In pet foods, choline is supplied as choline chloride. This salt is hygroscopic and makes food processing difficult. This has led to its replacement, in livestock farms, by chemically stable herbal additives rich in phosphatidylcholine. The herbal composition and the contribution of secondary metabolites give the additive nutraceutical properties that could benefit the animal’s health. In this sense, increased levels of a polyherbal additive were supplied in dog diets, and their effects were compared to diets with choline chloride and without extra choline. The results showed that the animal response was the same with the two sources of choline. However, the herbal additive at the gene level demonstrated properties to prevent cardiovascular and metabolic diseases, cancer prevention, inflammatory and immune response, and behavior and cognitive processes in dogs.Achyrantes aspera, Trachyspermum ammi, Citrullus colocynthis, Andrographis paniculata, and Azadirachta indica was evaluated in adult dogs through body weight changes, subcutaneous fat thickness, blood metabolites, and gene expression. Forty dogs (4.6 ± 1.6 years old) who were individually housed in concrete kennels were randomly assigned to the following treatments: unsupplemented diet (377 mg choline/kg), choline chloride , and polyherbal  for 60 days. Blood samples were collected on day 59 for biochemistry, biometry, and gene expression analysis through microarray assays. Intake, final body weight, and weight changes were similar for the two choline sources. Feed intake variation among dogs (p = 0.01) and dorsal fat (p = 0.03) showed a quadratic response to herbal choline. Dogs that received the polyherbal diet had reduced blood cholesterol levels . The gene ontology analysis indicated that 15 biological processes were modified (p ≤ 0.05) with implications for preventing cardiovascular and metabolic diseases, cancer prevention, inflammatory and immune response, and behavior and cognitive process. According to these results that were observed in a 60 day trial, the polyherbal form could replace choline chloride in dog diets at a concentration of 400 mg/kg.Choline chloride is used to provide choline in dog foods; however, in other domestic species, it has been replaced with a polyherbal containing phosphatidylcholine. A polyherbal containing  Choline chloride is commonly used as choline in dog food; however, its high hygroscopicity and acceleration of oxidative loss  have ledResearch in broilers and ruminants ,3,4,5 haThe understanding of the PtdCho as a key intermediate metabolite  raises tAchyranthes aspera, Azadirachta indica, Andrographis paniculata, Citrullus colocynthis, and Trachyspermum ammi) versus a diet with choline chloride or an unsupplemented diet.Therefore, the objective of this experiment was to evaluate the metabolism and health of dogs through measurements of blood chemistry, blood biometry, liver ultrasound, gene expression, and body weight changes, in dogs, supplemented with increasing dietary levels of a polyherbal compound (The experiment had a duration of 60 days and was conducted in the Centro de Investigación en Alimentos para Mascotas (CIAM), in Tepeji del Rio, Hidalgo, México . The average temperature is 15.2 °C (3 °C min–30 °C max), average humidity is 5% (0% min–10% max), and has light:dark cycles of 10:13 h.Canis lupus familiaris; 4.6 ± 1.6 years old) were individually housed in concrete kennels (2 m width × 5 m long). The breeds used were: Beagles, Schnauzer, Bichon Frize, Dachshund, Airedale Terrier, and Jack Russell. Dogs were fed individually and were randomly allocated to the following dietary treatments: (1) unsupplemented diet , (2) choline chloride , and (3) three levels of polyherbal supplement  with a contribution of 1.6% phosphatidylcholine.Forty  healthy adult dogs  that was formulated in accordance with the nutritional recommendations of NRC  and the 0.75) as recommended by NRC [Daily food intake was recorded, and consumption variation between days was evaluated as an indicator of stability and animal welfare . Animalsd by NRC , adjusti®; Chison Medical Technologies Co., Jiangsu, China) equipped with a multi-frequency linear transducer  was used. Dogs were prepared by cutting their hair prior to evaluation. To measure backfat, the equipment was used in the lumbar region because it has been reported to have less variation, and the results have been correlated with the body condition of dogs [On day 50 of the experiment, to measure backfat and to evaluate images of liver integrity, a portable ultrasound scanner , lactate dehydrogenase (LDH), aspartate amino transferase (AST), calcium, and phosphorus were determined with an autoanalyzer Kontrolab®  (2017). The blood sample collected in disodium–EDTA was used for hematocrit and blood count with a hematology analyzer EasyVet® .On day 59 of the experiment, pre-prandial blood samples of all dogs were collected from the jugular vein using vacutainer tubes with sodium citrate, EDTA, and without anticoagulant. The blood samples were transported under refrigeration (4 °C) to the veterinary laboratory Laclivet for immediate processing. Tubes without anticoagulant were centrifuged in a refrigerated centrifuge  at 3500× From dogs supplemented with 2000 mg synthetic choline and 800 mg polyherbal, RNA was extracted from whole blood following the protocol described by Winn et al. . By tripFor transcriptome analysis by DNA microarray, two pools of 30,000 ng of RNA were obtained. Each pool was made up of equal concentrations of RNA (3750 ng RNA) from each repetition  that made up the experimental group  to be evaluated. The transcriptome analysis was carried out in the Microarray Unit of the Institute of Cellular Physiology of the National Autonomous University of Mexico (UNAM), where a heterologous mouse chip (M22K) was used with 24,341 applications. The results of the microarray were analyzed with the GenArise software to identify differentially expressed genes . For the gene set enrichment, the DAVID 6.8 bioinformatics tool   was usedThe experiment was a completely random design with eight repetitions per treatment. A Shapiro–Wilk test was used to test the normal distribution of variables. Data were analyzed using R software  , and ortFor gene enrichment analysis, a minimum of 2 genes per biological group was considered. An EASE score (modified Fisher Exact) with a probability of ≤0.05 and the fold enrichment value were considered to identify the enriched annotation terms that belonged to the evaluated gene groups and biological closeness between the enriched processes , respectp = 0.75) in feed intake due to the type of choline supplement (p < 0.02) the lowest variation in consumption between days within each animal (p = 0.20), average daily gain (p = 0.62), or energy expenditure (p = 0.70). The polyherbal supplement showed a quadratic effect .No changes were observed  were observed in the liver enzymes ALP, LDH, and AST .The polyherbal affected genes that were related to cancer prevention  genes grouped in the biological process called nicotine addiction   stands oynthesis , respectThe polyherbal in intermediate doses improved body condition through the consideration of backfat as an indicator, which is within the values reported as healthy and below those of obese dogs . The lipThe effects of choline chloride or polyherbal on cholesterol may be due to the functions of phosphatidylcholine, which participates in the synthesis and export of triglycerides in very-low-density lipoproteins , and thiIn general, the blood count changes showed no immune response activation, and the results were within normal values . The NRCAn ultrasound indicated the absence of liver problems, a result confirmed by biochemical  and hematological data ,38. No pPolyherbal reduced the expression of genes that were involved in the renin–angiotensin system, whose high activity leads to vasoconstriction and sodium and water retention . This poG6PC and G6PC2 genes, which are involved in the digestion and absorption of carbohydrates , reduces the presence of abnormalities associated with Type II diabetes mellitus. This has been observed with the overexpression of G6PASE on hepatocytes under in vitro conditions [SLC37A4 gene [SLC2A2 gene [Another common condition in dogs is the incidence of Type II diabetes mellitus . The decnditions . Similar7A4 gene  and the 2A2 gene . This co2A2 gene .PLTP (phospholipid transfer protein) gene mediates the transfer of phospholipids and free cholesterol from triglyceride-rich lipoproteins into HDL. Although underexpression of PLTP has not yielded conclusive results in animal models, other functions have been recognized in vascular compartment lipid transport, impacting diseases such as atherosclerosis, cancer, and neurodegenerative disease [CYP8B1 gene codes for the cytochrome P450 protein, whose function is to catalyze the conversion of steroids determining the relative amount of bile acids (cholic acid and chenodeoxycholic acid); bile acids affect the solubility of cholesterol, promoting its absorption. The Cyp8b1–P450 pathway was also downregulated with the polyherbal. The reduced expression of the CYP8B1 gene has been of interest for pharmacological therapies since, at an experimental level, the incidence of fatty liver, lowered cholesterol absorption, prevention of atherosclerosis, hypercholesterolemia, and formation of gallstones in diabetic mice, as well as glucose homeostasis, have been reduced [Polyherbal affected the expression of genes involved in the PPAR signaling pathway, which is important for its cholesterol modulating role and its implications in dyslipidemia ,47. The  disease . The CYP reduced ,49. ThisCYP4A10 with the polyherbal could be beneficial since it is overexpressed in hepatic steatosis and steatohepatitis conditions. In mouse studies, CYP4A fatty acid ω-hydroxylase P450s has been shown to play an essential role in the development of steatohepatitis [The downregulation of epatitis . Howeverepatitis .The genes previously documented highlight that the polyherbal could reduce problems of metabolic syndrome, arterial hypertension, and obesity, as well as hypothetically impact the well-being and longevity of the dog.IL3, RUNX1T1, CD14, BCL2A1), B lymphoblastic lymphoma , T lymphoblastic lymphoma , Hodgkin lymphoma , and multiple myeloma ; epithelial cancers, such as prostate cancer ; neuroendocrine cancers, such as neuroblastoma , carcinoid (KMT2A), and Ewing’s sarcoma . Several herbs have been used to assist in cancer prevention in dogs [The impact of polyherbal on cancer pathway genes shows a potential that should be evaluated in terms of its preventive effects on cancer problems with long-term studies. Genes that were underexpressed by the polyherbal included: hematopoietic and lymphoid tissues, such as acute myeloid leukemia  [CDKN2C, Fc: 2.7).The polyherbal is composed of, among other plants, 4 (CDK4) , throughAzadirachta indica stimulates apoptosis [AKT1) through the AKT and ERK pathways  [The poptosis , which ekinases) . Inhibitkinases) ,57.NRAS, AKT1, and FKBP4, involved in the estrogen signaling pathway, could prevent the development of mammary tumors in female dogs, since high levels of expression of NRAS and AKT1 are related to carcinogenesis [FKBP4 is related to tumorigenesis, which is why it is common to observe them in hormone-dependent cancers, such as breast and prostate cancer [WNT1, WNT7B, APC, and AXIN genes with respect to choline chloride. Aberrant activation of Wnt/b-catenin signaling leads to the progression of canine malignant melanoma [CTNND1) was downregulated by the polyherbal.The under-expression of the genes ogenesis , while Fe cancer . Anothermelanoma , where 3melanoma . HoweverAzadirachta indicate. The MAPK pathway inhibits cell proliferation by the induction of protein kinases that are activated by AMP induction [Polyherbal stimulated the expression of genes in the MAPK signaling pathway, which could be explained by some of the effects of its herbs, such as nduction , as wellnduction . These anduction .BCL6 gene  has been identified as a proto-oncogene [FOXO genes. The expression of the MMP3 gene, which is related to cytokines, tumor promoters, and oncogenic products, was also decreased [Speculating on the effects of gene downregulation in the FoxO pathway is complicated. Still, it is recognized that FoxO transcription factors function as regulators of cellular homeostasis and are tumor suppressors . It is aoncogene , and itsecreased ; the effAFDN gene stands out , as wel tissues ), and thnd SAMD4 ).SMAD7 gene was downregulated by the polyherbal, which could be positive, since its overexpression is common in many types of cancer, and its abundance is positively correlated with malignancy [The underexpression of genes in the TGFβ pathway could be of interest, since this pathway is involved in gastrointestinal stromal tumor interactions from early to late tumor stages, creating a favorable microenvironment for tumor initiation and cancer cell growth. Although anticancer roles are recognized at the cellular level depending on the tumor’s stage and genetic alteration, clinical studies show that TGFβ inhibition has the potential to control and delay some tumors . The SMAlignancy .Therefore, all the data together indicate that the various genes that participate in cancer generation are affected by polyherbal consumption, especially those closely related to different types of cancer that occur more frequently in dogs and which cause high mortality . PolyherThe immune response involves a wide variety of cells, adhesion molecules, chemokines, and cytokines to defend itself against aggression from the external environment, such as infections, by exerting an inflammatory response to eliminate pathogens and harmful molecules and culminating in its regulation to maintain body homeostasis .IFN-γ gene is likely not positive, since this gene participates in diverse biological actions, including activating cells during the inflammatory process  with antiviral, immunomodulatory, and antiproliferative effects [IL-3-deficient mice had no defect in hematopoiesis [IL-3 contributes to leukocyte production, proliferation, and survival, and IL-3 potentiates inflammation in sepsis has been shown [Cytokines act as regulators of immune and inflammatory responses and intervene as growth factors of different cells, among which the hematopoietic ones stand out . The dow effects . The genopoiesis , even then shown .CXCL9 and CXCL11: Th1 response, Th1, CD8, and NK trafficking; CCL12: monocyte trafficking; CCL19: homing to lymph node), being able to modify the immunoregulatory processes of these cytokines [The polyherbal reduced the expression of some chemokines, CXC , and of some CC  . The recytokines .AFDN2, whose proangiogenic effects are of interest given that it contributes to physiological and pathological conditions such as atherosclerosis [AFDN gene participates in other processes, such as the hemostatic function of the endothelial barrier [Among the genes that were downregulated with polyherbal is clerosis . However barrier .TNF genes were downregulated by the polyherbal and participate in regulating immune cell functions and apoptosis [Some poptosis . The anspoptosis .BMP2 and IL3, identified by bioinformatics analysis [Among the genes that were downregulated with the polyherbal and were related to the development of glaucoma are analysis .Therefore, these findings indicate that the polyherbal exerts an anti-inflammatory effect and could contribute to the regulation of the immune response in different common inflammatory pathologies in dogs, such as inflammatory bowel disease.CHRNA7 gene could not be beneficial due to its neuroprotective, memory, and alertness functions [SLC genes may affect some central GABA neurons due to their specificity relating to membrane-bound transporters [The reduction of the expression of genes linked to the cholinergic synapse could have contrast effects. In the first place, it could have a beneficial effect on the stress levels of dogs. Stress impacts the GABAergic system and increases GABAergic transmission, elevates the baseline GABAergic response, and enhances the responsiveness of GABA receptors . Secondlunctions ,90,91,92sporters . Slc17 isporters  when Slcsporters . Howeversporters .The results of changes in weight, glucose, and blood cholesterol levels of the group without choline showed that it is required to incorporate phosphatidylcholine or choline chloride in the dog’s food and are similar to those of dogs with intakes below 40 mg/d . The herThe polyherbal at 400 mg/kg of diet, evaluated with phosphatidylcholine and other secondary metabolites in a 60-day trial, showed that its use is safe because no toxic effects were observed. At this dose, it can be used to replace choline chloride in adult dogs. The potential advantages over choline chloride in health effects must be evaluated in long-term studies and other life stages.


CTNS), encodes the lysosomal cystine carrier cystinosin.Cystinosis is an autosomal recessive disorder characterized by an accumulation of the amino acid cystine in lysosomes throughout the body. Cystinosis is an inherited disease resulting from the failure of lysosomal cystine transport. The responsible gene, Cystinosin, Lysosomal Cystine Transporter  and 11-year-old (patient B) symptomatic Iranian females with renal insufficiency, were diagnosed with cystinosis on the basis of their clinical features and laboratory tests. After genetic counseling, blood samples were obtained from the patients and their parents. Genomic Deoxyribonucleic Acid (DNA) was extracted from whole blood, and mutation analysis was performed using polymerase chain reaction and sequencing methods for all exons of the CTNS gene. At least 148 different pathogenic and deleterious mutations in the CTNS gene have been reported to date. Owing to our patient’s prominent clinical features of cystinosis, we carried out a targeted search for mutations in the CTNS gene.In this case report, we reviewed the genetic basis of cystinosis and investigated two Iranian cases affected by cystinosis, one of which revealed a rare mutation in the CTNS gene are frameshifts, and truncating mutations that affect product function result in relatively mild symptoms of cystinosis. The present finding confirms previous research and proves the importance of the association of this gene rare mutations with cystinosis. Since reported mutations are rare, their previous reports in Iranian patients indicate the high frequency of these mutations in our region.This led us to confirm the existence of a homozygous DNA variation c.257_258deletionCT (p.Ser86PhefsTer38) in exon 6 of the gene in patient A and another homozygous DNA variation, c.323delA (p.Q108RfsTer10), in the same exon in patient B. As expected, the mentioned mutation existed in both her parents in a heterozygous state. Variations c.257_258delCT and c.323delA reported in three Iranian patients in the  Cystinosis is an autosomal recessive disorder characterized by an accumulation of the amino acid cystine in lysosomes throughout the body , 2. LysoCTNS, encodes the lysosomal cystine carrier cystinosin and is located on the short arm of chromosome 17p13. Normal CTNS is 26 kb in length and has 12 exons with a coding region of 1104 bp [CTNS, is a 367-amino-acid peptide with seven transmembrane and two lysosomal targeting motifs, a 128-amino-acid N-terminal region bearing seven potential N-glycosylation sites, and a 10-amino-acid cytosolic C-terminal tail [The responsible gene, nal tail .The typical untreated child has short stature, light complexion, rickets, and photophobia. Failure to thrive is generally observed after the approximate age of 6 months; signs of renal tubular Fanconi syndrome  appear as early as age 6 months; corneal crystals can be present before age 1 year and are always present after the age of 16 months [Cystinosis occurs with a frequency of approximately one in 100,000 to 200,000 and has been found worldwide in all ethnic groups. The frequency of cystinosis in Brittany has been reported as 1 in 26,000 [CTNS gene have been reported. Pathogenic variants may consist of small intragenic deletions/insertions and missense, nonsense, and splice-site mutations [CTNS and the 5′ region upstream encoding the Carbohydrate kinase-like (CARKL) gene [CTNS have been reported in the Ensemble database. It is noteworthy that, according to recent studies, steroids can worsen the prognosis of cystinosis [Several benign variants in the utations . The mosKL) gene . Anotherstinosis , 9. Some missense, deletion, stop-gain, and insertion mutations are presented in Table CTNS gene mutations with cystinosis.In this study, we review the genetic basis of cystinosis and confirm the association of important CTNS gene. The exons were amplified by PCR using the primers listed in Table CTNS gene exons was performed. PCR products were purified on agarose gel and directly sequenced with the same PCR primers. The probands described in this study had clinical manifestations that conform to the diagnosis of cystinosis, including atrophy of the proximal convoluted tubules and renal stones, severe thirst and dehydration, failure to thrive, hematuria (+2), proteinuria (+1), glycosuria (+1), phosphaturia, decreased sodium, potassium, calcium, phosphorous, magnesium, and urea in blood, metabolic acidosis, and reduced skin and hair pigmentation.Clinical information was collected by reviewing the patients’ medical records. Evaluation of history, clinical manifestations, and laboratory findings of symptomatic patients A and B was performed. Patients A and B were 9-year-old and 11-year-old females, respectively, with renal insufficiency living in Tehran, the capital city of Iran. They were diagnosed with cystinosis on the basis of their clinical features and laboratory tests, and their parents were evaluated. After genetic counseling and assessing the familial pedigree  in exon 6 of the CTNS gene in patient A, and another homozygous DNA variation, c.323delA (p.Q108RfsTer10), in the same exon in patient B. These variations were detected in their parents in heterozygous states . Furthermore, both, unusually, did not show photophobia. Slit-lamp revealed cystine crystals in the cornea of the patients. In patient A, the mother noticed that she was vomiting a lot in the first months after birth and skeletal surveys showed mild rickets. Examinations conducted at 6 months of age showed that she is suffering from cystinosis. Both had been managed with growth hormone and vitamin D since 5 years ago, preventing dwarfism. Therefore dwarfism was not an acute sign. They both attended a regular school at a normal age level, so their intelligence was normal. Owing to our patient’s prominent clinical features of cystinosis, we carried out a targeted search for CTNS gene are frameshift and truncating mutations that affect product function and result in relatively mild signs and symptoms of cystinosis.Variations c.257_258delCT and c.323delA in the CTNS mutations, as well as the 57-kb deletion, result in severe classic (early-onset or infantile type) disease [CTNS. Patients with intermediate cystinosis  or non-nephropathic cystinosis  have one severe CTNS mutation, typical for nephropathic cystinosis, and one mild mutation [CTNS and its flanking genes may lead to contiguous gene deletion syndromes with more complex phenotypes than those of classic cystinosis [TRPV1, causing dysregulation of TRPV1 transcription in peripheral blood mononuclear cells [CTNS mRNA, while most other alleles produce some residual mRNA [CTNS are predicted to produce truncated cystinosin in the case of severely affected patients and produce cystinosis that retains some residual function in the case of mildly affected patients [A genotype–phenotype correlation related to the clinical forms of cystinosis is observed, with severe truncating mutations mostly seen in patients with the infantile form of the disease and some mutations allowing the residual function of cystinosin in patients with intermediate or adult cystinosis. However, several unexplained exceptions were reported . Within  disease , 12. Indstinosis . For exaar cells . The 57-ual mRNA . The mutpatients . et al. performed mutation analysis of 108 USA-based nephropathic cystinosis patients and found that 48 (44%) were homozygous for the European 65-kb deletion, 2 had a smaller significant deletion, 11 were homozygous, and 3 were heterozygous for 753G>A (W138X), and 24 had 21 other mutations. In 20 patients (19%), no mutations were found. Of 82 alleles bearing the 65-kb deletion, 38 were derived from Germany, 28 from the British Isles, and 4 from Iceland. According to a cystinosis clinical severity score, homozygotes for the 65-kb deletion and W138X had an average disease, whereas mutations involving the first amino acids before transmembrane domains were associated with mild disease [Shotelersuk  disease .et al. examined the predicted effects of mutations on cystinosin. They screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis, and patients whose phenotype did not fit the classic definitions. They identified 23 different mutations in the CTNS gene. Of 25 patients with infantile nephropathic cystinosis, 12 had two severely truncating mutations consistent with a loss of functional protein, and 13 had missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function. Mutations identified in two patients with late onset affected functionally unimportant regions of cystinosin, accounting for the patients’ milder phenotype. For three patients, the age of onset of cystinosis was under 7 years, but the disease course was milder than the infantile nephropathic form. This suggested that the missense mutations identified in these individuals allowed the production of a functional protein and may also indicate regions of cystinosin that are not functionally important [Attard et al. characterized two deletion breakpoints in the CTNS gene in affected individuals: one of approximately 65-kb, which was found in homozygous state in nearly one-third of individuals with cystinosis, and a smaller one of 9.5–16 kb, which a single family carried. They showed that the 65-kb deletion is present in either the homozygous or the heterozygous state in 76% of patients of European origin with cystinosis [Forestier stinosis . et al. stated that the most common CTNS mutation in cystinosis is the 57,257-bp deletion, which is found in a homozygous state in approximately 50% of patients of northern European descent. The deletion is an ancient founder mutation [Gahl mutation , 14. et al. detected a missense mutation in the CTNS gene (G110V) [In a patient with atypical nephropathic cystinosis, presenting with Fanconi syndrome and end-stage renal disease, but surprisingly without extrarenal symptoms even late in life, Kalatzis  (G110V) .et al. analyzed the CTNS gene in 42 Italian patients with nephropathic cystinosis and found that the mutation spectrum in this population differed from that previously reported for the northern European population: the 57-kb deletion was present in a lower percentage (17%), and splicing mutations accounted for 30% of the mutations detected [Mason detected . About 76% of cystinosis patients in the European and American population carried the common 57-kb deletion, while in Brittany, France, with particularly high incidence with cystinosis, the founder effects of 898_900+24 del27 and W183X mutation existed . CTNS mutations have been reported [Interestingly, in the Asian population, very few patients with cystinosis and reported , 18. In reported , 15.4% oreported . This mureported .The most common Egyptian mutation  was completely absent in other studies from the Middle East. Likewise, the most common mutation in the Saudi population , representing 34.6% of familial mutant alleles, was not detected in the three surrounding populations, and apart from the founder mutation , there were no other mutations detected in common among the Saudi, Turkish, and Iranian patients. This is quite remarkable considering the long history of commercial relations, invasions, and genetic contact between these four close countries over the last few thousand years .CTNS sequencing in populations outside its geographical distribution, at least in the region of the Middle East [CTNS gene mutations were studied in 28 Iranian patients aged 1–17 years, affected by nephropathic cystinosis. In 50% of patients , the mutations were identified in exon 6, among which 7.18%  were detected to have novel homozygous deletions, all of which cause the cystinosin protein to be truncated prematurely. The c.323delA (p.Q108RfsX10) mutation was also detected in three cases [The absence of the 57-kb deletion has been reported in the region of the Middle East in 13 Egyptian families with cystinosis, 13 from Saudi Arabia , 10 fromdle East . The CTNet al. reported a novel CTNS homozygous splice mutation, c.853-1G>A, in a rare nephropathic cystinosis case, initially presenting with features of Bartter syndrome [In 2018, Bastug syndrome .As far as we know, the identified mutation in our case can be considered a rare mutation. The detected mutation in our case B has been only reported in three other Iranian patients, showing that this mutation can be considered a specific one related to the Iranian population. The present finding will benefit the genetic diagnosis and carrier detection in the family and other patients with similar disease manifestations. Since reported mutations are rare, their previous reports in Iranian patients indicates the high frequency of these mutations in our region.


The relationship between radiation levels and mental health status after a nuclear disaster is unknown. We examined the association between individual external radiation doses and psychological distress or post-traumatic stress after the Fukushima Daiichi nuclear power plant accident in March 2011 in Japan.The Mental Health and Lifestyle Survey was conducted from January 2012. Based on the estimated external radiation doses for the first 4 months, a total of 64,184 subjects were classified into <1 mSv, 1 to <2 mSv, and ≥2 mSv groups. Odds ratios (ORs) and 95% confidence intervals (CIs) of psychological distress and post-traumatic stress, with the <1 mSv group as the reference, were calculated using logistic regression analysis adjusted for age, sex, evacuation, perception of radiation risk, and subjective health status.The prevalence of psychological distress/post-traumatic stress in the <1 mSv, 1 to <2 mSv, and ≥2 mSv groups was 15.1%/22.1%, 14.0%/20.1%, and 15.0%/21.7%, respectively. In women, although the ≥2 mSv group tended to have a higher risk of psychological distress with the age-adjusted OR of 1.13 , the adjusted OR decreased to 1.00  after controlling for all variables. On the other hand, there were no dose-dependent associations between radiation dose and post-traumatic stress.Although external radiation doses were not associated with psychological distress, evacuation and perception of radiation risk may increase the risk of psychological distress in women in the higher dose group. The accident was classified as a Level 7 emergency, the highest level on the International Nuclear Event Scale,3 Post-traumatic stress disorder (PTSD) and depression, which often coexist,4 are the most common mental health problems after disasters.5 In a previous study, the prevalence of general psychological distress was high 3 years after the Fukushima Daiichi nuclear power plant accident, although the prevalence of psychological distress and post-traumatic stress decreased over time.6 According to a cross-sectional survey conducted in 2020, the prevalence of psychological distress, post-traumatic stress, and radiation health anxiety was high among evacuees of the Fukushima Daiichi nuclear power plant accident.7A previous report on the health effects of the Chernobyl nuclear power plant accident by the World Health Organization concluded that mental health problems are the largest public health problem caused by the accident.8 showed that various factors were associated with mental health status after natural disasters, such as disaster-related factors, coping factors, health-related factors, and personal factors. Similarly, psychological distress among evacuees after the Fukushima Daiichi nuclear power plant accident was associated with disaster-related factors (experience of the nuclear power plant accident and loss of someone close due to the disaster), personal factors , health-related factors ,9 and perception of radiation risk.10,11 Moreover, the living area at the time of the disaster was also significantly related to psychological distress, and the distribution of psychological distress exhibited a pattern similar to that of the environmental radiation levels.9 However, the relationship between radiation levels and psychological distress or post-traumatic stress is unknown, and there are no well-designed studies regarding the association between radiation doses and psychological distress at the individual level. Therefore, we examined the association between individual external radiation doses and psychological distress or post-traumatic stress after the Fukushima Daiichi nuclear power plant accident using data from the large-scale Fukushima Health Management Survey.12,13A systematic review12–14 with a target population of 64,184 persons. Among them, we excluded 4,381 and 3,484 participants because of missing data on Kessler 6-item psychological distress (K6)15,16 (at least one missing item) and the PTSD Checklist Stressor-Specific Version (PCL)17 (at least two missing items) scores, respectively. Finally, 59,803 and 60,700 participants with available data on K6 and PCL scores were included in the analysis  of the evacuation zone designated by the Japanese government ,16 and PCL18 scale has been shown to have sufficient validity and reliability. The K615,16 is composed of six questions (score range: 0–24) that assess non-specific psychological distress during the previous 30 days. We adopted a cutoff score of ≥13, which is classified as indicating probable severe mental illness.19 The PCL was used to assess post-traumatic symptoms, which is a 17-item self-report measure, and the cut-off was set as a PCL score of ≥44.17 For perception of radiation risk to genetic effects, we asked participants about their beliefs on the potential risks of radiation exposure using the following question: “What is the likelihood that the health of your future  children and grandchildren will be affected as a result of your current level of radiation exposure?” on a four-point Likert scale and divided into two categories: “very unlikely” and “unlikely” or “likely” and “very likely”.11 Furthermore, subjective health status was obtained from five options and divided into three categories: “very good” and “good”; “passably” or “poor” and “very poor”.11 External radiation doses were estimated using data from the Basic Survey of the Fukushima Health Management Survey for the estimation of radiation dose, which was launched approximately 4 months after the nuclear power plant accident, and previous reports contain detailed descriptions on the methods.20,21 Briefly, it is a self-administered questionnaire survey that asked residents  of Fukushima Prefecture to record and send back information on their behavior  from March 11 to July 11, 2011. The respondents’ behavior records were digitalized, and a computer program calculated individual effective doses due to external exposure by superimposing the behavior records with daily gamma ray dose rate maps drawn after the nuclear power plant accident. In total, 568,632 (27.7%) residents responded to the questionnaire by March 31, 2020. In this study, 36,401 (56.7%) subjects responded to the questionnaire, and the distributions of individual external doses of the respondents for the first 4 months were as follows: <1 mSv, 69.8%; 1 to <2 mSv, 23.1%; and ≥2 mSv, 7.0%.The outcome of this study was the K6 and PCL scores. The Japanese version of the K622–24 In the imputation model, in addition to the estimated external radiation dose, the following variables were used as covariates: sex (no missing), age at disaster (no missing), area of residence at the time of the disaster (0.39% missing), and area of residence as half a year after the disaster (no missing). Odds ratios (ORs) and 95% confidence intervals (CIs) of psychological distress and post-traumatic stress, with the <1 mSv group as the reference, were calculated using logistic regression analysis adjusted for age at April 1, 2011 (continuous), sex, perception of radiation risk to genetic effects, subjective health status, and evacuation . The logistic regression analyses were performed for each imputed dataset, and the results were combined using Rubin’s rule.22 We also performed the same analyses using a non-imputed dataset for sensitivity analyses.The subjects were classified into three groups according to the estimated external radiation doses—<1 mSv, 1 to <2 mSv, and ≥2 mSv. Analysis of variance, Fisher’s exact test, and the chi-squared test were used to compare the characteristics of the subjects between the groups. Since information on external radiation doses was lacking for 27,783 participants (43.3%) because of the lack of response to the Basic Survey of the Fukushima Health Management Survey, we imputed external radiation doses to make 10 datasets using multiple imputation by chained equations with predictive mean matching methods under fully conditional specification.P < 0.05, and all tests were two tailed.Data were analyzed using SAS . The MI procedure was used to create multiply imputed datasets, and the MIANALYZE procedure was used to combine the results of the analyses of imputations. Significance was defined as 25 between the three external radiation dose groups in both men and women  and post-traumatic stress (PCL score ≥44) was 14.6% and 21.5%, respectively. The characteristics of each group according to the external radiation dose are shown in Table Table The ORs and 95% CIs for high risk of psychological distress (K6 score ≥13) according to external radiation doses are presented in Table Table For post-traumatic stress, the age- and sex-adjusted ORs for the 1 to <2 mSv and ≥2 mSv groups compared with the <1 mSv group were 0.94  and 1.07 , respectively Table . After cWhen imputation was not performed, the age- and sex-adjusted ORs for the 1 to <2 mSv and ≥2 mSv groups were 0.89  and 1.04 , respectively. After adjusting for all the above listed variables, the adjusted ORs for the 1 to <2 mSv and ≥2 mSv groups were 0.91  and 0.96 , respectively  before controlling variables, the adjusted OR decreased to 1.00  after adjustment for evacuation, perception of radiation risk to genetic effects, and subjective health status. Evacuation, perception of radiation risk, and subjective health status were associated with psychological distress in this study . It is s32 showed that chronic diseases , worries about livelihood, lost jobs, lost social ties, and concerns about compensation were significant predictors of probable PTSD 1 year after the Fukushima Daiichi nuclear power plant accident. In the present study, the proportion of subjects who experienced evacuation was significantly different among the three radiation dose groups, with the lowest in the 1 to <2 mSv group . Furthermore, evacuation was significantly associated with post-traumatic stress (PCL score ≥44)  . The dif22 the response rate to the self-administered questionnaire regarding external doses was relatively low (56.7%). There might be a possibility that some response bias could have an influence on this rate. However, the results without multiple imputation showed similar associations, which supports our findings. Second, external doses were estimated using a self-administered questionnaire, and it was possible that the inaccuracy of the answers affects classification in the external radiation groups. Third, there might be residents who were in a bad mental health status and could not answer the survey. Previous studies33,34 indicated that mental health status might affect the response rate to the survey, which might have occurred in the present study. Fourth, we did not examine the relationship between internal radiation doses and psychological distress or post-traumatic stress. Fifth, high level of psychological distress might affect subjective health status in the study participants. More frequent poor subjective health status could be a consequence of high level of psychological distress, which may lead to over-adjustment of ORs in the results of this study. Finally, insufficient information was available regarding pre-disaster factors, such as personality traits, social adaptation, traumatic experiences, and other mental health problems.11 Nevertheless, to our knowledge, this is the first large-scale and systematic survey among evacuees to investigate the association between external radiation doses and psychological distress or post-traumatic stress after a nuclear accident. Our findings of no associations between external radiation doses and psychological distress provide important evidence for evacuees after nuclear accidents.This study has some potential limitations. First, although we imputed external radiation doses using multiple imputation,In conclusion, although the women in the ≥2 mSv group tended to have higher risk of psychological distress, the adjusted OR decreased to 1.00  after controlling for variables. Although the 1 to <2 mSv group was associated with a lower risk of post-traumatic stress after controlling for variables, there were no dose-dependent associations between radiation dose and post-traumatic stress. Evacuation and risk perception of radiation may increase psychological distress in women in the higher radiation dose group. Since evacuees have different backgrounds and experiences after a nuclear disaster, careful assessment of various factors, including disaster-related factors and risk perception, is needed for individual interventions. Moreover, since mental health problems, such as psychological distress, post-traumatic stress, and radiation health anxiety, exist for a long time, further mid- and long-term studies are needed to confirm and extend the results.


Sesamin, a major phytochemical in sesame seeds and oil, has been reported to have effects on physiological and pathological angiogenesis in several studies. Nevertheless, the underlying mechanisms of sesamin’s effect on angiogenesis are not understood well enough. This study aimed to investigate its effect on both physiological and pathological angiogenesis using the in vivo chick chorioallantoic membrane (CAM) model and the in vitro human endothelial cell line, EA.hy926, model. Sesamin inhibited the VEGFA-induced pathological angiogenesis significantly, although no effect was seen on angiogenesis without induction. It reduced the formation of vascular branches in the VEGFA-treated CAMs and also the proliferation and migration of EA.hy926 endothelial cells induced by VEGFA. Sesamin impeded the VEGF-mediated activation of Src and FAK signaling proteins, which may be responsible for sesamin-mediated reduction of pathological angiogenesis. Moreover, the effect of sesamin on the expressions of angiogenesis-related genes was then investigated and it was found that both mRNA and protein expressions of Notch1, the key pathway in vascular development, induced by VEGFA, were significantly reduced by sesamin. Our results altogether suggested that sesamin, by inhibiting pathological angiogenesis, has the potential to be employed in the prevention or treatment of diseases with over-angiogenesis, such as cancers.  Angiogenesis is the process of developing new vessels from the pre-existing vasculature and it occurs throughout the entire lifespan of an organism under both physiological and pathological conditions . Since tAngiogenesis takes place in a number of sequential steps: endothelial cell activation, cell sprouting, proliferation, migration, lumen formation, cell remodeling, and maturation. It is initiated in normally quiescent endothelial cells (ECs) upon receiving pro-angiogenic factors such as Vascular Endothelial Growth Factor (VEGF), the principal angiogenic factor. When VEGF contacts the VEGF receptor (VEGFR), the receptor generates the signals to develop a vascular system in the embryo or to generate blood vessels and lymphatic vessels in adults . Among vVEGF/VEGFR signaling also has an influence on a variety of other pathways. Notch signaling is an evolutionally conserved pathway involved in the determination of fate of many tissues and cell types including endothelial cells. Notch receptors and their ligand, the delta-like ligand 4 (Dll4), are responsible for tip and stalk cell differentiation from endothelial cells (ECs) in nascent vascular sprouts, while the migration and proliferation of tip and stalk cells governs the formation, elongation, and maturation of new vessels eventually ,9. As angiogenesis is regulated by the balance between pro-angiogenic and anti-angiogenic signals, any disturbance in that balance would lead to pathological conditions. While an insufficient vascular supply results in ischemic diseases, excess neovascularization is a hallmark of solid tumors ,11. The Sesamun indicum L.), a lignan found in sesame seeds and oil, is reported to have biological effects including chemoprevention, anti-inflammation, and anti-oxidant properties [Sesamin   and in E® . Illustra RNAspin Mini RNA Isolation Kit and the primers for real-time RT-PCR were acquired from GE Healthcare Europe GmbH  and BioDesign , respectively. A Tetro cDNA Synthesis kit and a SensiFAST™ SYBR® No-ROX Kit were purchased from BIOLINE . Angiogenesis Antibody Sampler Kit, Notch1 (D1E11) and β-Actin Rabbit monoclonal antibody were procured from Cell Signaling Technology® .Methylthiazoletetrazolium (MTT) (PubChem CID: 64965), Dimethyl sulfoxide (DMSO) (PubChem CID: 679), and Resazurin (PubChem CID: 11077) were obtained from Sigma Chemical, Inc. . Recombinant human vascular endothelial growth factor A  was purchased from R&D systemsSesamin seeds were procured from the Lampang province of Thailand, and the voucher specimens (BKF no. 138181) were approved by the National Park, Wildlife and Plant Conservation Department, Ministry of Natural Resources and Environment, Bangkok, Thailand. Sesamin was extracted from the seeds by using the method reported in the previous study . The ses2 was gently opened on the wide end of the egg without damaging the embryo. A plastic ring was placed directly on the top of the CAM. Various concentrations of sesamin and/or 20 ng/mL of VEGFA were added directly onto the plastic rings. The eggs were transferred back into the incubator and the numbers of vascular branches were counted at day 10 of incubation by photographing the CAM area of each egg. Images were analyzed by counting the number of branching vessels in the plastic ring. Scores of primary-, secondary-, tertiary-, and quaternary-branching vessels are denoted by 1, 2, 3, and 4, respectively. Further branching vessels with more than quaternary-branching were scored as 5. Scores from each group were averaged by the number of eggs and the average score of each group was normalized to that of the control group [Fertilized chicken eggs were incubated at 37 °C and approximately 50–60% humidity. On day 3 of incubation, 5–6 mL of albumen was aspirated to detach the developing CAM from the top part of the shell. On day 8, a window of around 1.5 cmol group ,24.® (CRL2922™). This cell line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line, A549. This study used EA.hy926 because this cell line shows endothelial characteristics and more cells can be obtained than from the primary cells [2 at 37 °C. A cell passage of six or seven was used in the experiments.EA.hy926, a human umbilical vein endothelial cell line, was purchased from ATCCry cells . The celTo determine the toxicity on cells and select the optimal concentrations of sesamin, a MTT assay was performed. EA.hy926 cells were placed in a 96-well-plate (1000 cell/well) and incubated overnight. After cells were treated with various sesamin concentrations for 24 h, the culture media were discarded and replaced with 100 μL of MTT (0.5 mg/mL) solution for 4 h. Then, the MTT agent was discarded and 100 μL dimethyl sulfoxide (DMSO) was added into each well to solubilize the formazane crystals. The absorbance was measured at 540 nm using a microplate reader and the percentage cell survival compared to the controls was calculated as follows: To determine the migration ability of EA.hy926 cells, an in vitro wound healing assay was performed. EA.hy926 cells were seeded into 24 well plates  and incubated. After 24 h of incubation, a line was scraped through the monolayer of the cells using a 250 μL-pipette tip (wounding). The cells were then washed with PBS and treated with various concentrations of sesamin and/or 20 ng/mL VEGFA. The wounds were photographed at different time points under a light microscope (40× magnification). The measurements of the scratched region were calculated using AxioVision Analytic Software version 4.7 from Carl Zeiss . [2 for 24 h. The culture media was then discarded, and the cells were treated with various concentrations of sesamin and/or 20 ng/mL VEGFA for 1 week. Every 24 h, the culture media was discarded and replaced with 10% (v/v) Alarmar Blue fluorescent dye in media for 4 h at 37 °C. The absorbance of the media was read at 540 and 620 nm using a microplate reader spectrophotometer. The absorbance was measured at wavelengths of 540 and 620 nm. The percentages of the differences in reduction were calculated, and the data represented as percentages of the differences in reduction relative to those of the control group.The extent of cell proliferation was assessed using the AlamarBlue assay. EA.hy926 cells were seeded into 96 well plates (1000 cells/well) and incubated at 37 °C, 5% CO® No-ROX kit on Chromo4TM Four-Color Real-Time Detector from Bio-Rad  in a final volume of 20 μL. The primer concentration was 500 nM. Forty cycles of PCR amplification were performed at 95 °C for 5 s and 60 °C for 10 s. The primers used are shown in ΔΔCT method [Real time RT-PCR was used to examine the gene expression in EA.hy926 in response to treatment with various concentrations of sesamin and/or 20 ng/mL VEGFA. After 24- or 48-h incubation periods, cells were lysed, and the total RNA was isolated using the RNA extraction kit  following the manufacturer’s protocol. Samples were treated with DNase before washing and elution steps. After extraction, the amount of total RNA in samples was measured using nanodrop spectrophotometer , and 200 ng of total RNA were converted to cDNA using a Tetro cDN A Synthesis Kit in a final volume of 20 μL. Reaction conditions were set as suggested by the manufacturer. After reverse-transcription, the product cDNA was diluted with RNase free water in 1:10 dilution. Then, 8 μL of the cDNA mixture was used for real-time PCR experiments. Real time PCR was performed using a SensiFastTM SYBR2. At the indicated times, the cells were lysed with lysis buffer containing 50 mM Tris-HCL, pH7.4, 250 mM NaCl, 0.5% NP-40, 5 mM EDTA, and 50 mM NaF along with protease and phosphatase inhibitors . An equal volume of whole cell lysate was electrophoresed and transferred to a nitrocellulose membrane. After blocking with 5% skim milk in 0.05% PBS-TWEEN, the membranes were incubated with specific primary antibodies. After the incubation of the secondary antibodies and washing, specific protein bands were developed using Supersignal West Femto Substrate  and were photographed using the molecular chemidoc XRS system . The band density was analyzed using TotalLab TL120 software version 2006 and calculated in relation to the control sample. The beta actin was used as an internal protein control.EA.hy926 cells were treated with various sesamin concentrations and/or VEGFA and incubated at 37 °C, 5% COp < 0.05.All data are given as mean ± standard error of mean (SEM) from triplicate samples of three or two independent experiments. One-way analysis of variance (one-way ANOVA) and student’s t-test were used to compare the treatment and control conditions using data from three or two independent experiments, respectively. Statistical significance was assumed at This experiment was preliminary to the investigation of the effect of sesamin on both physiological and pathological angiogenesis in an in vivo model. The experiment focused on the number of vascular branches formed on the CAM. The results showed that various concentrations of sesamin  did not cause significant changes on the formation of new branching vessels in the CAM. Therefore, the concentrations of sesamin used did not affect in vivo angiogenesis under physiological conditions A. To mimic the pathological conditions with reported increased angiogenesis, such as cancer, the CAM was treated with the vascular endothelial growth factor A (VEGFA) . After 4Before the effects of sesamin on angiogenesis could be determined in the in vitro model, its cytotoxicity on the cell line was first examined. EA.hy926, a human endothelial cell line, was treated with sesamin, 1.6 to 25 μM concentrations, for 24, 48, and 72 h and the percentage of cell viability compared to the untreated control was determined by the MTT assay. The percentages of cell viability under all conditions were greater than 80% relative to the control, indicating that sesamin up to 25 μM had no significant cytotoxic effect on EA.hy926 A. DMSO, Since proliferation of endothelial cells is an important step in angiogenesis supporting the elongation of new blood vessels, the effect of sesamin on endothelial cell proliferation was investigated. EA.hy926, human endothelial cells, were treated with various concentrations of sesamin (1.5–10 μM), with or without VEGFA, and the cell proliferation was analyzed using the AlamarBlue assay at days 5, 7, 9, and 11. The percentage of proliferation of uninduced EA.hy926 cells was not significantly affected by the treatment with sesamin, compared with that of the control, indicating that sesamin has no effect on angiogenesis itself B. VEGFA expression is high in various pathological conditions such as cancer and inflammation. Moreover, previous studies have reported that in vivo induction with a single VEGFA treatment, VEGFA (164/5), can result in the formation of new blood vessels that differ from normal vessels . TherefoDuring angiogenesis process, endothelial cells migrate through the basement membrane, leading to the generation of new sprouting vessels. As the migration process is another crucial process in angiogenesis, the effect of sesamin on the migration of endothelial cells was also investigated in this study. After the monolayer of human endothelial cells, EA.hy926, was wounded using a 250 μL-pipette tip, they were treated with various concentrations of sesamin for 9, 12, and 24 h. After each incubation-time point, the size of the wound gap was calculated with Axio Vision analytic software to examine the migration ability of the cells. In un-induced EA.hy926, the migration ability was not changed significantly by the treatment with various concentrations of sesamin (1.5–10 μM) A. This rFollowing the pre-treatment with sesamin, EA.hy926 cells were induced with VEGFA to study the effect of sesamin on the activation status of signaling pathways such as ERK, p38, PLCγ1, Src, and FAK, which are reported to be responsible for the proliferation and/or migration of endothelial cells induced by VEGFA .The results of the western blot assay showed that VEGFA (20 ng/mL) activated all signaling pathways; the expression of phosphorylated forms of ERK, p38, PLCγ1, Src, and FAK proteins were obviously enhanced by VEGFA treatment in EA.hy926 cells. Interestingly, pre-treatment with sesamin  can diminish the expression of phosphorylated Src and FAK proteins augmented by VEGFA, although it had no effect on the VEGFA-induced expression of phosphorylated ERK, p38, and PLCγ1. Moreover, 10 μM sesamin alone slightly reduced the phosphorylation of ERK, p38, and Src compared to the untreated control, but had no effect on that of PLCγ1 and FAK . Taken tNOTCH1), delta like 4 (Dll4), vascular endothelial growth factor A (VEGFA), Kinase Insert Domain Receptor or VEGF receptor 2 (KDR), Angiopoietin-1 (ANG1), Angiopoietin-2 (ANG2), and Tie 2 receptor (Tie2), using real time RT-PCR. It is well known that VEGFA signaling has effects on the expression of the genes related to angiogenesis ,27. SincNOTCH1 significantly after 24 h of incubation, but not that of VEGF or Dll4 gene. In case of the VEGFA receptor, the KDR gene, VEGFA decreased the expression, but not to a significant extent  alone or together with various concentrations of sesamin  for 12 h and 24 h, the abovementioned genes were examined. For genes involved with specifying tip and stalk cells, VEGFA induced the expression of t extent A,B. Hereby VEGFA A,B.ANG1, ANG2 nor that of their receptor Tie2 were changed significantly by any of the treatments, either by VEGFA alone or cotreatment of VEGFA and sesamin  plays an important role in the sprouting step of angiogenesis. The balance of NOTCH1 activation status between tip cells and stalk cells determines the formation and branching of new vessels ,28. This experiment investigated the effect of sesamin on NOTCH1 expression in EA.hy926 using western blotting. After EA.hy926 cells were treated with 20 ng/mL of VEGFA for 24 h, the expression of NOTCH1 protein was increased and cotreatment with sesamin inhibited this induction significantly, corresponding to the result of mRNA analysis in the previous experiment . These rSesamun indicum L.), with known anti-inflammatory and anti-oxidant activities on the angiogenesis process. Nevertheless, their findings were contradicting as Tsai et al. reported sesamin as being anti-angiogenic while Chung et al. stated that sesamin induced angiogenesis [The angiogenesis process occurs throughout the entire life of vertebrates and plays an important role in both the physiological and pathological conditions ,6. Abnorogenesis ,20. MoreWe first investigated the effect of sesamin on in vivo angiogenesis in the CAM model and the formation of vascular branches in CAMs was not affected by sesamin, reflecting its lack of influence on the physiological angiogenesis. This finding is contrary to that of Chung et al., in which sesamin significantly increased neo-vascularization using the Matrigel model in male BALB/c mice . It appeNext, we further studied the effect of sesamin on angiogenesis and its underlying mechanisms, including its effect on NOTCH signaling, using the in vitro cell model, human endothelial cells EA.hy926. During the sprouting process of a new vessel, quiescent endothelial cells become activated and secrete enzymes that degrade extracellular matrix (ECM) proteins in basement membranes, allowing endothelial cells to migrate through. Then, endothelial cells in a nascent sprout are differentiated into two cell types, namely tip cells and stalk cells: tip cells are highly migratory and responsible for sensing the directional cues, thereby defining the route of the new sprouts and stalk cells, which highly proliferate and support sprout elongation ,30,31. SThe responsible signal transductions for the inhibitory effect of sesamin on VEGFA mediated angiogenesis were also investigated. We focused on the activation of p38, ERK, PLCγ1, Src, and FAK pathways, given that activation of ERK through PLCγ1 was reported to associate with the regulation of endothelial cell survival and proliferation while that of p38 and FAK was to control the migration of endothelial cells and that of Src was to regulate cell-cell contacts, proliferation, and migration ,32. AlthNOTCH1 expression was significantly induced, whereas Dll4, and VEGF expressions were not significantly changed. KDR expression was reduced, though not significant, by VEGFA treatment. This profile of high Notch and low KDR expressions proposed that endothelial cells in this study represent stalk cells, although it cannot be definitely distinguished since we used monolayer endothelial cell line lacking 3D interaction. Interestingly, sesamin was found to reduce the Notch1 induction by VEGFA significantly in both gene and protein levels. Previous studies found that inhibition of Notch signaling leads to an increased formation of non-functional vascular sprouts in tumor vasculature, which was explained by the de-repression of tip-cell behavior in endothelial cells [As mentioned above, endothelial cells in the form of tip or stalks cells work together in a harmonious way in sprouting angiogenesis. While the spear head tip cells migrate and lead the sprout towards the stimulant, a high VEGF gradient, they suppress the tip cell phenotype in the adjacent cells, which then become stalk cells. Stalk cells highly proliferate and facilitate the guiding of tip cells, resulting in elongation of the new vessels. Notch signaling pathway, which plays a critical role in cell fate determination, is involved in the specification of endothelial cells into tip and stalk cells . When VEal cells . Since sal cells . These cAng1, Ang2, and their receptor Tie2, in EA.hy926 cells. Nevertheless, no conclusive finding was obtained. Sesamin increased the expression of Ang1 under VEGF influence of 12 h-duration, suggesting its potential to help the newly formed vessels to become stabilized, but the finding was not confirmed as the effect was not significant and disappeared in the 24-h period. On the other hand, treatment with sesamin alone reduced the expression of Ang1 and increased that of Ang2 in EA.hy926 cells without VEGF induction, although it was not significant. Therefore, the effect of sesamin on angiopoietins is yet to be confirmed in future studies using other endothelial cell types or a different dose and duration of the sesamin treatment.Activation of alternative angiogenic signaling pathways is contributing to the resistance against VEGF blockade therapy in cancer patients. Angiopoietin 2 (Ang2), being the antagonist of Ang1 that stabilizes nascent vessels by recruiting mural cells, is implicated in the formation of unstable and leakier vessels and it is upregulated in many cancers . Hence, Summarizing our study, sesamin inhibited in vitro and in vivo VEGFA-induced angiogenesis, while there was no influence on angiogenesis without induction, and this effect was accounted for by the reduction of the activation of Src and FAK induced by VEGFA. In addition, inhibition of Notch signaling, which was crucial for the proliferation of stalk cells, may also be responsible for the inhibitory effect of sesamin on VEGFA-induced angiogenesis. These results altogether highlighted the potential of sesamin as an anti-angiogenic therapeutic adjuvant, nonetheless, confirming that its effect in therapeutic animal models for accessing functions of the vasculatures is an absolute must.


This study aimed to investigate multimorbidity patterns and their associated factors among elderly population in China.A total of 10,479 participants aged at least 60 years were drawn from the 2018 wave of the China Health and Retirement Longitudinal Study (CHARLS). Latent class analysis (LCA) was performed to identify distinct multimorbidity classes based on 14 self-reported chronic conditions. The multinomial logit model was used to analyze the associated factors of multimorbidity patterns, focusing on individuals' demographic characteristics, socioeconomic status (SES), and health behaviors.Among the 10,479 participants (mean age [SD]: 69.1 [7.1]), 65.6% were identified with multimorbidity. Five multimorbidity clusters were identified by LCA: relatively healthy class (49.8%), vascular class (24.7%), respiratory class (5.6%), stomach-arthritis class (14.5%), and multisystem morbidity class (5.4%). Multinomial logit analysis with the relatively healthy class as the reference showed that participants of older age and female sex were more likely to be in the vascular class and multisystem morbidity class. The probability of being in the vascular class was significantly higher for those with high SES. Ever smoking was associated with a higher probability of being in the respiratory class and multisystem morbidity class. Physical activity was associated with lower odds of being assigned to the vascular class, respiratory class, and multisystem class.The distinct multimorbidity patterns imply that the prevention and care strategy should target a group of diseases instead of a single condition. Prevention interventions should be paid attention to for individuals with risk factors.The online version contains supplementary material available at 10.1186/s12877-022-03154-9. Multimorbidity, the co-existence of two or more chronic conditions , has becGiven its importance, there has been a significant increase in studies on multimorbidity in both developed and developing countries, investigating the prevalence, risk factors, and/or outcomes. However, the measurement of multimorbidity varied in different studies. A large number of prior studies assessed multimorbidity by either the co-existence of two or more conditions or simply counting the number of concurrent conditions . As the Some scholars have tried to use cluster analysis approaches, such as latent class analysis (LCA), to investigate multimorbidity patterns. LCA is an appropriate tool to identify latent classes of observed variables based on structural equation modeling. It has been applied by some studies to examine how individuals are clustered according to patterns of chronic conditions. For example, by focusing on 10 self-reported conditions from the National Health and Aging Trends Study in the United States, Nguyen and colleagues  identifiHowever, the clustering patterns of chronic conditions were rarely examined in China using national-level data. Among the few exceptions, some studies used China Health and Retirement Longitudinal Study (CHARLS), a nationally representative dataset in China. A study on individuals aged 50 and older in China identified 76 dyadic and 169 triadic combinations using observed-to-expected ratios , which aThe multimorbidity concern could be a bigger problem for developing countries, as their healthcare systems may not be well prepared for the rapid transition from infectious diseases to non-communicable diseases . In addiData were from CHARLS, a nationally representative longitudinal survey in China. CHARLS, harmonized with the Health and Retirement Study (HRS) international family of surveys, is a nationally representative survey of people aged 45 years old or above in China. The strict adherence to stratified probability proportional to size (PPS) random sampling principles and strict quality control measures lead to its representativeness of China , as shown by the close match between the 2011 baseline wave and the 2010 census in demographics. CHALRS collects high-quality multidisciplinary data, including basic demographics, socioeconomic status (SES), and health information . The basn = 10,794) as the age of 60 is widely considered the onset of old age in China. To study multimorbidity patterns, we deleted those with missing information for any of the 14 chronic diseases (45 dropped), leading to a total of 10,749 participants for descriptive analysis and the identification of multimorbidity patterns , chronic liver disease , heart problems , stroke, kidney disease (except for tumor or cancer), stomach or other digestive diseases (except for benign tumor or cancer), emotional, nervous or psychiatric problems, memory-related disease, arthritis or rheumatism, and asthma. To provide a more comprehensive analysis of multimorbidity and be consistent with the literature, we included all 14 conditions in the assessment of multimorbidity , 14. EacGuided by the literature , 26, indWhen investigating the associated factors of multimorbidity patterns, we imputed all categorical independent variables by adding a missing category for each independent variable to retain the maximum number of cases. After the imputation, we excluded 7 participants with missing values for medical insurance and 3 on smoking from the descriptive sample, leading to a final sample of 10,739 respondents for the regression analysis of the factors influencing multimorbidity patterns.LCA was performed to identify the clustering pattern of different chronic diseases among the 10,749 participants based on 14 conditions. LCA is a useful tool for determining groups or subtypes of cases in multivariate categorical data. LCA assigns individuals into distinct and mutually exclusive multimorbidity classes based on the model-based posterior membership probabilities in our setting . We examAfter selecting the best fitting solution and individuals' classifications into different classes, multinomial logit analysis was used to examine the influencing factors of multimorbidity classes, with all selected sociodemographic and lifestyle characteristics entered into the model simultaneously. All analyses were performed using Mplus version 6.1 and Stata version 17.Table Table Table When identifying the clustering of diseases, two- to six-class models were examined using LCA. The five-class model emerged as the best fitting one  more likely to be classified into the vascular class . Individuals with higher education (primary and above) were associated with 1.20 times (95% CI: 1.08–1.34) higher probability being in the vascular class. The relationship was further evidenced by the significantly higher odds of belonging to the vascular class for those with higher income levels . Compared to having no insurance, all types of insurance were associated with an increased likelihood of being in the vascular class, with the highest RRRs for UEBMI . In addition, the probability of being classified into the multisystem morbidity class was 2.03 times (95% CI: 1.16–3.54) higher for those with URBMI.Lifestyle factors were also found to be important influencing factors. The probability of being assigned to the respiratory class increased by 2.07 times (95% CI: 1.62–2.65) for smokers compared with those who had never smoked. Also, smokers were more likely to be in the multisystem class . Physical activity decreased the probability of belonging to almost all other multimorbidity classes , except for the stomach-arthritis class. The results showed that intensive physical activity was associated with lower odds of being assigned to the vascular class, respiratory class, and multisystem morbidity class, with RRRs of 0.51 (95% CI: 0.43–0.60), 0.49 (95% CI: 0.36–0.65), and 0.40% (95% CI: 0.30–0.53), respectively. However, there were no significant influences of alcohol consumption on multimorbidity patterns.Using the latest wave of CHARLS, this study reported the prevalence of multimorbidity patterns and examined its associated factors among elderly population in China. About 65.6% of all participants had at least two chronic conditions. By applying LCA, all participants were assigned to five multimorbidity classes based on their shared disease pattern: relatively healthy, vascular, respiratory, stomach-arthritis, and multisystem morbidity. When examining the associated factors of multimorbidity patterns, we found that participants of older age and female sex were more likely to be assigned to the vascular class and multisystem morbidity class . A positive association was shown between SES and the vascular class. Also, the probability of being assigned to the multisystem morbidity class was significantly higher for those with UEMI or other MI. Smokers were more likely to be in the respiratory class, and multisystem morbidity class. Physical activity was found to decrease the probability of being in the vascular class, respiratory class, and multimorbidity patterns.Multimorbidity was highly prevalent (65.6%) among the studied population, which slightly differed from the prevalence reported in the literature , 27, 29,Using LCA, our study further identified five multimorbidity patterns among older adults in China, namely, relatively healthy 49.8%), vascular 24.7%), respiratory (5.6%), stomach-arthritis 14.5%), and multisystem morbidity (5.4%). Previous studies also studied multimorbidity patterns in China but more focused on disease similarities instead of categorizing all participants into exclusive multimorbidity classes , 31. The4.7%, res.5%, and 9.8%, vasBased on previous studies on the risk factors of multimorbidity, we extended the literature for multimorbidity patterns. Consistent with previous evidence , 26, 32,In contrast to previous studies in developed countries showing that people with low SES were more likely to belong to multimorbidity classes related to cardiometabolic conditions , 26, 32,In line with the existing evidence , we reveThe current study applied LCA to classify the participants into five latent classes and analyze the associated factors of multimorbidity patterns. Nevertheless, it has several limitations. First, most of the variables are self-reported, which may cause potential bias. For example, all 14 chronic conditions come from respondent self-reporting. Therefore, the multimorbidity prevalence was underestimated, and associated factors for multimorbidity patterns should be interpreted with caution regarding the problem of underdiagnosis. Second, the cross-sectional design of the current study made it impossible to draw confident causal conclusions. Third, other potentially influential factors such as the nutrition status were not involved due to the limitations of the secondary dataset. Our strengths include the provision of the recent picture of multimorbidity picture based on the latest data from a nationally representative survey in China, which could be generalized to the whole country, and more importantly, the examination of risk factors for different multimorbidity classes, which has great implications for both health policy and clinical practice.In conclusion, using nationally representative data of older adults in China, we revealed a multimorbidity rate of 65.6%. LCA analysis showed that different conditions clustered together in a predictable pattern and identified five multimorbidity classes: relatively healthy, vascular, respiratory, stomach-arthritis, and multisystem morbidity. Older age, female sex, high SES, smoking, and no physical activity were risk factors for certain multimorbidity patterns. The findings have significant policy and clinical implications concerning the choice of prevention interventions and treatment strategies to target a multimorbidity pattern instead of a single condition.Additional file 1: Supplementary Figure 1. Flow chart of sample selection. Supplementary Table 1. Characteristics of participants in the regression analyses by latent class membership A Comparison of the fit statistics of models of latent class analysis. Supplementary Table 2. Item response probability from the five-class model. Supplementary Table 3. Characteristics of participants in the regression analyses by latent class membership.


Surveillance of surgical site infection after cesarean section is challenging due to the high volume of these surgeries. A manual chart review of women undergoing cesarean section between January and June 2017  was compared to charts identified via an algorithm . The algorithm achieved 97.5% sensitivity and 83.9% specificity and reduced the workload of infection control personnel. Surgical site infection (SSI) surveillance is an essential tool for infection control. SSI rate is an important indicator of quality of care. Comparing institutional rates to a benchmark and providing feedback to surgeons have been shown to reduce infection rates. Chart review is less desirable for high-volume surgeries, such as cesarean section, because it is very time-consuming for infection control personnel (ICP). Thus, more efficient methods are needed, such as electronic surveillance systems, which rely on data collected automatically from hospital databases. In a semiautomated surveillance system, an algorithm identifies cases with high probability of infection, and subsequently, ICP verify or rule out infection based on manual chart review of identified cases. To reduce workload, an ideal semiautomated system would have high sensitivity with high negative predictive value (NPV)  and high specificity. Several electronic algorithms have been validated for various surgeries, but the patient population undergoing cesarean section is unique and thus it merits a specific algorithm. Our objective was to validate a semiautomated system for cesarean section SSI surveillance.Complete chart review is a commonly used surveillance method; it can be done retrospectively, and it is effective.We conducted a validation study on an historical cohort. The setting was a secondary-care, 740-bed, university-affiliated hospital in central Israel, with ∼6,000 births and ∼1,300 cesarean sections per year. In this study, we reviewed the electronic medical records of all women who underwent cesarean section between January 1 and July 30, 2017. Variables collected included demographics, gestational age at delivery, body mass index (BMI), comorbidities, previous cesarean section, antibiotic prophylaxis, cesarean section duration, American Society of Anesthesiologists (ASA) score, rupture of membranes, imaging, postoperative antibiotics, bacteriology results, wound class, diagnosis at discharge, length of hospital stay (LOS), readmissions or visits to the emergency department (ED) up to 30 days after cesarean section, ambulatory visits to family physician, and antibiotic prescription in the community within 30 days after cesarean section.All electronic medical records of study participants were reviewed retrospectively by one of the authors (R.M.) to assess the presence of SSI based on US Center for Disease Control and Prevention (CDC) definitions.In parallel, an algorithm was built to seek suspected cases for further manual review of the medical records by hospital ICP. The algorithm included at least 1 of the following criteria: (1) LOS >4 days, (2) fever during labor, (3) rehospitalization or return to any ED within 30 days of cesarean section, (4) blood or wound cultures performed during hospitalization, and (5) any antibiotic given during hospitalization, excluding prophylaxis. In all suspected cases, SSI was diagnosed independently by ICP according to CDC definitions.Because this was a quality intervention, it was not subject to approval by the institutional review board nor did it require written informed consent.Categorical variables were described as percentages and numerical variables as mean or median with standard deviation (SD) or range, as appropriate. For the validation phase, the complete chart review was used as the gold standard and the semiautomated surveillance system was compared to it. Sensitivity, specificity, positive predictive value (PPV), and NPV were calculated. These parameters were calculated for the entire tool and for each separate component.During the study period, 675 patients underwent cesarean section. The median age was 33 years . The median BMI was 27.2 , and the mean gestational age at delivery was 37.8 ± 2.8 weeks. The most common comorbidity was diabetes (9%), and 7% of the cohort smoked tobacco. The median duration of surgery was 39 minutes ; 61% were emergency operations; and most (88%) were clean/contaminated operations. The most common indication for surgery was “nonreassuring fetal heart rate.”Among the 675 surgeries, the complete chart review identified 40 SSIs (5.9%). Among them, 29 (4.3%) were superficial incisional SSIs, 1 (0.1%) was a deep incisional SSI, and 10 (1.5%) were organ-space SSIs.The automated surveillance method identified 141 suspected cases, 21% of the cohort. Among these, chart review identified 39 infections: 27% of the suspected cases and 5.6% of the entire cohort. The sensitivity of the semiautomated surveillance system was 97.5% ; specificity was 83.9% ; PPV was 27.6 ; and NPV was 99.8%   without missing any, thus increasing specificity to 89.3% and reducing ICP workload. and >20% in our medical center in Israel. These numbers make manual SSI surveillance with chart review incredibly challenging. In this study, we validated a semiautomatic tool that reduced ICP workload by 79% and had 97.5% sensitivity, missing only 1 patient with a superficial wound who did not return to the hospital.Cesarean delivery is a common procedure, accounting for >30% of births in the United StatesPatients undergoing cesarean section are typically healthy young women without comorbidities. Moreover, hospitalization after surgery is short and standardized. When a young woman returns to the ED after delivery, the visit is usually related to the delivery itself. As in other studies, we collected data on comorbidities, smoking status, duration of surgery, physician diagnosis, antibiotic use and more, but none of these data added sensitivity to our tool. In most cases, either the infection was evident early, causing prolonged hospitalization, or was evident late, resulting in return to the ED. The only case missed was a mild, superficial infection treated in the community, which would have been missed by any tool built on hospital-based data. Notably, the tool we created to identify cesarean section SSIs is specific to this patient population and would not be appropriate for an elderly, sick population undergoing orthopedic or colon surgeries. and Yokoe et al screened automated, ambulatory administrative claim data that were used to identify cesarean-section SSIs. The indicators they identified were diagnosis of infection, blood and wound culture, and antibiotics. The specific electronic database used should be tailored to each health system. Considering the high volume of cesarean section surgeries and the constraints in ICP personnel, an automated tool is essential for timely and efficient surveillance of cesarean-section SSIs.Few studies have specifically investigated an electronic tool for SSIs after cesarean section. Studies by Miner et alThis study has several limitations. It was a single-center study; the semiautomated system would miss patients who present for postoperative care at another hospital. Furthermore, in other countries, the LOS after cesarean section or the barriers to return to the ED might be different. However, compared to patients undergoing other surgeries, postdelivery women are a homogeneous population that require a focused approach. Our gold standard for SSI diagnosis was the complete chart review method and not prospective follow-up of patients, which is nearly impossible with such a high-volume surgery.In conclusion, this study validates a semiautomated tool with high sensitivity and high specificity that identifies SSIs after cesarean section. It can help provide valuable information and decrease the workload for ICP.