents with CHD previously undergoing a cardiac procedure, who were monitored in pediatric cardiology outpatient clinics in two reference hospitals in southern Brazil, from January to July 2017. ® 3.01 software  based on the study outcome variables , assuming a type 1 error (α) of 0.05, type 2 error (β) of 0.20, and 95% confidence interval. Considering that the highest prevalence among outcomes was 26.9% obesity, as assessed by air displacement plethysmography  in Brazilian children and adolescents with CHD [Sample size calculation was performed using the OpenEpiwith CHD , the stuInclusion criteria were: (I) age between 5 and 18 years; (II) diagnosis of CHD; and (III) after therapeutic catheterization or cardiac surgery for CHD. Exclusion criteria were: (I) secondary diagnosis of malignant neoplasm; (II) chromosomal anomalies; (III) primary or secondary familial dyslipidemia; (IV) diabetes mellitus or hypothyroidism; and (V) presence of acute or chronic inflammatory diseases in the last 15 days. The data are from the Floripa CHild study , a longitudinal study aiming to investigate risk factors for atherosclerosis in children and adolescents with CHD.This research was approved by the Research Ethics Committee at Joanna Gusmão Children’s Hospital, Brazil (no. 1.672.255/2016) and was conducted following the Declaration of Helsinki. The children and adolescents obtained written authorization from their legal guardians and agreed to participate in the study. The assessment of food consumption was based on three 24 h recalls on non-consecutive days (two on weekdays and one on weekend), using the multiple-pass technique ,23. ® (NDSR) software, 2017 version , which includes the name and meal times to obtain specific food consumption data by meals. This software uses the United States Department of Agriculture (USDA) database as the main database. Initially, the nutritional equivalences of the foods available in the software were checked on Brazilian charts [A photographic album to aid in reporting the portion sizes of food intake was used . The recn charts , and Bran charts . The datn charts ,29.This study adopted the concept of breakfast based on guardians of children and adolescents identification during the application of 24 h recalls . BreakfaThe breakfast patterns were generated only with data from participants who had this meal in the three 24 h recalls. One hundred twenty-seven breakfast food items were reported. Foods with consumption frequency lower than 5% were excluded from the analysis.Food items were grouped based on the similarity of nutritional composition and their respective degrees of processing, according to the NOVA  classifiThe breakfast patterns were derived by principal component analysis. The Kaiser–Meyer–Olkin (KMO) statistical test was performed to verify the applicability of the factor analysis, and a KMO value of 0.55 was considered acceptable . The eigFor each component retained, a score was generated for each participant. The score was calculated considering the amount in grams of each food group multiplied by the factor loading of this item in the pattern, with higher scores corresponding to greater adherence to a specific breakfast pattern.Sociodemographic data such as age (in years), sex , mother’s education (<10 years of schooling and ≥10 years of schooling), and family history for obesity (absent/present) were collected.Clinical information such as the classification of congenital heart disease (cyanotic/acyanotic)  and the Sedentary behavior was assessed by the number of hours spent leisurely in front of the television, computer/similar and/or electronic games and time spent sitting per day, categorized as no, <8 h/day, and yes, ≥8 h/day, and in hours/day .®, São Paulo, Brazil), by a trained nutritionist. The waist circumference percentiles for sex and age were calculated [Waist circumference (cm) was measured with the participant in the standing position on the iliac crest and at the end of a normal expiration , using alculated .® Body Composition System, COSMED, Concord, CA, USA) following the calibration procedures described by the manufacturer [Body composition was performed using air displacement plethysmography . The HDL-c concentration was obtained by the direct method, in vitro. Non-HDL-c values were obtained by subtracting total cholesterol from HDL-c. LDL-c concentrations were calculated using the Friedewald formula [®, Siemens Healthcare Diagnostics Inc., Newark, DL, USA), expressed in mg/L. The measurement of systolic and diastolic blood pressure was performed with a mercury sphygmomanometer and an appropriate cuff according to the arm circumference , following the protocol of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents (NHBPEP), expressed in percentiles and calculated according to sex, age, and height percentile [Fasting glucose concentration was assessed using the colorimetric enzymatic method and was expressed in mg/dL. Total cholesterol and triglycerides were determined by the enzymatic method  or median and interquartile range (IQR) and relative and absolute frequencies.t test were used. To assess the sociodemographic, clinical and lifestyle sample characteristics according to adherence to the breakfast pattern, the scores of each one of the four breakfast patterns were categorized as below or above the median, with children and adolescents with CHD above the median showing greater adherence to the breakfast pattern. In the bivariate analysis, between the breakfast consumption or the breakfast patterns and sociodemographic, clinical and lifestyle sample characteristics, the chi-square test and Student’s p < 0.20) and potential confounders for cardiometabolic and nutritional markers described in the literature [® software version 13.0 .Simple and multiple linear regression by the forward selection procedure was performed to investigate the association between breakfast skipping or factorial scores of each breakfast pattern (independent variables) and cardiometabolic and nutritional markers (dependent variables). Asymmetric variables were transformed into logarithmic and later into exponential numbers. The covariates considered in multiple analyses were based on the bivariate analysis , chromosomal syndrome (n = 7), under five years old (n = 4), over 18 years old (n = 12), and losses (nephrotic syndrome n = 1). Thus, 232 children and adolescents with CHD participated in the study. Of the 232 children and adolescents with CHD, 73% had breakfast consumption. The mean energy intake for breakfast among children and adolescents with this meal was 78.3 .p = 0.001) and those with acyanotic CHD (p = 0.01) were associated with breakfast skipping. Characteristics of the total study population according to skipping and breakfast consumption are shown in Among participants who skipped breakfast (27%), most were girls (57.1%), had a family history for obesity (52.4%), and 66.6% had acyanotic CHD. Bivariate analysis showed that participants with a family history for obesity  and to the breakfast pattern 4 of fruits/fruit juice, breakfast cereals, yogurts, and homemade cakes/pies and sweet snacks (p = 0.001), and older participants showed greater adherence to the breakfast pattern 3 of cold meats/sausage, cheeses and butter/cream (p = 0.003). Maternal education was associated with the breakfast pattern 1 of milk, UP bread, and chocolate milk (p = 0.01) and the breakfast pattern 4 of fruits/fruit juices, breakfast cereals, yogurts, and homemade cakes/pies and sweet snacks (p = 0.002), with participants whose maternal education level was greater than 10 years showing greater adherence to these patterns compared to participants with lower maternal education. In addition, the longer post-operative time of the participants was associated with greater adherence to the breakfast pattern 3 of cold meats/sausage, cheese, and butter/cream (p = 0.04)  .In the multiple linear regression analysis, after adjustments for confounding factors (Adjustments 1 and 2), no association was found between skipping breakfast and cardiometabolic markers and nutritional status markers .The cardiometabolic and nutritional markers were also not associated with any of the four breakfast patterns in the multivariate analysis adjusted for potential confounding factors  .To date, this is the first study that identified skipping, breakfast consumption and the breakfast patterns and assessed their association with sociodemographic, clinical and lifestyle characteristics as well as cardiometabolic and nutritional markers in children and adolescents with CHD. In this cross-sectional study carried out in southern Brazil with children and adolescents with CHD, 27% of the participants skipped breakfast. Breakfast skipping was associated with a family history for obesity and participants with acyanotic CHD. Among those who consumed breakfast, four breakfast patterns were identified. The first pattern characterized by the high consumption of milk, UP bread, and chocolate milk showed greater adherence in younger patients and between those with higher maternal education. The second pattern, constituted by the high consumption of margarine and processed bread, was not associated with any analyzed variables. The third pattern characterized by the high consumption of cold meats/sausages, cheeses, and butter/cream showed greater adherence among older patients and with a longer mean postoperative time. The fourth pattern characterized by the high consumption of fruits/fruit juices, breakfast cereals, yogurts, and homemade cakes/pies and sweet snacks showed greater adherence in younger patients and those with higher maternal education.The frequency of breakfast consumption among the participants in this study was 73%, similar to that found in previous studies carried out in Brazil (79 to 91%) ,48,49, JIn this study, no association was found between breakfast consumption and cardiometabolic and nutritional markers. This fact can be partially explained by our definition of breakfast, as we did not consider the occasional consumption of breakfast. In a study in Southern California  of overwThe breakfast pattern 1 “milk, UP bread and chocolate milk” identified in this study confirms the results of the research by Marchioni et al. 2015) , demonst , demonsThe breakfast pattern most consumed by children aged 9 to 11 years in France  includedThe breakfast pattern 3 of “cold meats/sausages, cheeses and butter/cream” in this study corroborates what was observed in a study carried out with schoolchildren aged 7 to 13 years in southern Brazil, in which the “Traditional Brazilian Pattern” was made up of bread, cheese, sausages, and coffee with milk, which was the most consumed among the three identified breakfast patterns . In addiA similar result to breakfast pattern 4, “fruits/fruit juices, breakfast cereals, yogurts, and homemade cakes/pies and sweet snacks”, was found in a study carried out in the United States  with heaIn the present study, the older age of the participants was associated with greater adherence to the “cold meats/sausages, cheeses and butter/cream” pattern, which is in line with a study carried out in Salamanca that identified an increase in the consumption of dairy products and fruits in adolescents . The youThe four breakfast patterns identified in this study can be considered mixed and were composed of UP and P products, which are formulations made mainly of substances derived from food and chemical additives, containing little or no whole food; they are more energy-dense, rich in saturated fats, trans fats and added sugar, and they are low in protein, dietary fiber, and micronutrients . FurtherSimilar to breakfast skipping, cardiometabolic and nutritional markers were not associated with any of the four breakfast patterns in the present study. Only one study carried out in the United States  investigThe main strengths of this study include, first, the originality of the study; second, the collection of food consumption data was obtained by three 24 h recalls on non-consecutive days; third, the sample size was representative of the population studied; finally, the use of an a posteriori approach to identify the breakfast patterns, which is a new and representative approach of the food consumption profile , making However, the present study had limitations: first, the cross-sectional design, which made it impossible to establish a causal relationship between breakfast consumption or identified breakfast patterns and cardiometabolic and nutritional markers; second, there is limited consensus as to what defines the breakfast meal. How eating occasions are defined has been shown to affect how eating patterns are quantitatively characterized. Leech et al. (2021)  suggesteIn the present cross-sectional study in children and adolescents with CHD, a high frequency of breakfast skippers and four breakfast patterns considered mixed and composed of ultra-processed foods were identified. These findings reinforce the need to promote intervention strategies to improve the lifestyle through food and nutrition education for a healthy breakfast, promoting the reduction in the consumption of ultra-processed foods and prioritizing fresh and minimally processed foods by this population. Thus, a longitudinal study with the addition of other biomarkers or a combination of inflammatory markers could contribute to the investigation of the association between breakfast patterns and cardiometabolic and nutritional markers.


PTEN) hamartoma tumor syndrome (PHTS) is a cancer predisposition syndrome characterized by an increased risk of developing benign and malignant tumors, caused by germline pathogenic variants of the PTEN tumour suppressor gene. PTEN gene variants often present in childhood with macrocephaly, developmental delay, and/or autism spectrum disorder while tumors and intestinal polyps are commonly detected in adults. PHTS is rarely associated with childhood brain tumors with only two reported cases of medulloblastoma (MB). We report the exceptional case of an infant carrying a germline and somatic pathogenic variant of PTEN and a germline and somatic pathogenic variant of CHEK2 who developed a MB SHH in addition to intestinal polyposis.Phosphatase and tensin homolog ( There aSusceptibility to develop intestinal polyps is one of the most distinctive features of PHTS, involving up to 95% of PHTS patients throughout life . Bowel pPTEN and in CHEK2, who presented a significantly early onset of MB SHH (15 months), in addition to a remarkably early picture of hamartomatous intestinal polyposis.Here we report the first pediatric case of PHTS with both a germline and somatic variant in 2.APC, BRCA2, PALB2, PTCH1, PTCH2, SUFU, PTEN, TP53, CHEK2, and GPR161), according to the manufacturer’s protocol . The presence of deletions and duplications in PTCH1 and SUFU genes on peripheral blood was also excluded by multiplex ligation-dependent probe amplification (MLPA) according to the manufacturer’s protocol .The patient and his legal guardians conferred informed consent for the study. A centralized review of histological characterization was performed. Molecular genetics studies were performed on genomic DNA extracted from peripheral blood using a next-generation sequencing (NGS) panel including medulloblastoma and cancer predisposition genes  and height  were normal, while his head circumference was above normal . On arrival to the hospital, at the age of 15 months, he presented with macrocephaly (+3.0 SD) and psychomotor delay with major weaknesses related to language skills as detected by Griffiths Developmental Scales.The surgical removal was fraught with difficulty, despite neuroimaging suggested a superficial, almost extra-axial lesion. The tumor was in fact very hard and bled profusely, to the point of reminding more of a hemangioblastoma, with a complex pattern of intratumoral vessels, than of an MB, which was moreover completely isodense at the pre-operative computed tomography (CT) scan. Complete resection was confirmed by postoperative magnetic resonance imaging (MRI) . CerebroThe histological examination revealed an embryonic neoplasm characterized by the presence of nodular and internodular areas. The nodular areas showed elongated aspects and consisted of neurocytic-type cells immersed in a fibrillar stroma. In the internodular areas, the cells were markedly hyperchromic with frequent mitosis. The immunohistochemical investigation showed a pattern coherent with MB SHH. The cells were positive for synaptophysin in the nodular areas; positivity was observed for GAB1, YAP1, and Filamin A. The proliferation index evaluated with Ki67 was high in the internodular areas (about 30%).APC, BRCA2, PALB2, PTCH1, SUFU, and TP53) (PTEN gene (NM_000314.6) determining the missense change p.Tyr27Asn (rs746128825), previously reported in association with PHTS   were fouith PHTS . This side novo nature of the variant.Segregation analysis performed on the parents confirmed the CHEK2 gene (NM_007194.3). The analysis of this gene was recently included in the panel of genes studied in patients with MB at our center, as an association between CHEK2 and MB is reported in the literature, although not well established  in the ablished . The varablished .PTEN, c.388C > G (p.Arg130Gly) in addition to the germline change p.Tyr27Asn . This variant has been reported in the literature in individuals with clinical features characteristic of a PTEN-related disorder and identified as somatic variant in multiple malignancies  in gnancies . The p.Ativities . The varPost-surgical chemotherapy was performed according to the Italian Association of Pediatric Hematology and Oncology MB infant reccomandations. It consisted in three courses of induction chemotherapy  and two courses of high-dose thiotepa  followed by autologous hematopoietic stem cell transplantation . Four yeAt 3 years of age, the patient presented with blood and mucus in stools, inappetence, recurrent abdominal pain and weight loss. For these reasons a colonoscopy was performed, and colic polyposis was found . Some skin lesions compatible with PHTS  were alsPTEN regulation of the PI3K-AKT/mTOR pathway  was diagnosed and removed. The following year (at 5 years of age) severe hypoglycaemia was found and the patient needed to positionate a sensor and initially started feeding with the nasogastric tube. Severe hypoglycemia, although not always present in PTHS, is described in the literature as part of the clinical picture [always linked to  pathway ].4.PTEN associated with a germline and somatic variant of CHEK2, with a phenotype characterized by macrocephaly, DD, skin lesions, and very early onset of MB SHH and intestinal polyps.We present the first known case of a child carrying a germline and somatic pathogenic variant of PTEN low allele frequencies, in whom genetic analysis was performed only at the somatic level. Several studies have shown that PTEN variants are associated in 5% of cases with the development of CNS tumors : if large deletions encompass these genes the phenotypic expression can include features of both PHTS and JPS, most typically with juvenile polyposis of infancy (JPI), an aggressive subtype of JPS characterized by severe gastrointestinal symptoms, including diarrhea, intestinal bleeding, rectal prolapse, protein-losing enteropathy with a high risk of intussusception and consequently high infant mortality  and is considered to be likely pathogenetic for cancer predisposition syndromes. However, the role of this variant is not yet fully understood, and it might be speculated that it elicits its effect in a context of polygenic inheritance, contributing to cancer risk in association with other susceptibility alleles and increasing the oncological recurrence risk in the family (The frameshift variant in the e family .5.PTEN. The role of CHEK2, instead, remains uncertain at this time. The discovery of a PTEN germline mutation, even if in childhood, should induce the clinician to promptly provide genetic counseling in order to assess and monitor the occurrence of other PHTS clinical features and set up careful surveillance.Although the association is rare, the panel of genes to be tested in the presence of an MB SHH could be extended to AMC, AM, EA, and LB: conceptualization. AMC and SR: investigation and writing—original draft preparation. AMC, SR, LB, EA, MM, and AntC: data curation. AM, LB, EA, MM, FT, GSC, and AT: writing—review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thymallus thymallus. The testes were examined microscopically with a transmission electron microscope to find out details of the structure and morphology of the grayling germ cells, spermatozoa and some somatic cells. The grayling testis has a tubular shape, with cysts or clusters of germ cells within seminiferous lobules. The spermatogenic cells, including spermatogonia, spermatocytes, and spermatids, can be found along seminiferous tubules. There are electron-dense bodies in germ cells from the primary spermatogonia to secondary spermatocyte stages. These undergo mitosis to reach the secondary spermatogonia stage, when they form primary and secondary spermatocytes. Spermatids undergo three different stages of differentiation during spermiogenesis, characterized by the level of chromatin condensation, elimination of cytoplasm, and the occurrence of the flagellum. The midpiece of spermatozoa is short and contains spherical or ovoid mitochondria. The sperm flagellum has an axoneme with nine doublets of peripheral microtubules and two central microtubules. The result of this study is valuable to be used as a standard reference for germ cell development, which is of great importance to get a clear insight into the process of grayling breeding practice.This study aimed to examine the ultrastructure of spermatogenic stages and mature spermatozoa in the European grayling,  The finCyprinus carpio  is a key native freshwater rheophilic fish species in central, northern, and eastern Europe. This species is considered an ecologically valuable bioindicator of water contamination, local habitat, and food supply . Testes samples were carefully washed with de-ionized water and kept in ice box prior to transfer to the laboratory. Then in the laboratory, the gonads were fixed with Bouin’s solution. After 24 h, the specimens were transferred to 70% ethanol, and the usual histological method was used. Specimens were dehydrated in ascending series of ethanol solutions and then cleaned in a series of xylene solvents (three times). The specimens were then embedded in paraffin and cut into 5 μm sections. The slides containing the samples were stained with hematoxylin and eosin (H&E) and then observed under a light microscope .2.2.To produce tissue specimens for TEM observation of the testicular structure of grayling, the gonads were minced and fixed in 2.5% paraformaldehyde/glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.4) at 4°C for 2 h. Subsequently, the samples were washed thrice with the same buffer solution and postfixed in 2% osmium tetroxide solution in the buffer solution at 4°C for 1 h. The samples were then dehydrated in ascending acetone concentrations  for 15 min each and embedded in resin . Ultrathin sections (0.07–0.08 μm) were sliced using a UCT ultramicrotome . These were mounted on copper grids, double-stained with uranyl acetate  for 30 min, and counterstained with 1% lead citrate for 20 min. A 1010 transmission electron microscope  operated at 80 kV was used to examine the samples .2.3.This study was conducted in compliance with the Czech Republic’ regulations (law nos. 166/1996 and 246/1992). Further, it was granted permits (nos. 58672/2020-MZE-18134 and 33446/2020-MZE-18134) under the NAZV Project QK22020144. Sampling was carried out with the permission of the Departmental Expert Committee for the Authorization of Experimental Projects of the Ministry of Education, Youth and Sports of the Czech Republic (permit no. MSMT-8155/2022-4).3.3.1.Macroscopic examination of grayling testes showed a similar to that of other teleost fishes. Their testes are paired, elongated, and attached to the dorsal wall of the body by a mesorchium. The grayling testis is tubular, with germ cells arranged in clusters inside the seminiferous lobules. Each tubule contains numerous cysts with different germ cells, and spermatogenesis occurs at multiple sites within these cysts. Spermatogonia are in peripherical locations along the length of the lobule, whereas spermatocytes, spermatids, and spermatozoa are found in the interior . Sertoli3.2.TEM observations revealed that, according to their pattern and level of chromatin condensation, germ cells in the spermatogenetic process of the grayling could be classified into ten steps. Primary spermatogonia contain a large spherical nucleus, whereas secondary spermatogonia have small heterochromatin clumps both on the inner surface of the nuclear membrane and in the central area of the nucleus. Primary spermatocytes can be categorized into four subcellular stages, including leptotene, zygotene, pachytene, and diplotene spermatocytes that have thick and long heterochromatin blocks or cords. Secondary spermatocytes contain a nucleus with large blocks of heterochromatin along the inner facet of the nuclear envelope. Spermatids can be divided into three stages.Primary spermatogonia (Sg1) contain a large spherical nucleus with very few blocks of euchromatin . Each nuPrimary spermatocytes are categorized into leptotene (LSc), zygotene (ZSc), pachytene (PSc) and diplotene DSc) spermatocytes based on their distinct patterns of chromatin organization. The early LSc is spherical in shape, with a large spherical nucleus . Small lSc spermaThere are three stages in spermatid differentiation during spermiogenesis, i.e., spermatid I (St1), spermatid II (St2), and spermatid III (St3), which are classified according to the elimination of organelles and cytoplasm, flagellum formation, patterns of chromatin condensation, and the occurrence of the flagellum. The nuclei of successive spermatid stages vary from round to oval and finally, cylindrical. St1 are grouped in cysts that lie close to the lumen . Each ceMature spermatozoa (SZ) are characterized by their elongated oval nuclei composed of a head, short midpiece, and single flagellum . The matSertoli and Leydig cells, which are somatic cells, are observed within the tubules. Sertoli cells surround the spermatogonia and spermatid cells . The nuc4.Liposarcus anisitsi , and was granted permits (nos. 58672/2020-MZE-18134 and 33446/2020-MZE-18134 under the NAZV Project QK22020144). All samples were collected with the permission of the Departmental Expert Committee for the Authorization of Experimental Projects of the Ministry of Education, Youth and Sports of the Czech Republic (permit no. MSMT-8155/2022-4).HD: conceptualization, investigation, and writing – original draft preparation. HD and AI: methodology. HD, AI, OM, TPe, JK, and TPo: sample collection. HD, FD, and AI: formal analysis and data curation. HD: re-sources. HD, FD, and TPe: writing – review and editing. TPo: project administration and supervision. All authors contributed to the article and approved the submitted version.This research was funded by the Ministry of Agriculture of the Czech Republic, Project NAZV QK22020144.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


High-risk newborns are exposed to neonatal conditions such as prematurity, very low birth weight, and congenital malformations that can affect development and behavior. Coronavirus disease 2019 (COVID-19) restraint and control measures have been identified as important stressor events and cumulative risk factors for behavioral changes in these children. This study examined social isolation-related factors that contribute to internalizing and externalizing behavior problems in children already at risk for neurodevelopmental disorders. This cross-sectional, multicenter study included 113 children (18 months to 9 years) who were followed in reference services for neonatal follow-up in tertiary units of the public health system in the city of Rio de Janeiro, Brazil. Behavior was assessed using the child behavior checklist, and a structured questionnaire was used to assess sociodemographic aspects. In the bivariate analysis, prematurity was associated with externalizing problems and change in eating habits with internalizing problems. The logistic model indicated that both parents having completed high school and both sharing care of the child were protective factors for behavioral problems; however, reports of sleep problems and living with another child were risk factors. In conclusion, the study identified internalizing and externalizing behavior problems related to prematurity and aspects of family structure and routine in children at risk. The findings confirm the importance of family functioning for child health and family-centered interventions. The corMany studies have described the emotional health of specific population subgroups, such as those with autism spectrum disorder, attention-deficit hyperactivity disorder, cancer and other neurodiverse conditions, or chronic physical health conditions . We founIn addition to the aforementioned biological risks, social issues such as low socioeconomic status and low maternal education are also risk factors for neurodevelopment. According to the cumulative risk model, when an individual is exposed to more than one risk factor, these factors have complex interacting effects . The greIdentifying factors associated with behavioral problems during the COVID-19 pandemic may facilitate the design of mental health programs in critical situations. Therefore, this study aimed to analyze factors contributing to behavioral problems in children already at risk for neurodevelopmental disorders, considering biological, sociodemographic, and sociofamilial routine aspects of social isolation.2.2.1.This multicenter cross-sectional study was conducted in three tertiary hospitals of the public health system in the city of Rio de Janeiro, Brazil, including children at risk admitted to the NICU of these hospitals and followed up in the respective outpatient clinics. The study was conducted between September and December 2020.2.2.The sample was composed of all children between 18 months and 12 years of age who were followed up in these units and who attended the consultations when they resumed regular care during the study period, following the recommended contingency measures. The exclusion criteria were genetic syndromes and cerebral palsy with greater impairment  .2.3.The following blocks of variables were considered:Demographic and perinatal aspects: sex, age at assessment, gestational age, birth weight, bronchopulmonary dysplasia, intracranial hemorrhage, retinopathy of prematurity, perinatal asphyxia, and necrotizing enterocolitis.Sociodemographic and economic aspects: parents’ schooling, the presence of the father living with the child, main caregiver of the child, living with another child, employment status of the parents, unemployment of the mother or father because of the pandemic, government assistance , COVID-19 diagnosis in the family nucleus, and attendance at daycare or school before the pandemic was declared. Bolsa Família is a government cash transfer program for poor and extremely poor families in Brazil , and Auxcaregiver’s perception of worsening sleep quality and change in eating habits. Exclusively for preschool and school children, the following were also included: receiving educational activities from daycare/school to be done at home, online classes (synchronous or asynchronous), maintaining communication with friends, and leisure activities at home such as using electronic games, listening to music and dancing, and playing games with the family (such as board games).Child’s routine: Behavioral assessment: child behavior checklist [CBCL] syndromic categories.2.4.Information on birth and perinatal morbidity was obtained from the medical records. A structured questionnaire was used with items about social, family, and routine aspects of the child.To assess the child’s primary caregiver, the respondent was asked to indicate whether it was the mother, father, both (mother and father), or someone else. It was explained that the person responsible for the child’s care, such as feeding, education, guidance on hygiene habits, behavior, and social interaction, is considered a caregiver.To assess changes in eating habits, the respondent should consider changes in schedules, increased consumption of snacks or processed foods, and skipping one or more meals during the pandemic.To assess children’s behavior, we used the CBCL instrument , which wThe CBCL scores generate syndrome scales, which refer to a set of items that tend to occur together. In CBCL-1, four syndromes are characterized as internalizing problems , two as externalizing problems , sleep problems, and stress. The CBCL-2 scores generate three syndrome scales to characterize internalizing problems  and two for externalizing problems (rule-breaking behavior and aggressive behavior), in addition to social problems, thought problems, attention problems, and stress. Regardless of which CBCL model is being scored, the total behavior problem score is the sum of all items on the instrument. The score is categorized as normal, borderline, or clinical. For the syndrome scales, scores of 65–69 points are considered borderline, and > 70 points are considered clinically significant. For the internalizing, externalizing, and total problem scales, scores of 60–63 points are considered borderline, and ≥ 64 points are clinically significant. For analysis, participants were grouped into clinically/borderline (>60 points) and normal according to their CBCL scale scores.2.5.The database was prepared using the EPIINFO program version 7.2.4.0, and statistical analyses were performed using IBM SPSS Statistics version 23 . The children were divided into three age groups: toddlers (up to 35 months), preschoolers (36 months to 71 months), to whom the CBCL-1 was administered, and schoolchildren (≥6 years), to whom the CBCL-2 was administered. Data from toddlers were analyzed separately from preschoolers and schoolchildren because of expected differences in feeding, play, and socialization routines. Categorical variables were described by frequency of occurrence and numerical variables by median and interquartile range. Pearson’s chi-squared test, with Fisher’s correction when appropriate, was used to compare demographic, perinatal, sociofamilial assessment, and child routine variables among behavior categories found on the CBCL, considering a significance level of <0.05. A multivariate logistic model was used to evaluate the association between the independent variables of demographics, perinatal, sociofamilial, and child routine changes with the dependent variables of the internalizing scale and the externalizing scale. Both regression models included age and all variables with a value of p of <0.20 in the bivariate analyses. Borderline and clinical scores on the CBCL were combined into a single dependent variable. A significance level of <0.05 was considered in the final model.2.6.This study was approved by the ethics committees of the participating institutions , and children were included in the study after their legal guardians signed the informed consent form.3.Most questionnaires were answered by the mothers (90.3%), while another 6 (5.3%) by the fathers and 5 (4.4%) by the grandmothers.During the study period, 133 children were eligible, of whom six were excluded . The parents of the three children refused to participate, and 11 children did not complete the tests. Thus, 113 children were evaluated and distributed as follows: 48 (42.5%) up to 3 years of age, 31 (27.4%) preschoolers, and 34 (30.1%) schoolchildren. The mean age of the participating children was 4 years, ranging from 18 months to 9 years. a Brazilian regular assistance, and 55.8% had access to Auxílio Emergencial, an emergency government assistance due to the pandemic. Regarding COVID, 24.8% of the families reported that a family member living in the same house had been affected. In this sample, 18.8% of toddlers, 90.3% of preschoolers, and all schoolchildren attended daycare or school before the pandemic. Parents reported that 31.9% of children’s sleep quality was affected by the pandemic, and 42.5% had a change in eating habits.In the sample, the children were born preterm (74.3%), were small for gestational age , and had bronchopulmonary dysplasia (15%), intracranial hemorrhage (12.4%), necrotizing enterocolitis (6.2%), perinatal asphyxia (8.8%), and other associated comorbidities (23%). Regarding the mothers’ education, 17.7% had completed elementary school, 9.7% had incomplete high school, 61.1% had completed high school, and 11.5% had a college degree. As for the fathers, 21.2% had completed elementary school, 17.7% had incomplete high school, 47.8% had completed high school, and 6.2% had a college degree. Regarding financial support from the government, 29.2% reported receiving funds from Bolsa Família, Toddlers and preschoolers have a comparable distribution of the proportions of normal, borderline, and clinical scores on the internalizing scale, with 20.8% of toddlers and 22.6% of preschoolers scoring in the clinical range. A high percentage of school-aged children (44.1%) scored in the same range. On the externalizing scale, a higher proportion was noted in the clinical range in toddlers (31.3%) compared with preschoolers (9.7%) and schoolchildren (20.6%) .On the internalizing scale, a higher prevalence in the clinical range for toddlers was found for emotional reactivity (18.8%) and for preschoolers for anxiety/depression and withdrawal/isolation (both at 12.9%). Among schoolchildren, 35% scored outside the normal range for the anxiety/depression component . On the externalizing scale, the most common component in the clinical range was attention problems for toddlers (27.1%) and preschoolers (9.7%), and it was aggressive behavior for schoolchildren (8.8%) .The variable relating to maternal schooling was significant in both the internalizing and externalizing scales for toddlers. In the externalizing scale in the same age group, prematurity was also significant .In preschoolers and schoolchildren, paternal schooling was related to the internalizing and externalizing scales. Changes in eating habits were associated with internalizing problems .The logistic model indicated that having both parents who completed high school was associated with a lower chance of clinical or borderline problems on the internalizing scale of the CBCL; however, the presence of sleep problems was associated with a higher chance of such problems. Children whose parents took care of the children together were less likely to report clinical or borderline problems on the externalizing scale of the CBCL; however, living with another child contributed to a greater chance of externalizing problems .4.This study analyzed factors that contribute to behavioral problems in at risk children, taking into account biological, sociodemographic, and routine-related aspects in social isolation. In the bivariate analysis, prematurity was associated with externalizing behaviors in toddlers, and changes in eating habits were associated with internalizing problems in preschoolers and schoolchildren. The model indicated that having both parents who completed high school and both sharing childcare were protective factors for behavioral problems; however, living with another child and reporting sleep problems were risk factors.The association between prematurity and externalizing behavior in toddlers was attributed to the high prevalence of attention problems, which comprise the externalizing scale. The findings are consistent with studies conducted before the pandemic. In this study, frequencies in the clinical range for externalizing problems were higher in toddlers and internalizing problems in schoolchildren. Social isolation limited families’ access to support networks and allowed for a broader observation of family functioning. In the present study, results showed that children whose parents shared childcare had fewer externalizing problems, confirming the importance of the presence of support in the family nucleus. The division of child-related care is one of the components of coparenting, defined as the joint and reciprocal involvement of both parents in the education, and decisions about their children’s lives . GiannotIn this study, an association was found between parents’ education  and children’s behavior. Higher education is likely to enhance various skills, including problem-solving and coping skills , which mContrary to our expectations, the results indicated a greater chance of externalizing problems for those living with another child. These findings corroborate reports from studies of family dynamics during the pandemic. According to Changes in family routines affected behaviors. Children with sleep problems were twice as likely to have internalizing problems, confirming other findings that sleep quality affected children’s psychological well-being during the pandemic . Sleep dThis study has some limitations. First, it has a cross-sectional design; therefore, it lacks information on the pre-pandemic behavior of these children. Second, appointments were resumed with a limit on the number of children treated per shift, which reduced the number of participants. Third, families more affected by the pandemic may not have had the health or financial conditions to attend the hospital, as well as it is possible that parents concerned about their children’s behavior might have sought care more often, which may have led to a selection bias, but not a systematic one.Despite these limitations, this study stands out because it adds to our knowledge of the behavior of children considered at high-risk in the context of a developing country, where many accumulate biological risk and social vulnerability. In addition, the study was conducted in reference hospitals for neonatal and follow-up care, with trained teams and in full compliance with the measures recommended by health authorities during the pandemic. The face-to-face evaluation allowed the inclusion of families with limited access to the Internet or electronic devices who are often not represented in surveys that use online questionnaires.In conclusion, the results found have implications for professionals who care for these children because the identification of factors allows for preventive intervention and early guidance of families. The study identified internalizing and externalizing behavior problems in at risk children and that these were related to prematurity, family structure, and routine. Risk factors identified included parental educational disadvantage, number of children in the home, and lack of a structured daily routine, whereas the presence of reciprocal parental caregiving support was a protective factor. The findings confirm the importance of understanding family structure to design policies and interventions that reduce negative influences on behavior and promote positive family experiences.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by (1) Instituto Fernandes Figueira - Fundação Oswaldo Cruz, (2) Hospital Universitário Gaffrée e Guinle - UNIRIO and (3) Hospital Universitário Pedro Ernesto - Universidade do Estado do Rio de Janeiro. The patients/participants provided their written informed consent to participate in this study.MMé and MV contributed with the conception of the study. MMé was responsible for the coordination of the study, contributing with the analysis and interpretation of the data, the writing of the manuscript, and its critical revision. MV, AR, LV, MR, FA, RC, EM, and CC contributed with material preparation and data collection. AR, RC, EM, and CC applied the CBCL. LP and SG-J performed the statistical analysis and contributed with the interpretation of the results. MV wrote the first draft of the manuscript. AR and MMé contributed with the subsequent revisions. LV, MR, FA, RC, EM, CC, and MMo contributed with the critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Programa INOVA FIOCRUZ (grant no. VPPCB-005-FIO-20-2-67 / 48400640976574), and by FAPERJ (project no. 210.925/2021).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Conclusions: CBD may improve patients’ ability to differentiate taste strengths during chemotherapy.Introduction: Taste alteration is a common adverse effect of chemotherapy. This study aimed to investigate the effect of cannabidiol (CBD) on Lean Body Mass (LBM), and taste alterations during oxaliplatin- or paclitaxel-based chemotherapy. Methods: LBM was estimated by bioelectrical impedance analysis (BIA), and taste perception was evaluated by a randomized sensory test of six samples: sweet, salt, and umami, all in weak and strong concentrations. Taste perceptions were scored on visual analog scales. Patients in the intervention group received oral CBD 300 mg/day for 8 days; patients in the control group did not. Patients were followed for three cycles of chemotherapy. Results: Twenty-two/ten patients (intervention/control group) were eligible. No effects on LBM were demonstrated. At baseline, the control group was able to differentiate between weak and strong saltiness and weak and strong sweetness but lost this ability after three cycles of chemotherapy. At baseline, the intervention group was unable to differentiate between the concentrations but gained the ability to significantly differentiate between weak and strong sweetness ( Chemotherapy-induced taste alteration is a common adverse effect of cancer chemotherapy. Taste alterations and other adverse effects such as constipation and nausea often result in malnutrition leading to weight loss, decreased quality of life, and increased risk of treatment toxicity ,2,3,4,5.The use of medical cannabis and cannabinoids in the management of chemotherapy-induced side effects has a significant global interest . Still, Although cannabinoids have been used for the treatment of nausea, arthritis, and epilepsy, adverse effects limit treatment possibilities ,10,11. TThe effect of CBD on symptoms and disease has primarily been investigated in animals and in patients diagnosed with epilepsy ,16. ThisThe exact pathophysiological mechanisms of taste alterations are unknown, but increased production of inflammatory cytokines and denervation of taste bud sensory afferents have been implicated ,2. A proThe effect of CBD on chemosensory perception has, to the best of our knowledge, not been investigated in humans previously. This pilot study investigates the potential effect of CBD on taste alteration in patients receiving chemotherapy.A non-randomized, controlled design was used to investigate if CBD has an effect on lean body mass (LBM) and perception of taste in patients with cancer receiving oxaliplatin or paclitaxel-based chemotherapy. The study was carried out at the Department of Clinical Oncology, Zealand University Hospital, Roskilde, Denmark.The intervention group enrolled patients already included in the phase II CINCAN-2 study . The CINEligible patients were 18 years or older, diagnosed with cancer, chemotherapy-naïve, and scheduled to receive at least 4 cycles of paclitaxel- or oxaliplatin-based chemotherapy. Patients unable to answer patient-reported outcome (PRO) measurements, unable to cooperate with the study procedures and patients already using cannabis were ineligible.The study was not randomized but used a control group of patients receiving the same kind of chemotherapy. The control group fulfilled the same inclusion and exclusion criteria as the intervention group but did not receive CBD.n = 10) were included from December 2020 to March 2021. Patients in the intervention group (n = 22) were included from March 2021 to August 2021. Patients in the control group did not receive CBD.Patients in the control group . Paclitaxel (intravenous infusion) was given in combination with carboplatin (intravenous infusion). Both regimens were administered on a thrice-weekly schedule. Chemotherapy dose, dose reductions, and adverse events were registered and prescribed by an oncologist not involved in this study.The intervention group self-administered an oral dose of 150 mg CBD twice a day , starting the day before chemotherapy and continuing for eight days totally in every cycle of chemotherapy. This dose schedule was chosen because animal models suggest that neuroinflammatory changes in nerve cell ganglia occur in the first 24 h and up to 6 days following injection of classical chemotherapeutic agents . Since pThe dose of CBD was apportioned by the patients using a syringe. The CBD oil was manufactured, packaged, and labeled by Glostrup Pharmacy adhering to GMP standards, as described earlier . PatientPatient LBM was estimated by bioelectrical impedance analysis (BIA) using the Medical Body Composition Analyzer Seca 515/514. Estimates were carried out before each of the patients’ first four cycles of chemotherapy. The first estimate, before the first round of chemotherapy, is considered baseline. Before BIA, the patient’s age, height, and sex were verified from the patient’s medical journal and entered into the device. During measurements, the patients stood barefoot with hands and feet placed on electrodes while being measured with frequencies of 5 and 50 kHz. BIA was displayed as fat-free mass, in this study used as an equivalent to LBM.Patients were followed until before their fourth course of chemotherapy. Patients receiving neoadjuvant chemotherapy had surgery scheduled after the third course of chemotherapy. As surgery has an impact on cancer patients’ metabolic response and constitutes a risk of nutritional decline , we deciChange in patients’ ability to differentiate taste was measured by a single-blinded (patients were blinded), randomized sensory testing before each cycle of chemotherapy. The first test, before the first chemotherapy, is considered baseline.Patients tasted six different taste samples in random order: weak and strong sweet, weak and strong salt, and weak and strong umami. The weak tastes were above the detection threshold, and the strong tastes were above the recognition threshold for patients with cancer ,24,25.Patients were asked to taste the numbered samples 1–6 and indicate, on a 100 mm long visual analog scale (VAS), how strong they perceived each of the six different tastes. A value of 0 mm indicated weakest; 100 mm indicated strongest. Baseline measurements were performed before the first chemotherapy. In this study, we wanted to test if CBD had an effect on taste alterations in patients receiving chemotherapy. Participants were patients without any formal training in sensory tasting. Consequently, we limited the taste material to consist of three of the “basic” tastes only, namely sweet, umami, and salty . Bitter ®) was prepared following directions on the package. One of the six tastes  in each. The samples were sealed individually with VitawrapBefore the sensory test, one of each of the six samples was collected from the freezer. The samples were numbered 1–6. The randomization was carried out by rolling a die and allocating each number (1–6) to a new glass. If the die showed a number already picked, it was rolled again until 6 glasses had a number. The same sequence was not repeated on the subsequent measurements.The frozen samples were transferred to a plate with numbers according to the randomization. They were then heated in a microwave oven at 800 W for 60 s, ensuring a temperature at the center of each sample of at least 75 °C. The samples were cooled for 1 min and transferred back to the polystyrene glass. All six samples were served on a serving tray with a single-use plastic teaspoon and a napkin.t-test was used to compare doses of chemotherapy. Analyses were performed using Rstudio . A p-value < 0.05 was considered to be statistically significant.The Mann–Whitney U test was used to compare data from the sensory test. A two-sample Sample size was calculated with the primary outcome being an effect of CBD on LBM. The power was 0.20, and the minimal relevant difference in change in LBM was set as 5% since this is the diagnostic criteria for cachexia . The numclinicaltrials.gov (NCT04585841). All patients gave written informed consent before enrolment in the study, and the study was conducted in accordance with the principles of the Helsinki Agreement.The protocol was approved by the Regional Scientific Ethics Committee (Zeeland Region) (SJ-860) and the data protection agency (REG-141-2020). The study was registered at The intervention group consisted of 22 patients. The control group consisted of 10 patients. Patients in the control group were included from December 2020 to March 2021 and patients in the intervention group from March 2021 to August 2021. Standard regimes of chemotherapy remained the same in these periods. Baseline patient characteristics are provided in p = 0.78/0.88), starting dose of capecitabine (p = 0.38) or starting dose of carboplatin (p = 0.22).There was no significant difference between the two groups as concerns the starting dose or cumulative dose of oxaliplatin after three cycles of chemotherapy (p = 0.029), a higher starting and cumulative dose of paclitaxel (p= 0.01/>0.01), as well as a higher cumulative dose of carboplatin (p = 0.01).The intervention group received a larger cumulative dose of capecitabine (The starting dose of chemotherapy in the control and intervention groups as well as the cumulative dose during the three cycles of chemotherapy can be seen in No significant difference was found in LBM between the two groups (results not presented).p = 0.01) and weak and strong salty taste (p = 0.03). Prior to the fourth cycle of chemotherapy, only the intervention group had the ability to differentiate between weak and strong sweet taste and weak and strong salty taste.At baseline, the control group had the ability to significantly differentiate between weak and strong sweet taste  . A previTaste receptor cells have a turn-over of approximately 10 days . During Chemotherapy-induced taste alterations could be caused by intravenous chemotherapy entering the oral cavity via tears and saliva, but this should decrease with the clearance of the drug ,35. The p = 0.03) as well as weak and strong sweet taste (p = 0.01). This ability disappeared at test four (p = 0.14 in both tests). The intervention group could not distinguish between salty (p = 0.39) and sweet tastes (p = 0.20) at baseline but could significantly distinguish at test four for both salty (p = 0.04) and sweet (p = 0.03).At baseline, the control group had the ability to distinguish between strong and weak salty taste , as compared with six men in the intervention group (27%). The sex difference is not considered to affect the results of taste perception. Although some studies report taste perception to be affected by sex, other studies do not find this difference .The control group is a little younger than the intervention group  and younger than the target population . Though Development of paclitaxel- or oxaliplatin-induced neuropathy is more likely to increase with increasing age , and thiIn the sensory test, more patients in the intervention group than in the control group did not answer all sample questions  ,43, and In this study, we investigated the short-term effect of CBD on the gustatory effect in patients receiving chemotherapy. Future studies should include larger patient samples, use a randomized design, and also investigate the long-term effect of CBD to explore the delayed regeneration of taste buds. Including more tastants, such as sour and bitter tastes, would also be relevant.We have solely focused on the ability to differentiate taste, not on the differences of hypogeusia (decline in taste sensation), hypergeusia (increase in taste sensation), augusia (complete lack of taste functions in the tongue), parageusia (perversion of the sense of taste), cacogausia (unpleasant taste that does not originate from food or beverage), phantageusia , xerostomia, oral ulcers, or oral hygiene in general or their potential effect on the results ,42,43. TIn summary, this study indicates that CBD might have an effect on patients’ ability to differentiate between weak and strong sweet and salty tastes during chemotherapy. Before the fourth course of chemotherapy, the intervention group had gained the ability to differentiate between weak and strong salty and sweet tastes, whereas the control group lost this ability during chemotherapy.If CBD helps diminish adverse effects of chemotherapy such as taste alterations, it would be relevant to also investigate the prevalence of malnutrition and the effect on patients’ perceived quality of life during and after chemotherapy. Data from this study will assist in the future design of studies to explore the effect of CBD on the preservation of taste during chemotherapy.


Colorectal cancer (CRC) is the third most common cancer in Iran. Inflammation plays an essential role in developing CRC. A dietary pattern called the empirical dietary inflammatory pattern (EDIP) has recently been designed based on the inflammatory potential of the diet. Therefore, the present study aimed to investigate the impact of EDIP on the risk of CRC.The current case-control study was conducted on 142 controls and 71 CRC cases in three general hospitals and Hospital Cancer Organization in Tehran, Iran. We calculated EDIP by a semi-quantitative food frequency questionnaire. The association between EDIP and CRC were evaluated by logistic regression. The level of significance was p < 0.05.The results revealed that people who were in the highest tertile of the EDIP had higher odds of CRC (in the adjusted model: odds ratio (OR) = 3.74; 95% confidence interval (CI): 1.38–10.14; P = 0.011).The present study demonstrated the potential role of dietary-induced inflammation in developing CRC. In the current study, an increase in the intake of red meat, processed meats, and refined grains was observed in the higher EDIP tertiles compared to the lower tertiles. Consequently, to decrease the risk of CRC, it is recommended to reduce the consumption of these foods. Colorectal cancer (CRC) is slow-growing cancer that begins as a tissue growth or tumor in the inner lining of the colon or rectum . CRC is Inflammation plays an essential role in developing CRC . SpecifiThe relationship of EDIP with metabolic syndrome , cognitiEpidemiological studies have not consistently shown an association between the levels of inflammatory markers and the risk of CRC . To the The present case-control study was conducted on three hospitals that included Shariati, Imam Hossein, and Ayatollah Taleghani and 19 CRC surgery departments of Imam Khomeini Hospital Cancer Organization in Tehran. The study sample size was calculated based on the odds ratio (OR) = 0.45, α = 0.05, and β = 0.20 based on Terry and et al. study .The newly diagnosis cases with CRC were those who were 40 to 75 years old at the time of study, their diseases were confirmed pathologically. Characteristics of the control group included random selection from the same hospitals and hospitalization for acute and non-neoplastic conditions at the same time, without diet-related chronic diseases. The most common reasons for hospitalization included fractures and sprains, bone and joint disorders, and disc disorders. Each patient with CRC was matched in age and gender with two patients in the control group.At first, 267 patients (89 cases and 178 controls) were assessed, and 24 participants were excluded by inclusion and exclusion criteria (8 cases and 16 controls). In addition, 10 cases and 20 controls were excluded for incomplete food frequency questionnaire (FFQ), unwillingness, and total energy intake (out of mean ± 3 standard deviations (SDs)). Finally, 71 participants in the case and 142 participants in the control group were included for statistical analyses. This study was approved by the Medical Research and Ethics Committee of Shiraz University of Medical Science (IR.SUMS.SCHEANUT.REC.1401.011). Details of the current study have been previously published .To evaluate dietary intake, we used a semi-quantitative FFQ that included 168 foods and drinks (with standard portion sizes). Based on previous studies, it has been determined that this FFQ has good reliability and reproducibility , 29.The participants stated the frequency of consumption of one meal of each food item daily, weekly, monthly and yearly before CRC diagnosis. Then, the data obtained from their answers were converted into daily consumption frequency. After the portion size of each food item consumed was converted to grams, the consumption amount of each food item in grams was obtained by multiplying the portion size by the number of daily consumption. The edible portion of foods was determined using household measurement guidelines . The nutTabung et al.‘s study was used to calculate the EDIP score . This stThe case and control groups were interviewed by skilled interviewers. Information obtained from this interview included socio-demographic characteristics, physical activity, family history of CRC, medication information , vitamin/mineral supplements), smoking habits, cooking methods, and dietary intake.To accurately measure the participants’ weight, they were asked to wear minimal clothing and without shoes, and they were weighed with an accuracy of 0.1 kg. SECA scale (Germany) body measuring device with 0.1 cm accuracy was used for height measurement. Their height was measured while standing without shoes. Recumbent length was acquired for hospitalized patients.The body mass index (BMI) was calculated as weight (in kg) divided by the square of height (in meters) and classified according to the World Health Organization standard for adults . A validThe Kolmogorov-Smirnov test was utilized to evaluate the normality of variables. The chi-square test was used to assess the relationship between classified variables. Mann-Whitney or independent samples T-test was used to determine the association between continuous variables. Also, the Kruskal-Wallis test was used to evaluate food group intake across the tertiles of EDIP. The association between EDIP and CRC were evaluated by logistic regression and adjusted for energy and fiber intake, smoking, physical activity, BMI, family history of CRC, household income, education level, the common way of cooking meat, and taking ibuprofen, aspirin, acetaminophen and mineral supplement use. SPSS software (version 26.0) was used for statistical analysis. The level of significance was p < 0.05.The baseline characteristics of study population are shown in Table Based on Table Table In the current case-control study, we examined the association between the inflammatory potential of the diet, as measured by EDIP, and developing CRC odds. The findings showed that greater adherence to EDIP was associated with a higher odds of CRC. Also, the results indicated that more adherence to this dietary pattern was associated with more intake of red meat, processed meats, and refined grains. Therefore, limiting the consumption of the mentioned foods may play a role in reducing the odds of CRC.EDIP was recently developed, and its validity was evaluated in two independent cohorts of men and women . It has As previously mentioned, the findings showed a positive association between EDIP and CRC odds. Our findings are in line with previous research. Tabung et al. demonstrated that the higher EDIP was positively related to the CRC risk . Also, aThe precise mechanisms by which inflammation leads to CRC are gradually becoming elucidated . The traAs shown in the current study, higher EDIP scores were related to more intake of red meat and processed meats. Processed meats and various components of red have been proposed to contribute to chronic inflammation and the risk of disease, such as nitrosamines, heme iron, and advanced glycation end products . Also, fAlso, more intake of refined grains was observed in people who had more adherence to EDIP. An association between increased consumption of refined grains and the risk of CRC has been shown . ChatenoThe role of other dietary patterns on inflammation has also been investigated. A systematic review study on interventional and observational studies showed that there is an inverse relationship between dietary patterns such as Dietary Approaches to Stop Hypertension (DASH) diets, and the Mediterranean diets and pro-inflammatory biomarkers . Also, iThe strengths of the present study are that we controlled for several potential confounding factors for examining the relationship between EDIP and CRC risk. A valid and reliable questionnaire was also used to collect data. However, although the effect of several confounding variables was adjusted, there might be other confounding variables that we did not consider in the study. Also, like other case-control studies that examine dietary intake before diagnosis, this study may have recall bias.This case-control study demonstrated the potential role of dietary-induced inflammation in developing CRC. In the present study, an increase in the intake of red meat, processed meats, and refined grains was observed in the higher EDIP tertiles compared to the lower tertiles. Consequently, to decrease the risk of CRC, it is recommended to reduce their consumption. Also, it is possible participants change their diet after diagnosis of each diseases and improve their usual diet, for this reason, dietary intake assessed after CRC diagnosis and newly CRC diagnostic cases were selected. Since this study was conducted in Iran, the results cannot be generalized to other populations. Therefore, more research is necessary to clarify the role of this dietary pattern in CRC risk.


Results: After screening and inclusion using a machine language, the results showed that RBM10 was significantly highly expressed in the lung adenocarcinoma tissues. The related signaling pathways were mainly concentrated in ncRNA processing, rRNA metabolic processes, ribosome biogenesis, and the regulation of translation. The qRT-PCR for 20 lung adenocarcinoma tissues showed that the expression of RBM10 in these tissues was significantly different from that in normal tissues (p = 0.0255). Immunohistochemistry analysis and cell immunofluorescence staining also confirmed that RBM10 was involved in the immune regulation of lung adenocarcinoma tissues, and the number of immune cell aggregations was significantly higher than that of the control group. RBM10 regulates B cell memory-CIBERSORT (p = 0.042) and B cell memory-CIBERSOTRT-abs (p = 0.027), cancer-associated fibroblast-EPIC (p = 0.001), cancer-associated fibroblast- MCPCounter (p = 0.0037), etc. The risk score was significantly associated with the sensitivity of patients to lapatinib (p = 0.049), nilotinib (p = 0.015), pazopanib (p = 0.001), and sorafenib (p = 0.048). Conclusions: RBM10 can inhibit the proliferation and invasion of lung adenocarcinoma cells through negative regulation and promote the apoptosis of lung adenocarcinoma cells through immunomodulatory mechanisms. The expression level of RBM10 affects the efficacy of targeted drug therapy and the survival prognosis of lung adenocarcinoma patients, which has a certain guiding significance for the clinical treatment of these patients.Objective: The RNA-binding protein RBM10 can regulate apoptosis during the proliferation and migration of pancreatic cancer, endometrial cancer, and osteosarcoma cells; however, the molecular mechanism underlying lung adenocarcinoma is rarely reported. Recent studies have detected multiple truncated and missense mutations in RBM10 in lung adenocarcinoma, but the role of RBM10 in lung adenocarcinoma is unclear. This study mainly explored the immune regulation mechanism of RBM10 in the development of lung adenocarcinoma and its influence on sensitivity to targeted therapy drugs. Methods: The transcriptome data of CGAP were used to analyze the RNA-seq data of lung adenocarcinoma patients from different subgroups by using the CIBERSORT algorithm to infer the relative proportion of various immune infiltrating cells, and Spearman correlation analysis was performed to determine the gene expression and immune cell content. In addition, this study utilized drug trial data from the GDSC database. The IC50 estimates for each specific targeted therapy were obtained by using a regression method, and the regression and prediction accuracy were tested via ten cross-validations with the GDSC training set. An immunohistochemical test was performed on the samples of 20 patients with lung adenocarcinoma in the subcomponent analysis of immune cells, and the protein expression of RBM10 in lung adenocarcinoma tissues was verified by cellular immunofluorescence assays. Nucleic acids were extracted at low temperatures, and qRT-PCR was used to verify the expression levels of the mRNA of RBM10 in lung adenocarcinoma tissues and normal tissues ( In cell mitosis, chromosomal separation errors lead to whole chromosome aneuploidy mutation in daughter cells, or DNA damage leads to chromosomal structural changes, resulting in phenomena such as gene translocation, deletion, inversion, and fracture, which are all referred to as genomic instability ,2. RBM10A related study performed exon and transcriptome sequencing on 98 lung adenocarcinoma precursor lesions and 99 invasive adenocarcinoma tissue specimens and identified RBM10 as a significantly mutated gene in the progression of lung adenocarcinoma . It is cTargeted drug therapy is currently one of the effective means used for the treatment of lung cancer, and it is widely used in the systemic treatment of advanced patients, preoperative neoadjuvant therapy, and postoperative adjuvant therapy, for example . In the In this study, we obtained sequencing data from the TCGA database of lung adenocarcinoma patients with high expression of RBM10 in order to analyze patient sensitivity to targeted drugs. Through statistical analysis of the data, for the lung adenocarcinoma patients with positive RBM10 expression, the survival prognosis and drug sensitivity of the lung adenocarcinoma patients showed statistically significant differences. This also suggests that RBM10 is involved in the regulatory process for the mechanisms underlying resistance to targeted drugs for lung adenocarcinoma, which has important clinical significance for the effective survival of patients . A numbehttps://portal.gdc.cancer.gov/, accessed on 1 January 2022), GEO , ImmPort , and GDSC (Genomics of Drug Sensitivity in Cancer)  databases to download transcriptome data on lung adenocarcinoma and clinical data. These data include gene expression data, miRNA expression data, copy number variation data, and so forth. The downloaded data were transformed and organized. Perl  was used to run scripts (merge.pl and move.pl) to convert gene IDs into gene names in the data, and tumor tissue samples were grouped with normal tissue samples according to the instability of the genome. The corresponding mutation data were obtained.We used information from the TCGA , while 25% of the genes with the lowest mutation frequency were included in the low mutation group . RBP genes were extracted from the downloaded data and analyzed at the same time to obtain the prognostic RBP-related genes of lung adenocarcinoma. In addition, we also downloaded the subcomponent data of immune cells from ImmPort and corrected the data.In R software (V3.6.0), the “edgeR” package was used to identify the downloaded TCGA data of 361 samples of gene transcription and 107 clinical samples’ data, adjusted|logFC|>2 and corrected https://cibersortx.stanford.edu/, accessed on 1 January 2022) to screen the samples by transforming the gene expression matrix into the matrix of immune cells, that is, the composition of the various subtypes of immune cells in the tumor tissue. For genomics data based on the largest drug database (GDSC—Genomics of Drug Sensitivity in Cancer) , we used R software package “pRRophetic” to predict each tumor sample for targeted drug sensitivity. The IC50 estimate for each specific targeted therapy was obtained through regression analysis.CIBERSORT is an R or web version tool for deconvolving the expression matrix of human immune cell subtypes based on the principle of linear support vector regression. For more chip expression matrices, to determine an unknown mixture containing similar cell types to the expression of the matrix, deconvolution analysis is superior to other methods . This method is based on a known reference set and provides a gene expression signature set for 22 immune cell subtypes. We used CIBERSORT  and low-frequency mutation genomes (the gene stability group). In this study, TCGA data and GEO data were used for double validation. At the same time as constructing and verifying the prognostic model of the unstable genome, experimental group A, validation group B, and group C were used as the statistically comprehensive results of the two groups.p < 0.05 indicated that the difference between the two groups was statistically significant. An AUC curve was frequently used to evaluate the feasibility and accuracy of the model, which was constructed with big data, and AUC > 0.6 indicated that the model had a good ability for prediction and evaluation.A Kaplan–Meier curve was used to evaluate the survival time of unstable genomes, and The core module can be determined by calculating the correlation coefficient between the feature vector gene of each module and the sample feature information. Genomically unstable genes are a series of genes with high connectivity and modular connectivity. One of the goals of WGCNA is to find the key genes of the target modules. In general, the key genes in this submodule are biologically more important than in the global network. Module identity can be used to measure the importance of genes in a module, and it has been shown that M is positively related to module connectivity. Therefore, the major genes of a specific module are selected based on the M value. If the value of M in a specific module is the largest, the gene is considered to be a key gene.https://david.ncifcrf.gov/summary.jsp, accessed on 1 January 2022), an online database, to carry out gene ontology for target mRNA genes in the ceRNA network. We also used GO annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, with p < 0.05 as the screening condition. GO analysis included biological process (BP), cell component (CC), and molecular function (MF) analysis.We used the application DAVID , a PPI network was constructed, medium confidence was taken as the truncation standard, and the nodes without mutual relationships in the network were hidden. The PPI network information obtained from the STRING database was imported into Cytoscape software (V3.8.2). The MCODE plug-in Cytoscape was used to screen the key modules and identify the seed factors at the same time, and the MCC algorithm in the CytoHubba plug-in was used to screen the hub genes.Target mRNA genes in the chromosome instability group gene network were uploaded to the STRING online database  extracted from the tissue were dewaxed in xylene and rehydrated in graded alcohol, and endogenous peroxidase activity was blocked by infiltration into 0.3% hydrogen peroxide. Next, the sections were rinsed with phosphate-buffered saline (PBS) (PH 7.2), and all of the sections were treated with immunoglobulin IgG as a secondary antibody. After being washed with PBS, the sections were incubated with DAB  to observe the peroxidase reaction. The final sections were stained with hematoxylin, dehydrated with graded alcohol, and mounted on resin supports. The stained sections were examined with a microscope.Isolated and cultured cells were fixed with 4% paraformaldehyde for 15 min. Next, the cells were blocked with 2% BSA in PBS and then treated with 0.2% triton for 15 min. The diluted primary antibodies were added as follows: anti-RBM10  and then the secondary antibody . In addition, the cells were stained with DAPI  and observed under a fluorescence microscope.The data in this study were obtained from the TCGA and GEO databases. After screening and identifying the intersection of the lung adenocarcinoma gene set and the chromosome gene instability group gene set, a separate network was constructed to analyze the prognosis model of the lung adenocarcinoma chromosome instability gene set, and an ROC curve was used to evaluate the prediction accuracy of the prognosis model. In addition, univariate and multivariate analyses of gene clinical risk and functional enrichment analysis of related signaling pathways were further analyzed. In this study, another risk model of lung-adenocarcinoma-associated RNA-binding protein was constructed to analyze the distribution of upregulation and downregulation of differential genes and the interaction analysis of the PPI network. The second model was also evaluated by an ROC curve and a survival curve. In the later stage of this study, qRT-PCR assays, immunohistochemistry analysis, cell immunofluorescence assays, and pathological characteristics analysis were performed to investigate the mechanism of drug sensitivity and the immune microenvironment .p < 0.001)  c.p < 0.05)   d–f. In a= 0.750) g–i.The validation of the prognosis model of lung adenocarcinoma is mainly divided into two parts: One is the validation of the training group, and the other is the validation of the test group. The “Pheatmap” package in R software was used to organize the downloaded TCGA data and GEO data, calculate the risk coefficient of lung adenocarcinoma patients, calculate the distribution matrix of surviving and dead patients, analyze the differential expression of mutant genes in the prognostic heatmap related to immune infiltration, and successfully construct the prognosis model of lung adenocarcinoma. From left to right, we found that, with an increase in the mutation rate of mutated genes, the prognostic risk coefficient of lung adenocarcinoma patients also increased, and deaths were concentrated in the high-risk population area on the right, suggesting that the high mutation rate of prognosis-associated unstable genes may be associated with the high prognostic risk and mortality of lung cancer .p < 0.001), gender , M0 (p < 0.001), N1–3 (p = 0.001), stage I–II (p = 0.050), stage III–IV (p < 0.001), T1–2 (p = 0.002), and T3–4(p = 0.020)  were divided into high- and low-risk groups according to the expression level of RBM10, and the independent predictive efficacy was evaluated. The survival curve results showed that there were statistically significant differences in the risk of some clinical factors in patients with RBM10 (+) lung adenocarcinoma, including age (= 0.020) I–VI.p = 0.044). A nomogram model of related RBM10 was constructed and showed that it can inhibit the occurrence and development of lung adenocarcinoma  item in ncRNA processing, rRNA metabolic processes, rRNA processing, the regulation of cellular amide metabolic processes, RNA catabolic processes, and RNA changes such as modification and RNA splicing increased significantly. CC: preribosome, nucleolar part, Cajal body, ribosome, P granule, etc. MF: catalytic activity, acting on RNA, mRNA3′-UTR binding, translation regulator activity, ribonuclease activity, single-stranded RNA binding, ribonucleoprotein complex binding, etc. .In this study, the high expression of RBM10 was classified into a low-risk group, and the low expression of RBM10 was classified into a high-risk group. Univariate and multivariate independent analyses of survival and prognosis of lung adenocarcinoma were conducted using the data of the test group and the control group, respectively, and the results of both groups of data included RBM10 a,b.p < 0.05)   c,d, and = 0.705) e,f.The “Pheatmap” package in R software was used to organize the downloaded TCGA data, calculate the risk coefficient of lung adenocarcinoma patients, calculate the distribution matrix of surviving and dead patients, analyze the differential expression of RBM10 in the immune-infiltration-related prognostic heatmap, and successfully construct the prognosis model of lung adenocarcinoma. From left to right, we found that, with the decrease in RBM10 expression, the prognostic risk coefficient of lung adenocarcinoma patients also increased, and deaths were concentrated in the high-risk population area on the right, which also proved that its prognostic correlation with low expression of RBM10 may be associated with the prognostic risk and high mortality of lung adenocarcinoma .p < 0.05)  a,b.Nomogram analysis was also used in this study, and the results also showed that SMAD9, KHDRBS2, and RBM10 had a certain risk correlation with the survival rate of lung adenocarcinoma patients c. FinallThe immunohistochemistry analysis showed that the cell nuclei of the carcinoma tissues were significantly enlarged and deeply stained, and RBM10 was expressed in the nucleus and cytoplasm a–f. The Microscopically, adenocarcinoma is composed of new cubic and columnar cells, tending to form an adenoid structure supported by a fibrous matrix, with large or irregular nuclei, distinct nucleoli and mucin in the cytoplasm g–i.We identified RBM10 as a tumor suppressor gene through database screening and PCR assays, and it plays a negative regulatory role in the occurrence and development of lung adenocarcinoma. In this study, RBM10 inactivation was classified as indicating high risk, and high expression of RBM10 was classified as indicating low risk. Based on this feature, we performed a subgroup analysis of clinically relevant factors  for lung adenocarcinoma. The results indicated that male patients with RBM10 inactivation aged over 65 years with G3 tumor stage were at higher risk I. Some sp < 0.05)  f–i.Further verification in the A549 lung adenocarcinoma cell line showed that the mRNA expression level of RBM10 was lower than that of the control group a. AccordLung adenocarcinoma (LUAD) is the most common histological subtype of non-small-cell lung cancer (NSCLC), accounting for approximately 40% of lung cancers. RBM10 belongs to the RNA-binding motif protein family, which plays an important role in a variety of biological processes . More trIn this study, we collected the expected lung cancer tissues and normal paracancerous tissues of 20 patients who had not received any treatment before surgery, as well as relevant clinical data such as the age, gender, and smoking history of these patients, and further analyzed the risk correlation of independent clinical factors. The results showed that the age of the patients in the high-risk group with RBM10 deletion, their gender, and their tumor stage and grade have certain risk correlations. In the in vitro experiments conducted in this study, we first verified that RBM10 was expressed in the nucleus and cytoplasm of lung adenocarcinoma A549 cells in equal amounts by using an immunofluorescence method, and qRT-PCR showed that the expression of RBM10 in the A549 cell line was significantly lower than that in the control group. Studies ,32,33,34The mutation of RBM10 co-exists with the known lung cancer target genes KRAS, EGFR, and PIK3CA, among others . Current