The multifaceted activity of vitamin D in patients with inflammatory bowel disease (IBD) presents a challenge for further research in this area. Vitamin D is involved in the regulation of bone mineral metabolism, it participates in the regulation of the immune system, and it is an underlying factor in the pathogenesis of IBD. Additionally, vitamin D affects Th1 and Th2 lymphocytes, influencing the release of cytokines and inhibiting tumor necrosis factor (TNF) expression and the wnt/β-catenin pathway. As far as IBDs are concerned, they are associated with microbiota dysbiosis, abnormal inflammatory response, and micronutrient deficiency, including vitamin D hypovitaminosis. In turn, the biological activity of active vitamin D is regulated by the vitamin D receptor (VDR) which is associated with several processes related to IBD. Therefore, in terms of research on vitamin D supplementation in IBD patients, it is essential to understand the metabolic pathways and genetic determinants of vitamin D, as well as to identify the environmental factors they are subject to, not only in view of osteoporosis prevention and therapy, but primarily concerning modulating the course and supplementation of IBD pharmacotherapy. Vitamin D has been of particular interest to researchers in recent years. It has been emphasized that it presents a multidirectional effect, in terms of both bone tissue and several extraskeletal activities, such as affecting cell proliferation and differentiation, as well as regulating gene expression. All the abovementioned functions are crucial in inflammatory bowel diseases, i.e., systemic diseases with multifactorial etiology .The prevalence of both Crohn’s disease and ulcerative colitis in industrialized societies is increasing, particularly among young people. Moreover, vitamin D deficiency, caused by nutritional deficiencies, vitamin and microelement malabsorption, and avoidance of sun exposure, is also a common issue, especially in patients with inflammatory bowel disease (IBD). Vitamin D metabolism and intestinal microbiota are closely intertwined, which is particularly important in this group of patients . VitaminVitamin D, which serves as a prohormone, is a fat-soluble vitamin in two forms: ergocalciferol vitamin D2) and cholecalciferol (vitamin D3). The primary sources of vitamin D in the body include dermal synthesis induced by sunlight and dietary intake . Initial and cholVitamin D is transported to the liver in order to be metabolized into 25(OH)D by 25-hydroxylase (CYP2R1); 25(OH)D is the inactive form of vitamin D that is subsequently converted in the kidneys by the 1-α-hydroxylase enzyme (CYP27B1) into its active form 1,25(OH)2D. This process, in turn, is closely regulated by the parathyroid hormone (PTH), calcium and phosphate levels, and the fibroblast growth factor.Then, 25(OH)D and 1,25(OH)2D are both degraded by 24-hydroxylases (CYP24A1) as a feedback loop negatively induced by 1,25(OH)2D. This results in calcitriol and calcitroic acid production, which are excreted in the urine or in the bile through the intestine. In vitro, calcitroic acid in higher concentrations has also been reported to bind the vitamin D receptor (VDR) and affect gene transcription. Although these studies were not conducted on the basis of intestinal tissue , the preThe primary food sources of vitamin D include fish oil , cod liver oil, liver and organs, and egg yolk, although vitamin D has also been found in certain mushrooms . Generally, animal products contain cholecalciferol , whereasNevertheless, vitamin D content in various products is low. For instance, in the UK-based McCance and Widdowson’s Food Composition database, the percentage of products containing vitamin D above 10 µg/100 g is only 1%. Thus, in order to prevent vitamin D deficiency, in some countries traditional foods  are fortified with vitamin D . HoweverNearly 80% of dietary vitamin D is absorbed in the jejunum and the terminal ileum. It is subsequently released from the enterocytes through the formation of chylomicrons and transported through the lymphatic system to the muscles and adipose tissue. Vitamin D as a prohormone undergoes activation .Ultraviolet-B (UVB) radiation (with energy of 290–315 nm) is absorbed in the epidermis by 7-dehyrdrocholesterol and subsequently converted to previtamin D3, isomerizing into vitamin D3 . AfterwaInflammatory bowel diseases comprise a group of chronic inflammatory conditions of the gastrointestinal tract, which include Crohn’s disease and ulcerative colitis . InflammInflammatory bowel diseases are found globally. As pointed out in a systematic review by Molodecky et al., the highest incidence and prevalence has been reported in Western countries, particularly, in northern Europe and Canada . It is eThe main factors contributing to an increase in the incidence of IBDs include socioeconomic trends and the resulting lifestyle changes, dietary habits, hygiene standards, and other factors comprising the so-called “Western lifestyle” . Crohn’sThe etiology of IBDs has not been entirely determined, although the researchers claim that it involves such factors as inadequate regulation of the immune system , genetic and environmental factors  leading to the chronic inflammatory process of the alimentary tract ,41,42. IVitamin D deficiency appears to be a worldwide problem. According to estimates, nearly one billion people worldwide suffer from vitamin D deficiency . A meta-A group particularly prone to vitamin D deficiency are patients with inflammatory bowel diseases. According to the available data, 22–70% of patients with Crohn’s disease and about 45% of patients suffering from ulcerative colitis show vitamin D deficiency ,49,50. LIBDs are associated with microbiota dysbiosis, abnormal inflammatory response, and micronutrient deficiency, including vitamin D hypovitaminosis. Thus, vitamin D deficiency may constitute a triggering factor and a consequence of IBD. Serum 1,25(OH)2D is involved in immune cell differentiation, gut microbiota modulation, gene transcription, and barrier integrity. Furthermore, it has additional vital roles in the intestines, including maintaining the integrity of mucosal tight junctions, enhancing folate absorption, and activating cytochrome P450 3A4 expression . SeveralIt has been demonstrated that VDR showed extensive interactions at the genomic level and that VDR binding depended on the cell type. However, only one of these studies was performed on a cancer cell line delivered from the colon, which might not have been the most suitable model for VDR genome occupancy in IBD .kB) which induces the expression of the gene encoding antimicrobial peptide defensin β2 (DEFB2/HBD2) [Binding of 1,25(OH)2D to VDR leads to hetero-dimerization with the retinoid X receptor (RXR), as well as with other co-factors. Subsequently, the VDR complex translocates to the cell nucleus where it binds to vitamin D response elements (VDREs), mainly present in promoters of target genes, and either promotes or suppresses gene transcription . In addiB2/HBD2) . FurtherB2/HBD2) .A substantial genetic component predisposes to the development of UC and CD, and a number of single nucleotide polymorphic (SNP) variants are common to both diseases, hence, resulting in several pathways potentially similar . HormonaIn the studies concerning cell cultures obtained from biopsies of healthy colon tissue treated with 1,25(OH)2D, Alleyne et al., identified 465 upregulated and 417 downregulated genes across the entire genome . AdditioThe VDR regulates the biological action of active vitamin D. The interaction between vitamin D and VDR is involved in the genetic, environmental, immune, and microbial aspects of IBD . VDR is The human VDR is a known IBD risk gene encoding six domains of the VDR protein. Data demonstrate that VDR plays a vital role in the epithelium, which in turn suggests that it either protects against the induction of colitis, or enables effective damage repair . In contMoreover, single-nucleotide polymorphisms in the human VDR gene were reported to be associated with an elevated susceptibility to IBD ,78. The Free 25OHD, mainly linked to the GC vitamin D-binding protein (DBP), is transported in the blood. DBP encoding gene, i.e., GC gene  polymorphism leads to differences in affinity for both 25(OH)D and 1,25(OH)2D. The GC gene sequence variants were correlated with DBP protein and circulating 25(OH)D concentrations. Moreover, higher DBP concentrations were found to be positively correlated with the exacerbation of IBD, and inversely correlated with inflammation in pediatric IBD patients ,68.Comparing the frequency of two SNPs in the GC gene between IBD patients and controls, Eloranta et al., found that the haplotype consisting of wild type alleles, 416 Asp and 420 Lys, was less frequent in the IBD cases .Vitamin D is an essential regulator of calcium-phosphate metabolism, although it also performs immunomodulatory functions . It has As already mentioned, a Th1 response is predominant in Crohn’s disease, whereas a Th2 response is dominant in patients with ulcerative colitis . AdditioIt is also worth noting that vitamin D affects the regulation of B lymphocyte activity. The inhibition of their activity and maturation towards plasma cells contributes to a decrease in autoantibody production, which, in turn, may lead to a decreased risk of IBD. Additionally, 1,25(OH)2D stimulates B lymphocytes to produce anti-inflammatory cytokines, mainly IL-10 . A decreDue to the significant effects of vitamin D on the immune system in patients with IBD, it has been suggested that 25(OH)D levels are associated with disease activity in this patient group. According to a meta-analysis by Gubatan et al., who comprised 27 studies including 8316 patients with IBD, vitamin D levels were associated with an increase in disease activity, relapses, and reduced quality of life in these patients . MoreoveIn a study conducted by Ananthakrishnan et al., on 2809 patients with IBD, reduced plasma 25(OH)D levels were associated with an increased risk of cancer, particularly colorectal cancer . VitaminCurrently, there is an on-going debate as to whether vitamin D deficiency is a cause, or a consequence of IBD. Some researchers have referred to a link between the highest incidence of IBD and the latitude of areas with the least amount of sunlight . As outlA key pathophysiological element of inflammatory bowel disease is the cytokine response, which is responsible for inflammation regulation, evolution, and occurrence. It consists of two different levels of cellular response. The first comprises the process of cytokine differentiation from Th1 and Th17 cells, where the central cytokines are IFN-γ and IL-2 and IL-17. These cytokines, in turn, support the cellular response and are particularly involved in Crohn’s disease, in which IFN-γ plays a significant role Conversely, in ulcerative colitis, a Th2-like differentiation process is most relevant, in which NKT cells that produce IL-13 and IL-5 are crucial.These aforementioned IBD-specific cytokine patterns contribute to the second level of cytokines response, involving Th1, Th17, and Th2 differentiation processes, conditioning the humoral response and mediating the inflammatory process. These cytokines are TNF-α, IL-1β, IL-6, IL-4, IL-5, and IL-10 . In factStudies regarding the intestinal epithelial cell line Caco-2 have shown that 1,25(OH)2D regulates the expression of zonula occludens 1 (ZO-1) and occludin, which are vital compounds of the intestinal barrier . These pThe intestinal microbiota is a group of microorganisms (microbiome), mainly bacteria, that form a complex ecosystem in the gastrointestinal tract. The human gastrointestinal microbiota is composed mainly of bacteria, although it also contains Archaea, microeukaryotes, and various viruses, mainly bacteriophages. Nearly 2000 metagenomic species of bacteria have been described, which are distributed throughout the gastrointestinal tract. Interestingly, the density of the populations is not constant, and the highest density has been observed in the large intestine . The intFirmicutes, Bacteroidetes, and Proteobacteria are the most abundant bacteria in the human intestinal microbiome composition [Proteobacteria  has been described in IBD patients. Moreover, the composition of the microbiome may also differ between CD and UC patients. It has been shown that, in patients with CD, the number of bacteria of the genus Firmicutes, in particular Faecalibacterium prausnitzii, was significantly reduced, whereas the numbers of bacteria of the genus Proteobacteria and Bacteroidetes was increased. In contrast, in patients with ulcerative colitis, the number of butyrate-producing bacteria in the intestinal microbiota was significantly reduced [position . However reduced .The multipotential activity of vitamin D has been known for several years, however, its effect on autoimmune diseases has not been fully understood. There are reports according to which vitamin D may regulate gastrointestinal inflammation by binding to the intracellular receptor VDR, affecting the transcription of relevant genes. Nevertheless, the main effect of vitamin D on the intestinal microbiome is altering the transcription of the immune proteins as cathelicidin and defensin and enhancing intestinal barrier function . In turnVitamin D increases transepithelial resistance and decreases intestinal mucosal permeability, thus, preventing endotoxins and LPS from entering the bloodstream. Moreover, vitamin D deficiency leads to NF-kB activation and increases the transcription of cathelicidin and defensin . The losProteobacteria. Chlamydia trachomatis infection affects the gut microbiota and causes a decrease in VDR activity [Lactobacillus reuteri increased serum 25(OH)D levels [Vitamin D is also involved in detoxifying lithocholic acid (LCA) produced by the intestinal microbiome, which may adversely affect intestinal and liver cells . Kong etactivity . In contD levels . MoreoveD levels . All theOsteoporosis is defined as a systemic skeletal disease with low bone mineral density and a disruption of bone tissue microarchitecture, leading to an increased risk of fracture . The WorAs it has already been mentioned, generalized inflammation constitutes a significant risk factor for bone mineral density loss in patients with IBD . InflammThe Wnt/β-catenin  pathway also plays a vital role in the regulation of bone metabolism. Wnt glycoproteins bind to the receptor complex, which includes the receptor for very-low-density lipoproteins LRP5 or LRP 6. Activation of the signal transduction pathway leads to stabilization of β-catenin, and the effect role of the Wnt/β-catenin pathway is stimulation of bone formation. A critical regulator of the Wnt pathway is sclerostin, a glycoprotein produced by mature osteocytes in response to bone-damaging stimuli . ScleroskB to the nucleus. This, in turn, resulted in a decrease in osteoclast precursors in bone marrow and in peripheral blood [Another significant risk factor for osteoporosis in patients with IBD is the use of glucocorticosteroids (GCS) in the course of treatment. These drugs contribute to inhibition of the inflammatory response; nevertheless, they also negatively affect bone tissue, and their administration results in a significant decrease in bone mineral density and an increased risk of fracture, since they stimulate osteoblast apoptosis and decrease the number of osteoblast precursors. Interestingly, the Wnt/β-catenin pathway is partially involved in this process along with increased expression of sclerostin, which antagonizes Wnt signaling. The Wnt/β-catenin pathway, as a pathway which promotes osteoblastogenesis, may also be important in bone mineral density disorders in IBD patients. Ohnaka et al., indicated that Wnt3a increased T-cell factor (Tcf)/lymphatic enhancer factor (Lef)-dependent transcriptional activity in primary cultured human osteoblasts, whereas glycocorticosteroids were a significant inhibitor of this process. The researchers also showed that 1,25-dihydroxyvitamin D3 stimulated this activity. Hence, Ohaka et al., speculated that glucocorticosteroids inhibited the Wnt pathway in cultured human osteoblasts, partly by increasing the production of Dickopf-1 . This pral blood . Throughal blood . In viewal blood . The mecDue to its direct and indirect involvement in a number of metabolic pathways that regulate bone tissue metabolism, vitamin D is of particular importance in patients with IBD. However, further research involving this selected group of patients is necessary, with a particular need for randomized studies on large groups of subjects. Furthermore, understanding the molecular mechanisms determining the effect of vitamin D on bone tissue in patients with CD and UC is of clinical importance, since it may improve clinical interventions.The importance of vitamin D in patients with IBD implies that maintaining adequate vitamin D levels in these patients constitutes an important clinical issue. However, no clear guidelines on vitamin D supplementation in patients with IBD have been developed. According to the European Crohn’s and Colitis Organisation (ECCO) guidelines concerning the management of the extraintestinal complications in IBD, a preventive vitamin D supplementation is recommended in patients receiving systemic steroid therapy, as well as in patients with osteopenia and osteoporosis. Thus, the most appropriate management seems to be evaluating vitamin D levels in patients with IBD and the supplementation/treatment of the deficiency given the guidelines for the general population . FurtherFurthermore, Arihiro et al., demonstrated that the incidence of upper respiratory tract infections was significantly lower in a group of IBD patients who supplemented with vitamin D at 500 IU/day as compared with a placebo group, and this effect was most prominent in patients with low baseline 25(OH)D levels . In factPatients with inflammatory bowel disease are a group particularly susceptible to vitamin D deficiency and the associated implications. However, it is not entirely clear whether vitamin D deficiency in these patients is the cause or the result of IBD. It has been suggested that avoiding UV exposure may account for vitamin D deficiency in patients with CD and UC, which may be related to the need to stay indoors due to the disease. Another reason for avoiding sun exposure in this patient group may be excessive photosensitivity of the skin associated with the use of certain medications .Due to its multifunctional effects, vitamin D plays a significant role in patients with IBD. Vitamin D influences the expression of known IBD risk genes and modulates immune function by influencing the activation and differentiation of immune cells. Additionally, it also affects the intestinal microbiome and maintains the integrity of the intestinal barrier, as well as contributes to normal bone mineralization. The potential therapeutic value of vitamin D in patients with IBD and, consequently, the improved clinical status of patients is also emphasized. Therefore, further research is necessary to understand, in detail, the pathways of vitamin D activity in relation to the immune system, bone tissue, and gene expression regulation. Moreover, there is also a need to establish guidelines concerning vitamin D supplementation and treatment for patients suffering from IBD. The most significant information and studies presented in the paper are showed in


Soil salinization become worse in the last decades, leading to reduced crop yields, especially in the Mediterranean basin. Eruca sativa is a common species cultivated in this area with remarkable economic importance. This study aimed at investigating the effect of salinity on this plant, focusing on (i) seedling development in terms of variations in germination and growth parameters and (ii) anatomical and ultra-structural changes in the morphology of cotyledons. For this reason, seeds were treated with different salinity levels ranging from 137 to 548 mM NaCl. Seed germination was delayed by all the concentrations tested, but only above 137 mM seedling growth was impaired. Results showed a high occurrence of lipid bodies within the mesophyll cells of cotyledons of seedlings exposed to salt concentrations above 137 mM, suggesting an impairment in lipid mobilization caused by salinity during plant development. The cotyledons of treated seedlings showed reduced intercellular spaces and ultrastructural changes in chloroplasts and peroxisomes. Moreover, salt-induced autophagic processes were present in samples grown at the highest NaCl levels. Interestingly, at 137 mM NaCl, seedlings showed the highest values of mesophyll thickness and fresh weight, implying a possible mechanism of salt adaptation during germination. A soil  or more .Soil salinization can be the result of natural conditions or human activities. Natural reasons comprise weathering of rocks or salt deposits due to precipitation , while a+ and K+ to lower cellular osmotic potential [+ and Cl− accumulation, causing a disorder in nutrient uptake and toxicity effects [+ and Cl− ions can negatively interfere with many biochemical processes and can impair lipid reserve mobilization during germination that is a fundamental process for the subsequent seedling development [The salinization of soil heavily affects agriculture due to the decline in crop yield . Salinitotential . Moreoveotential . Insteadotential . Regardiotential ,12,13. I effects ,15,16. Ielopment ,18,19. Telopment ,20. In bIn addition, Cui et al. (2016)  reportedMoreover, salt stress also affects the anatomy and the cell structure of plant at every developmental stage . RegardiEruca sativa Hill (or E. sativa Miller or E. vesicaria (L.) Cav. Ssp. Sativa (Miller) Hegi), also known as arugula or rocket, is an annual herbaceous species that grows in the Mediterranean region and is widely cultivated, especially in the vicinity of coastal areas. Rocket leaves are commonly eaten raw, accompanying a large number of local dishes, and for this reason, its cultivation has a remarkable economic importance in many coastal regions [E. sativa under salt stress conditions, Miceli et al. (2003) [−1 with no remarkable morphological variation in seedling plants. Fallahi et al. [E. sativa under different salinity levels, observed a reduction of the germination rate by 16% and 29% under 100 and 150 mM of NaCl, respectively. In addition, the authors showed a germination percentage of 38% for E. sativa under salinity stress of 200 mM. Other plant species as Brassica oleracea, Cynara scoolymus, and Echinacea purpurea, subjected to the same salt stress level, showed germination percentages of 5%, 6%, and 2%, respectively [E. sativa germination was observed by mathematical models in which Bakhshandeh et al. [E. sativa a salinity threshold of 257 mM NaCl for seeds and 247 mM NaCl tolerance threshold for seedlings, on the basis of a 50% reduction of seed germination and normal growth seedling percentage.Since salinization of soil is becoming worse in countries of the Mediterranean basin , studies regions . Regardi. (2003)  recordedi et al. , testingectively , thus shh et al.  estimateE. sativa affected by salt excess at various development stages, studies on the anatomy and ultrastructure of E. sativa seedlings under salinity stress are still missing. Moreover, although lipids are the main energy source for seedling development after germination in oil seeds plants as rocket [E. sativa have been performed. Obtaining this information could help to understand the response of E. sativa under salinity in order to set strategies aimed to improve the tolerance of the species against this abiotic stress.Despite the wide literature about several aspects of s rocket ,32, no mE. sativa. In particular, the study focused on seedling lipid reservoirs, hypothesizing the detrimental effect of salt on the mobilization of this source of energy during the first stages of plant development. In the light of above, the aims of this paper were (i) to investigate how salinity affected seedling development, considering the growth parameters, and (ii) to provide, for the first time, anatomical and ultrastructural observations of the changes of both tissues and cells cotyledons in the cultivated plant of E. sativa is shown in The effect of different NaCl concentrations on the germination of As shown in Root length decreased significantly with the increase in salt concentration, except for the 137 mM NaCl treatment group, where the value was not statistically different with respect to that of the control group A,B. Regarding stem length values, both control and 137 mM NaCl-treated groups showed significant differences compared to the other tested concentrations A,C. The The fresh weight (FW) of seedlings is reported in In the mesophyll cross-sections stained with Sudan III–IV , an incrPlants exposed to 137 mM and 274 mM NaCl B,C treatStatistical analysis  revealedThe percentage of intercellular spaces in the control plants was 0.12%, while already in plants treated with 137 mM NaCl solution, a drastic reduction of this parameter was observed. In particular, in the 137 mM NaCl group, the occupied area was 0.07%, and in the three higher concentrations, the intercellular area was under 0.03% A. IntereMoreover, observing the semi-thin sections of cotyledon mesophyll, a significant reduction of intercellular spaces was noted in salt-treated samples. In particular, a drastic reduction of the area occupied by the substomatal chamber of the spongy tissue was observed A,B. In the control group, chloroplasts were adjacent to the plasma membrane, and the morphology of their section was oblong with well-developed grana and thylakoids A,B. The Plants grown with 137 mM NaCl showed chloroplasts with starch grains in higher quantity and dimensions with respect to the control C. The thAt 411 mM NaCl, starch grains were absent, while lipid droplets in the cytoplasm were abundant G. A resiStatistical analysis on the percentage of the area occupied by starch grains within the chloroplast in mesophyll cells of cotyledons was performed . A signiE. sativa germination under salinity conditions [E. sativa as “marginally tolerant” since the seeds can germinate at a salt concentration between 125 and 500 mM. In addition, for the first time to our knowledge, we found the concentration of 548 mM as the salinity level beyond which the seed germination of E. sativa is totally inhibited. Actually, this salt concentration simulates the salinization of some coastal soils in India during the dry season, confirming that this level of salinity totally inhibited seed germination, affecting agriculture [The results revealed that germination was strongly delayed by the different salt concentrations, excluding the lowest one of 137 mM . Similarnditions . Moreovenditions  on maizenditions  for halonditions , our resiculture .+ and Cl− toxicity, in agreement with Abogadallah and Quick [The root development was delayed with the increase in salt concentration . The recnd Quick , Amiri end Quick , and Falnd Quick .+ and Cl− ions from the root and the reduced water translocation [Regarding plant development, the root elongation was negatively affected by salt stress above 274 mM, whereas shoot length was significantly reduced only over 343 mM . Comparilocation ,41,42. Salt stress induced a reduction in the fresh weight, except for the concentration 137 mM where an increase in the seedling weight was observed A. This i2 absorption, as reported by Shannon et al. [In light of these findings, a possible stimulatory effect of salt in the low-dose zone can be hypothesized at least in the first stages of seedling development. A paradoxical effect of increase in biomass with low NaCl concentrations was observed also in other plants, such as Eucaliptus camaldulensis, Dalbergia sissoo, Sesbania grandiflora, and Casuarina spp. ,56,57. Tn et al. . 2 conductance as an effect of the intercellular space reduction generated by salt stress. In addition, the mesophyll thickness was comparable among the different salt treatments, except for the 137 mM NaCl samples, where this parameter was higher  were treated with several saline solutions, with reference to Bakhshandeh et al. (2019) [w/v (137 mM); 1.6% w/v (274 mM); 2% w/v (343 mM); 2.4% w/v (411 mM); 3.2% w/v (548 mM), in addition to distilled water (as control). To evaluate germination responses under different salt concentrations, . (2019) , at the −2 s−1. Before treatments, seeds of E. sativa were sterilized in ethanol 70% (v/v) for 20 min and washed 3 times with distilled water. Then, 25 seeds were placed in 9 cm diameter Petri dishes on a filter paper moistened with 2 mL of saline solutions and put inside the thermostatic chamber. A total of 1 ml of water was added after 48 and 96 h. Each treatment was conducted in quadruplicate.Salt treatments were carried out for 120 h in a thermostatic chamber with a temperature of 21 °C, a photoperiod of 18/6 h (light/darkness), and light radiation of 200 µmol mThe germination, considered as the number of seeds with a 2 mm long geotropic rootlet, was recorded every 12 h up to 120 h (5 days). At each time step, the number of germinated seeds with roots was recorded and, at a more advanced development stage, the number of seedlings with cotyledons was evaluated as well. By these data, the percentage of plants with developed cotyledons, the percentage of seeds with only emerged root, and the percentage of no germinated seeds was measured at every time step. At the end of the experiment, the developed seedlings of each treatment were collected to assess the length of stems and roots using a grid paper. Stem length was considered as the distance between the base of the cotyledons and the beginning of the root. Moreover, the inhibition of salt on root and stem length was measured using the following formulaThe plants were weighted with a precision scale  to record the fresh weight and then dried into an oven at 60 °C for 72 h to measure the dry weight.Cross-sections of fresh cotyledons of developed plants for each treatment were obtained by using a vibratome . The cross sections had a thickness of 50 µm. Sections were stained with Sudan III–IV a selective hystochemical dye for neutral lipids  and then observed through a Leitz DM-RB “Fluo” light/fluorescence microscope  equipped with a digital camera  to detect the amount of lipids in the plant tissues.v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) and stored at 4 °C for 24 h and then fixed in 1% OsO4 in 0.1 M phosphate buffer (pH 6.8). Afterwards, an ethanol series dehydration was performed, followed by a propylene oxide step. Lastly, samples were embedded in Spurr’s epoxy resin [Cotyledon samples for each treatment were collected and immediately fixed in 1.25% (xy resin .A Reichert–Jung ULTRACUT ultra-microtome with a diamond knife was used to cut cross-sections about 70 nm thick that were subsequently stained with uranyl acetate  and leadThe observations were performed with a Philips 201 Transmission Electron Microscope (TEM) Koninklijke Philips N.V., Amsterdam, Netherlands at 80 kV.Semithin cross-sections (thickness 1–5 µm) were also stained with toluidine blue 0.1% and observed with a light microscope. By image analysis, the measurement of the intercellular area, considered as the percentage of area occupied by intercellular space within the area of the whole section, was carried out, together with the measurement of the mesophyll thickness. The evaluation of lipid droplets was also carried out, calculating the percentage of area occupied by oil bodies within the cell. Lastly, the estimation of the quantity of starch in the chloroplasts was measured as the percentage of the area occupied by the starch grains within the organelle.All the images were analyzed with ImageJ . E. sativa seeds was performed with GerminaQuant for R (https://flavjack.shinyapps.io/germinaquant/ accessed on 29 December 2022). Time evolution of germination under different saline treatments was assessed, and the following three germination indexes were estimated.Germination analysis for Germination rate percentage (GRP): defined as the percentage of seeds that germinate developing the cotyledons.Mean germination time (MGT) expressed in hours:Germination speed percentage (GSP): The statistical analysis of the intercellular space, stem length, and root length were performed with Graph Pad Prism (version 8.0.1) using the one-way ANOVA test  and the Tukey–Kramer test for multiple comparisons. E. sativa showed the capability to germinate until a salinity level of 548 mM. Moreover, in comparison with the control (E. sativa seedlings, thus ensuring the recycling of damaged organelles and cytosolic components ( control A, the lo control B stress  control C. It is mponents D. The remponents . The adamponents .


In addition, the incidence of bradycardia was significantly lower in the sugammadex group (10.2%) compared with the neostigmine group . However, the total costs were significantly lower in the neostigmine group (50.6 ± 21.4 United States dollars) compared with the sugammadex group (212.0 ± 49.5 United States dollars). Despite improving postoperative recovery, the benefits of sugammadex did not outweigh its higher costs compared with neostigmine, possibly due to the low costs of labor in Taiwan’s healthcare system.Sugammadex has several pharmacological advantages over neostigmine, including faster reversal of neuromuscular blockade and fewer adverse effects. However, the economic impact of sugammadex remains controversial due to the considerable heterogeneity of study designs and clinical settings in previous studies. In a post-hoc analysis of a randomized controlled trial, we evaluated patients who underwent elective surgeries and general anesthesia with endotracheal intubation in a medical center in Taiwan between March 2020 and August 2020. Patients were divided into either the sugammadex or neostigmine group based on the neuromuscular blocking drug used. Propensity score matching was used to balance the baseline patient characteristics between the two groups. The patient’s recovery from anesthesia and the putative cost-effectiveness of sugammadex versus neostigmine was assessed. Derived cost-effectiveness using personnel costs in the operating room and the post-anesthesia care unit was estimated using multiple linear regression models. A total of 2587 and 1784 patients were included before and after matching, respectively. Time to endotracheal extubation was significantly shorter in the sugammadex group (mean 6.0 ± standard deviation 5.3 min) compared with the neostigmine group (6.6 ± 6.3 min;  The European Union first approved the use of sugammadex for reversing neuromuscular blockade in 2008. This modified γ-cyclodextrin encapsulates free molecules of certain types of muscle relaxants, forming stable complexes and, thus, more rapidly reversing neuromuscular blockade compared with traditional reversal agents, such as neostigmine ,2.n < 1000) [The clinical benefits of sugammadex versus neostigmine have been evaluated across a range of features, including rates of postoperative pulmonary complications, residual paralysis, postoperative nausea and vomiting (PONV), 30-day readmission, and cognitive function ,14,15,16 < 1000) ,18,19,20 < 1000) ,21. In a < 1000)  and thre < 1000) ,23). TheIn this study, we sought to clarify and investigate the potential cost-effectiveness of sugammadex versus neostigmine using real-world data in Taiwan. We reasoned that the use of sugammadex could accelerate the patient’s emergence from general anesthesia, increasing operating turnover rates and, therefore, reducing total medical costs compared to neostigmine. Based on current evidence ,21,22,23This study was a retrospective sequential analysis of a published randomized controlled trial registered on Clinicaltrials.gov (identifier: NCT04272177) . The two−2, and aspartate aminotransferase or alanine aminotransferase >2 times upper limits of normal), planned postoperative admission to the intensive care unit (ICU) for mechanical ventilation, myasthenia gravis, refusal to participate, and incomplete medical records . The Institutional Review Board waived written informed consent due to the retrospective nature of this research. All methods were performed following the Declaration of Helsinki 2013. Inclusion criteria were (1) patients who were admitted for elective surgeries requiring general anesthesia with endotracheal intubation at Shuang Ho Hospital, Taipei Medical University, from 1 March 2020 to 31 August 2020, (2) ≥20 years old, and (3) participated in pre-anesthesia assessments. Patients were excluded for the following reasons: inability to communicate in Mandarin or Minnanese, contraindications to neostigmine or sugammadex . After surgery, a modified Aldrete score >9 was required for discharge from the PACU.All patients received propofol 1–2 mg·kgThe electronic medical database was used to evaluate patient and clinical characteristics, including age, sex, body mass index (BMI), and serum creatinine levels. We also collected data on types of surgery performed, intravenous and inhalational medications used, duration of surgery and anesthesia, length of PACU stay, postoperative adverse events, and oxygenation supply techniques at the PACU based on the electronic anesthesia and PACU records. This data was extracted by an independent physician (W.L.), who was not involved in the data analysis. The quality of the datasets was validated using random samples by other authors.The primary outcome was time to extubation, which was defined as the interval from the administration of neuromuscular blockade reversal agents to endotracheal tube removal. The secondary outcomes were the duration of surgery and anesthesia, length of PACU stay, and adverse events in the PACU. Adverse events included PONV, bradycardia (a resting heart rate of <50 beats per minute), oxygen desaturation , use of a nasal cannula, a simple mask, or continuous positive airway pressure therapy for supplemental oxygen, and the need for tracheal re-intubation. In the PACU, the data of heart rate and peripheral oxygen saturation were recorded in 5-min intervals. In the original randomized trial, the adverse events were assessed by a certificated nurse anesthetist blinded to the choice of a reversal agent. It was thought that the reduced amount of time spent in the operating room (OR), as caused by sugammadex, could create cost savings for our healthcare system and patients. As detailed by prior studies ,18,20, pThe Department of Pharmacy in our hospital provided information regarding the unit price of neuromuscular blocking and reversal agents, which were used for general anesthesia on a self-paid basis. The total costs of each drug were calculated based on the dose used for each individual. Economic benefits were defined as the difference in personnel costs derived from the mean difference in time to endotracheal extubation between the two groups. The associated costs of medication-related adverse events  were not included in our analyses due to the relatively low incidence rate, considerable interindividual variation in relevant medications and interventions and insufficient evidence in estimating the potential costs.p-value < 0.05 was considered statistically significant. A Bonferroni correction to the significance criterion was applied for multiple testing adjustments.Based on a previous study, at least 84 patients are needed in each group to detect a mean difference of 2.7 min in time to extubation between the sugammadex and neostigmine groups, accepting a type I error of 5% and a type II error of 10%, with an anticipated mean time to extubation of 9.0 ± standard deviation (SD) 5.4 min in the neostigmine group ,26. It sA total of 2689 consecutive patients were screened for eligibility, and 2587 were selected for propensity score matching . Before p = 0.0032), respectively (p = 0.0108), arrive in the PACU , and the duration of anesthesia  were all significantly shorter in the sugammadex group compared with the neostigmine group. There was no notable difference in the length of PACU stay or the duration of surgery between the two groups. In the matched cohort, the use of sugammadex significantly reduced the time to extubation compared with neostigmine, with means of 6.0 ± SD 5.3 and 6.6 ± 6.3 min (ectively . In addip < 0.001) (The incidence of bradycardia was significantly lower in the sugammadex group (10.2%) compared with the neostigmine group  . There wp = 0.0032; 27.2 ± 18.7 USD, p = 0.0130) (p < 0.0001). The total costs in the neostigmine group were significantly lower compared with the sugammadex group, 50.6 ± 21.4 and 212.0 ± 49.5 USD (p < 0.0001), respectively.The time to extubation costs and personnel costs were significantly lower in the sugammadex group  compared with the neostigmine group  . HoweverIn this study, we found that the use of sugammadex was associated with a reduced time to extubation compared with neostigmine, although this difference was less than one minute. In addition, patients receiving sugammadex had a shorter time to leave the operating room and duration of anesthesia and a lower incidence rate of bradycardia in the PACU. However, the cost-effectiveness analyses demonstrated that the time-saving benefits of sugammadex could not compensate for its higher price compared with neostigmine. Our results did not support the cost-effectiveness of sugammadex for reversing neuromuscular blockade compared with neostigmine in Taiwan’s healthcare system. Several previous studies have examined the economic impact of sugammadex in various clinical settings. A simulation model by Insinga et al. showed that sugammadex could reduce the risk of residual neuromuscular blockade and improve the efficiency of the OR . This bePrevious studies have shown that the cost-effectiveness of sugammadex compared with neostigmine can only be established when two premises are met ,21,22,23In our hospital, more than 100 surgical procedures were performed each weekday, and we included both the duration of OR and PACU stay in the cost-effectiveness analyses, as our compact surgical scheduling required both OR and PACU staff to work overtime routinely. Our analyses did not demonstrate the cost-effectiveness of sugammadex, possibly due to the relatively low labor costs of our OR staff. According to open government data, the salaries of consultants and registered nurses in the United States are nearly 3 and 4 times higher than those in Taiwan, respectively . AnotherSome previous studies compared the cost of postoperative adverse events between sugammadex and neostigmine, such as unplanned postoperative ICU admission , 30-day Compared with previous studies, our cost-effectiveness analyses were based on a large patient sample using real-world data ,18,19,20We found that the use of sugammadex was associated with shorter operating room turnover times compared with neostigmine, including time to extubation, time to leave the operating room, and duration of anesthesia. In addition, the incidence of bradycardia was significantly lower in the sugammadex group. However, the time saving of sugammadex versus neostigmine was only modest, and the faster patient recovery of sugammadex did not outweigh its higher price. Our real-world data did not support the cost-effectiveness of sugammadex, possibly due to the relatively low labor costs in Taiwan’s healthcare system. Further studies are needed to calculate the derived costs of adverse events related to reversal agents and to build personalized decision-making models based on different patient and surgical risk groups.


As a rare tumor disease, only single case reports and small case series have been published on meningeal melanocytomas. In the case of complete surgical resection, there is a shallow risk of recurrence, whereas the benefit of radiotherapy or chemotherapy, both as single or combined therapy, is unclear. This work aims to analyze and summarize previously published cases and proposes a therapeutic algorithm.Background: Meningeal melanocytomas (MM) are rare primary melanocytic tumors of the leptomeninges with an incidence of 1:10,000,000. Until now, there has been only sparse information about this tumor entity. Here, we provide a meta-analysis of all cases published in the English language since 1972. Methods: A literature review was performed using PubMed and Web of Science. All published cases were evaluated for location, sex, age, therapeutic approach, and outcome. In total, we included 201 patient cases in our meta–analysis. Results: The majority of MM was diagnosed more frequently in men between the third and fifth decade of life. Surgery is the preferred therapeutic approach, and total resection is associated with the best outcome. Patients with partial resection or tumor recurrence benefit from adjuvant radiotherapy, whereas chemo- or immunotherapies do not improve the disease course. Malignant transformation was described in 18 patients. Of these, 11 patients developed metastasis. Conclusions: We present the first retrospective meta-analysis of all MM cases published in the English language, including an evaluation of different treatment strategies allowing us to suggest a novel treatment guideline highlighting the importance of total resection for recurrence–free survival and characterizing those cases which benefit from adjuvant radiotherapy. Meningeal melanocytomas (MM) are rare primary melanocytic tumors of the leptomeninges. They can be divided into circumscribed or diffuse, benign or malignant lesions. Well–differentiated, circumscribed tumors are called meningeal melanocytomas, while their malignant counterparts are meningeal melanomas. Additionally, MM with increased mitotic activity or invasion of the CNS parenchyma are considered intermediate-grade lesions. Furthermore, diffuse melanocytic tumors are characterized by invasion into subarachnoid space. Depending on the dignity of the histological phenotype, the lesion is called meningeal melanocytosis or meningeal melanomatosis . Since tDepending on the amount of melanin , MM appeMacroscopically the tumor is encapsulated without infiltration of the surrounding tissue ,7. The cAlthough several case reports have been published, so far, it remains unclear which is the best treatment approach for MM, in particular for those cases in which only incomplete surgical resection could be performed. Therefore, the aim of this study was to reanalyze all published cases, as well as one unpublished case from our department, concerning the applied treatment strategies and their outcome.MM cases were identified by using PubMed (Medline) and Web of Science (Clarivate). As the keyword, “meningeal melanocytoma” was used. In total, 219 (PubMed) and 247 (Web of Science) results were found, which were published between 1972 and 2022. After subtracting duplicates, the total data covered 312 items. Articles were excluded if they did not provide a novel case report, if the abstract did not include MM as a topic (n = 77), or if written in another language than English (n = 35), so in total, 201 cases were included in this analysis, as we also included one unpublished MM case from our department . All pubp values less than 0.05 were considered statistically significant.All patient cases were included in our analysis, of which at least one of the above variables could be collected. For this reason, the population groups for the different analyses also differed in size (indicated each time in the text). Histological diagnoses of tumors described here were adopted from the original manuscripts. No reevaluation of histological specimens was performed by local neuropathologists according to the 2021 WHO classification. Therefore, the lesions depicted here were classified according to the current WHO classification in each case. Statistical analysis was performed using the statistics software SPSS . Descriptive data included the calculation of the mean or median and standard deviation. Pearson Chi–Square testing was performed to compare categorical variables. Since 1972, 201 cases of MM were reported . In 89.6The location of the tumor was reported in 189 out of 201 cases. About half of the MM were found intracranially . The predominant location was the posterior fossa , followed by the middle cranial fossa with 11 cases  and the sellar region with 9 cases . Within the posterior fossa, MM occurred most often at the cerebellopontine–angle (CPA) . In the spine, MM occurred predominantly in the thoracic spine  and cervical spine . Furthermore, in eight cases, tumors grew in orbit. Two cases reported multifocal location within the spine as well as intracranially.Treatment strategies were reported in 186 out of 201 cases (92.5%). As the primary therapeutic approach, surgery was performed in 179 out of 186 patients (96.2%) of the cases. In the remaining cases, radiotherapy was used in two, and no therapy was applied in five others. Total resection was achieved in 89 out of 179 (49.7%) of cases and partial resection in 73 out of 179 cases (40.7%), while in 9.5% of surgical procedures, the extent of resection was not documented (17/179).Total resection was performed without any further therapy in 81 out of 179 patients (45.3%). The combination of total resection and radiotherapy was applied in 8 (4.5%), partial resection alone in 45 cases (25.1%), and adjuvant radiotherapy after partial resection in 24 out of 179 cases (13.4%). In two cases, radiosurgery was used after partial resection so that the remaining tumor could also be targeted ,16. Due Chemo- or immunotherapy such as Temozolomide , CisplatDue to tumor localization in an eloquent region, resection could not be performed in three patients. In two of these patients’ definite radiotherapy was chosen as a treatment option with a tumor-free follow-up of 42 months in 1 patient . The othRe–operation was the main therapy chosen in cases of tumor regrowth in 14 out of 44 cases (31.8%), followed by combination of re-operation and radiotherapy, applied in 7 (15.9%), re–operation combined with radiochemotherapy in another 4 (9.1%), while radiotherapy alone was used in 4 out of 44 cases (9.1%). In addition, three cases in which radiosurgery was used as follow-up therapy after recurrence of the tumor were also reported ,33,34.p = 0.001). The median tumor-free interval was 18 months, with a minimum of 1 month and a maximum of 35 years. If, alternatively, only partial resection was performed, a better outcome was shown in 61.9% of the patients by the additional use of adjuvant radiotherapy . Total resection was the most efficient therapy and showed a tumor-free interval without recurrence in 68.1% of cases . In total, 16 intermediate cases were reported, 14 showed a high recurrence rate without adjuvant radiotherapy after resection, and 2 were treated by radiotherapy without recurrence .Furthermore, another 17 cases of MM reported malignant transformation ,34,43,44Regarding the temporal component at follow–up, we were able to highlight a median of 18 months (range: few days–35 years).Since its first description in 1972 by Limas and Tio [Interestingly, our data demonstrated that MM occurs at a younger age in females than in males and is more frequent in males than in females, which contradicts the impression from smaller studies where female patients were thought to develop MM more often ,51. We cFurthermore, our analysis shows that the predominant location is the posterior fossa, as well as the cervical and thoracic spine, which is in line with the embryological development of melanocytes . MelanocWe evaluated treatment strategies and their outcome, showing the best recurrence-free follow–up after total resection . In thesOnly in those cases that first received a total resection but developed MM recurrence, radiotherapy appears to be an advantage for recurrence-free survival .In patients where only partial resection was achieved, radiotherapy showed a clear beneficial effect, while other therapeutic approaches, such as chemotherapy or immunotherapy, did not show any advantages. Furthermore, some case reports indicate a potential benefit of radiosurgery.When MM is considered as a diagnosis, we do not suggest deciding on conservative follow-up observations, as we learned that MM appear capable of developing metastases. When the tumor is inoperable, definite radiotherapy has also been used successfully.Therefore, we suggest the following treatment approach for patients that are diagnosed or considered to have MM :PLCB4 and CYSLTR2 [SF3B1–, EIF1AX–, or BAP1–mutation or complex copy number variations indicate aggressive behavior consistent with meningeal melanoma [Although chemo– and immunotherapy has failed so far, several mutations, such as in Guanine-nucleotide binding protein G subunit alpha GNAQ, GNA11), have been identified . Both GN CYSLTR2 ,60,61 an1, have b CYSLTR2 . The premelanoma ,63,64. Emelanoma ,66,67.Beyond that, novel immunotherapies applied for malignant melanoma therapy may be beneficial also for MM. Both share common antigens, such as Melan-A or S–100 ,12,13. TAs MM is a rare entity, the composition of our multi–center derived data set was very inhomogeneous due to different authors and unrelated case reports. For that reason, this study can only serve as a meta–analysis of all published case reports, which intuitively cannot fulfill the criteria for a real comprehensive study. Thus, future multi–center prospective studies with fixed follow-up periods are now needed in order to achieve precise outcome and prognosis and verify our treatment suggestions derived from our retrospective analysis.The fact that we were unable to reanalyze the tissue samples in this study is a methodological limitation of studies such as this and can bias conclusions. Hopefully, future reports will be able to use more up–to–date histopathological classifications.Based on the first retrospective meta–analysis of all MM cases published in the English language, we propose a novel guideline for the treatment of MM, highlighting the importance of total resection for recurrence-free follow-up and suggesting in which cases adjuvant radiotherapy may have beneficial effects of the patient’s disease course.


Cervicogenic headache (CEH) has long been recognized as a referred pain deriving from pathological changes in the upper cervical nerves. However, previous clinical studies found that anterior lower cervical discectomy for the treatment of cervical myelopathy and/or radiculopathy can also help relieve associated headaches. To date, there is still a lack of large sample and prospective study to investigate the effect of anterior cervical decompression and fusion (ACDF) on CEH associated with cervical spondylosis.A total of 656 patients with cervical radiculopathy and/or myelopathy were enrolled in three spinal centers. Among them, 221 patients who were diagnosed with CEH were collected in this study, and 204 completed a 1-year follow-up. The primary endpoint was headache intensity during a 12-month follow-up period measured by the numeric pain rating scale (NPRS). The secondary outcome measures included headache frequency, headache duration, and the neck disability index (NDI).p < 0.05 for all). There were statistically significant lower NDI, less frequent headaches, and lower headache duration in the surgery group during follow-up .Among all 204 patients with CEH who completed a 1-year follow-up, 166 received anterior cervical surgery (surgery group) and 38 received conservative treatment (conservative group). There were statistically significant lower NPRS in the surgical group during follow-up. Between-group differences showed that NPRS in the surgery group was significantly greater improvement at 1 month , 3 months , 6 months , and 12 months  (This study indicates that ACDF can effectively relieve CEH associated with cervical myelopathy and/or radiculopathy. Cervicogenic headache (CEH) is a common chronic and recurrent headache . It is tpost-hoc analysis from a multicenter randomized clinical trial  . Howeveral trial , found tal trial  and 10-yal trial . Howeveral trial . It is u . Howeveal trial . Accordial trial , 9. Howeal trial  and a smal trial , the incRecently, a retrospective study found that anterior lower cervical discectomy can also relieve the accompanying CEH based on the criteria of the ICHD-3 beta version . So far,The study was approved by the Ethics Committee of the Third Medical Center of Chinese PLA General Hospital and written informed consent was obtained from each participant.We conducted a multicenter, prospective, cohort clinical trial in three centers from August 2017 to August 2018. This trial was prospectively registered in the Chinese Clinical Trials Registry (ChiCTR-ONC-17012027). Originally, the purpose of our clinical trial was to observe the effect of ACDF on tinnitus in patients with cervical spondylosis. During this period, we simultaneously observed the effect of ACDF on CHE in the same group of patients. A total of 656 patients with cervical radiculopathy and/or myelopathy were enrolled in three spinal centers. They did not respond to conservative treatment for at least 3 months and were candidates for ACDF surgery due to severe neurological dysfunction or intolerable symptoms. Among them, 221 patients who were diagnosed with CEH according to the ICHD-3 beta version criteria  (3) wereThe inclusion criteria were patients between the ages of 18 and 55 years with typical signs and symptoms of myelopathy and/or radiculopathy; those with objective signs of the spinal cord and/or nerve root compression as shown on magnetic resonance imaging (MRI); and those having concomitant CEH with or without other symptoms, such as dizziness, tinnitus, blurred vision, and nausea.Exclusion criteria included patients with a history of neck trauma or surgery; those suffering from neurological disease or any other possible treatable causes for headache; and those with congenital or developmental cervical malformations and those unable to follow the study.Decompression and fusion segments depended on clinical manifestations and corresponding nerve root and/or spinal cord compressions shown on MRI. The choice of surgery levels was determined by a senior spine surgeon at each center . Anterior cervical interbody fusion was performed with a cage that was filled up with autogenous bone obtained by local decompression and anterior plate fixation. The operative segments ranged from C2/3 to C7/T1 . ConservThe primary efficacy endpoint was the mean change in headache intensity from baseline to 12 months in the surgery group, as measured by the Numeric Pain Rating Scale (NPRS), compared with the mean change in the conservative group. At baseline, 1, 3, 6, and 12 months after treatment, patients were asked to use an 11-point scale ranging from 0 (“no pain”) to 10 (“worst pain imaginable”) to express the average headache intensity in the past week . The NPRThe study was also powered for the assessment of secondary efficacy endpoints: the neck disability index (NDI), headache frequency, and headache duration. The NDI is the most widely used tool to assess self-rated disability in patients with neck pain , a self-Baseline data included age, gender, NPRS score, NDI score, headache frequency, headache duration, diseased disc segments, and classification of cervical spondylosis were collected before treatment. Re-evaluation was performed at 1, 3, 6, and 12 months, respectively, after treatment.t-test. Separate Mann–Whitney U tests were performed with the headache frequency and headache duration between the two groups at baseline, 1, 3, 6, and 12 months. SPSS version 23.0  was used for all analyses. The significance level was set at p < 0.05.Descriptive statistics, including frequency counts for categorical variables and measures of central tendency and dispersion for continuous variables, were calculated to summarize the data. Comparisons of NPRS score and NDI score between the two groups were at baseline, 1, 3, 6, and 12 months conducted with a one-way analysis of variance. Comparisons of NPRS score and NDI score at baseline, 1, 3, 6, and 12 months within groups were conducted with paired-sample p < 0.001) and lasted for 12 months . After conservative treatments, NPRS of the conservative group also decreased  and lasted for 12 months . At 1, 3, 6, and 12 months after treatment, NPRS of the surgery group was significantly lower than that of the conservative group (p < 0.05). The improvement of NPRS in the surgery group was significantly greater than that in the conservative group. The difference in NPRS improvement between the two groups was 2.8 at 1 month, 2.6 at 3 months, 2.4 at 6 months, and 1.5 at 12 months (p < 0.05). Besides, in order to explore the influence of surgical segments on headache improvement, we classified the surgery patients into two subgroups, namely, single segment  and multisegment  and compared the NPRS between the two subgroups at baseline and 12 months. There was no significant difference in NPRS between the two subgroups .After cervical decompression, NPRS of the surgery group decreased immediately . The NDI in the surgical group was 20.9 and 7.2 at baseline and 12 months, respectively, while that in the conservative group was 19.1 and 13.5 at baseline and 12 months, respectively. Between-group differences showed a significantly greater improvement in the NDI at 1 month , 3 months , 6 months , and 12 months  in the surgery group (p < 0.05).During the follow-up, the NDI in the surgical group was significantly lower than that in the conservative group . The duration of headache in the surgery group was significantly lower at 1, 3, 6, and 12 months (p < 0.001).The Mann–Whitney A total of 11 patients had mild dysphagia and nine patients had hoarseness immediately after the operation, but all of the symptoms disappeared within 1 month. Notably, five patients developed C5 nerve root paralysis, and the symptoms disappeared within 3 months after the operation. There were no other surgical complications, such as aggravation of neurological symptoms, implant loosening and loss, and infection.Numerous studies have identified the short-, mid-, and long-term effects of anterior cervical surgery on headaches associated with cervical myelopathy and/or radiculopathy , 13, 14;This study has also shown that CEH in patients with cervical radiculopathy and/or myelopathy significantly improved or disappeared after ACDF, which was consistent with previous studies , 17, 22.Currently, less is known about the pathogenesis of CEHs, and their anatomical pain generators are even more difficult to define . Bogduk The most typical pathological change in cervical spondylosis is intervertebral disc degeneration . Degenerpost-hoc analysis of a prospective, multicenter study with a 10-year follow-up conducted by Lombardi et al.  or sympathetic nerve irritation at the uncovasculoradicular junction, anterior dura mater, or cervical posterior longitudinal ligament (PLL). The cervical dura and PLL have different sympathetic innervation and may induce sympathetic reflexes . The actIn addition, Schrot et al.  found thThis study has several limitations. First, in this study, the patients were selected for their radiculopathy and/or myelopathy rather than headaches. Therefore, the results cannot be interpreted as all patients with cervical spondylosis complicated by CEH should be treated with anterior cervical decompression surgery. Second, the pathogenesis of CEH may be multifactorial. Most of the studies related to CEH, including this study, are clinical studies and cannot provide direct and convincing evidence to confirm the pathogenesis of CEH. However, this study provides a direction for relevant basic research. Finally, this study only completed a 1-year follow-up, and further long-term follow-up is needed to confirm the efficacy of ACDF in the treatment of CEH.This study indicates that both ACDF and conservative treatment can improve CEH associated with cervical myelopathy and/or radiculopathy, but ACDF is better than conservative treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Local Ethics Committee of the Third Medical Center of Chinese PLA General Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.BP and XC designed the study. LY, YL, and BP drafted the manuscript. LY, XP, YW, and XC carried out data analysis. LY, CD, XP, DL, YW, and XC performed data collection. YL, XP, YW, XC, and BP revised the manuscript. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chersina responded to these conditions by having a lower rate of growth as compared with their modern counterparts, which thrive in the current prevailing more favourable Mediterranean type of climate.Here we examine the tibial microstructure of modern and fossil angulate tortoises to assess the histology and growth from the late Miocene–early Pliocene, Pleistocene through to modern forms. The cross-sections of all the tibiae sampled revealed highly vascularized, uninterrupted, fibrolamellar bone tissue during early ontogeny, which suggests that early growth was fast. However, later in ontogeny, growth was slower, as indicated by the deposition of parallel-fibred bone tissue in the outer cortex, and even ceased periodically, as indicated by lines of arrested growth. Comparative analyses of the growth rates of the tortoises from different time periods showed that the tortoises from the late Miocene–early Pliocene Langebaanweg locality and from Diepkloof Rock Shelter had relatively slower growth rates under less optimal growth conditions. Additionally, these prehistoric specimens show extensive remodelling, and several generations of secondary osteons further suggest functional and/or metabolic stresses on the skeleton. Palaeoenvironmental reconstructions suggest that it was mostly cooler and drier with seasonal fluctuations in late Miocene–early Pliocene, and it is likely that  Althou5.15 Ma; ,15,18), 5.15 Ma; ,20. Angu5.15 Ma; ,22, and 5.15 Ma; ,24. Thei5.15 Ma; ,17 and i; [sensu ) and for; [sensu . In SoutChersina angulata from the Miocene to present, to assess whether their growth dynamics and life-history patterns changed over the past 5 million years.Long bone histology has long been used to infer growth dynamics and life history of extant and extinct vertebrates  is located approximately 105 km northwest of Cape Town in the Western Cape Province of South Africa . In the . 2.2The archaeological site at Diepkloof Rock Shelter (DRS) is located approximately 180 km north of Cape Town ,72. Alon. 2.3Another archaeological site, Dunefield Midden (DFM) is a late pre-colonial campsite located 2 km north of the mouth of the Verloren Vlei river in Western Cape Province, South Africa, and displays a series of brief occupations between 900 and 600 years ago, a time when the Holocene sea level was beginning to drop to its current level . The bas. 2.4Modern tortoise skeletons studied were collected post natural fires in the Cederberg (approx. 300 km north of Cape Town) and Onderplaas, Worcester (approx. 110 km from Cape Town).. 2.5Prior to the preparation of the histological thin sections, all the bones were measured and photographed. Standard measurements such as total length, mid-shaft diameters, and widths of proximal and distal ends were measured using Mitutoyo digital calipers with 0.01 mm precision . Differe. 2.62O3) solution. The final thickness of the sections was approximately 30–35 µm. All the thin sections of the bones were studied and photographed using digital compact cameras Canon Power Shot D10 and Nikon DS-Fi1 mounted on Nikon Eclipse E200 and Carl Zeiss Axio Lab A1 polarizing microscopes, respectively. Bone wall thickness measurements were made using NIS Elements Microscope Imaging Software (v. 3.22.14), and the relative bone thickness (RBT), that is, the mean thickness of the cortical bone wall divided by the mean diameter of the total bone [et al. [For studying tortoise bone histology, thin sections were generated using cutting and grinding techniques following . Thin setal bone ,85, was  [et al.  and Chin [et al. .. 2.7sensu [et al. [sensu [Skeletochronology is a technique in which the age of an individual is determined by counting the number of lines of arrested growth (LAGs) within the skeletal tissue ,83,86–91sensu ). Indeedsensu . However [et al.  reported [et al. ,43,55,93 [et al. . In some [et al. ,39,55. H. [sensu ) and sec. [sensu ,83,95–97. [sensu . It is p. [sensu . Althoug. [sensu , their a. [sensu –102.et al. [et al. [et al. [In addition to estimation of the age of individuals ,83,86–91et al. , Woodwar [et al. , Orlandi [et al.  and Chin [et al. . To visu. 3. 3.1a). At mid-shaft, the medullary cavity is centrally located (figure 1a–e), but in a few specimens, it is slightly displaced toward the lateral region of the cross-section (figure 1f–g). In most of the individuals , the medullary margins are irregular and highly resorptive, which contrasts with that of the largest individual in which the medullary cavity is small and sub-circular to circular, and there are fewer resorption cavities present (figure 1g). In several of the sections, the medullary cavities are surrounded by a thin layer of centripetally deposited lamellar bone . The inner and outer cortex is almost entirely composed of parallel-fibred tissue with fibres concentrically oriented with respect to bone axis (figure 1a–c). However, in the perimedullary regions, small areas of primary woven bone tissue occur (figure 1b). The osteocyte lacunae are more abundant in the perimedullary regions, and they are less dense toward the periosteal margin (figure 1d). However, their density varies between individuals. In a few specimens, most of the cortex exhibits simple globular lacunae but they are flattened towards endosteal regions (figure 1e). In the largest individual , the cortex is thickest . In this region, the tissue is also more vascularized, and the canals tend to have a radial organization (figure 1f). The medial margin also shows similar features, but to a lesser extent. Fibrolamellar bone tissue has a patchy distribution in the perimedullary regions  with longitudinally oriented primary osteons. Several LAGs occur throughout the cortex, and they interrupt the deposition of the parallel-fibred bone tissue ; however, it is evident that in some individuals the earlier LAGs were erased due to remodelling (figure 1g). Eight LAGs are present in the largest individual . Isolated radial canals are present in the cortex , and in a few instances they extend deep into the cortex. Several of the bones show secondary reconstruction, but in the largest individual (figure 1g), the oldest part of the cortex shows a few secondary osteons, several erosion cavities in the process of being infilled by endosteally formed lamellar bone, and very few open (unfilled) erosion cavities (figure 1g).Histological analysis of all the tibial specimens from Cederberg reveal cross-sections with cortical regions composed of a thick compact bone walls  and a ring of circumferentially formed lamellar bone tissue lines the medullary cavity (b). In general, the bone walls are composed of poorly vascularized parallel-fibred bone tissue  which enclose a central open medullary cavity . However, in a female tortoise  as well as in a few males , there are long radial canals that extend through the entire cortex. In a tibia of a female, the medullary cavity is small, and there are few enlarged erosion cavities present (figure 2b). Although, patches of fibrolamellar bone occur in some areas of the inner cortex (figure 2h), parallel-fibred bone tissue is the dominant component of all the cortices (figure 2g). They are flattened in the regions of endosteal bone tissues (figure 2h). Primary and secondary osteons are generally scarce in the compacta; however, few large erosional cavities occur in the perimedullary regions , a maximum of 15 LAGs, periodically interrupting parallel-fibred bone tissue, are noted (figure 2d). A male individual with the largest carapace length of 17 cm has eight LAGs in the compacta  and is surrounded by a thin layer of endosteal lamellar bone (figure 3e). The thickness of the cortex varies around the bone cross-section , often with the medial side having the thicker compacta. The cortex of the bones is predominantly composed of parallel-fibred tissue, although there are a few enlarged erosion cavities concentrically arranged in the perimedullary region . There is a distinctive lack of secondary osteons throughout the compacta, although occasionally radial vascular canals traverse the bone wall (figure 3a–c). The osteocyte lacunae are abundant with a globular shape (figure 3c), preferentially arranged parallel to one another, whereas those in the endosteal lamellar bone appear to be more flattened (figure 3e). Several LAGs are evident in the cortex . The outer cortex is predominantly composed of parallel-fibred bone (figure 4a), but parts of the compacta are more woven textured with numerous primary and secondary osteons (figure 4b–e). The cross-sectional histology reveals that there was a distinctive drift towards the posterior region of the cross-section, and this results in a distinctive asymmetrical distribution of the bone tissues (figure 4a). In general, parallel-fibred bone increases its thickness throughout the ontogeny , whereas the woven bone region appears to be early formed and less dominant in the cortex (figure 4d). Although secondary reconstruction is mostly restricted to the perimedullary region, in a few specimens, it extends mediolaterally, right to the periosteal margin (figure 4e). Several LAGs interrupt the deposition of the parallel-fibred bone tissue (figure 4a), and it is likely that LAGs formed during earlier stages of ontogeny may have been erased by remodelling (figure 4e). Ten LAGs were counted in two specimens, whereas the smallest specimen in the sample has seven growth marks , but in a few specimens, the entire medullary region is infilled with secondary remodelled bone, and in some cases trabecular bone (g). The transition from the medullary region to the outer cortex is indiscrete. Instead, a large distinctive transitional region of compacted coarse cancellous bone occurs between the inner and the outer cortex (figures 5c and 6e–h). In a few specimens, compacted coarse cancellous bone occurs and is restricted to the medial region of the inner cortex. The primary cortex is made up of woven bone tissue richly vascularized by large, primary osteons, which are often eroded to form enlarged erosion cavities, some of which are lined by lamellar bone tissue. Several generations of secondary osteons and enlarged cavities are present in the inner regions of the compacta, although in some tibiae they reach the medial peripheral margin. In addition, their density is much higher in areas where the primary cortex grades into the coarse cancellous bone . Overall, the outer cortex is dominated by parallel-fibred bone tissue with globular osteocyte lacunae arranged parallel to the peripheral margin (figures 5a and 6a–c). In a few specimens, long radial canals penetrate almost the entire outer compacta , where there is a marked decrease in the density of vascular canals. A maximum of 20 LAGs were counted in the outer cortex, and given the extensive secondary reconstruction, it is likely that earlier formed LAGs were removed (a). It may be noted that the largest specimen  along with the modern specimens from Cederberg and Onderplaas showed much slower growth patterns, as illustrated by the predominance of slowly forming parallel-fibred bone tissue with LAGs in the outer cortex. LAGs are also present in the outer cortex of DRS and LBW tortoises, although they exhibited different bone tissue types in their compacta. The occurrence of multiple LAGs in the cortex indicates a cyclical or flexible growth strategy –89,107. et al. [Overall, the DRS and LBW tortoises show similar histological pattern to the tortoises from the 600–900-year-old DFM site, and the two modern sites (Cederberg and Onderplaas) , suggesting fast growth early in ontogeny followed by slow growth until the animals died e.g. ,48,49). ,49. 48,4et al. ,108–110.et al.  as they et al. . This alet al. . In modeet al. [During late stages of ontogeny, in the tortoises from Dunefield Midden (DFM) and the two modern sites (Cederberg and Onderplaas), there is a marked increase of the organization in the collagen fibres with the deposition of poorly vascularized parallel-fibred bone in the outer cortex. In South American long-necked chelid turtles, Pereyra et al.  identifie; [et al. [Another interesting finding is that the tortoise remains from archaeological site DRS and palaeontological site LBW showed intense secondary reconstruction ,32,97 infigure 4e; ). Generae; ,112,113 e; ,113–116.e; ,117,118. [et al.  suggestef). Based on the histological observations, we propose that this tissue represents a periosteal reactive growth. Similar pathological conditions were observed in the ribs of Proneusticosaurus silesiacus and have been attributed to tuberculosis-like respiratory infection [In one individual Ad-01, , in the figure 1nfection . Such renfection , sauroponfection  and extanfection . HoweverC. angulata (irrespective of the age of the sample) is comparable to that reported for the aquatic turtle Podocnemis expansa [Pelomedusa subrufa as well as the tortoises Homopus femoralis [Testudo hermanni, Testudo graeca, Astrochelys radiata, Geochelone carbonaria [Stigmochelys pardalis [Dermochelys coriacea [et al. [Stigmochelys pardalis [Stigmochelys pardalis [Overall, the bone microstructures of  expansa  and Peloemoralis ,56, Testrbonaria  and Stigpardalis . The precoriacea  as an ad [et al.  reportedpardalis  and proppardalis . This copardalis , and is pardalis , althouget al. [C. angulata. Such histological features have also been noted in naked mole-rats, Bathyergidae [C. angulata described in this study and suggest that the overall asymmetrical growth of the compacta might be related to the digging behaviour.In the modern angulate tortoise, Bhat et al.  reportedet al. ), which yergidae  and in m. 4.2et al. [a), but it must be noted that this reflects a minimum estimation of age [g and a). It is evident that the 8th–9th growth mark distinctly shows a transition to narrow growth rings (d), in general the spacing of the 8th–9th growth marks typically marks the transition to a slower pace of growth irrespective of the geological age.) Based on these findings, we propose that these tortoises reach sexual maturity in the 8th year [et al. [Branch  reportedet al.  reportedn of age ,91. It ith rings . .The data retrieved for modern tortoise remains from Cederberg and Onderplaas follow similar growth trajectories early in ontogeny; however, Cederberg tortoises show a steeper growth rate later in ontogeny , and it . 4.4Aldabrachelys gigantea; Geochelonegrandidieri; [The cooling effect from the Benguela current, as well as the latitudinal, seasonal movement of the South Atlantic high-pressure system, have a significant role on the present climate of southwest South Africa . Summersdidieri; ), anguladidieri; ,141. Thididieri; .The climate at the time of occupation at DFM is assumed to have been relatively similar to the present with hot, dry summers and cool, moderately wet winters . Our hisThe Diepkloof Rock Shelter document the occupations from the MSA, but some LSA components are also recorded in the form of superficial archaeological layers, pits excavated into the MSA deposits, and paintings on the wall of the shelter ,78. At pca 5.15 Ma), the local environment at LBW remained C3-dominated canopy [Dental mesowear and stable isotope studies of fossilized animals at the Langebaanweg fossil site show that during the early Pliocene , through several highly significant rock shelters, including provincial heritage site of Diepkloof Rock Shelter (DRS), to late pre-colonial site of the terminal Holocene at Dunefield Midden (DFM) and modern sites at Worcester and Cederberg provided relevant information about the life history of these tortoises over a considerable period of time. Our results revealed that these tortoises experienced cyclical growth dynamics with faster rates of bone deposition during early stages of ontogeny, and slower rates during later stages of ontogeny. Here for the first time we document an abundance of fast-growing fibrolamellar bone tissue and profuse secondary remodelling in the inner cortices of tuations ,102. We tuations ,148–150)tuations ,127,151.


LPL, APOC2, LMF1, and APOA5 genes. Case: We present a clinical case of severe primary hypertriglyceridemia (TG level > 55 mmol/L in a 4-year-old boy) in a consanguineous family. The disease developed due to a previously undescribed homozygous deletion in the APOA5 gene (NM_052968: c.579_592delATACGCCGAGAGCC p.Tyr194Gly*68). We also evaluate the clinical significance of a genetic variant in the LPL gene (NM_000237.2: c.106G>A (rs1801177) p.Asp36Asn), which was previously described as a polymorphism. In one family, we also present a different clinical significance even in heterozygous carriers: from hypertriglyceridemia to normotriglyceridemia. We provide evidence that this heterogeneity has developed due to polymorphism in the LPL gene, which plays the role of an additional trigger. Conclusions: The homozygous deletion of the APOA5 gene is responsible for the severe hypertriglyceridemia, and another SNP in the LPL gene worsens the course of the disease.Background: Hypertriglyceridemia (HTG) is one of the most common forms of lipid metabolism disorders. The leading clinical manifestations are pancreatitis, atherosclerotic vascular lesions, and the formation of eruptive xanthomas. The most severe type of HTG is primary (or hereditary) hypertriglyceridemia, linked to pathogenic genetic variants in  Hypertriglyceridemia (HTG) is a lipid metabolism disorder occurring when the level of triglycerides (TG) in the blood plasma exceeds 1.7 mmol/L 150 mg/dL). Average TG levels range from 1.45 128 mg/dL) to 1.25 mmol/L (110 mg/dL) for adult men and women, respectively 8 mg/dL t. Accordi0 mg/dL. According to the Framingham Study, and other publications, a TG level over 1.7 mmol/L is associated with a significantly higher risk of cardiovascular events ,9,10,11.The main mechanism underlying atherosclerotic lesions during HTG is the overproduction of very-low-density lipoproteins (VLDL) in the liver. In atherogenic combined dyslipidemia, it has been noted that there is a transfer of TG from VLDL to low-density lipoproteins (LDL), and at the same time, a transfer of cholesterol esters from LDL to VLDL. This phenomenon leads to the appearance of an additional number of atherogenic lipoproteins: VLDL have lost some TG and become smaller, thus increasing atherogenicity .In addition, the leading clinical manifestation of familial HTG is acute pancreatitis ,15,16, wThe etiology of HTG varies. Primary or hereditary HTG are genetically determined and marked by clinical variability and heterogeneity. The clinical case studies a male child, four years old, Avar by nationality, born from a third pregnancy in the family from a consanguineous marriage: father and mother are fourth cousins . He has During a routine examination of the proband at the age of 1.5 years, serum lactescence was revealed with a TG level up to 55 mmol/L (4850 mg/dL). Later, while dyslipidemia remained, an increase in liver enzymes was found . Ultrasound examination revealed hepatomegaly, splenomegaly, and pancreatic inflammation. During several hospitalizations, serum lactescence and HTG have been reported and have not been eliminated by a low-fat diet . In addiThe patient was referred to the Moscow Research Center for Medical Genetics, named after the academician N.P. Bochkov, to clarify the diagnosis. On examination, the patient did not have any stigmas of dysembryogenesis, but on the skin of his hands, there was a light rash, and on his legs, there was a reddish rash of the xanthoma type, in total more than 10 elements. On the skin of the dorsum of his hands and in the area of the first finger, whitish elements of a rounded shape (draining in places) up to 5 mm in diameter, resembling xanthomas, were noted. In the sacral region and on the inner surface of the legs, there were single reddish elements, also resembling xanthomas .LPL gene was carried out because genetic variants in this gene are associated with impaired metabolism of TG. We found an exonic variant: LPL (NM_000237.2: c.106G > A (rs1801177) p.Asp36Asn), in a heterozygous state. This variant is described in HGMD (CM910259) and ClinVar as associated with an increased risk of cardiovascular disease and is noted in the UniProtKB database as associated with familial type 3 hyperlipidemia (P06858). The frequency of this variant according to GnomAD data is 0.014733.Initially, an analysis of the APOA5 gene (NM_052968: c.579_592delATACGCCGAGAGCC) was suggested, leading to the formation of a premature stop codon, p.Tyr194Gly*68.This genetic variant was initially regarded as a polymorphism, which should not have led to clinical manifestations of HTG. To verify the genetic substrate, an additional analysis of 60 genes responsible for the development of hereditary dyslipidemias was carried out  due to a previously undescribed homozygous deletion in the Ion S5  sequencing systems were used. Genomic DNA (gDNA) was extracted from whole blood with the use of a Diatom DNA Prep reagent kit  according to the manufacturer’s protocols. The concentration of gDNA as well as DNA concentrations of the libraries afterwards were determined using a Qubit™ Fluorometer .DNA libraries were constructed with the Ion AmpliSeq™ targeted custom panel and the Ion AmpliSeq™ Library kit 2.0 . The panel contains exons of 60 genes, including genes for primary monogenic hypertriglyceridemia and hypercholesterolemia see .DNA quality was confirmed by the final stage of library preparation using the PCR test, performed with the manufacturer’s primers to adaptors’ sequences. The thermocycling conditions used were as follows: 95 °C for 40 s, 68 °C for 35 s, and 72 °C for 75 s, and the number of cycles used was dependent on the library concentration. PCR results were visualized on silver-stained 8% acrylamide gel (staining time: 10 min at 4 °C). Massive parallel sequencing of pooled libraries with a loading concentration of 75 pmol was performed using an Ion 540™ Chip kit  and Ion 540™ Kit-Chef , with an average amplicon length of 175 bps. Variant validation was performed by PCR direct sequencing with the Applied Biosystems 3500xL Genetic Analyzer.APOA5 gene (NM_052968). There were signs of a homozygous deletion of no more than 40 nucleotides (approximate coordinates: c.450_590del). Direct sequencing analysis revealed a homozygous frameshift deletion, c.579_592delATACGCCGAGAGCC, resulting in the premature stop codon p.Tyr194Gly*68.Targeted DNA sequencing of the proband supposed the presence of a homozygous deletion in exon 4 of the Bioinformatics Genome Reference Consortium Human Build 37 (GRCh37/hg19) was used for data analysis. The home program for assessing the quality of NGS and variant annotation was used.https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV000995944.1 (accessed on 27 May 2022), rs1940989106). Location: chr11: 116661353–116661366 (GRCh37) UCSC NC_000011.10:116790636:GGCTCTCGGCGTATGG:GG; NG_015894.2:g.6771_6784del; NM_052968.5: c.579_592delATACGCCGAGAGCC; NP_443200.2:p.(p.Tyr194Gly*68).The pathogenicity of the variants was assessed in accordance with the recommendations of ACMG Guidelines, 2015 . AccordiAPOA5 gene, located on chromosome 11q23, encodes one of the proteins that builds up lipoproteins and is expressed mainly in the liver and small intestine [The ntestine . Apolipontestine . Howeverntestine , activatntestine ,30. It intestine  and accentestine . Thus, antestine ,31,32,33APOA5 gene consists of 4 exons and 3 introns, with a total protein length of 366 amino acids. The signal sequence is located at the beginning of the protein, at amino acid positions 1 to 23, followed by a long polypeptide chain from amino acid position 24 to 366. Intracellularly, the protein is predominantly located in endosomes and the Golgi apparatus.The APOA5 gene are described in HGMD, with the stop codons at positions from 95 to 313 (CM151055).ApoA5 is a hydrophobic protein with well-defined functional domains. The extreme C-terminus (mature sequence positions 295–343) facilitates lipid binding, while the N-terminal domain (amino acids 1 to 146 of mature apoA5) adopts a water-soluble helix bundle conformation. The intervening central segment (residues 147–294) possesses a positively charged region (residues 186–227) that is functionally important for lipolysis. The truncated pathogenic variants of the APOA5 gene, is described in OMIM (145750) and HGMD as an autosomal dominant disorder. At the same time, according to ClinVar, only 18 pathogenic/probably pathogenic variants are described in this gene, while according to HGMD, there are 82 variants, and most of them are associated with an increased risk of myocardial infarction and cardiovascular diseases [HTG, caused by pathogenic variants in the diseases ,35,36.APOA5 gene range from severe hyperchylomicronemia to normotriglyceridemia [APOA5 gene in two sisters born from a consanguineous marriage in Chile, whose blood TG level was up to 112 mmol/L [According to the existing published research, clinical manifestations in patients with heterozygous pathogenic variants in the eridemia ,37. In s2 mmol/L . In thisAPOA5 gene that we discovered leads to the formation of a premature stop codon in the region of amino acid 262 and is localized in the fourth and last exon of this gene. Based on this, the mutant protein most likely avoids NMD (nonsense-mediated mRNA decay) and may be present in the cell. The APOA5 variant described here is completely lacking the C-terminal lipid-binding motif. This loss of function results in reduced lipolysis and remnant clearance, with the subsequent manifestation of HTG.The previously undescribed deletion of 13 nucleotides (c.579_592delATACGCCGAGAGCC) in the APOA5 gene was inherited from heterozygous parents in a consanguineous marriage, which led to a severe case of homozygous HTG with a 32-fold increase in TG serum concentration. The 5-year-old boy did not develop classical clinical manifestations of severe HTG, such as atherosclerotic vascular lesions, fatty hepatosis, and pancreatitis. However, diffuse changes in the pancreas revealed by ultrasound examination, as well as an increase in hepatic transaminase level, can be regarded as an early onset of damage of the pancreas and liver.In our proband, a homozygous deletion in the LPL gene, which was initially considered as an insignificant finding. This variant was identified in the father of the proband and was absent in his mother and brother. Considering that the main effect of the ApoA5 protein is its activating effect on lipoprotein lipase, we assume that even a slight polymorphism in the LPL gene, which reduces the activity of this enzyme, in the presence of a pathogenic variant in the APOA5 gene, will significantly worsen the course of HTG. In the family, the polymorphism in the LPL gene seems to be the additional trigger that is necessary for the development of HTG in heterozygous patients. For the proband, this polymorphism has an additional effect, worsening the course of HTG.At the same time, the mother of the proband, a carrier of the deletion, has no clinical picture or changes in the lipid profile, while the father of the proband has severe HTG (6.8 mmol/L), and mild HTG (2 mmol/L) is present in the 11-year-old sibling, who is also a carrier of a heterozygous deletion. The diet of different family members does not differ, and the proband’s parents do not have endocrinology or other disorders. No secondary factors that could have led to such different clinical pictures in heterozygous carriers of the mutation have been identified, and therefore we have put forward the concept of the additional influence of the genetic environment. For the parents and siblings, we searched for the variant found in the proband in the APOA5 gene ((NM_052968: c.579_592delATACGCCGAGAGCC p.Tyr194Gly*68) which, in a homozygous form, leads to severe HTG and early onset of clinical symptoms. In the presence of this variant in a heterozygous state, clinical manifestations vary from average HTG to normotriglyceridemia, which corresponds with published studies. Additionally, we have shown that the presence of a relatively frequent polymorphism in the LPL gene worsens the clinical course of primary HTG caused by a mutation in the APOA5 gene. These results convincingly show that genetic diagnosis is indicated for all patients with suspected hereditary HTG and should include all major genes responsible for lipid metabolism disorders. This approach will make it possible to correctly assess the patients’ risks and prescribe adequate therapy. In addition, we have clearly demonstrated incomplete penetrance in autosomal dominant hypertriglyceridemia caused by a pathogenic variant in the APOA5 gene.Using our unique diagnostic panel for 60 genes, we have discovered a new pathogenic variant in the