To evaluate the effectiveness of transarterial chemoembolization (TACE) combined with lenvatinib and sintilimab in treating patients with midstage hepatocellular carcinoma (HCC).  Sixty-two patients with midstage HCC were enrolled in this study. All of them were firstly treated in our hospital between September 1, 2019, and March 1, 2020. According to different treatment regimens, they were divided into the control group  and the observation group . Each patient was followed up for at least 30 months to compare the short-term clinical efficacy and survival rate between the two groups. P < 0.05). The 2-year cumulative overall survival rate of the observation group was 64.5%, which was significantly higher than that of the control group (P < 0.05). The survival curve of the disease-free survival rate in the observation group was higher than that in the control group, and the difference was statistically significant .  The objective response rate (ORR) and disease control rate (DCR) of the observation group at 3 months were 77.4% and 93.5%, respectively, which were higher than those of the control group ( TACE combined with lenvatinib and sintilimab in the treatment of Barcelona Clinic Liver Cancer (BCLC) stage B hepatocellular carcinoma can effectively control the tumor progression and prolong the survival time of patients. Those preliminary findings need validation in larger studies, with a prospective design and longer follow-up. Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed and fourth most lethal neoplasm worldwide. The incidence of HCC has doubled over the past two decades, especially in the United States, Europe, Japan, and China . AccordiAt present, both the European Association for the Study of the Liver (EASL)  and the Combination therapies have been researched for liver cancer, with synergistic effects , includiIn this retrospective study, sixty-two patients with HCC admitted to the Department of Minimally Invasive Interventions and the Department of Oncology of Ganzhou Hospital affiliated to Nanchang University between September 1, 2019, and March 01, 2020, were included. All patients were required to complete clinical information, and both patients and families cooperated with the follow-up. All patients were informed verbally and in writing on the study and provided their informed consent; this procedure and the whole study protocol has been reviewed and approved by the local ethics committee. Based on the Chinese Hepatocellular Carcinoma Diagnosis and Treatment Standard, all patients were diagnosed with HCC by histopathological or clinical diagnosis. The inclusion criteria for patients in this study were (1) aged between 18 and 75 years; (2) patients with pathologically histologically or clinically confirmed HCC; (3) BCLC staging B; (4) ECOG performance status less than 2; (5) Child-Pugh class A or B; no extrahepatic metastases and/or macrovascular invasion; (6) no prior TACE/Hepatic Artery Infusion Chemotherapy (HAIC) and I-125 particle implantation or systemic therapy , and PD-1/PD-L1); (7) intrahepatic reproducible lesions according to the modified Response Evaluation Criteria In Solid Tumors (mRECIST) [μm PVA particles were slowly injected until the blood flow stopped, so as to achieve complete tumor embolization or staged compression embolization. Postoperative hepatoprotective support and symptomatic management were provided. The interval of TACE treatment was determined by imaging review.TACE was performed using the Seldinger technique, and a catheter was placed in the celiac artery or common hepatic artery for angiography. After acquiring images of the arterial phase, parenchymal phase, and venous phase, we can figure out the anatomical shape of hepatic artery, the presence or absence of vascular variation, and the location, size, quantity, blood supply type, and presence of arteriovenous fistula of then hepatic parenchyma tumor. Then, super selective cannulation to the tumor-feeding arteries for angiography was performed. Depending on the size of the tumor, the appropriate amount of chemotherapeutic drugs  was mixed with 10 ml of poppy lipiodol to embolize the tumor blood vessels. After the tumor vessel was saturated and the portal vein branches around the lesion were stagnant, 300-500 Patients received lenvatinib  orally within one month after the first TACE, at a dose of 12 mg/d (weight ≥ 60 kg) or 8 mg/d (weight < 60 kg) until tumor progression, patient death, or intolerable adverse drug reactions. Adverse drug reactions include the following: increased blood pressure, gastrointestinal reactions, thyroid dysfunction, and albuminuria, which are graded according to NCI-CTCAE version 4.03 . For graPatients should receive the first injection of sintilimab  within 1 week before discharge and then every 3 weeks thereafter. The dose and method of each administration is sintilimab 200 mg with 100 ml of saline and intravenous infusion for 30 minutes. Adverse drug reactions include macular papules, itchy skin, gastrointestinal reactions, hepatic impairment, thyroid dysfunction, rash, immune pneumonia, reactive capillary hyperplasia, and discontinuation of dosing if still intolerable after symptomatic treatment.Follow-up visits were required after the TACE procedure. The patients were followed up by inpatient or outpatient during 1 month. The deadline for follow-up was March 1, 2022. The follow-up included physical, laboratory, and imaging examinations, as well as adverse reactions. Laboratory examinations included blood routine tests, blood biochemistry tests, alpha-fetoprotein (AFP), coagulation function tests, liver and kidney function tests, thyroid function tests, and other tests. Imaging examinations include enhanced MRI of the upper abdomen and chest CT. For patients with residual tumor or recurrence detected by imaging and AFP, follow-up interventional therapy was considered. The indications for re-TACE were as follows: Child-Pugh class A or B, ECOG score 0 or 1, prothrombin prolongation time < 4 s, and platelet count > 109/l.The efficacy of cases was evaluated after each treatment and follow-up with reference to the mRECIST . Complett-test and chi-square test. The Kaplan-Meier method was used for survival analysis to plot survival curves, and log-rank test was used for comparison of survival rates. P < 0.05 means the difference is statistically significant. All statistical analyses were performed with SPSS version 20.0.The differences in the clinical characteristics between two groups were assessed by Student P > 0.05). In the observation group, 4 patients stopped taking medicine due to drug reaction during the study period.In total, 62 HCC patients were enrolled in this study, including 31 in the observation group and 31 in the control group. Each patient was followed up for 24 months. The comparison of baseline data between the two groups is shown in All 62 patients completed interventional surgery successfully. One patient developed liver abscess 1 week after operation and 42 patients developed postembolization syndrome such as abdominal pain, fever, nausea, and vomiting, while the other patients did not have obvious complications.P < 0.05).According to mRECIST, CR, PR, SD, progressive disease (PD), ORR, DCR, and other efficacy indicators of patients in the observation group and the control group at 1 and 3 months after surgery were counted and compared . The ORRP < 0.05). The progression-free survival curves of the observation group and the control group are shown in X2 = 4.313, P < 0.05).All 62 patients completed 2-year follow-up and the follow-up span was 30 months. Disease progression occurred in 46 patients during follow-up (20 in the observation group and 26 in the control group). Thirty patients died (11 in the observation group and 19 in the control group), including 25 deaths due to tumor progression, 3 deaths due to liver failure, 1 death due to upper gastrointestinal bleeding, and 1 death due to cerebrovascular accident. The six-month, 1-year, and 2-year cumulative overall survival rates of the observation group are shown in In this study, we investigated the feasibility and efficacy of TACE combined with lenvatinib and sintilimab in the treatment of intermediate-stage HCC. It was found that the one-year survival rate and two-year survival rate of patients treated with this regimen could reach 80.6% and 64.5%, respectively. This is a significant improvement compared with TACE alone. It can be a reliable option for HCC patients. This result is determined by the role of interventional therapy, molecular targeted therapy, and immunotherapy and can be deduced from the perspective of pathophysiology and pharmacology.α, KIT, and RET [TACE is an effective treatment option for intermediate stage (BCLC B) HCC , 17. How and RET , 26. We PD-1 receptor expressed by T cells can inhibit T cell proliferation and cytokine production by binding to its ligands, PD-L1 and PD-L2. For some tumor cells, when PD-1 ligand is upregulated, the immune monitoring of activated T cells can be inhibited through this signal transduction pathway –29. SintP > 0.05). With the extension of time, there was significant difference between the two groups at 3 months (P < 0.05). This result may be attributed to the respective pharmacological mechanism of TACE, lenvatinib, and sintilimab. TACE alleviated the disease mainly by blocking the blood supply of the tumor, and it played a key role in the early stage, so there was no significant difference in ORR and DCR between the two groups in 1 month. With the emergence of neovascularization and the recovery of tumor microenvironment, lenvatinib and sintilimab gradually took the lead in treatment [In this study we can find that short-term efficacy indexes such as ORR and DCR of the observation group in each period were higher than those of the control group. There was no significant difference between the two groups at 1 month (reatment , 35, so There were several limitations in this study. Firstly, this was a retrospective trial with a limited sample size, which may lead to potential bias. In the absence of a sample size calculation, and according to the unclear robustness of the conclusions in this study, the findings must be seen as preliminary results. Secondly, the treatment options in this study were determined according to the preferences of doctors or patients, which may lead to the selection bias of our study population. Thirdly, follow-up period was relatively short and the outcome for some patients has not been obtained, so it was impossible to fully evaluate PFS and OS. Fourthly, in this study, the combination regimen was only compared with TACE alone. Next, we will conduct a multivariate analysis of the efficacy among TACE, lenvatinib, and sintilimab. In addition, there are some limitations regarding the inclusion and exclusion criteria. Thereby, patients with an age over 75 years were included to eliminate high age as a potential confounder. Overall, as mentioned above, this is a preliminary study, which requires further validation in larger studies with higher follow-up periods.Within the limitations of this preliminary study, the blood flow of the tumor can be blocked by TACE to the greatest extent. Lenvatinib can effectively inhibit neovascularization and reduce collateral circulation; at the same time, sintilimab can effectively activate T cells and enhance immune response. TACE combined with lenvatinib and sintilimab appears to be an effective method for the treatment of intermediate HCC.


This study, as part of a study on the dietary lives of elderly people, investigates the association between plant food (fruit + non-starchy vegetable) intake and the prevention of periodontal disease among elderly people aged over 65 years. A total of 4514 subjects over 65 years of age participated in a dental survey, health behavior interview, and 24 h dietary recall test for the Korea National Health and Nutrition Examination Survey (KNHANES). Subjects with energy intake of less than 500 kcal or more than 5000 kcal were excluded. The results showed that plant food intake was inversely associated with the prevalence of periodontal disease in the elderly Korean population. The prevalence of periodontal disease decreased with increasing plant food consumption. Compared with those in tertile 1 (T1), subjects in tertile 3 (T3) showed a decrease in periodontal disease of about 26.7% (OR = 0.733). In conclusion, the consumption of plant food lowers the risk of periodontal disease, suggesting that it should be encouraged among elderly Koreans. Periodontal disease (PD) is characterized by microbial-associated host-mediated inflammation and is a major risk factor for tooth loss, as it reduces alveolar bone density and damages the bone structure due to plaque on the dental surface ,2,3,4. TIt is reported that 85% of the adult population over the age of 35 has PD , and accDespite the lack of previous studies on PD mechanisms, it has been reported that nutrition intake is significant in maintaining healthy teeth and oral tissues and is closely related to oral health . It is kVarious domestic and international studies have shown the relationships between the consumption of various food groups and nutrients and PD; however, the association in the elderly has not yet been investigated. Therefore, as South Korea is becoming an aged society, this study examined the correlation between diet and oral health in the elderly population. In particular, the prevalence of PD in people over 65 and its relationship with plant-based food (fruits and vegetables) were investigated. This study can contribute to the prevention of PD in elderly people and may provide basic data for dietary educational programs and policy content to improve older people’s dietary habits in preparation for a super-aged society.This study was based on the fifth and sixth Korea National Health and Nutrition Examination Survey (KNHANES) conducted in 2012 and 2015. The KNHANES is a nationwide, population-based, cross-sectional study that aims to assess the health and nutrition status of the Korean civilian, noninstitutionalized population. The KNHANES administered by the Korean Ministry of Health and Welfare and a stratified multistage probability design were used with subject selection from sampling units using household registries. It also provides basic data for framing a health policy such as for improving the nutrition of the people, preventing diseases, and developing health promotion programs . The samThis study selected elderly people over 65 years old who participated in the nutritional survey and health interview of the KNHANES 2012–2015. To examine the intake of plant food (fruit and non-starchy vegetable) of these subjects, adults who were 65 y of age or older who participated in the 24 h recall method were first selected as subjects (5893 persons). Some of the subjects who consumed less than 500 kcal or more than 5000 kcal (77 persons) were excluded. Outlier data on subjects who did not participate in the dietary survey  were also excluded 250 persons). A total of 4514 people were selected in the study . The KNH0 personsPeriodontal examination was performed using the community periodontal index (CPI) certified by the WHO  as the cSix segments were evaluated for each mouth. Upper right second molar (#17), upper right first molar (#16), upper right central incisor (#11), upper left first molar #26), upper left second molar (#27), lower right second molar (#47), lower right first molar (#46), lower left central incisor (#31), lower left first molar (#36), and lower left second molar (#37) were designated as index teeth. The CPI scores range from 0 to 4 and are defined as: 0, healthy; 1, bleeding on gentle probing; 2, dental calculus and bleeding; 3, shallow pockets of 4 or 5 mm; and 4, deep periodontal pockets of 6 mm or more. The lower the value, the better the periodontal condition [, upper lA previous study reported that a CPI score of 3 or 4 indicates clinically significant periodontitis . As withGeneral characteristics of the subjects, such as gender, age, marital status, education level, residential area, and occupation, were analyzed. Among these characteristics, age was classified as 65–74 years or ≥75 years, and marital status was classified as unmarried or married. The education level was classified as less than high school graduate (<12 years), high school graduate (12 years), or college degree or higher (>12 years). Residential area was classified as city area or rural area, and occupational status was classified as occupied or unoccupied. Finally, household income level was classified using the household income level variables in KNHANES.For health behavior and weight status, the parameters of smoking, alcohol consumption, stress, exercise, and body mass index (BMI) were analyzed. Smoking status was divided into three groups: current smokers, ex-smokers, and nonsmokers. Alcohol consumption status was divided into more than four times a week, 2–3 times a week, 1–4 times a month, and less than once a month. The stress status was categorized into “feel it very much”, “feel it a lot”, “feel a little”, and “feel it rarely”. Exercise was classified into <1 day/week, 1–2 days/week, 3–4 days/week, and ≥5 days/week. The BMI variable used for weight status was based on the Asia-Pacific Obesity Criterion.Dietary behaviors were analyzed for breakfast, snack, location of daily meal, and eating-out frequency. Food insecurity is a dietary survey questionnaire that has been included since the 2005 KNHANES, and the question, “Which of the following best represents your family’s eating habits over the past year?” was selected based on previous studies ,31. FoodFood intake was categorized into 17 food groups  using food classification code  and food intake (variable name: nf_intk) in the food  data; individual intake was obtained for each survey subject. In addition, energy, macronutrient , and micronutrient  intake and contribution ratios were calculated using the daily intake for each nutrient.Data on the intake of fruits or non-starchy vegetables among plant foods were obtained to calculate the total intake of fruits or/and non-starchy vegetables per day using the food classification code (variable names: n_fcode2 and n_fcode3) and food intake (variable name: nf_intk) variables among the variables of the dietary intake survey  of the KNHANES. Among fruits and non-starchy vegetables, based on previous studies , non-stat-test and a general linear model were conducted for unadjusted and adjusted cases, respectively, and gender, age, and energy intake were used as adjusted variables. Finally, plant food intake was categorized into three stages . A logistic regression analysis through SURVEYLOGISTIC was conducted to determine the relationship between the prevalence of periodontitis and the groups . In the groups, their intake was divided by the third quartile, and the odds ratio (OR) and the 95% confidence interval (CI) were presented. Furthermore, the influence of the variables, such as gender, age, energy intake, sodium intake, smoking, alcohol consumption, exercise frequency, stress perception, dairy intake, frequency of eating out, snack intake, food stability, and breakfast intake, was stepwise adjusted in performing multiple logistic regression analysis before conducting analysis. The p for trend value for evaluating the tendency according to the tertile was calculated using SURVEYLOGISTIC by setting the median of the third quartile as a continuous variableBecause the KNHANES data are based on stratified multistage probability sampling rather than simple random sampling data, this statistical analysis included the weight, stratification variable (KSTRATA), and colony variable (PSU: primary sampling unit). All analyses were conducted using SAS ver. 9.4 . Frequency analysis was conducted to determine the general matters related to the prevalence of periodontitis and the matters related to eating habits to show a percentage (weighted %) considering the frequency and weight. Furthermore, the intake of food and nutrients according to the prevalence of periodontitis was represented as mean and standard error using the SURVEYMEANS procedure. The significance test for this analysis was performed using SURVEYREG. A p < 0.0001). The average age was 72.15 years in the normal group and 72.06 years in the periodontal group, indicating similar age distribution. In terms of the education level and income, the proportions of those who were less educated than high school graduates and those with low income were higher in the periodontal group than in the normal group. In terms of occupation, the proportion of employed individuals was significantly higher by about 6.43% in the periodontal group than in the normal group (p = 0.0003). In terms of region, the proportion of city dwellers was higher than that of rural area dwellers (p = 0.0233).p < 0.001). In terms of alcohol consumption, the proportion of those drinking ≥4 times a week was by approximately 2.4% higher in the periodontal group (9.78%) than in the normal group (7.38%) (p < 0.001), and regarding stress, the proportion of those who “feel it very much” was lower in the periodontal group than in the normal group (p < 0.05). Regarding the parameters of exercise and weight status, there was no difference between normal and periodontal groups, with the two groups showing similar ratios.p = 0.0031). Regarding serving location of daily meals, the proportions of home and institution location were similar in both groups, whereas the proportion of commercial location was 4.29% lower in the periodontal group than in the normal group (p = 0.0317).p-value = 0.0171; potassium, adjusted p-value = 0.0009), and conversely, niacin intake was higher in the periodontal group than in the normal group . Vitamin C intake was higher in the normal group than in the periodontal group, and there was a significant difference regardless of the adjusting . There were no significant differences in the remaining nutrient intake and energy contribution of carbohydrate, protein, and fat.p-value = 0.0260). The intake of cereals and grain products, potatoes and starches, fruits, and milk and dairy products was significantly higher in the normal group than in the periodontal group, regardless of the adjusting . There were no significant difference in other food intakes between the normal and periodontal groups.p-value < 0.01, adjusted p-value < 0.01).p for trend = 0.001). In Model 2, which was adjusted for gender, age, and energy intake, the prevalence of periodontitis in the T3 group decreased by 25.4% (OR = 0.746). In Models 3, which were stepwise adjusted for smoking, alcohol consumption, stress status, weight status, intake of milk and dairy products, food security, snack, eating out frequency, breakfast, education, household income, and marital status, the prevalence of periodontitis, compared with the T1 group, was lower by 21.5% (OR = 0.785) only in the T3 group.p for trend = 0.0072). In Model 2, which was adjusted for gender, age, and energy intake, the prevalence of periodontitis in the T3 group tended to decrease by about 30.5% (OR = 0.695). In Model 3, which was additionally adjusted for alcohol consumption, stress status, and weight status, the prevalence of periodontitis, compared to the T1 group, was lower by 26.7% (OR = 0.733) only in the T3 group (p for trend= 0.0101).Furthermore, on studying the relationship with the periodontitis group according to the third quartile of fruits + non-starchy vegetable intake, which was related to the prevalence of periodontitis, the prevalence of periodontitis tended to decrease by about 25.6% (OR = 0.744) as it moved toward the T3 group (fruits + non-starchy vegetable intake range: 409.23–5899.35 g) based on the T1 group (fruits + non-starchy vegetable intake range: 0–77.13 g) in Model 1 without adjusting  classifies societies according to the proportion of elderly people: aging society (more than 7%), aged society (more than 14%), and super-aged or post-aged society (more than 20%). By 2026, it is forecasted that elderly people will make up 20.8% of South Korean society, making it a super-aged society in which one in every five people is elderly .This rapid aging of the population has raised social concerns such as health issues, the social and economic burden of elderly support, the prolongation of healthy lives, and improvement of quality of life . FurtherPD is caused by and progresses by complex risk factors . In thisThis study investigated the relationships between PD prevalence and various dietary-related factors, such as the breakfast frequency, snacking habits, serving location, and frequency of dining out. Among these factors, the frequency of snacking was negatively correlated with PD prevalence. A previous study suggested that elderly people with snacking habits have better nutritional status, which may explain this phenomenon ,46. In rAccording to previous studies, PD and obesity have a close relationship. Analyses of South Korean adults who participated in the 2013 KNHANES revealed that the odd ratio of PD in overweight subjects with a high BMI was 1.68 times higher than that in normal-weight subjects, and those with high waist circumference had a 1.37 times higher odds ratio (OR) than those with a normal waist circumference, showing a positive correlation with obesity . FurtherIn previously discussed nutritional factor effects on PD, it has been reported that calcium , proteinAmong the food groups, the consumption of milk and dairy products significantly lowered periodontitis prevalence in elderly people. This result is consistent with that of previous research using KNHANES data, which has shown milk and dairy product consumption to decrease the prevalence of PD . FurtherIn this study, the amount of fruit and non-starchy vegetable intake of South Korean elderly people was 364.15 g, which is approximately 36 g less than 400 g, which is the recommended intake by the World Health Organization (WHO) and World Cancer Research Fund (WCRF) ,34. The In non-starchy vegetable consumption, none of the three subdivided groups showed any significance regarding PD prevalence, which indicates that fruit consumption was the main factor. However, when fruits and vegetables were consumed together, the OR was reduced when compared with only fruit consumption. The combined consumption of vegetables and fruits is considered to be an important factor that reduces PD, which supports the plant-based diet (fresh fruits and non-starchy vegetables) recommendation from the WHO/WCRF ,34.The examination of the results of previous studies and this study shows that increased fruit intake and combined vegetable and fruit intake are effective in reducing PD risks in South Korean elderly. Further studies, such as research on diets for disease prevention and intervention, clinical research on fruit and vegetable ingredients that influence disease control, research on the causal relationships between nutrients and food groups and PD, randomized clinical trials, and cohort studies are required. Furthermore, from the public health viewpoint, the significance of fruit and vegetable consumption needs to be emphasized not only to the elderly population but also to younger generations to prevent and manage PD, and diet education and public health policies need to be established accordingly.There are several limitations to this study, First, the results cannot prove a causal relationship between plant-based food (fruit and non-starchy vegetable) intake and PD because the study was designed cross-sectionally. Additionally, it used only 24 h recall data, which makes it difficult to determine the general daily food intake due to the information sampled only covering one day . FurtherNext, the prevalence of PD varies depending on the different diagnosis criteria, which have different standards such as probing pocket depth and clinical attachment loss. This study has the limitation of using Community Periodontal Index (CPI)-diagnosed PD, which only considers probing pocket depth. Despite these limitations, this study is significant in that it investigated a national sample size, which enabled multivariate analysis and stratification analysis by providing information on potential confounding factors. Furthermore, the KNHANES, which represents South Korean standards, was used to investigate the relationship between periodontitis in South Korean elderly and plant-based food (fruits and non-starchy vegetables) consumption. In future studies, human application tests or long-term follow-up cohort studies are required to clarify the effects of fruit and vegetable consumption on PD from a long-term perspective.This study aims to investigate the relationship between the intake of plant foods (fruit + starch-free vegetables) and the prevalence of periodontal disease in elderly people 65 years of age or older as part of a study on the dietary lives of the elderly. A total of 4514 subjects over the age of 65 participated in dental surveys, health behavior interviews, and 24 h diet recall tests. As a result, it was shown that the consumption of plant foods was inversely related to the prevalence of periodontal disease in the elderly in Korea. The prevalence of periodontal disease decreased with the increasing consumption of plant foods. T3 (fruit + non-starchy vegetable intake range: 409.23–5899.35 g) showed an approximately 26.7% (OR = 0.733) reduction in PD in Model 3 compared with the T1 (0–23.7 g) criterion. In conclusion, this suggests that plant food (fruits and non-starchy vegetables) consumption should be recommended in order to prevent periodontal disease in elderly people. Further research should be performed to reveal the mechanism behind the effects of plant food (fruits and non-starchy vegetables) on periodontal disease. Randomized clinical trials and cohort studies should also be conducted to confirm the effects of plant food (fruits and non-starchy vegetables) on periodontal disease. It will be necessary to establish nutrition education and policies.


The average administration of micronutrients and antioxidants  was significantly less than the dietary reference intake (p < 0.01). Conclusion: ENIs lead to substantial caloric, protein, and antioxidant deficits.Background and Aim: Feeding interruptions in critical care patients are often unjustified. We aimed to determine the causes, duration, and frequency of enteral nutrition interruptions (ENIs) and to assess macronutrients and antioxidant deficits according to European Society of Parenteral Enteral Nutrition (ESPEN) guidelines. Methods: We prospectively enrolled Intensive Care Unit (ICU) patients admitted for more than 48 h with an inability to orally eat from April to December 2019. The type of enteral nutrition, the number of calories administered, the time of feeding initiation, the reasons for delaying feeding, and the causes for ENI were recorded. Results: 81 patients were enrolled, with a median duration of ENIs of 5.2 (3.4–7.4) hours/day. Gastric residual volume (GRV) monitoring—a highly controversial practice—was the most common cause of ENI (median duration 3 (2.3–3) hours/day). The mean energy intake was 1037 ± 281 kcal/day, while 60.5% of patients covered less than 65% of the total energy needs ). The median daily protein intake did not exceed 0.43 ± 0.3 gr/kg/day of the actual body weight (BW), whereas ESPEN recommends 1.3 gr/kg/day for adjusted BW ( Critically ill patients need the utmost medical nutrition therapy and specialized feeding protocols because critical illness is associated with increased catabolic stress, systemic inflammatory responses and, therefore, energy and protein requirements . CriticaGlucose is the preferred energy substrate in the acute phase of illness . The glu2 production as part of EN. Additionally, they provide the building blocks of the cell membrane structure and essential fatty acids, thereby preventing essential fatty acid deficiency and allowing for fat-soluble vitamins delivery [Lipids are used primarily due to their high caloric content as an energy-dense substrate, lowering the required number of carbohydrates and COdelivery . During delivery . Among odelivery . The magdelivery . The incdelivery . Serum cdelivery . These ldelivery . Metaboldelivery . During delivery .Critical illness leads metabolism to a dynamic state divided into different stages. During the acute phase, the patient goes through the ebb phase, which includes the initial 24 to 48 h, and the flow phase, which lasts until seven days. After the acute phase, some patients may enter the recovery stage, while others remain in a prolonged critical illness. After the ebb phase, the catabolic response increases, and the human-stored components, including proteins, are reduced . SkeletaOxidative stress, caused by an imbalance among increased reactive oxygen , nitrogen species, and endogenous antioxidant mechanisms, is associated with protein and lipid oxidative damage and usually coexists with septic shock, severe pancreatitis, acute respiratory distress syndrome (ARDS), major burns, and trauma . AntioxiThe primary aim of the present study is to determine the causes, duration, and frequency of ENI episodes and to assess the impact on macro- and micronutrient deficits (mainly antioxidants). The secondary endpoints will evaluate the nutrient intake in ICU patients fed with EN and examine the possibility of EN sufficiency in reaching the metabolic requirements of the critically ill without supplementation.A single-center, prospective, observational study was performed in a mixed 19-bed ICU in a tertiary hospital  in Athens, Greece. Participants were enrolled between April and December 2019. Patients with an ICU stay of more than 48 h and an inability to feed orally  were inThe initial sample size was calculated to be 93 subjects, based on one of the primary aims to determine the proportion of patients with low energy intake , with a confidence interval of 95%, expected subjects with a low energy intake of 60%, and a margin of error of 10%.2)) was calculated [2 were classified as underweight; those with a BMI from 18.5kg/m2 to <25 kg/m2 were classified as normal; those with a BMI from 25 kg/m2 to <30 kg/m2 were classified as overweight; and those with a BMI ≥30 kg/m2 were classified as obese [Demographic data, weight, and height were recorded upon admission to the ICU, and Body Mass Index (BMI) (weight (kg)/height  of energy and 1.2 g/kg of proteins daily [Energy, macro-, and micronutrient daily intake  were calculated based on the labels of commercial formulas (various types and brands) and were collected for a seven-day follow-up period. The adequacy of the macro- and micronutrient intake was assessed according to the most recent European Society for Clinical Nutrition and Metabolism (ESPEN) guidelines . In the ns daily . The calns daily ,30.The frequency, the causes, and the duration of ENIs were recorded. The causes of ENIs were classified into three groups: diagnostic procedures, interventional procedures, and patient-related factors. Diagnostic procedures included GRV management (measured by aspiration using a syringe)  and bodyNutritional and ENIs data were collected from the first day of admission. However, the average nutrition intake and the duration of ENIs recorded in the first two days of the ICU stay were not included in the final results, reclaiming time in order to reach the desirable feeding levels.n) and relative frequencies (%), while the ENIs duration and nutrient intake were presented as the median  due to their non-normal distribution. Whether these variables were normally distributed was tested graphically, through a histogram and a P-P plot, and statistically, with a Shapiro–Wilk test. A Mann–Whitney U test was used to assess the median hours of ENIs among sex, age groups (adults versus older adults), BMI classification , comorbidities, the need for mechanical ventilation, and energy intake (low versus high). The Wilcoxon signed-rank test was used to compare the median micronutrient intake to Dietary Reference Intake (DRIs). Beta coefficients and their corresponding 95% Confidence Intervals for the association of the daily average duration of ENIs (in hours) with their daily mean macro- and micro-nutrient intake were evaluated throughout multivariable linear regression analysis. We added three different models  for energy intake and each macro- and micronutrient intake separately to estimate the effects of the duration of ENI on these intakes. All models were tested for collinearity. Two-tailed hypothesis tests were performed. Stata SE 16.1 software  was used for the statistical analysis, and a p-value ≤ 0.05 was regarded as statistically significant.Categorical variables were presented as absolute .A total of eighty-one patients who met the eligibility criteria were included in the cohort. Patients were classified according to the primary diagnosis at the time of ICU admission, with cardiovascular disease complications being in first place (27.2%), followed by respiratory insufficiency (19.8%), sepsis or septic shock (18.5%), trauma (17.3%), and neurologic or oncologic conditions (8.6%) .p < 0.05) (p < 0.05). The median duration of ENIs  did not differ significantly between age, sex, the presence of sepsis, and comorbidities. In addition, the mNUTRIC score was available for 45.7% of the sample, indicating that 56.8% were at a high nutritional risk (score > 5).Patients’ characteristics and median duration of ENIs by every single characteristic are presented in  < 0.05) . The meaENIs were observed in all patients, with a median duration of 5.2 (3.4–7.4) hours . The mosp-values ≤ 0.01)  . In all p-values < 0.05) in both crude and adjusted models. For every lost hour of feeding, a mean of 60.3 kcals, 7.5 g of carbohydrates, 2.75 g of protein, and 2.33 g of fat were lost, respectively, according to multi-adjusted models . Similarly, for micronutrients and antioxidants, every lost hour was connected with a significant quantitative loss of 55.5 μg RE/day for vitamin A, 3.61 μg/day for selenium, 0.13 mg/day for manganese , 0.81 μg/day for vitamin D, 0.59 mg/day for zinc, 83.4 mg/day for omega-3 fatty acids, and 0.07 g/day for arginine .In The current study sheds light on the causes, frequency, and duration of ENIs, while macro- and micronutrient deficits in ICU patients were highlighted as a subsequent effect. The most frequent causes of ENI episodes were diagnostic procedures, specifically GRV monitoring  h). Mechanically ventilated patients had the longest ENIs due to GRV management. Documentation and medical orders were frequently missing, and a nutrition support team was absent, especially the dietitian. The interruption of EN as an unavoidable reality led to substantive energy and protein deficits. Finally, for every lost hour of feeding, a significant number of calories and nutrients was estimated to be lost .Interestingly, the reasons for ENIs in the present study seem to be different from those published in other papers. The primary cause we identified was GRV monitoring, a common practice in ICU patients. Onuk et al., in a recent study, mentioned that radiological procedures were the most common reason for ENIs, with the longest ENIs being caused by tube feeding malfunctions during the first week of the ICU stay . These fThe median daily protein intake did not exceed 0.43 ± 0.3 gr of the actual body weight, whereas the ESPEN recommendation is 1.3 g/kg for adjusted body weight/d (limitations included in the same section) , and theOur results demonstrate that patients received lower amounts of protein but match with current data indicating that ICU patients receive an average of 0.6 g/kg/day for the first two weeks ,45,46,47The energy deficit for the daily caloric goal was 38.7% (±19.7). It was surprising that the prevalence of energy deficits was one of the highest in the literature; Adamo et al. reported caloric deficits of 24% , Uozumi The restriction of ENIs, attempted for avoidable purposes, seems to be of vital importance. Despite GRV management being a common practice in ICU patients and the main reason for ENIs in our study, in another study, the removal of GRV monitoring resulted in a significantly increased EN provision . Accordip-values < 0.01). Starting with arginine, in healthy situations, it is considered a nonessential amino acid, but in critical illness, it becomes an essential component for protein synthesis and immunomodulation. While arginine supplementation in sepsis remains controversial, on the other hand, it is recommended for wound healing and postoperative surgical and severe trauma patients [In all patients, the median daily intake was significantly less in all micronutrients, except for manganese, compared with DRIs  of energy and 1.2 g/kg of proteins daily, independent of the stage and disease progression. On the one hand, body weight was used to estimate protein goals, not adjusted for fluid retention or positive fluid balance, which could lead to the overestimation of protein goals. On the other hand, all patients received significantly less protein than recommended, with an average daily intake equal to one-third of the goal, something that could not change even with the presence of oedemas. Consequently, we hypothesized that this could be attributed to the combination of under-prescribing by physicians and the limited involvement of dieticians in the daily monitoring and evaluation of critical care patients. This is a single-center non-controlled study, leading to a potential bias in the results due to the lack of a control group. However, the results of this observational study could be used as pilot data that could inform future well-designed studies. Finally, due to the pandemic, the entrance in the ICU was forbidden, and all studies stopped; hence, the sample size of our study was smaller than calculated (81 from 93). Nevertheless, we found highly significant differences in the energy, protein, and nutrient intakes of our subjects compared with the recommended levels and demonstrated a significant impact of ENIs, indicating a major problem related to micronutrient and antioxidant deficiencies in ICU patients.The main cause of ENIs was diagnostic procedures—specifically, GRV monitoring. The ENIs resulted in approximately 60.5% of patients failing to meet the calculated caloric requirements. Similarly, the results revealed that ENI episodes could lead to substantial protein and antioxidant deficits. Even though these results were suboptimal compared with the guidelines, they are keeping up with general international trends.Uozumi et al. supported the development of a feeding protocol for ENIs management that could possibly prevent energy deficits at an early stage . Based o


Sclerotinia sclerotiorum strains from sclerotium samples in Quincy, Washington State, USA, and found that four single-sclerotium-isolation strains  had almost identical genomes to the reference strain 1980 isolated in the west of Nebraska 40 years ago. The genome of strain PB4 sequenced by the next-generation sequencing (NGS) and Pacific Biosciences (PacBio) sequencing carried only 135 single nucleotide polymorphisms (SNPs) and 18 structural variations (SVs) compared with the genome of strain 1980 and 48 SNPs were distributed on Contig_20. Based on data generated by NGS, three other strains, PB273, PB615, and PB623, had 256, 275, and 262 SNPs, respectively, against strain 1980, which were much less than in strain PB4 (532 SNPs) and none of them occurred on Contig_20, suggesting much closer genomes to strain 1980 than to strain PB4. All other strains from America and China are rich in SNPs with a range of 34,391–77,618 when compared with strain 1980. We also found that there were 39–79 SNPs between strain PB4 and its sexual offspring, 53.1% of which also occurred on Contig_20. Our discoveries show that there are two types of genomes in S. sclerotiorum, one is very stable and the other tends to change constantly. Investigating the mechanism of such genome stability will enhance our understanding of heredity and variation.Heredity and variation are inherent characteristics of species and are mainly reflected in the stability and variation of the genome; the former is relative, while the latter is continuous. However, whether life has both stable genomes and extremely diverse genomes at the same time is unknown. In this study, we isolated  Heredity and evolution are the most concerning issues of mankind. Every living individual inherits the genome of its parents, that is, a set of genetic instructions. Except for non-cellular organisms, such as RNA viruses, life has a DNA genome. Because of different genomes, species show unique biological characteristics, and all species form a colorful life world. Each species has a relatively stable genome ,2, in thHeredity and variation are the core characteristics of life. The modern evolutionary theory believes that heredity is relative while variation is continuous and never stops. It is generally believed that variation is neutral, but evolution is directed by environmental factors and even affected by non-DNA sequences ,19,20. VFungi are eukaryotes whose cell walls are mainly composed of chitin and glucan. Usually, their vegetative bodies are branched tubular filaments and their chromosomes are haploid, and most fungi can reproduce both sexually and asexually. Fungi have become an important model organism for studying eukaryote genetics, variation, and evolution due to their small genome size, short life cycle, ease of cultivation, and ease of genetic manipulation ,33,34,35Sclerotinia sclerotiorum is a notorious phytopathogen for its broad host range [Sclerotinia sclerotiorum is typical necrotrophic and leads to significant yield loss and economic damage on many economically important crops worldwide each year. It overseasons in the soil as sclerotia, the dormant body. Sclerotinia. sclerotiorum could not produce any asexual spores and use ascospores to initiate its infection on plant petals and senescent or wounded tissues [Sclerotinia sclerotiorum is homothallic [S. sclerotiorum strain 1980 UF-70, a single-ascospore-isolation offspring of strain 1980 [st range ,41,42. Mst range . Sclerot tissues ,44. Scleothallic  and has othallic ,46,47, sothallic . The genain 1980 , was firain 1980 , then thain 1980 , and recain 1980 . It is nS. sclerotiorum from pinto bean seeds at Central Bean Company, Quincy, Washington (WA) in 2018 to isolate and screen beneficial mycoviruses and mycovirus-mediated hypovirulent strains. Meanwhile, we found that some single-sclerotium-isolation strains, such as strain PB4, had very similar biological phenotypes to strain 1980 which was isolated by Dr. Jim Steadman from bean culls in western Nebraska in 1980 [S. sclerotiorum, reminding us that there may be unknown mechanisms to balance the inheritance and variation in one specific species.We collected sclerotia of  in 1980 . Thus, wBrassicae napus) in a single field in Xinyang, Henan Province, China in 2020. Strain Ep-1PN was isolated from a diseased eggplant in Jiamusi, Heilongjiang Province, China in 1992, and strain Ep-1PNA367 is an ascospore progeny of strain Ep-1PN which was isolated in 1997 [The wild-type (WT) PB strains were isolated from sclerotia mixed in seeds of pinto beans at Central Bean Company, Quincy, WA in 2018. The XY series of WT strains were isolated from diseased rapes  on PDA plates with a membrane. The genomic DNA was extracted according to the previous protocol  and the de novo assembly.The DNA concentrations and purity were tested by a NanoDrop™ 2000 Spectrophotometer . The DNA integrity was determined by electrophoresis analysis in 1% agarose gel. The average insert size of the library for NGS  is 200–400 bp. The reads with length > 2000 bp were maintained for SMRT . For quality control, the raw data of NGS were treated as follows: (1) removing the reads with a certain proportion (40% as default) of low quality (≤20) bases; (2) removing a certain proportion (40% as default) of Ns (unknown or unspecified nucleotides) and (3) removing adapter contamination and duplication contamination. The raw data produced by SMRT was treated: (1) the PacBio subreads were filtered from polymerase reads and (2) adapters and sequences less than 2000 bp were removed before de novo genome assembly of strain PB4 with default settings. To estimate the assembly quality, high-quality reads were aligned onto the genome draft using BWA v.0.7.17 [de novo assembly draft was polished by Pilon v.1.23 [http://gitlab.com/ezlab/busco/, accessed on 3 September 2020) was used to evaluate the completeness of the de novo genome assembly with default parameters [The genome draft of strain PB4 was generated by a hybrid and hierarchical assembly strategy using all clean reads from both NGS and SMRT. Flye v.2.8.3  was usedv.0.7.17  with defv.0.7.17  was usedv.0.7.17  by defaun v.1.23  with rearameters .http://www.blast2go.com/b2ghome/, accessed on 3 November 2020). Homology of the pathogenicity, virulence, and effector-related proteins, cytochrome P450 proteins, and CAZymes were identified by BLASTP against the PHI database v.3.6 , CYP database  and CAZY database , respectively. The effector-like proteins were identified by the following steps: (1) signal peptides (SP) were identified by SignalP v.5.0 ; (2) proteins containing putative glycophosphatidylinositol membrane-anchoring domains or transmembrane domains were identified by GPIsom  and TMHMM v.2.0 , respectively and (3) EffectorP v.1.0  was used to identify potential effectors from the proteins with SP but without glycophosphatidylinositol membrane-anchoring domains and transmembrane domains. The genome of S. sclerotiorum strain PB4 was visualized by Circa .The PB4 genome was annotated by MAKER v.2.30  accordinhttps://github.com/tseemann/snippy/, accessed on 24 March 2021) was used to identify core SNPs and InDels between de novo assembled genome of PB4 and 1980 with default parameters. Assemblytics  was used to analyze the alignments obtained by MUMmer’s nucmer program [The genomic collinearity between strains PB4 and 1980 (GCA_001857865.1) was analyzed by MCscanX [ program  to ident program  was used program .A genome-wide SNP-based phylogenetic tree was constructed for all 34 strains. All sites with SNP were used for the phylogenetic analysis. MEGA v.6  was usedS. sclerotiorum putative effectors (SsPEs) genes and 102 cytochrome P450 (CYP) genes were identified in the strain PB4 genome using our genome annotation pipeline  was generated from strain 1980 UF-70, a single-ascospore-isolation offspring of strain 1980, by using Sanger technology, while the one released in 2017  was directly generated from strain 1980 by using PacBio technology. Version 2 was used here as a reference.The genomic synteny of strains PB4 and 1980 was quite good B, indicaRegardless of the different false positive rates caused by sequencing reads coverage, data quality, filter criteria, and so on. SNPs (532) identified using the data of NGS were 3.94-fold of those (135) identified by the combination of SMRT and NGS between strain PB4 and 1980. The MUMmer confirmed that there were 18 SVs (11 insertions and 7 deletions) between the two strains, a total of 39,420 bp length in the genome was found to be involved in .To further investigate the variation among the genomes, 14 more strains isolated from sclerotia mixed in bean seeds of America were sequenced by using NGS, and SNPs were identified by comparing with the genome of strain 1980. We found that three strains, PB273, PB615, and PB623, had even fewer SNPs than strain PB4 (532 SNPs), by 256, 275, and 262, respectively A. These Compared with the genome of strain PB4, there were 139, 142, and 80 SNPs in strains PB273, PB615, and PB623, respectively B, among S. sclerotiorum strains at different locations, we compared the data generated by NGS of 18 Chinese strains (14 from a single rapeseed field at Xinyang city and 4 from different locations) with strain 1980 .Thirty-one single-ascospore-isolation offspring of strain PB4 were obtained. The growth rate, virulence, and colony morphology of the offspring did not change significantly from the parent strain PB4 (Data not displayed). All 31 offspring were subjected to genome sequencing analysis by using NGS and compared with strain PB4. The results showed that SNPs were found in 435 loci on 21 contigs and the number of SNP in the offspring was 39–79 with 59 on average . InteresAs a hotspot, the mutation position within Contig_20 was counted. The mutation sites in strain PB4 were concentrated on nt 20,000 to 40,000, nt 80,000 to 90,000 and nt 100,000–110,000 compared with strains isolated in the field . NotablyS. sclerotiorum isolated from a sclerotium mixed in seeds of pinto bean collected by Central Bean Company, Quincy, WA, and unexpectedly found that the genome of strain PB4 is almost identical to that of the reference strain 1980 isolated from diseased bean culls nearly 40 years ago in western Nebraska, USA. We further found that three other strains isolated from the same sclerotium mixture are much closer to strain 1980 even than strain PB4 at the genome level. Comparing the strains from China and the USA, it was found that Chinese strains, even from a single small rapeseed field, have much richer SNPs than the USA strains, suggesting that S. sclerotiorum population of China and the USA carried out independent evolution routes. The considerable SNP diversity of strain Ep-1PN and its sexual offspring Ep-1PNA367 which was isolated 25 years ago showed that the genome represented by strain Ep-1PN had an extremely fast variation speed even under lab conditions on PDA medium routinely. Our study here may provide a new vision for the understanding of the genome evolution of S. sclerotiorum and even of other organisms that in a specific species, some groups are constantly changing, while others tend to be stable. In this study, we sequenced and assembled a genome of a strain PB4 of Sclerotinia sclerotiorum is rich in genetic diversity worldwide [S. sclerotiorum population was found to have a greater genetic variance for most of the traits than the US population and they did not share any mycelial compatibility groups or multilocus haplotypes [S. sclerotiorum should not be difficult to obtain in the field [S. sclerotiorum in the sampled place, which is highly consistent with the previous findings in New York State, USA [Sclerotinia, such as beans and cruciferous crops, are also major traditional food and vegetable crops, which gives S. sclerotiorum the opportunity to spread in China year after year.orldwide ,74,75,76plotypes . Our stuhe field ,77. In tate, USA . The bigS. sclerotiorum spread by mixing in the seed of beans for a long distance. However, this event is unlikely to have occurred in the near recent past since strain 1980 was isolated 40 years ago. Considering that ascospores are the main inocula on beans and other crops, strain PB4 and other strains should have performed sexual reproduction alone at least 40 times in the field. Thus, descendants of strain 1980 should have accumulated more SNPs than those in the genome of strains PB4, PB273, PB615, and PB623. In fact, this may not be the case. Strain 1980 and PB strains have established their own communities at a distance of 2000 km from each other. From the rich SNP diversity of the offspring, it can be seen that strain PB4 may have a similar rate of variation to other strains. Of course, there is also a possibility that the sclerotia of S. sclerotiorum can stay awake for a long time and can germinate myceliogenically to infect directly plants. However, unlike ascospores with super quantity and convenience for air transmission [The fact that strains are very similar to those they were 40 years ago Indicates that the genome of these strains is very stable and the population represented by strain 1980 is widely distributed. The best explanation is that sclerotia of smission , sclerotsmission  and be iEscherichia coli cultures maintain a stable subpopulation structure during long-term evolution [S. sclerotiorum strain PB4. When compared with strains 1980, PB273, PB615, and PB623, most SNPs in strain PB4 focused on nt 30,000–40,000, nt 80,000–90,000, and nt 100,000–110,000 within Contig_20. Among sexual offspring of strain PB4, 53.1% of SNPs occurred on Contig_20, which is also significantly distinguished from the overall mutations distributed in other contigs. However, for another pair of strains, Ep-1PN and its sexual progeny Ep-1PNA367, it is hard to identify a mutation hotspot. Even though strains 1980 PB273, PB615, and PB623 might share the same recent ancestor with strain PB4, it is surprising that PB4 could maintain such a stable genome for more than 40 years despite having a mutations hotspot during sexual reproduction.Genome stability is crucial to maintain the characteristics of species, while the acquisition of mutations plays a key role in species adaptation . There avolution . In thisSaccharomyces cerevisiae [Sixteen strains isolated from sclerotium mixture in pinto bean seeds might share the same recent ancestor with strain 1980 while with different mutation rates. Obviously, the genome represented by strain 1980 and four PB strains is very stable. However, other sets of genomes phylogenetically distant from that of strain 1980 are also found in the field. Therefore, there might be two types of genomes, one is very stable, like strain 1980 and PB strains; another is unstable and tends to mutate. The forces driving genomic mutation include endogenous DNA polymerase errors, endogenous DNA damage, DNA damage caused by exogenous agents, and the activity of error-prone polymerase, among which oxidative DNA damage and repair significantly affect the mutation rate or spectra ,81,82. Rrevisiae . On the revisiae . DNA misrevisiae . Some strevisiae ,80. Becarevisiae ,86,87. Brevisiae ,89. BothS. sclerotiorum strain PB4 was assembled using NGS and SMRT technology. We found that the genome of this strain and the other three strains isolated from the same group of sclerotia was almost identical to the genome of the reference strain 1980, though they were isolated apart for almost 40 years and at a distance of 2000 km from each other. This phenomenon is rather rare since it exhibited a scenario where extremely stable genomes and rapidly mutated genomes coexist in the same species. This indicates that there may be an unknown mechanism in the field to stabilize some genomes of species. Here we report the existence of genomic stability in the USA; whether it is universal in S. sclerotiorum population in other regions or in other life species is to be further investigated.In this study, a high-quality genome of a new


Neuronal Ceroid Lipofuscinosis (NCL) comprises a clinically and genetically heterogeneous group of 13 neurodegenerative lysosomal storage disorders. Neuronal Ceroid lipofuscinosis type 2 disease (NCL2), caused by the deficient lysosomal enzyme tripeptidyl peptidase 1 (TPP1), is the only one with an approved enzyme replacement treatment (ERT). Early initiation of ERT appears to modify significantly the natural history of the disease. We aimed to shorten the time to diagnosis of NCL2.In March 2017, we started per first time in Spain a selective screening program, the LINCE project, in pediatric patients with clinical symptoms compatible with NCL2 disease. The program covered the whole country. We distributed kits to pediatricians with the necessary material to assess patients. All samples in this study were received within one week of collection. Enzymatic activity determined on dried blood spots was the main method used to screen for TPP1 and palmitoyl protein thioesterase 1 (PPT1) for the differential diagnosis with neuronal ceroid lipofuscinosis type 1 (NCL1).Over a period of three years, we received 71 samples. The analysis was minimally invasive, relatively cheap and fast-executing. Three cases identified as a direct result of the selective screening strategy were confirmed by genetic study of NCL2 disease with a median age of 4.5 years. Our screening method has a specificity of 100%, and, with the absence to date of false negatives. We did not detect any NCL1-positive cases.LINCE proved to be a simple, useful, and reliable tool for the diagnosis of NCL2, enabling clinicians to diagnose NCL2 faster. The presence of NCL2-positive cases in our population and availability of treatment may facilitate the inclusion of NCL2 in neonatal screening programs for early diagnosis. Neuronal Ceroid Lipofuscinoses  comprise a group of progressive encephalopathies that typically present during childhood and are characterized by intralysosomal accumulation of an autofluorescent lipopigment, lipofuscin, in various tissues. All except one (NCL4B) have an autosomal recessive inheritance pattern. Although NCLs are the most common cause of neurodegeneration during infancy and childhood, they are rare even as a combined group.Since they were first described in 1903 by the British pediatrician and neurologist Frederick Batten, NCLs were originally classified according to the age of onset of clinical symptoms into four categories: infantile (NCL1), late infantile (NCL2), juvenile (NCL3), and adult (NCL4)  is one of the most frequent NCLs and is caused by autosomal recessive mutations in the TPP1 gene resulting in a deficiency of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1). Clinical manifestations start between 2 and 4 years of age, with epilepsy that becomes resistant to multiple antiepileptic drugs, myoclonic ataxia, pyramidal signs, and developmental regression –5. The dPrompt diagnosis is critical for optimal disease management and appropriate genetic counseling because of rapid disease progression after the onset of the first symptoms , 14. HowMedical management has relied on symptomatic treatment and supportive and palliative care. Fortunately, since 2017, intraventricular enzyme replacement therapy (ERT) with cerliponase alfa has been approved in the USA by the Food and Drug Administration and in Europe by the European Medical Agency for the treatment of NCL2 . Early aIn March 2017, the Unit for the Diagnosis and Treatment of Congenital Metabolic Diseases at our Center started the LINCE project with the endorsement of the Spanish Federation for Rare Diseases (FEDER) and the Spanish Society of Pediatric Neurology (SENEP) to identify possible cases of NCL2 throughout Spain. This study has been approved by the Territorial Research Ethics Committee with the register number 2017/185.LINCE is a selective screening program aimed at pediatric patients (0–15 years) with clinical signs and symptoms compatible with NCL2 disease. Specific kits were designed with the necessary material, as follows: WhatmanTM 903 analytical paper for the collection of dried blood spots (DBS), an informed consent form to be signed by the parents of the participating children, a guide with indications on how to collect the samples, a contact telephone number, a contact e-mail address, and a clinical guide showing the signs and symptoms to be considered .Pediatricians interested in participating contacted us via email in order to obtain the LINCE kit. After reception, the enzymatic activities of TPP1 and palmitoyl protein thioesterase 1 (PPT1) were measured .This nationwide selective screening project targeted an at-risk pediatric population, defined as patients whose pediatricians observed clinical signs and/or symptoms compatible with a diagnosis of NCL2. The alert to the main symptoms was taken into account, including: early language delay, other developmental delays as motor difficulties or ataxia; seizures including generalized tonic-clonic, absence, myoclonic, atonic, clonic and tonic, febrile seizures; myoclonic seizures, both epileptic and non-epileptic; dramatic loss of previously attained skills as loss of voluntary movements and ability to walk and language regression; movement disorders such as myoclonus, spasticity, dystonia and chorea; visual deterioration, blindness. Samples were received with a brief description of the patient's symptoms. All probands were phenotyped using human phenotype ontology (HPO) terms extracted from the records of the medical history sent.4-Methylumbelliferone sodium salt (4-MU), 7-amino-4-methylcoumarin (4-MC), 4-MU-β-D-galactoside, dimethyl sulfoxide, Triton X-100, DL-dithiothreitol, sodium azide, β-glucosidase, pepstatin A, E64 (trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane), and ethylenediamine were purchased from Sigma-Aldrich Corp. . 4-MU-6-thio-palmitate-β-D-glucopyranoside was purchased from Carbosynth (United Kingdom). Ala-Ala-Phe-7-amido-4-methylcoumarin was purchased from Bachem (United Kingdom). Sodium carbonate, glycine, sodium hydroxide, acetic acid, sodium acetate, citric acid, sodium phosphate, sodium chloride, chloroform, and methanol were from Merck . Bovine serum albumin (BSA) was purchased from ICN Biomedicals . Ethylenediaminetetraacetic acid (EDTA) was purchased from Panreac (Barcelona).As main screening method, enzymatic activity was determined on DBS for TPP1 (EC 3.4.14.9) and PPT1 (EC 3.1.2.22) for the differential diagnosis with NCL1. Beta-galactosidase (EC 3.2.1.23) was also measured as a sample quality control. Metabolomics has demonstrated that DBS are stable where collected and transported within 28 days at room temperature . All samWe adapted the methods of van Diggelen et al. , 23 and For the biochemical confirmatory test, leukocytes were isolated from blood in EDTA tubes using the Wizard® Genomic DNA Purification Kit (Promega) and stored at −20°C until use. The leukocyte samples were diluted in 0.9 % sodium chloride solution and sonicated in an Ultrasonic Sonicator Processor (Bandelin Sonopuls HD 2070). The Bradford method was used to quantify protein in leukocytes . For theFluorescence  was measured on a BMG Labtech spectrofluorometer (FluoStar Optima). The readings were corrected for blanks and compared with 4-MU calibrators in the case of PPT1 and β-galactosidase, and with 7-amino-4-MC calibrators for TPP1. Enzyme activities were expressed in micromoles of 4-MU or 4-MC of product formed per h/L of blood (DBS samples) or nanomole per h/mg of protein (leukocytes) for PPT1 and TPP1, respectively.A total of 143 kits were distributed between March 1, 2017 and May 1, 2020. Samples from 71 patients  were received from 21 of the 17 regions of Spain. Madrid accounted for 24.4% and Valencia 13.4%. It is a minimally invasive test with a total cost per sample of around €50, compared to €80 to €100 for leukocytes and €300 for fibroblasts.The main manifestations reported were language delay (15.5%), psychomotor delay (13.5%), and epilepsy (13.5%) .Analysis of the DBS enzyme enabled identification of three patients with absent activity in TPP1 but normal PPT1 activity ; Table 3All patients had similar ages at diagnosis , rangingPatient 1, a male whose symptoms first manifested at 12 months of age as language delay followed by full stagnation of development at 15 months and cognitive deterioration at four years of age, as reported by the parents. His motor development was reported as normal. He experienced his first seizure at 3.5 years, followed by refractory epilepsy at age five despite treatment with valproate, clobazam, and levetiracetam. He presented tonic, atonic, tonic-clonic, and myoclonic seizures, together with myoclonic and atonic absences. He showed myoclonus (3.5 years), spasticity, and dystonia (4–4.5 years) with motor regression and total loss of gait at the age of five. Visual impairment was observed at 3.5 years, progressing to total blindness at five. Intraventricular ERT was not started after the diagnosis, as the child did not meet the inclusion criteria because of the severity of his disease. He died at the age of 6 years old.Patient 2, a female whose symptoms first manifested at 12 months of age as language delay followed by cognitive deterioration starting at 2.5 years of age and coinciding with the first seizures. She experienced absences and right focal tonic-clonic seizures, requiring three antiepileptic drugs due to refractory epilepsy. She also had axial tremor, myoclonus, oro-facial dyskinesias, and oculomotor apraxia. No visual deficit was recorded. ERT was initiated at the age of diagnosis (five years and six months), although it was interrupted after six months due to lack of response and deterioration.Patient 3, a male who showed the first symptoms of the disease at 3.5 years of age, namely, mild language delay and clumsiness. Cognitive decline was observed at four years of age, coinciding with the onset of epilepsy. He experienced his first seizure at three years and 11 months of age, followed by refractory epilepsy at age five. He currently requires treatment with three anticonvulsants: valproate, clobazam, and levetiracetam. His seizures are tonic, atonic, tonic-clonic, and myoclonic, together with absences. He also has myoclonus and spasticity, together with ataxia, tremor, and choreic movements of the upper extremities. He has needed a wheelchair since the age of five years, although he is able to crawl. Visual deficit has not been evidenced. Intraventricular ERT was started at the age of diagnosis (five years). He remains stable after one year on therapy. Currently, at six years of age he has a comprehensive language, and although expressive language is mostly unintelligible, he tries to communicate and says some words with meaning.Clinician awareness and regional differences in availability of TPP1 testing may affect prompt diagnosis. Furthermore, ensuring that clinicians are aware of NCL2 during the differential diagnosis may facilitate early treatment with ERT. LINCE is an acronym for Spanish Neuronal Ceroidea Lipofuscinosis. Our goal is not only to speed up the diagnosis of NCL2 disease for physicians who suspect a NCL, but also to create awareness of these dramatic, debilitating diseases. In this study, we demonstrated that diagnosis of NCL2 is possible using a simple, selective screening method. The use of DBS as a first-tier sample is very convenient, since it is easy to collect and remains stable at room temperature; therefore, it can be sent by ordinary mail –28. It iUnfortunately, since NCL2 and NCL1 are among the few NCLs with lysosomal soluble proteins that can be measured to support a diagnosis, genetic analysis is the only diagnostic option for the remaining NCLs. Two of the patients in the present series were compound heterozygous, the first was homozygous, and the variants had already been described and classified as pathogenic . Of the Several therapeutic approaches for NCLs are currently under investigation, including gene therapy, neuronal stem cell therapy, immunomodulation, small molecule therapy, and ERT –41, whicThe study has been carried out in patients with symptoms and with voluntary participation, because of this does not reflect the real incidence.In summary, the LINCE project is a simple, useful, not very expensive, and novel tool that speeds up the diagnosis of NCL2. All positive cases experienced a rapid decline in their medical condition, which is characteristic of NCL2 patients. An early diagnosis is key to accessing treatment, but diagnosis is challenging because of similarities with other disorders and limited awareness of NCL2 disease because of its rarity. Since therapy is now available, screening for NCL2 should be implemented in neonatal screening programs for early detection, ultimately leading to better outcomes.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee Santiago-Lugo with the register number 2017/185. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CC, MJC, and MLC contributed to design of study. DR, PC, and CC contributed to analysis and interpretation of data. AD-R, AM, and MT contributed to acquisition of clinical data. All authors have participated and/or reviewing, the manuscript and have approved its submission.This study received funding from Grant Biomarin Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.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.


Castanopsis hystrix is a valuable native, broad-leaved, and fast-growing tree in South China. In this study, 15 phenotypic traits and 32 simple sequence repeat (SSR) markers were used to assess the genetic diversity and population structure of a natural population of C. hystrix and to construct a core germplasm collection by a set of 232 accessions. The results showed that the original population of C. hystrix had relatively high genetic diversity, with the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon’s information index (I), and polymorphism information content (PIC) averaging at 26.188, 11.565, 0.863, 0.897, 2.660, and 0.889, respectively. Three sub-populations were identified based on a STRUCTURE analysis, indicating a strong genetic structure. The results from the phylogenetic and population structures showed a high level of agreement, with 232 germplasms being classified into three main groups. The analysis of molecular variance (AMOVA) test indicated that 96% of the total variance was derived from within populations, which revealed a low differentiation among populations. A core collection composed of 157 germplasms was firstly constructed thereafter, of which the diversity parameters non-significantly differed from the original population. These results revealed the genetic diversity and population structure of C. hystrix germplasms, which have implications for germplasm management and genome-wide association studies on C. hystrix, as well as for core collection establishment applications in other wood-producing hardwood species. Information on genetic diversity is important for understanding the extent of genetic variability in existing plant material and the breeding and conservation of genetic resources ,2. HowevCryptomeria japonica [Robinia pseudoacacia [The collection and preservation of germplasm resources are of great significance to genetic improvement, new variety breeding, and germplasm innovation of forest trees . Brown ,8 and Frjaponica  and Robidoacacia . Recent doacacia ,19,20,21Castanopsis hystrix is an evergreen broad-leaved tree in the Fagaceae mainly distributed in Guangdong, Guangxi, Fujian and other southern provinces of China. It is widely used in furniture, construction, and shipbuilding due to its fast growth and good adaptability to various materials. C. hystrix has been identified as a precious species in the Guangdong province. Since 1999, Guangdong has continuously collected germplasm materials of C. hystrix from different habitats. Aimed at developing effective and efficient conservation practices for plant genetic resources, understanding the genetic diversity between and within the population is important [C. hystrix. In the past, molecular markers, i.e., random amplified polymorphic DNA (RAPD) [C. hystrix resources. Molecular markers provide a powerful tool for genetic diversity analysis and core collection establishment. Of the marker types, SSR markers consist of 2–6 bp nucleotide repeat motifs, which are considered to be one of the most effective molecular markers for studying genetic diversity. Its codominance, abundant polymorphism information, and genomes specify and show a fairly even distribution over the genome [C. hystrix, and there is no relevant research on the construction of a C. hystrix core collection.mportant ,23,24. Amportant . The anaA (RAPD)  and inteA (RAPD) ,28,29 hae genome . SSR mare genome ,32,33,34C. hystrix accessions from 17 provenances were analyzed using 32 SSR markers distributed throughout the C. hystrix genome and using 15 phenotypic traits in the whole distribution area. Our objectives were to estimate the levels of genetic diversity and to characterize the population structure of the C. hystrix germplasm collection. The results are intended to provide a molecular basis for understanding C. hystrix genetic diversity, effectively preserving and utilizing C. hystrix germplasm resources, which provide better materials for C. hystrix breeding and ensure the population inheritance of important traits.In this study, to ascertain the genetic diversity and population structure and to construct a core collection, a total of 232 C. hystrix Genetic Improvement Research Collaboration Group, composed of the Guangdong Academy of Forestry and other institutions, has systematically investigated, selected, and sampled superior trees from 17 provenance areas with relatively concentrated distributions of C. hystrix germplasm resources according to the distribution of existing C. hystrix resource. Furthermore, the group has also carried out the collection of C. hystrix germplasm resources. Superior trees were selected by the method of five dominant trees, and one tree was selected for every 30–50 m. The number of superior trees selected from each producing area was generally 10–30 trees according to the distribution area. After selecting excellent trees, we collected seeds and the sunny branches of the specific year in the south side of the middle and upper part of the crown from October to November 2001. After sending them directly to the grafting site, 30–50 plants were grafted on each excellent tree. The grafted trees were transplanted in the gene bank in the spring of 2003, and five plants were planted in each excellent tree; the plant row spacing was 3 m × 5 m, and the size of the hole was 60 cm × 60 cm × 40 cm.The germplasm gene bank was established in 2003. Since 1999, the C. hystrix were mainly collected from the whole range (C. hystrix germplasm gene bank (113° 46′ E and 23° 29′ N), Baiyun District, Guangzhou City, Guangdong Province. From November to December 2018, 3 trees were selected for each clone, and 15 phenotypic traits were investigated, including 5 growth traits, 2 morphological traits, and 8 wood properties ) was used for data visualization. Additionally, to analyze the relationships of the 232 germplasms, a genetic distance matrix between the clones was generated, and an unrooted phylogenetic tree was constructed using the neighbor-joining method in PowerMarker 3.25 software [An analysis of molecular variance (AMOVA) test was carried out to determine the relative partitioning of the total genetic variation among and within different groups of genotypes by using GenAlEx 6.5. The principal coordinate analysis (PCoA) was also performed using GenAlEx v6.5. The genetic structure of unique genotypes was investigated using STRUCTURE v2.3.4 software  using ansoftware .QGAStation v2.0 software was used to construct the core collection based on phenotypic data, combining three systematic clustering methods , two genetic distances , three sampling methods  and seven sampling ratios  .t-test was used to analyze the significance of differences in genetic diversity parameters between the core collection and the original collection [Core Finder v1.1  and Corellection . If the C. hystrix had a high level of diversity. The average polymorphism information index (PIC) was 0.889, ranging from 0.744 to 0.958. According to the standard of PIC ≥ 0.5 [C. hystrix populations. The results showed that the genetic diversity of these SSR loci was generally high, and the highest genetic diversity was SSR04 while the lowest genetic diversity was SSR10, observed by combining the values of each genetic parameter.A total of 335 alleles were detected by 32 polymorphic microsatellite markers, with an average of 10.458 alleles detected at each locus, among which 15 alleles were detected at the SSR04 locus. The alleles of SSR02, SSR10, SSR15, SSR18, SSR23, and SSR27 were the same and least, having only eight alleles . The aveIC ≥ 0.5 , all of C. hystrix populations, the genetic parameters, containing Na, Ne, Ho, He, and I were calculated, respectively  was identified based on the maximum likelihood and DK values; the results showed that the DK value reached the highest when K = 3, which indicated that the whole population was divided into three subgroups . The thrC. hystrix germplasms to visualize the relationships between genotypes. A three-dimensional graph was created based on the value of each sample in the first (PC1), second (PC2), and third (PC3) principal components , and an AMOVA analysis of molecular variance , whereas 4% of the variation was between populations.In order to understand the level of genetic differentiation and reflect the source of variation among variance  was perfUsing QGAStation software to construct core collection based on phenotypic data, a total of 126 core collections were constructed. Among the 126 core collections, three with CR < 80% were removed ; three ct-test results showed that the six genetic diversity parameters of core collection Mc1 and Mc2 were not significantly different from those of the original collection, indicating that both core collections could be a good representation of the original population. Based on the values summarized in both Core Finder software was used to analyze the SSR data with the M strategy based on the principle of maximizing alleles. The core collection Mc1 having a sample size of 158 retained 100% of the allele number of the original collection and had increased genetic parameters ; thus, Mc1 with its strong genetic diversity should be a good representation of the original collection. In addition, Core Hunter 3 was used to determine the optimal size of the core collection using 10 preset sampling ratios . The sixFinally, a total of 157 clones were obtained by combining the phenotypic core collection (B2C3D3-15) and molecular core collection (Mc1) into the final core set (BM). In order to check if the BM could effectively represent the genetic diversity of the whole germplasm, a principal coordinates analysis (PCoA) was used to generate a distribution map of the core and original collection with SSR data . The resPinus leucodermis, Eucalyptus globulus, Swietenia macrophylla, and Populus deltoides [Characterizing breeding collection germplasms is crucial in plant breeding as the genetic advancement of economically valuable traits relies on the genetic diversity available within the breeding gene pool. Learning about genetic diversity also assists in minimizing the use of closely related clones as parents in breeding programs. Genetic diversity is an integral part of all biological diversity; it is the basis of biological evolution and species differentiation and is of great significance for population maintenance, reproduction, and adaptation to habitat changes. The higher the genetic diversity, the more likely a population is to adapt to different environments, and variations in DNA sequences are the primary drivers of such diversity . Moleculeltoides ,52,53,54eltoides ,56. SSRseltoides  or phenoeltoides . ConsequC. hystrix is a precious local wood and an efficient multi-purpose fast-growing tree species in South China. Genetic diversity, population structure, and molecular markers knowledge may accelerate the selection of desirable traits in C. hystrix. Nei’s gene diversity, the observed heterozygosity and expected heterozygosity, the Shannon–Wiener index, the polymorphism information content, etc., have all been used to evaluate the level of genetic diversity of plant species [Xanthoceras sorbifoliai, and higher PIC and genetic diversity scores were reported in studies using SSRs [C. hystrix populations, the P2 population  had the highest genetic diversity, while the P13 population  had the lowest genetic diversity. The genetic diversity of the P13 population was significantly lower than that of the other populations, which may be due to the small number of samples. As previously demonstrated, the SSR assay approach is appropriate for genetic relationship studies [C. hystrix and identification of its populations in China. species . The hig species ,59 as we species ,61. In o species . This reing SSRs . Accordi studies ,64, and C. hystrix germplasm at the genomic level, the SSR data of 232 germplasms, STRUCTURE analysis, UPGMA cluster analysis, and PCoA analyses were used to thoroughly investigate the genetic structure of C. hystrix. Based on the SSRs and multiple analyses, including population structure and phylogenetic analyses, it was confirmed that the within population was clearly clustered to three groups, which is more than previous studies using SSR and ISSR molecular marker analysis [Xanthoceras sorbifolium accessions.Population structure is an important component in association mapping analyses between molecular markers and traits. Differences in population genetic structures reflect genetic diversity and convey the adaptation potential of a species to its changing environment . To undeanalysis ,66. Bothanalysis  reportedp < 0.001), the results indicated a greater within population variation (96%) than between populations (4%), and the genetic variation within populations was the primary source of the total variation. This indicates that there is little genetic differentiation among the populations, which matches the results recorded in previous studies [C. hystrix is an outcrossing plant mainly pollinated by wind, its wide distribution provides opportunities for gene recombination and produces rich genetic diversity. At the same time, the gene exchange between populations was promoted and the differentiation between populations was reduced. (2) Plant genetic diversity is related to environmental adaptation [C. hystrix has a wide distribution range and strong adaptability. Under the action of long-term natural selection, a wide range of genetic variation has been produced, resulting in the formation of geographical provenances with different phenotypes and different requirements for environmental conditions. (3) The level of genetic diversity within populations was also influenced by the number of samples [The AMOVA analysis is a satisfactory grouping criterion for evaluating the variation within and among populations. Most scholars generally believe that the level of genetic diversity in woody plants with wide distribution, perennial, outcrossing, wind-borne seeds, or feeding by birds and animals is higher and that the genetic diversity within the populations is richer than that between populations ,69,70. I studies ,28. The  studies  and Bela studies ,72. In captation . The C.  samples ,75, and  samples .C. hystrix resources have been evaluated using SSR molecular markers, which identified rich genetic diversity among the C. hystrix germplasm within populations. Although the core collections of many trees have been established, the construction of a core collection of C. hystrix had not been conducted. According to the method of Hu [C. hystrix with a 100% allelic representation based on 15 phenotypic traits and 32 SSR markers.In this study, the genetic diversity, population structure, population differentiation and, core collection of od of Hu , when MDod of Hu ,78. We sC. hystrix phenotype core collection was 15%, which is consistent with previous studies [The rich diversity of different germplasm resources is detailed, the sampling ratio of core collection bank varied, and the sampling ratio of woody plants ranged from 10.00% to 45.00% ,79,80. I studies . While p studies ,83,84 caC. hystrix core collection was constructed using Core Finder and Core Hunter 3 software. The results showed that the retention rates of Core Finder in the four genetic parameters Na, Ne, Ho, and I were higher than those of Core Hunter 3 (C. hystrix core collection. Consistent with this study, Gong et al. [C. hystrix germplasm set, BM, was constructed based on 15 entries of phenotypic data and 32 SSR markers, which were composed of 157 C. hystrix accessions. The results of the principal component analysis showed that some of the core collection overlapped with the original collection and some were scattered around. The reason may be that when using Core Finder software to analyze SSR data and extract the core collection, the amount of data was too large. In the future, we can try to use other software to construct a core collection based on the SSR data and compare the results.Based on the M strategy, Core Finder software selects core collections by maximizing the number of alleles at each locus, which can eliminate genetic duplication in materials during construction and screen materials with a large number of alleles and low redundancy. Core Hunter 3 mainly screens the core collection based on maximizing genetic diversity and allele richness, and different sampling ratios can be set. In this study, a Hunter 3 . Moreoveg et al.  construcC. hystrix resources have been evaluated using SSR molecular markers. The results showed that the genetic diversity of these SSR loci was rich. Moreover, C. hystrix samples were grouped into three clusters. We successfully established a core collection, BM, by combining 15 phenotypic data and 32 SSR molecular markers. We demonstrated that SSR markers were successful and effective for the assessment of the genetic diversity and structure of the C. hystrix populations. The established core collection can be used for future genome association analysis and breeding program research. This study provided a theoretical basis for germplasm resource management as well as the conservation and utilization of C. hystrix germplasm resources.In this study, the genetic diversity, population structure, population differentiation, and core collection of


BackgroundVenous and arterial thrombotic conditions are the two types of thromboembolic events. Main venous thromboembolism (VTE) includes deep vein thrombosis (DVT) and pulmonary embolism (PE), while arterial thromboses include ischemic stroke and ischemic heart disease (IHD).AimThis study aimed to assess the prevalence of thromboembolic events among intensive care unit (ICU) patients in Al-Qassim region, Saudi Arabia.Patients and methodsThis is a retrospective chart review of ICU patients diagnosed with thromboembolic disease who were seen at the intensive care unit of King Fahad Specialist Hospital between July 2020 and June 2022. Data were obtained from hospital medical files and gathered into an Excel sheet . All data analyses were carried out using Statistical Package for the Social Sciences (SPSS) version 26 .ResultsOf the 38 patients included, 52.6% were males : 23.9). The most common risk factors for thromboembolic events were immobilization (23.7%) and major surgeries (18.4%). The incidence of DVT was 42.1%, while PE was 39.5%. Seven patients were detected with combined incidence (DVT and PE). Mortality rates accounted for 39.5%. It is interesting to note that the prevalence of patients who use heparin treatment was statistically significantly higher among DVT patients (p=0.043).ConclusionThe incidence of deep vein thrombosis was 42.1%, while pulmonary embolism occurred in 39.5%. However, 18.4% of the ICU patients had an occurrence of both DVT and PE. Furthermore, immobilization was identified as the most common risk factor for thromboembolic events, followed by major surgeries. More research is necessary to determine the incidence and prevalence of thromboembolic disease and its manifestations. Thromboembolic events are a major complication among hospitalized patients and are associated with a high rate of morbidity and mortality . ThromboDVTs and PEs affect between 300,000 and 600,000 Americans each year . The incAn old cross-sectional retrospective study conducted in Jeddah, Saudi Arabia, examined all cases of DVT and PE admitted to King Abdulaziz University Hospital from January 1994 to March 1999 to assess the incidence, risk factors, diagnostic modalities, and treatment of DVTs and PEs. A total of 75 patients with a mean age of 44.16 years (standard deviation (SD): 14.5 years) and a male/female ratio of 1:2 were diagnosed with DVT. Doppler ultrasound (US) was used for diagnosis in 56 (75%) of the 75 patients. PE as a complication of DVT developed in 24 (32%) patients. The most common risk factor (prolonged immobilization) was present in 17 (23%) patients. All patients were treated with a conventional course of heparin, followed by warfarin. They concluded that subcutaneous heparin should be administered as preventive anticoagulation to postoperative patients who are expected to be immobilized for a prolonged period of time. Also, people with recurrent deep vein thrombosis or those with a positive family history should undergo thrombophilia screening [We maintain that it would be beneficial to further test the prevalence, diagnostic modalities, management plan, causes, and risk factors of thromboembolic events. This study aims to assess the prevalence of thromboembolic events among ICU patients in the Al-Qassim region of Saudi Arabia.This is a cross-sectional retrospective observational study of patients admitted to King Fahad Specialist Hospital in Buraydah, Saudi Arabia, between July 2020 and June 2022. To assess the prevalence, diagnostic modalities, management plan, causes, and risk factors of thromboembolic events, all patients admitted to the ICU with a thrombotic event were included. Patients with an ICU stay of less than two days were excluded.Inclusion criteriaThe inclusion criteria were as follows: ICU patients, male or female, patients hospitalized for two or more days in the ICU, and Saudis and non-Saudis.Exclusion criteriaThe exclusion criteria were as follows: non-ICU patients and patients hospitalized for less than two days in the ICU.We collected data on age, gender, nationality, height, weight, and body mass index (BMI). We also measured the length of ICU stay, type of thrombotic event, diagnostic modalities used, risk factors, type of prophylaxis used, and treatment. Factors such as mechanical ventilation status, Glasgow Coma Scale (GCS) score, diagnosis, associated comorbidities, and mortality were also recorded. Data were collected from the clinical notes and patient files of King Fahad Specialist Hospital.All data were analyzed using Statistical Package for the Social Sciences (SPSS) version 26 . We applied descriptive statistical methods using continuous variables such as mean, standard deviation (SD), range, and median, as well as categorical variables of proportion and frequency. Furthermore, we tested statistical significance among various cross-tabulated groups by employing well-known statistical tests, including Fisher’s exact test and the chi-squared (χ2) test. We utilized the independent parametric t-test and the nonparametric Mann-Whitney U test for continuous variables to determine associated factors. We set the value of statistical significance to p<0.05. Ethical consent was acquired from the Qassim Region Research Ethics Committee (QREC) with the following approval code: 1443-830910. We guaranteed the anonymity and the protection of privacy of all study participants.2, while the median value of ICU stay was five days. The most common type of thrombotic event was DVT (42.1%), and the most common diagnosis was respiratory distress (28.9%).This study analyzed 38 patients Table . The meaIn Figure In Figure Regarding the treatment and outcome of the patients Table , the preWhen measuring the relationship between the type of thrombotic events and the baseline characteristics, treatment, and outcome Table , it was This study investigates the prevalence of thromboembolic events among ICU patients in the region of Al-Qassim, Saudi Arabia. Of the 38 patients included in the study, DVT occurred in 42.1%, PE occurred in 39.5%, and DVT and PE occurred together in 18.4%. These results are higher than those of the study conducted in Jeddah , where fAfter excluding patients with the combined disease (DVT and PE), the data in this study suggest that only the use of heparin medication was found to have a direct relationship with thrombotic events. The prevalence of patients using heparin medication was significantly higher in DVT patients (p=0.043). Other studied variables, including age (in years), gender, BMI, length of ICU stay, associated risk factors, associated comorbidities, mechanical ventilation status, mortality rates, and GCS score, had similar incidences of DVT and PE. In China , among eMoreover, a study by Minet et al.  found thAspirin was the most commonly used prophylactic for thrombotic events. In India , two of Regarding the treatment of patients with thromboembolic events, apixaban was the therapeutic of choice for treatment. Doppler ultrasound was assumed to be the most trusted imaging method (55.3%).Immobilization was identified as the most common risk factor for thromboembolic events. Major surgery was identified as the second most common. Doppler ultrasound was the most effective imaging method for the detection of the disease, while aspirin was the most commonly administered thromboprophylaxis. More research is warranted to determine the incidence rate of thromboembolic disease and its manifestations.