It is essential to diagnose and treat this disease early to prevent permanent neurological damage secondary to this neurotransmitter disorder. We present the first two cases of PTPSD in Romania that were genetically confirmed and treated late. Improving the diagnosis and monitoring procedures in Romania with correct metabolic management will prevent severe neurological impairment from PTPSD or other BH4Ds. BecDs ,2,3. PTPSD has clinical manifestations in early childhood that include neurological signs  and hyperphenylalaninemia (HPA) ,11.In Caucasians, the prevalence of BH4 deficiency is 1–2% of all HPA , and PTPThe incidence of HPA in Europe is approximately 1:10.000, and BH4Ds are 1–2% of all cases . PTPSD iIt is essential to early diagnose children with PTPSD to prevent them from developing symptoms. After HPA is detected in NBS by dried blood spot (DBS) testing, the next step is pterin analysis and CSF studies ,13,14,15We present the first two cases of PTPSD in Romania that were genetically confirmed and treated late.Improving the diagnosis and monitoring procedures in Romania with correct metabolic management will prevent severe neurological impairment from PTPSD or other BH4Ds.Patient 1 is a girl diagnosed late, at nine months of age.She first presented at our hospital at two months due to a positive newborn screening of HPA. Her first value on DBS was 331 µmol/L  < 126 µmol/L).She was born to consanguineous parents (both parents and grandparents are cousins). Her family history revealed that an aunt died in early childhood because of an unknown neurological disease. She is the second child of the family and she was born at 38 weeks of gestation (WOG), from a natural birth; her birth weight was 2300 g (1st percentile) and her length was 48 cm (27th percentile). The clinical exam was normal, except for a small umbilical hernia.The serum phenylalanine (Phe) was 237.77 µmol/L (NV36.3–66.55 µmol/L). We then determined pterins on DBS, revealing a low biopterin concentration, indicative of PTPSD. We also determined the dihydropteridine reductase (DHPR) enzyme activity on DBS to exclude dihydropteridine reductase deficiency (DHPRD), which was normal.The family did not bring her for a follow-up for seven months. At 9 months, she returned to our clinic because of hypotonia and neurological developmental delay .We determined the serum Phe level, which was slightly elevated (75.6 µmol/L), and the patient still had low biopterin on DBS, and the neopterin/biopterin ratio was low .The first neurological exam performed at two months was normal. The head circumference was within the normal range for her age. At the second admission, at nine months of age, she had severe hypotonia, poor head control, progressive motor impairment (loss of head control at six months), and mild hypokinesia. The patient also experienced oculogyric crises, interpreted as dystonia, dystonic postures in the upper limbs, and tremors triggered by psychomotor agitation  She also encountered a daily fluctuation of movement symptoms . We performed an electroencephalogram (EEG) to exclude other neurological diseases, showing a regular awake pattern without epileptiform activity. At 11 months, she had a 48 h BH4 loading test that lowered the Phe levels to 53% after administration of Sapropterin dihydrochloride (10 mg/kg BW/day).We then introduced treatment with L-Dopa/Carbidopa, starting at 0.5 mg/kg BW/day, and increasing to the current very low dosage of 2.5 mg/kg BW/day.We could not establish the severity form of PTPSD because we could not perform CSF studies. The administration of neurotransmitters was based on the clinical response.DNAJC12, GCH1, PAH, PCBD1, PTS, QDPR, SLC25A13, SPR) showed a homozygous genotype for the pathogenic c.84-3C > G variant of the PTS gene.At the age of 1 year, we performed genetic testing, and a hyperphenylalaninemia NGS (next generation sequencing) panel . The blood phenylalanine concentrations decreased after BH4 supplementation, and the patient had normal plasma phenylalanine levels and regular protein intake.We tried two types of 5-hydroxytryptophan (5-HTP) treatment, but the patient did not tolerate it because of gastrointestinal adverse reactions; she had vomiting and diarrhea, and we had to stop the treatment.After the start of specific treatment (Sapropterin dihydrochloride and L-Dopa/Carbidopa), the evolution indicated a progressive recovery of motor skills, improved head control and sitting position without support. The hypotonia, hypokinesia, and dystonia were ameliorated. She had mild cognitive impairment, with a developmental quotient (DQ) using the Portage Assessment Scale of 54 at the first assessment, which decreased to a DQ of 45 (moderate cognitive impairment) without treatment and increased to a DQ of 58 after the specific treatment was started. She also had kinesiotherapy almost every month since the diagnosis, and she walked at two years with unilateral support and spoke a couple of meaningful words.To exclude other neurological diseases, we performed cranial magnetic resonance imaging (cMRI) at one year and four months, which was normal.As a less expensive alternative for clinical monitoring, we determined blood prolactin serum levels, which were high , meaning that the metabolic disease was not adequately controlled.Patient 2 is a boy, also born from consanguineous parents (third cousins), from a pathological and incorrectly monitored pregnancy, with the threat of miscarriage, born with c-section, with an average birth weight . The clinical exam indicated facial dysmorphism , seborrheic dermatitis, heart murmur, ankyloglossia, and generalized hypotonia.The first value of Phe at NBS was 348 µmol/L, and the serum Phe levels at six weeks were 312.4 µmol/L (NV 36.3–66.5 µmol/L). The DBS attested a high neopterin and low pterin ratio, indicating PTPSD. DHPR enzyme activity in the DBS was normal.The heart ultrasound detected a patent foramen ovale; the abdominal ultrasound revealed minimal right hydronephrosis; and the head ultrasound normal, ophthalmological consult were normal.The routine lab tests indicated hypercholesterolemia, positive COVID IgG antibodies, hyperammonemia, positive IgG anti-CMV antibodies, slightly elevated liver enzymes, and iron deficiency anemia.PTS gene, in homozygous state; this could be due to the patients belonging to the same ethnicity and the same geographical area.At the age of four months, we collected DNA from peripheral blood cells to conduct genetic testing, and a hyperphenylalaninemia NGS panel was performed revealing the same mutation as the first patient, the c.84-3C > G variant of the At the age of 5 months, our patient’s cerebrospinal fluid (CSF) was analyzed, and neurotransmitter metabolites 5-hydroxindolacetic acid (5-HIAA) and homovanillic acid (HVA) were measured to determine disease severity. The patient had low HVA and 5HIIA in the CSF, so we could include the patient in the severe form of PTPSD.We did not perform a BH4 loading test. Still, he received donation treatment at five months with Sapropterin dihydrochloride (10 mg/kg BW/day) until he was included in the National Treatment Program.The therapy with Sapropterin dihydrochloride was discontinued because of the latency period until the introduction into the National Treatment Program at eight months. We also introduced L-Dopa/Carbidopa treatment (0.5 mg/kg BW/day).An initial neurological assessment was performed at two months for the second patient. The head circumference was within the normal range for his age. The patient had mild axial hypotonia with asymmetric postures. No movement disorders were reported at the first assessment, but they occurred in the upper limbs by nine months of age.An electroencephalogram was performed, and no epileptic activity was recorded during the tracing. The developmental quotient (DQ) was 90, indicating that the patient had moderate cognitive development.At the age of 10 months, after one month from the start of the specific treatment, the hypotonia and dystonic postures had significantly improved, and he could stand without support. Fine motor development and cognitive development were within normal limits. We also reported excessive salivation since the age of 5 months, that improved after treatment.We determined plasma prolactin levels for this patient, which were still higher than the normal range, indicating that we still had not reached the proper dosage of neurotransmitters to control the disease.Phe levels on follow-up were within the normal range, and he was not on a Phe-reduced diet.Regarding the treatment, both patients received Sapropterin dihydrochloride (10 mg/kg BW/day) and L-Dopa/Carbidopa (dose range 2 mg/kg BW/day), with an increasing dose over the next months. None of the patients is taking 5-HTP because of the gastrointestinal adverse reactions; both had vomiting and diarrhea and we had to stop the treatment.Both children have developmental delays, and treatment was introduced late (at one year and four months for patient 1 and at eight months for patient 2).Follow-up was done every three months since Sapropterin dihydrochloride and L-Dopa treatments were introduced.The data about the demographic, biochemical, and genetic descriptions of our two patients are available in In our case presentation, consanguinity was reported in both of our patients. Similarly, Almannai et al. reported consanguinity rates as high as 89% in Arab subjects, mostly from Saudi Arabia. Carducci et al. reported a 57% rate in Jordanian patients. Bozaci et al. reported 77% consanguinity in Turkish patients. Blau et al. concluded that the highest incidence of PTPSD in Saudi Arabia is probably due to the high consanguinity rate ,16,17,18As cited in the literature, our patients had neurological and cognitive impairments. Most patients with BH4 deficiency have hypotonia, impaired motor and cognitive development, movement disorders, parkinsonism, and rest tremors ,19.Truncal hypotonia, postural instability, dystonic limb movements, hypersalivation, and oculogyric crises were reported in a long-term follow-up of Chinese patients who received delayed treatment for PTPSD, as was also seen in our two patients that also received late treatment. Oculogyris crises were reported in 5–15% of PTPSD patients, and were observed in patient 1 ,19.One of our patients reported excessive salivation. Lee et al. identified hypersalivation in 40% of their 10 PTPSD patients .Our two patients revealed improvements in the DQ  scale scores after receiving neurotransmitter treatment, as cited in the literature. Wang et al. indicated that language, adaptability, and at a later age, mathematics were fragile areas for PTPSD patients. Lee et al. also used DQ as a tool to establish neuropsychological impairment. They observed improvement after administering neurotransmitters in the 15 years of follow-up of 10 PTPSD patients, even in severe forms ,21.One of our patients had intense irritability. Only 10% of patients with PTPSD have reported this symptom in the literature ,16.Prematurity was not found in our patients ,16, but The pattern of pterins in PTPSD in DBS and urine consists of highly elevated neopterin and low biopterin, similar to our patients ,13.We selected the DBS sample for measuring pterins because our hospital does not have the instrumentation to perform the measurement of pterins in urine. Transportation to an external laboratory was not the best option because the pterins in urine are more susceptible to degradation by light and high temperatures .Levels of 5-hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA) in CSF are usually low in BH4Ds; our second patient had his CSF analyzed, and we could include him in a severe phenotype of PTPSD ,5,9,10.We elected serum prolactin as a marker to evaluate the L-Dopa dose. Dopamine is an inhibitor of prolactin secretion, and serum prolactin can be elevated in dopamine deficit disorders. The serum prolactin was elevated in both patients, indicating that we must increase the L-Dopa/Carbidopa dosage ,23.Ogawa et al. described a case of PTPSD with a more significant correlation of L-Dopa dosage with serum prolactin levels than CSF homovanillic acid levels .Mutation detection is the preferred method for diagnosis ,24.The c.84-3C > G variant is considered pathogenic in the ClinVar database (Variation ID: 553378), and was previously reported to be associated with as a severe form of PTPSD in Albania, Italy, and Iran ,18,25,26Patients with PTPSD may have abnormal brain imaging results such as delayed myelination, periventricular hyperintensities, and brain atrophy. We performed a brain MRI for one of our patients, and we did not find hyperintense lesions in the periventricular white matter ,19,21,27Patient 1 had a BH4 loading test. As there is no uniform procedure available, we opted for the 48 h test with a dosage of 10 mg/kg BW/day, and the levels of Phe were lowered by 53% ,29.Sapropterin dihydrochloride treatment was preferred instead of the Phe-reduced diet to normalize the levels of Phe, first because the studies revealed that this is an easier way to monitor Phe intake and second because of lack of cooperation for a Phe-reduced diet regarding our patients’ families .The combined treatment with Sapropterin dihydrochloride and L-DOPA/Decarboxylase inhibitor improved the clinical manifestations of our patients, consistent with the results of other studies ,30,31,32We were not inclined toward a monotherapy of Sapropterin dihydrochloride because our patients had developmental delays and clinical neurological signs. Our patients had no adverse reactions secondary to Sapropterin dihydrochloride administration .We used an L-DOPA/Carbidopa 10:1 preparation with an extemporaneous prescription because of our country’s lack of suspension treatments and since the literature did not report any side effects compared to the 4:1 preparation. Our two patients did not reach the target treatment dose of L-Dopa/Carbidopa of 10 mg/kg BW/day ,33.A study indicating a discontinuity of 5-HTP treatment registered no signs of neurological deterioration. Our two patients did not tolerate the 5-HTP treatment provided in Romania because of gastrointestinal side effects (vomiting and diarrhea) ,34.Patients from Taiwan with PTPSD discontinued 5-HTP treatment for 15 years and have remained free of recurrent neurological or psychiatric symptoms [The long-term outcome of the disease is influenced by the time of the initiation of treatment ,35,36,37Kao et al. mentioned that even PTPSD patients treated early have subtle neurological dysfunctions .PTPSD is rarely diagnosed in our country because of the absence of a standard evaluation protocol after identifying HPA during NBS. Just two centers in Romania are evaluating pterins on DBS to investigate BH4Ds further.


Diabetes mellitus is a significant health problem for medicine and economics. In 80–90% of cases, it is type 2 diabetes (T2DM). An essential aspect for people with T2DM is to control blood glucose levels and avoid significant deviations. Modifiable and non-modifiable factors influence the incidence of hyperglycemia and, sometimes, hypoglycemia. The lifestyle modifiable factors are body mass, smoking, physical activity, and diet. These affect the level of glycemia and impact molecular changes. Molecular changes affect the cell’s primary function, and understanding them will improve our understanding of T2DM. These changes may become a therapeutic target for future therapy of type 2 diabetes, contributing to increasing the effectiveness of treatment. In addition, the influence of external factors  on each domain of molecular characterization has gained importance towards a better understanding of their role in prevention. In the current review, we aimed to collect scientific reports on the latest research about modifiable factors connected with the style of life which affect the glycemic level in the context of molecular discoveries. Diabetes mellitus (DM) is a fundamental health problem for medicine and socioeconomics. According to The World Health Organization data, the number of patients who suffer from DM is consistently growing—from 108 million in 1980 to 422 million in 2014 .Type 2 diabetes (T2DM) accounts for 80–90% of diabetes cases . This tyEnvironmental factors such as the patient’s lifestyle, smoking, lack of physical activity, and improper diet lead to obesity ,5. This The factors affecting glucose fluctuations can be divided into modifiable and unmodifiable. Many modifiable factors influence increases and decreases in blood glucose levels, which is positive information for clinicians dealing with T2DM . In thisIn addition, in the latest literature, there are modifiable factors such as the model of care, insurance structure, or drug therapy. These factors affect the course of the disease but are not measurable in the molecular context. Therefore, despite their importance, they will not be included in this work .The non-modifiable factors in the current literature include age, longer duration of diabetes, sex, family history, or gestational diabetes ,9,10. InThe low-grade inflammation in metabolic diseases, including obesity and diabetes, is widely described in the literature . There iOne of the newest obesity treatment interventions is bariatric surgery . Zuo et On the other hand, bariatric surgery such as Roux-en-Y gastric bypass (RYGB) surgery, sleeve gastrectomy, and fundoplication contribute to the occurrence of hypoglycemia . The occAdipose tissue is now recognized as a vital endocrine organ that contributes to the energy homeostasis of the whole body. The role of calcium homeostasis in metabolic disorders has become a subject of interest in recent years. During fasting and obesity, a calcium-sensitive kinase, calcium/calmodulin dependent-protein kinase II (CaMKK), becomes activated in the liver and plays a critical role in glucose homeostasis ,19,20.Deletion or inhibition of hepatic CaMKK in obese mice protects against glucose intolerance and hyperinsulinemia . This meSimilarly, CaMKK deficiency enhances skeletal muscle insulin receptor (INSR) signaling and glucose transport in diabetic mice . Wen DaiUsing cultured CaMKK-deficient adipocytes and an inducible mouse model of CaMKK deficiency in adipose tissue, he demonstrated that the CaMKK adipocyte regulates insulin signaling and glucose homeostasis throughout the body. In addition, CaMKK is involved in lipolysis and adipocyte inflammation, which may further contribute to the pathogenesis of insulin resistance. These data identify the CaMKK adipocyte as a critical component of metabolic regulation in obesity . Thus, a2+ and reactive oxygen species (ROS) levels were significantly elevated in β-TC-6 cells; therefore, nicotine exposition might lead to ROS accumulation in pancreatic islet cells. The expression of senescent markers and accumulation of ROS in β cells revealed that nicotine intake enhances the senescence of β cells. These changes may contribute to the progressive loss of islets and progressively more frequent fluctuations in blood glucose levels and gradually approaching diagnosis of T2DM [Cigarette smoking is another modifiable factor influencing glucose levels. On the one hand, smoking is a risk factor for the onset of T2DM. In the meta-analysis, Willi et al. show clear evidence that active smoking is related to up to 30–40% increased risk of T2DM  in activ of T2DM . β cell  of T2DM . Interes of T2DM . Medical of T2DM . Smoking of T2DM , althoug of T2DM , althoug of T2DM . Unfortu of T2DM . It shou of T2DM . It is u of T2DM . Unfortu of T2DM . Gene TC of T2DM . This fiThe following recognized modifiable factor for glucose control is physical activity. It is recommended for people of all ages, regardless of their health condition, as it is perceived as a factor leading to organism homeostasis. Undoubtedly, physical activity reduces the probability of T2DM by increasing insulin sensitivity and glucose tolerance , leadingNowadays, the molecular background of this relationship is investigated. Heat shock proteins (HSPs) play an essential role in the pathogenesis of insulin resistance, which leads to hyperglycemia, for example, Heat shock protein 70 (HSP70) ,85. HSP7The investigation in well-characterized animal models to represent the human condition with T2DM showed that long-term exercises ,87 increIn recent decades, microarray analysis has allowed the consideration of many potential therapeutic targets. In human skeletal muscles, researchers noted two proteins of the orphan nuclear receptor family, Nur77 and NOR1 , which iDiabetic patients suffer from anxiety and depressive symptoms more often and are more likely to develop dementia than healthy people of the same age and gender ,92,93. TAt this point, attention should also be paid to another effect of sugar levels on the nervous system. Hypoglycemia also has an adverse effect on its operation. A study presented by Zhou et al. found disruption of mitochondrial homeostasis in the hippocampal tissue of diabetic mice experiencing repeated mild hypoglycemia. Hypoglycemia disrupted the fine mitochondrial structure, reduced the number of mitochondria, and upregulated the expression of mitochondrial dynamics and mitophagy markers, including dynamin-related protein 1 (Drp1), Bcl-2/adenovirus E1B 19-kDa-interacting protein-3 (BNIP3), and microtubule-associated protein 1 light-chain 3 (LC3) in the hippocampus of T1DM mice . In subsThe last featured factor that influences glucose variability is diet. The goals of introducing a diet in patients with T2DM are based on well-introduced dietary habits, which can eliminate the primary symptoms of the disease, reduce the risk of glucose fluctuations, and thus eliminate micro and macrovascular complications . The metAs mentioned earlier in this paper, diabetes is a factor in Alzheimer’s disease. A HFD contributes to the development of glucose homeostasis disorders . HFD stiA high level of SFA is a pro-inflammatory factor and leads to impairment of β-cells function and, as a result, loss of glycemic control ,105,106.2+ dynamics, and regulating insulin secretion. Therefore, limiting the caloric supply may be a factor that will prevent patients from developing metabolic diseases, including obesity and T2DM, and in people with diabetes, it can decrease glucose variability [In addition to the Mediterranean diet, there are various dietary ideas to improve health. Bearing in mind that T2DM is also influenced by changes in the composition of macronutrients, regulating metabolic functions, glucose homeostasis, and influencing the biology of pancreatic cells ,118, Heriability .The current review of the latest research proved that the modifiable factors affecting glucose variability, hypo-, and hyperglycemia are significant in the development and progression of T2DM. The interventions in body mass, smoking, diet, and physical activity in diabetic patients impact the course and prognosis of the disease or even stop diabetes development. Nowadays, medicine emphasizes the need to understand the pathophysiological background of the disease in order to target treatment. There is a strong relationship between molecular pathophysiology and modifiable factors of glucose level—summary in In type 2 diabetes, patients mainly struggle with the problem of hyperglycemia, however, an equally important and often overlooked aspect is the presence of hypoglycemia in them. As this work shows, so far, few studies have focused on the impact of too-low blood sugar levels on changes taking place in the cell, despite the significant clinical problems presented by patients with hypoglycemia, ranging from impaired consciousness and cognitive functions to even death. This indicates the need for further research on this aspect.


Cri du Chat syndrome (CdCS) is a genetic disorder resulting from a variable size deletion of the end of the short arm of chromosome 5 (5p), including a critical region located at p15.2. It represents one of the most frequent chromosomal deletions, with an incidence in the general population of 1/20,000 to 1/50,000.Through this observation we update the scientific news of this rare syndrome and present an observation of a Cri du Chat syndrome confirmed by metaphasic karyotype (p13) de novo) with autism spectrum disorder.Description a case with cat cry syndrome seen in child psychiatry consultation in our institutionDiscussion through articles published on pubmed, googlescholar and science directTypical features of CoCs present in the subject include intellectual disability, psychomotor acquisition delays, language delay, and dysmorphic features . Expected features of CoCs that are not present are: growth retardation, microcephaly, round facies, micrognathia, epicanthal folds and characteristic high-pitched cry. Behavioral features in this subject include symptoms of autism spectrum disorder.The deletion of the short arm of chromosome 5, when it includes a critical region located at p15.2, is responsible for a well-characterized syndrome, Cri-du-Chat disease, including a characteristic craniofacial dysmorphia that evolves with age, the mental handicap in the characteristic form is very severe. Visceral malformations are relatively rare and not very specific.None Declared


Einkorn breads were bigger than the control (736 vs. 671 cm3); crumb pores percentage was similar, but medium-size pores were scarcer. Finally, a 52-h shelf-life trial demonstrated that einkorn bread had a softer texture, maintained for a longer time, and a slower retrogradation than the control. Therefore, choice of appropriate varieties and process optimisation allows the production of excellent einkorn breads with a superior nutritional value and longer shelf life.Einkorn flour, rich in proteins, carotenoids, and other antioxidants, generally has poor breadmaking value. In this research, the composition and technological characteristics of the flours and breads of two elite einkorns (Monlis and ID331) and a bread wheat (Blasco), cropped in four different environments, were evaluated. The einkorns confirmed better flour composition than bread wheat for proteins , soluble pentosans (1.03 vs. 0.85 g/100 g), and yellow pigment (10.0 vs. 1.0 mg/kg). Technologically, they had better SDS sedimentation values (89 vs. 66 mL), lower farinographic water absorption (52.6 vs. 58.8%), and a similar development time, stability, and degree of softening. Viscoelasticity tests showed lower storage and loss moduli and more prevalent elastic behaviour for Blasco, while rheofermentographic tests showed an anticipated development time (120.8 vs. 175.0 min), higher maximum height (73.0 vs. 63.0 mm), and superior retention coefficient (99.1 vs. 88.7%), but a lower CO Triticum monococcum L. subsp. monococcum), a diploid hulled wheat (2 n = 2 x = 14) with high protein [Einkorn  and, as control, of one bread wheat cultivar (Blasco); the two einkorn accessions were selected because in previous unpublished tests repeatedly showed the best breadmaking attitude. Hence, all-around information about the breadmaking properties of these two elite einkorns will allow the baking industry to develop new leavened products through adopting the most appropriate processing approaches. To this end, the composition and technological and rheological properties of their flours, doughs, and breads were assessed throughout the breadmaking process; furthermore, the shelf life of the bread loaves was investigated.2 plots according to a Randomized Complete Block Design with three replications. For weed control, the herbicide Ariane II  was applied just before heading to the conventional management plots, while the organic trials were manually weeded. After machine harvesting, by mid-July  and a breadmaking-quality bread wheat (cv. Blasco) were cropped in four different environments, two under conventional management  and two under organic management . Some relevant agronomic parameters of the four environments are summarized in mid-July , the Monw/w) was computed.Kernel moisture was determined by a GAC2000 moisture analyzer  and corrected to 15% for the einkorns and 16% for Blasco (harder texture) by overnight tempering. Afterwards, the hectoliter weight (kg/hL) was determined with a GAC2000 instrument . The seeds were milled with a Bona–GBR laboratory mill , which separates flour from bran and germ, and the milling yield , starch2 total , CO2 loss , CO2 retained , and retention coefficient .Dough leavening properties were assessed by rheofermentographic tests and were performed with a Chopin F3 Rheofermentometer , according to . The indTo better understand the fundamental properties of the doughs, rheological analyses were carried out according to  with a P3) by rapeseed displacement (method 10-05.01 [3/g) was computed as volume-to-weight ratio.Two breads per accession were produced according to method 10-10 B  with min10-05.01 ), and br0: 0 h, t1: 24 h, t2: 30 h, t3: 52 h), two bread loaves of each accession were weighed to evaluate weight decrease during storage, then they were transversely cut to obtain three uniform 25-mm-thick slices. Crumb moisture was assessed on two slices while water activity (wa) analysis was performed on the third central slice with an AQUALAB apparatus .Bread quality changes during storage were monitored on eight breads per accession, prepared only from SAL trial flours. Two breads were immediately analyzed, while the others were packaged in food paper bags and stored at 25 °C and 60% relative humidity in an HC 0020 air-conditioned cell . Four times  with a standard illuminator C. Additionally, images were captured using a flatbed scanner  in 256 grey level at 300 dots per inch and were processed using a dedicated software . The parameters evaluated were density red (R), density green (G), density blue (B), and density mean. At the same time, crumb porosity was determined by assessing the number, area, diameter, and shape of the pores, their size distribution , and % of pore area.Crust color and crumb color of the three slices of each sample at t2), hardness (N), springiness or elasticity , cohesiveness , and chewiness .Crumb texture characteristics were assessed by Texture Profile Analysis on the three central slices from each sample at each storage time with an HD.plus Texture Analyzer TA double-column dynamometer  connected to a Texture Exponent 32 recording system version 4.0.8.0 . The operating conditions adopted were: load cell 500 N, crossbar speed 2 mm/s, compression plate diameter 36 mm, sample compression up to 40% thickness, and 25 s waiting time between 1st and 2nd compression cycles. The parameters derived from the Force/Time curve were: young modulus (N/mmp ≤ 0.05) were found, Fisher’s lowest significant difference (LSD) was computed. Both ANOVA and LSD testing were performed with the software STATGRAPHICS plus v.4 . Means and standard errors were determined using the software Excel® .A two-way analysis of variance (ANOVA) was carried out for most parameters, using environment and genotype, or genotype and storage time, as factors. A one-way ANOVA was performed on the results of the color and porosity parameters, assessed on the breads prepared from the SAL flours. When significant differences . However, the amylose proportion of total starch was similar between Monlis and Blasco, and was only slightly inferior in ID331 . On the aestivum ,41,42. Tf starch .A peculiar characteristic of einkorn flour is its yellowness, due to the abundant presence of carotenoid ,5. This The two-way ANOVAs  verifiedAs already hinted by the protein content, the dry gluten content was higher in ID331 and Monlis than in Blasco . FallingT. monococcum to prepare leavened products, and their SDS quality was superior even to the good breadmaking wheat variety Blasco.The SDS sedimentation test underlined the good breadmaking propension of the two einkorns tested and of the bread wheat control. Einkorn generally has a poor breadmaking capacity ,33, but The farinographic results  suggeste2 loss, which determined a significantly higher CO2 retention coefficient, probably attributable to a more compact gluten network due to the higher protein content of the two einkorn flours.The unreplicated results of the rheofermentographic parameters are summarized in The two-way ANOVA  of the vThe evaluation of the region of linear viscoelasticity of a sample is an important step: when materials are tested in the linear range, their viscoelastic behaviors do not depend on the magnitude of the stress, the magnitude of the deforming strain, or the rate of application of the strain , but on The two-way ANOVA for bread volume, height, and specific volume  scored sw (0) than the einkorn samples. Monlis exhibited the highest loaf volume , followed by ID331 (732 cm3) and Blasco (671 cm3). Similar values for loaves obtained from selected einkorn flours are reported [Due to the higher amount of water added following indication of the farinographic test (56.0% vs. 53.4% to reach 500BU), the bread produced from Blasco flour had a significantly higher loaf weight , crumb hw , t0 thana* (crust and crumb) and b* (crumb). L*, a*, and b* evaluated on the crust and crumb of each bread sample. The crust color showed a low variation, with very similar L*, a*, and b* values among samples, as observed also by D’Egidio et al. [a* values, but a much inferior b* (yellow index) than both the einkorn genotypes. This reflects the differences in the yellow pigment content of the raw materials  as the einkorn breads, while the number of medium-sized pores (1–5 mm2) was significantly higher (p ≤ 0.05). Among einkorns, Monlis bread porosity was characterized by a greater number of large pores (>5 mm2), with an average area of 9.21 mm2, significantly higher than the Blasco and ID331 pores of the same class.Bread can be considered a solid food foam because the gas developed during fermentation is trapped in its solid matrix. Information about shape, size, and distribution of the pores is very important in order to identify the influence of processing conditions on the quality of the final products and to devise methods to manufacture products with peculiar textural characteristics . Image AInformation about the shape and regularity of the pores is obtained by considering that the larger the value, the greater the irregularity. Indeed, the smallest pores had the most regular and uniform shape (range: 1.78–1.89), while the large pores ranged from 2.34 to 2.60, indicating that as their size increased, their form became more irregular and less homogeneous. In our case, ID331 bread had the most regular pores for all the three size classes: its bread presented a porosity similar to Blasco, but was characterized by a more regular shape, thus making the porosity of the crumb more homogeneous.2), mainly because of the greater number of medium-sized pores. In Monlis bread, the large pores contributed considerably to the total porosity, as also confirmed by the less homogeneous internal loaf structure. Nevertheless, the loaves obtained from Monlis were the ones with the largest specific volumes, probably due to the high presence of these large pores. Despite the distribution of pores in the three classes being different among the three samples, the percentage of pores, computed as ratio between total alveolate area and total bread slice area, was similar among the three accessions.The number of pores and their average area is reflected in the percentage of relative area occupied in the slice and, ultimately, in the total porosity area of the bread. For Blasco, the surface occupied by the small pores was similar to that found in the einkorn samples, while the surface associated with medium-sized pores was significantly higher. Overall, pores area was highest in Blasco bread (810.57 mmp ≤ 0.05) between accessions only for young modulus (consistency index), hardness, gumminess, and chewiness. Storage times were always significant and so was the genotype x time interaction (except for elasticity). At t0, Blasco showed the highest consistency, hardness, gumminess, and chewiness, and Monlis exhibited the lowest values, while in general ID331 had results in line with Monlis. These data indicate how the bread loaves obtained from Monlis were the softest ones and were characterized by the best chewability, despite the finer and more regular porosity and the higher moisture content of Blasco breads, which are factors that are conventionally related to a softer structure. Possibly, the superior softness of the einkorn breads may be related to the lower starch content of their flours.The mechanical and geometrical characteristics of bread influence its behavior during oral processing . To assew, weight, young modulus, and texture parameters of the breads’ slices during storage. Blasco bread lost most of its moisture in the first 24 h after the production, while the decrease was more progressive for the einkorns. Interestingly, Blasco bread did not present significant water activity changes during storage, while the einkorn breads showed a significant increase after the first 24 h, followed by a plateau. This increase could be due to a reorganization of starch molecules to a different composition of starch with particular reference to the amylose content whose molecules, involved in the phenomenon of retrogradation, have a greater tendency to associate with each other and form hydrogen bonds, releasing water, or even to a reorganization of the protein matrix [n matrix , more ab0 was higher than that of the einkorn breads. During storage, this index increased for all samples, but was faster in the bread wheat. This behavior is also reflected in the hardness: in fact, the loaves from einkorn flours showed, already at t0, lower hardness values  compared with Blasco (4.35 N), and this difference amplified over time, as the kinetics of the bread from bread wheat were more accelerated than those of einkorn breads. The speed of the process, as measured by the value of the angular coefficient of the regression curves, was 0.24 N/h for Blasco and 0.12–0.14 N/h for the two einkorn samples. Hence, the einkorn breads maintained their softness for a longer time and their retrogradation was slower than that of the bread wheat.The Young modulus of the Blasco bread at tElasticity and cohesiveness decreased with similar trends in einkorn and wheat breads over the storage time. For that reason, bread chewiness  was mainly related to the hardness values, which was much higher for wheat bread, particularly during storage. Since the superior softness of the einkorn breads cannot be attributed to a higher moisture content , such behavior may be related to the lower starch content of einkorn flours and/or to the lower presence of amylose in the samples. Additionally, the slightly higher amylase activity of Monlis and ID331 may have contributed because amylases decrease starch retrogradation, diminish rigidity of the starch gel network, and limit starch–protein interactions ,53. TheiThis study demonstrates the possibility of obtaining einkorn breads with technological properties comparable to those of breadmaking-quality bread wheat. However, to achieve this result, high-quality genotypes are necessary. Furthermore, as the quality characteristics of the breads are influenced by processing conditions, einkorn bread manufacturing should utilize short mixing and leavening times to avoid overstressing the doughs. The choice of appropriate einkorn varieties and optimization of the breadmaking process will allow the production of breads with appropriate technological characteristics, endowing the market with an innovative cereal-based food possessing a superior nutritional value and longer shelf life.


To date, several epidemiologic and observational studies have evaluated the possible impact of BCG vaccination on T1D development, but the results are controversial. To elucidate this issue, we aimed to conduct a systematic review and meta-analysis of published cohort studies in this field. (2) Methods: A systematic search was performed for relevant studies published up to 20 September 2022 using Pubmed/Medline, Embase, and Scopus. Cohort studies, containing original information about the association between T1D and BCG vaccination, were included for further analysis. Pooled estimates and 95% confidence intervals (CI) for the risk ratio of T1D in BCG-vaccinated individuals compared to unvaccinated ones were assessed using the fixed effect model. (3) Results: Out of 630 potentially relevant articles, five cohort studies met the inclusion criteria. The total population of all included studies was 864,582. The overall pooled risk ratio of T1D development in BCG vaccinated and unvaccinated individuals was found to be 1.018 (95% CI 0.908–1.141, I Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by progressive and irreversible autoimmune destruction of pancreatic beta cell islets, resulting in absolute insulin deficiency. The incidence rate of T1D has been increasing globally by 2–3% yearly ) NOS score was 9 (0.0), which is suggestive of a high methodological quality and a low risk-of-bias of the included studies .n = 3), and the rest were conducted in Germany (n = 2)  of participants were female, and 51.23%  of participants were male. All the subjects of the two studies were newborns, and the mean age of other individuals with available data was 18.5 years , while others were vaccinated after the first year of age  or after three months of age . An amount of 94.46% of our studied population were given only one dose of the BCG vaccine , and 5.54% of individuals were vaccinated with two doses or more of the BCG vaccine . Our cohort populations were followed up for a duration of nine months to 18 years in different studies (The study period ranged from 1974 to 2014. Most of the studies were conducted in Canada ( (n = 2) . The tot (n = 2) , of whic.5 years . Two stu studies . VariousOnly one study confirmed the association between type 1 diabetes and BCG vaccination. It reported that BCG vaccination before one year of age had a preventive role after 30 years old. Four other studies refused the preventive role of BCG vaccination in T1D .Two studies evaluated the immunological markers in their study cohort. Islet cell antibodies increased in one study; however, in another study, they remained unchanged. Anti-glutamic acid decarboxylase antibodies and insulinoma-associated protein-2 were also unchanged in one study .2: 0%), indicating no impact of BCG vaccination in T1D development (p > 0.05).The overall pooled risk ratio of T1D development in BCG vaccinated and unvaccinated individuals was found to be 1.018 . Despite the existence of mechanisms justifying the protective role of BCG vaccination in T1D development, our meta-analysis did not show any decreased risk, which concurs with Chang et al.’s meta-analysis study of randomized controlled trials .Different mechanisms, involving the innate and adaptive immune system, have been suggested for the protective or therapeutic role of the BCG vaccine in autoimmune disorders, such as T1D. In the T1D process of autoimmunity, the imbalance between T-reg cells and cytotoxic T lymphocytes (CTLs) leads to the destruction of pancreatic islet cells . Both BCAfter BCG vaccination, a metabolic switch from overactive oxidative phosphorylation to aerobic glycolysis is induced in immune system cells. As a result, the following effects occur: increase in early glycolytic enzymes, up-regulation of glucose uptake that results in lowering of blood glucose, shunting of the accelerated glucose utilization through the pentose phosphate pathway, decrease in utilization of the late glycolytic steps, including the Krebs cycle, increase in lactate production, and decrease in oxidative phosphorylation ,36,37,38Regarding the mechanisms mentioned above, this vaccine seems to have some immune-modulatory properties. Still, our data suggest that, concerning T1D, these properties cannot prevent disease development in susceptible individuals. However, in the cohort study conducted by Corsenac et al., there were some protective effects after 30 years in participants who were vaccinated before one year of age . Most ofAnother critical point is that the immune system response contains humoral immunity, which produces auto-antibodies that may precede T1D’s clinical manifestation onset for years. The main autoantibodies detected in patients with T1DM are those against GAD65, zinc transporter (ZnT8), tyrosyl phosphatase (IA-2), and insulin (IAA) . HoweverMycobacterium avium subspecies paratuberculosis (MAP) in T1D pathogenesis [M. bovis, a different species from MAP, and our conclusion does not rule out the potential of MAP to cause T1D. Further investigations are required to clarify this issue.Growing evidence supports the involvement of ogenesis ,44,45. Sogenesis ,49,50,51ogenesis ,53, BCG ogenesis . HoweverPreviously, the duration of BCG-induced protection has been considered limited to the first few years of life. However, recent studies revealed that BCG is effective for at least 20 years against tuberculosis (TB) when given at birth or school ages ,55. AddiBCG vaccine induces regional inflammatory responses with granulocyte influx in the preliminary phase, followed by CD4+ T-cells, as well as rising levels of pro-inflammatory Th1-type cytokines, i.e., IL-2, IL-12, and IFN-γ . In the In terms of sex difference’s impact on BCG vaccination outcome, Koeken et al. found a significant sex-differential effect, with non-negligible reductions in inflammatory proteins after BCG administration in males only . This waIn the studies included in our review, there was no comparison between different administered BCG strains. In some studies, it is manifested that, from those who were vaccinated with varying BCG vaccine strains, different degrees of T-cell responses are induced in in vitro peripheral blood mononuclear cell (PBMC) cultures ,65. HoweIn our study, the vast majority (94.46%) of the vaccinated population received only a single dose of the BCG vaccine, along with the standard required regimen for the prevention of TB . HoweverOur study has some limitations. First, the scarcity of cohort studies and the probability of overlap between studied populations is the major limitation of our study. Three out of our five included studies were from Canada, and the other two were from Germany. Despite the differences in age groups, study periods, and the number of cohort populations of each study, there might probably be some overlap between study populations in articles published from the same country. We could not confirm or ignore this issue. However, it will challenge our study conclusion, and further cohort studies from different countries are greatly required to make any solid conclusion in this regard. Secondly, although our included studies for meta-analysis were cohort studies with large samples and long follow-up durations, most did not follow the cases until adulthood. This may conceal the effect of vaccination at older ages. Thirdly, 94.46% of our vaccinated cases received a unique dose of BCG, and the impact of multiple dosing vaccinations has remained unclear. Fourthly, due to the unavailable data of our included studies, we could not conduct any subgroup meta-analysis to evaluate the effects of age, sex, age at vaccination, immunization dose, etc. in conclusion. Fifthly, most of our reviewed studies complained about the lack of adjustment for potential confounders, which may affect our analysis. Sixthly, different studies conform to varying criteria for T1D diagnosis of their cases. Additionally, they used different follow-up techniques, as the studies from Germany only considered autoimmunity and immunologic modifications among their cohort patients, while the studies from Canada followed clinical improvements in their patients. These issues may lead to heterogeneity for a definite conclusion. Seventhly, some of our included studies used self-reported data instead of registered information about BCG vaccination status, which has poor validity and may conform to recall and misclassification bias.Based on our study, it seems that there is no protective or facilitative effect of prior BCG vaccination in T1D development. Although some studies showed that BCG vaccination causes a delay in T1D occurrence or has a late outgrowth in adulthood, these studies are limited. There is little evidence on this topic, and more extensive observational studies with long-term follow-up duration are required to delineate these issues. Additionally, further studies are warranted to compare the effect of multiple dosing vaccination and different time of exposure on the possible preventive role of BCG in T1D development.


Vaccines against coronavirus disease 2019 (COVID-19) have been in use for over two years, but studies that reflect real-world vaccination coverage and demographic determinants are lacking. Using a multistage stratified random cluster sampling method, we planned to directly explore vaccination coverage and the demographic determinants of different doses of COVID-19 vaccines in Beijing, especially in older populations. All 348 community health service centers in 16 districts were involved. We performed multivariable logistic regression analyses to identify demographic determinants of different coverage rates via adjusted odds ratios (aORs) and 95% CIs. Of the 42,565 eligible participants, the total vaccination coverage rates for ≥1 dose, ≥2 doses, ≥3 doses, and 4 doses were 93.3%, 91.6%, 84.9%, and 13.0%, respectively, but decreased to 88.1%, 85.1%, 76.2%, and 3.8% in the older population. Among all participants, younger , male , and better-educated residents  were more likely to be fully vaccinated. People who lived in rural areas  and held the new rural cooperative health insurance  established a higher rate of full vaccination coverage. No history of chronic disease was positively associated with a higher coverage rate . Occupation also affected vaccination coverage. Demographic factors influencing the rate of vaccination with at least one or three doses were consistent with the results above. Results remained robust in a sensitivity analysis. Given the highly transmissible variants and declining antibody titers, accelerating the promotion of booster vaccination coverage, especially in high-risk groups such as the elderly, is a top priority. For all vaccine-preventable diseases, rapidly clarifying vaccine-hesitant populations, clearing barriers, and establishing a better immune barrier can effectively safeguard people’s lives and property and coordinate economic development with epidemic prevention and control. Twenty optimized measures”, “Ten new measures”, and “Class-B management of COVID-19” have been announced successively in China [Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, coronavirus disease 2019 (COVID-19) has ravaged the world for more than three years . It has in China ,8. With Vaccination is considered to be the most effective and economical strategy to prevent and control the epidemic of infectious diseases ,12,13,14To our knowledge, the vast majority of previous studies on vaccine acceptability have focused on vaccination intentions and influencing factors, including age, gender, income level, marital status, education level, rural environment, occupation, health insurance, etc. ,40,41,42This cross-sectional study was conducted from 26 December to 31 December 2022, in collaboration with the Beijing Municipal Health Commission and 16 district-level health commissions. The inclusion criteria were as follows: (1) ≥18 years old; (2) living in Beijing for more than 6 months; and (3) at least one family member signed a family doctor service contract with CHSCS. PASS software 15.0  was used to calculate the minimum sample size with a coverage rate ranging from 10% to 100%, an α of 0.05, and a two-sided confidence interval width of 0.02, using the exact (Clopper–Pearson) method. The minimum sample size was 9701. Considering the nature of cluster sampling, we expanded the sample size.We used a multistage stratified random cluster sampling method to recruit a representative sample of approximately 43,000 individuals. In the first stage, we selected all 16 districts in Beijing, and the number of recruits was proportional to the number of permanent residents in each district. In the second stage, the target survey population of each CHSC was assigned based on their service circle. All 348 CHSCs in 16 districts were involved. In the third stage, households were randomly selected from the CHSC family doctor contract system. All members in the selected households who met the inclusion criteria were consecutively enrolled. Electronic questionnaires were distributed through WeChat groups established in advance at each community health service center. For those who could not respond independently via the online survey , phone interviews were conducted by trained community health workers. Respondents who submitted questionnaires agreed to participate in the study. Finally, 42,565 eligible respondents were included in the analysis, yielding an overall response rate of 99.0%.This study met the requirements of the Declaration of Helsinki and was approved by the Ethics Committee of Peking University (IRB00001052-21126).Gender, age, location, education level, health insurance, occupation, and self-reported chronic disease history  were all investigated.We set separate questions to ask about COVID-19 vaccination status, including “unvaccinated”, “1 dose”, “2 doses”, “3 doses”, and “4 doses”. The inactivated vaccines  are the most widely used COVID-19 vaccines in China, covering almost all recipients .The COVID-19 vaccination coverage rate was the primary outcome of this study. Based on the results of the questionnaire on vaccination status, we divided COVID-19 vaccination coverage into four categories: ≥1 dose; ≥2 doses; ≥3 doses, and 4 doses. Taking the coverage rate of ≥1 dose of COVID-19 vaccines as an example, the numerator was the number of residents who received at least 1 dose, and the denominator was the number of residents who did not. The other three categories of vaccination coverage rates were calculated with similar formulas separately. Notably, the coverage rate of 4 doses only refers to the proportion of people who have received a total of four doses of COVID-19 vaccines in the whole population. Completion of ≥2 doses of COVID-19 vaccines was considered to finish the primary vaccination series (full vaccination), and completion of ≥3 doses was considered to complete the first booster immunization. The fourth dose usually means a second booster dose.p ≤ 0.05 were considered to be statistically significant.Frequencies and percentages were used to describe the basic characteristics of the study population. We calculated the COVID-19 vaccination coverage rates  and 95% confidence intervals (CIs) for the overall population, for persons 18 to 59 years of age, and for persons 60 years of age or older. Independent chi-square tests and Cochran–Armitage tests for trends were used to compare the vaccination rates among groups stratified by demographic characteristics. We performed multivariable logistic regression analyses to identify demographic determinants of different coverage rates. Adjusted odds ratios (aORs) and 95% CIs were both calculated. The Hosmer and Lemeshow test was used to assess the goodness of model fitting. Age was considered a continuous variable in sensitivity analyses to verify the robustness of the results. All statistical analyses were performed using SPSS 26.0 . Two-sided Among all participants, 12,530 (29.4%) people were aged 60 years and above. For the elderly population (≥60 years), 37.0% (4638) were at least 70 years old, and the difference between the proportions of males and females was significantly smaller than that of the whole population (17.2% vs. 31.6%). The overall education level of the elderly is relatively lower, and nearly half of them have only a junior high school education or below. Interestingly, the number of people with chronic diseases was almost the same as those without chronic diseases in all participants, but the former accounted for 83.8%  of the elderly population.Four types of vaccination coverage rates are shown in trend < 0.05) and was higher among men than among women , the coverage rate increased with education , male  and better-educated residents  were more likely to be fully vaccinated. People who lived in rural areas  and held the new rural cooperative health insurance  established a higher rate of full vaccination coverage than those who lived in urban areas. No history of chronic disease was positively associated with a higher coverage rate . Civil servants and employees of enterprises/institutions, pleasant, retirees, and freelance workers had lower vaccination coverage (≥2 doses) than those in the service industry, whereas the reverse was true for healthcare workers. The demographic factors influencing the rate of vaccination with at least one or three doses were consistent with the results above. In addition, all demographic characteristics included in the model, except gender and chronic disease history, affected the fourth injection rate of residents. It is worth noting that civil servants and employees of enterprises/institutions  showed a higher four-dose vaccination rate than service industry personnel. The vaccination rate of residents protected by the new rural cooperative medical insurance was lower than that of residents protected by urban medical insurance .For the vaccination rates  in the older population, lower age, living in rural areas, better level of education, and healthcare workers were all significant positive demographic determinants. Compared with participants with chronic disease, those without chronic disease who received at least one dose , at least two doses , and at least three doses  increased significantly, but this result did not apply to the coverage of the fourth injection. The type of health insurance was not associated with the four-dose coverage but with the coverage of the other three vaccination groups. The vaccination coverage rate of the unemployed/freelance was lower than that of service workers . Men were 1.38 (95% CI: 1.15–1.67) times more likely to receive the fourth dose than women.Demographic determinants associated with COVID-19 vaccination coverage among people aged 18–59 years old are presented in To our knowledge, few studies have directly reported vaccination coverage and the demographic determinants of different doses of COVID-19 vaccines, especially in the older population. According to our results, 91.6% of the 42,565 eligible participants were fully vaccinated (≥2 doses), and 84.9% were at least administered the first booster dose (≥3 doses). For the 12,530 older people, the coverage rates of full vaccination and the first booster dose decreased to 85.1% and 76.2%, respectively. The second booster vaccination is in the early stages of being rolled out. Age, location, education, health insurance, occupation, and chronic disease history were all demographic determinants of vaccine coverage rates. Gender played a more significant role in all populations than in the elderly population, and older men were more likely to receive a second booster dose. Our study reveals the real-world vaccination coverage and demographic distribution of COVID-19 vaccines in Beijing two years after vaccine introduction, which may clarify the publicity and implementation direction for the next dose of COVID-19 vaccines or future vaccination programs for other pandemics in priority populations. The demographic characteristics that affect the actual vaccine coverage rate are universal and can be used as a reference for other similar vaccine-preventable diseases and countries with similar national conditions to promote vaccination from the simplest and most direct perspective. Additionally, our results may also provide basic data support for modeling studies based on the current status of real-world vaccination or correlation with disease outcomes after SARS-CoV-2 infection.Vaccination is considered to be the most effective and economical strategy to prevent and control the epidemic of infectious diseases ,12,13,14According to our results, age was negatively associated with COVID-19 vaccine coverage in both the whole population and the elderly population. It is well known that old age is a high-risk factor for poor prognosis of various diseases, including severe illness and death after SARS-CoV-2 infection ,54. AnotThe limitations of this study are as follows. First, although this study adopted multistage stratified cluster sampling to cover all the CHSCs in all 16 administrative districts of Beijing, China, bias is hard to avoid. Therefore, we chose to expand the sample size as much as our financial and human resources allowed. More than 42,000 people were eventually enrolled. Second, because the proportion of females in our included population was significantly higher than that of males, we may have underestimated the vaccine coverage in the total population to some extent based on the analysis of the effect of demographic characteristics on vaccination rates in this study. In addition, caution is needed when extrapolating the results. Third, the vaccination status and demographic characteristics were obtained through self-report of the respondents, which may cause recall bias. Fourth, given that information about infection status was not available at that time, we have not yet further explored the association between vaccination status and COVID-19 outcomes in such a large population. Nevertheless, this is the first real-world study of a large sample in a contracted population of family physicians in a community health service center in China. The results filled the research gap, that is, few large sample studies have published the status of residents’ vaccination and its determinants of demographic characteristics in such detail. Because this is a quick survey with a large sample size, more demographic characteristics, such as income, marriage, and childbirth, were not examined, which further research should take seriously. In addition, studies on the association between vaccination and infection outcomes based on real-world data are also feasible research directions.In conclusion, the population coverage rate of the primary vaccination series has reached a high level in China, while the booster coverage still needs to be improved, especially in the older population. Age, location, education, health insurance, occupation, and chronic disease history were all demographic determinants of vaccine coverage. Gender played a more significant role in all populations than in the elderly population. It is more targeted and practical to carry out vaccination promotion based on demographic characteristics according to local conditions. For all vaccine-preventable diseases, rapidly clarifying vaccine-hesitant populations, clearing barriers, and establishing a better immune barrier can effectively safeguard people’s lives and property and coordinate economic development with epidemic prevention and control.


Background: Lumbar degenerative disc disease (LDDD) is a leading cause of chronic lower back pain; however, a lack of clear diagnostic criteria and solid LDDD interventional therapies have made predicting the benefits of therapeutic strategies challenging. Our goal is to develop machine learning (ML)–based radiomic models based on pre-treatment imaging for predicting the outcomes of lumbar nucleoplasty (LNP), which is one of the interventional therapies for LDDD. Methods: The input data included general patient characteristics, perioperative medical and surgical details, and pre-operative magnetic resonance imaging (MRI) results from 181 LDDD patients receiving lumbar nucleoplasty. Post-treatment pain improvements were categorized as clinically significant  or non-significant. To develop the ML models, T2-weighted MRI images were subjected to radiomic feature extraction, which was combined with physiological clinical parameters. After data processing, we developed five ML models: support vector machine, light gradient boosting machine, extreme gradient boosting, extreme gradient boosting random forest, and improved random forest. Model performance was measured by evaluating indicators, such as the confusion matrix, accuracy, sensitivity, specificity, F1 score, and area under the receiver operating characteristic curve (AUC), which were acquired using an 8:2 allocation of training to testing sequences. Results: Among the five ML models, the improved random forest algorithm had the best performance, with an accuracy of 0.76, a sensitivity of 0.69, a specificity of 0.83, an F1 score of 0.73, and an AUC of 0.77. The most influential clinical features included in the ML models were pre-operative VAS and age. In contrast, the most influential radiomic features had the correlation coefficient and gray-scale co-occurrence matrix. Conclusions: We developed an ML-based model for predicting pain improvement after LNP for patients with LDDD. We hope this tool will provide both doctors and patients with better information for therapeutic planning and decision-making. Lumbar degenerative disc disease (LDDD) is a leading cause of lower back pain ,2 and ofMRI is a non-invasive method and represents the gold standard for detecting and diagnosing LDDD. Axial T2WI during lumbar MRI (L-MRI) improves tissue contrast and provides higher sensitivity than traditional computerized tomography (CT) imaging. Axial T2WI is used as an image target to provide richer information on the status of the nucleus pulposus of the intervertebral disc; the nucleus pulposus is mainly composed of 66% to 86% water, with the remainder consisting primarily of type II collagen and proteoglycans. The matrix of cartilage (soft bone) is made up of glycosaminoglycans, proteoglycans, collagen fibers, and sometimes elastin. The similarities in materials between the intervertebral disc and soft bone construct the intervertebral fibrocartilaginous joint. To better interpret the differences between them and surrounding tissues in various characteristics, such as collagen content, texture characteristics, annular tearing, degree of degeneration, flavum ligament status, nerve impingement, canal stenosis, soft bone, and porosity tissue, T2WI axial MRI is most helpful. The differentiation of these tissues in image presentation is relevant to the improvement of patients’ surgical pain ,20,21. TRadiomic feature analysis refers to a quantitative approach for converting images into parameters that can be used in advanced mathematical analyses, and it has been adopted for a wide range of medical applications, including the evaluation and interpretation of radiologic and pathologic images . To analThe technological analysis of medical imaging has rapidly advanced over recent years , accompaThe present study represents the first attempt at developing a predictive model using AI and radiomic feature analysis for LNP prognosis in patients with LDDD. The model provides predictions for therapeutic prognosis to support clinicians’ decision-making about treatment strategies for LDDD. In addition, the model also has the potential to reduce patients’ unnecessary suffering resulting from treatment delays. It could also lead to a more personalized approach to patient care, optimized treatment plans, and ultimately improved patient outcomes.This retrospective study enrolled 181 patients diagnosed with LDDD who received LNP. Information was obtained from the China Medical University Hospital Information System, including pre-operative L-MRI results, sex, age, height, weight, body mass index (BMI), and medical history data, such as perioperative medical and surgical details and clinical outcomes.The exclusion criteria included patients younger than 20 years and patients lacking L-MRI data within 180 days before the operation. A total of 181 de-identified patients  were enrolled in this study . The datThe outcome of interest was the visual analog scale (VAS) value, including the values recorded within 1 month before surgery (pre-VAS) and 1 month after surgery (post-VAS). In addition, sex, age, height, weight, BMI, and disc level were recorded . All axiAll L-MRI data were performed within 180 days before operation. These images were acquired by two 1.5T MR scanners  in China Medical University Hospital. The parameters loaded from these two MRIs were the following: (1) Signa Excite 1.5T  with the following parameters: axial T2-weighted frFSE  Toshiba MRT 200 1.5T  with the following parameters: axial T2-weighted  models, including sex, age, operative level, pre-VAS, and post-VAS . Prior tImage size varied as images were acquired using multiple MRI scanners. The original DICOM image pixel sizes were 320 × 320, 320 × 356, 512 × 512, and 512 × 568. We collected the pixel spacing value in every DICOM file and counted the plural of these figures. We rescaled all images to display at the same scale by adjusting the images based on the pixel spacing value . After rescaling, the image size changed to 511 × 511, 512 × 512, 540 × 540, 568 × 511, 568 × 512, 568 × 568, 625 × 625, and 654 × 654. These images were obtained from multiple MRI scanners, and differences in image scaling can affect feature calculations. We then extended outwards from the critical hot spots of disc degeneration and cropped the images to a size of 224 × 224 to facilitate focused calculation of critical features in subsequent analyses. We used the radiomic toolkit to extract features from the images, including descriptors of the intensity histogram, size- and shape-based features, voxel relationship descriptors, e.g., size zone matrix (SZM), gray-level co-occurrence matrix (GLCM), etc.; there were 91 features. Finally, we standardized the clinical and radiomic feature data by transforming them from their natural range  into a standardized range of 0 to 1.In order to focus on disc degeneration of the lumbar spine. Our study cropped the MRI image size to 224 × 224 by extension from the image center, which contained the critical hot spots of disc degeneration.Radiomic features can be classified into five categories: descriptors of the intensity histogram, size, and shape-based features, voxel relationship descriptors , and fractal-based textures. In this study, 91 radiomic features were extracted from these five categories—please see .Normalization is widely known to improve the performance and training stability of the model. Our study converted clinical data and radiomic feature data from their natural range  into a standard range of 0 and 1.To avoid model training bias, we take the K-fold cross-validation widely used to abstain the specific data separation in the training and testing dataset. First, split the training dataset into K pieces, taking one piece as the validation set each time, and the remaining data as the training set, so we can obtain the results of K validation sets. The results obtained by averaging multiple test sets are often more representative. The advantage of this step is that when there is not much training data, the model’s generalization ability is tested multiple times in the limited training set, and the results obtained by averaging over multiple validation sets are usually more representative. When separating the data, we avoided repeated patient data from being mixed in the training and test sets.SVM maps the training data to the hyperplane to maximize the margin of the gap between the two categories. The farther the classification boundary is from the nearest training data point, the better because this can reduce the generalization error of the classifier. In cases where the data is not linearly separable, we need to transform the data to a higher dimensional space to accommodate the support vector classifier.LGBM uses a histogram algorithm to segment continuous features to speed up the training process and reduce memory load. In addition, LBGM takes advantage of the leaf-wise best-first method to determine DT growth, which reduces losses and avoids overfitting.XGB is a DT-based boosting algorithm that utilizes a new generalized gradient boosting DT algorithm to speed up model construction.XGBRF is an ensemble learning method for classification, regression, and other tasks, which operates by constructing a multitude of decision trees (DTs) during training. The advantage of this algorithm is the ability to accommodate a high noise margin and high-dimensional feature data.CatBoost is a gradient boosting library based on binary decision trees. Target leakage and prediction shifts are avoided by grouping categories with target statistics (TS). The log loss and zero-one loss were better than the traditional greedy algorithm.The random forest used in this study underwent some optimizations. Random forest is an ensemble machine learning algorithm that uses multiple decision trees to make predictions. Each decision tree in the random forest is trained on a random subset of the available data and features. The final prediction is made by aggregating the predictions of all the individual trees. The essential advantage of the random forest algorithm is that it reduces the risk of overfitting by introducing randomness to the training process.In this study, we used optimization parameters to control the leaves and depth of the decision trees. They experimented with different values of the maximum depth of the trees, and a minimum number of samples were required to split an internal node. These parameters have a significant impact on the performance of the random forest model as they control the complexity of the decision trees and the degree of overfitting.We used a cross-validation approach to select the optimal values of these parameters. We split the available data into training and validation sets. We then trained multiple random forest models on different subsets of the training data set with different combinations of the optimization parameters. The models were then evaluated using the validation data set, and the combination of parameters that performed the best was selected as the optimal configuration.In addition to the optimization parameters, we used a random seeding mechanism to choose the best model after a certain number of random seeds. Random seed ensures that the random forest model is trained on a different subset of the data and features each time it is run, thereby introducing additional randomness to the training process. By running the model multiple times with different random seeds and selecting the best-performing model, we ensured that the final model was not overly sensitive to the initial random seed and was robust to variations in the training data. Overall, the combination of optimization parameters and the random seeding mechanism used in the study allowed us to build a high-performing random forest model robust to overfitting and variations in the training data .Most of the models were generated using default parameters in Scikit-learn. The SVM algorithm used radial based function kernel, the complexity of model was 100, and the gamma was 0.6. For the LGBM algorithm, the number of DTs was 202, the maximum tree depth was 40, learning rate was 0.05, the number of leaves was 25, and the proportion of features used when built per tree was 0.53. For the XGB algorithm, 305 DTs were used, the depth was 29, learning rate was 0.6, the number of leaves was 46, and the proportion of features used when built per tree was 0.52. For the XGBRF algorithm, the number of DTs was 451, the maximum tree depth was 29, learning rate was 1, the number of leaves was 46, and the proportion of features used when built per tree was 0.52. The iRF algorithm was implemented using MATLAB; the maximum tree number was 20, the number of leaves was 20, and the learning rate was 1.All models were trained on the NVidia DGX-2 system : central processing unit, Dual Intel Xeon Platinum 8168 , 2.7 GHz, 24-cores × 2; system memory, 1.5 TB RAM; storage, 960 GB solid-state drive (SSD) × 2, 30 TB internal SSD; graphics processing unit (GPU), 16-slice NVIDIA Tesla V100 ; GPU memory, 512 GB; software: Ubuntu Linux operating system , Red Hat Enterprise Linux OS (version 9.2); the total training time was about 12 h. The software versions and environment packages are detailed as follows: python 3.10.9, pandas 1.5.3, tensorflow 2.12.0, matplotlib 3.7.0, scikit-learn 1.2.1.To ensure the fairness of the model, the performance of each model was evaluated using the same testing set. We split the modeling dataset into training and testing sets using the K-fold cross-validation method, which assigns 80% of data points to the training set and the remaining 20% of data points to the testing set. As mentioned above, we avoided repeated patient data from being mixed in the training and test sets. Evaluation indicators, including the confusion matrix, accuracy, sensitivity, specificity, F1 score, and area under the receiver operating characteristic (ROC) curve (AUC), were used to measure model performance. The Scikit-learn suite was used to establish the derivation among evaluation parameters and verify each model.TP, True Positive; TN, True Negative; FP, False Positive; FN, False Negative.This study developed various ML models for predicting pain relief following LNP based on the pre-operative L-MRI T2WI and physiological parameters of patients with LDDD and verified the predictive efficacy of each developed model. Physiological parameters included sex, age, disc level, and VAS. Both L-MRI T2WI and physiological data were preprocessed before model training. Six types of ML models were trained and compared, including SVM, LGBM, XGB, and XGB, based on RF and iRF, and the results are shown in . The besThe iRF model was the best performing in comparison with four other machine learning models. The iRF model was trained using a combination of L-MRI T2WI and physiological parameters to develop a predictive model of postoperative pain improvement.To obtain texture information from T2WI and volumes of interest in L-MRI, the Pyradiomics suite was used to extract five sets of radiomic features, resulting in 91 radiomic features used for training the ML models.We ranked the top five influential factors after analyzing the influences of the various features used to train the ML models . The preBased on these findings, we concluded that the patient’s physiological parameters had a more significant impact on the accuracy of the ML models than radiomic features. This highlights the importance of incorporating physiological parameters into predictive models for postoperative pain improvement. Overall, the iRF model trained using the combination of L-MRI T2WI and physiological parameters proved to be a highly accurate and effective predictive model of postoperative pain improvement.The optimization strategy used in this study was control of the depth of the tree and the number of leaves in the iRF model. The depth of the tree refers to the number of feature segmentation points used in the decision tree, while the number of leaves determines the granularity of the tree structure. We aimed to achieve an optimal balance between model complexity and accuracy by adjusting these parameters.Once the iRF model was trained, we used it to analyze the feature importance of the data set. We did this by computing branch prediction performance from the last leaf of each decision tree in the forest. By examining the feature segmentation points associated with the best-performing leaves, we determined the influence of each feature on overall model performance.The iRF algorithm was particularly useful because it allowed us to identify the data set’s most essential features while accounting for their interactions with other features. We obtained a more accurate and interpretable model that provided valuable insights into the underlying data. Overall, the iRF model proved to be a robust tool for feature selection and analysis in this study.Our iRF model had the best performance among all evaluation indicators, with accuracy, F1 score, sensitivity, and specificity values of 0.76, 0.73, 0.69, and 0.83, respectively. Traditionally, the decision of who receives or when to receive LNP relies solely on the experience and suggestions of physicians. Most prognostic and outcome assessments are based on three factors: (1) general patient data such as age, sex, and body mass index; (2) physicians’ subjective reading of images, e.g., of the positive hyperintensity zone, black disc, and Modic changes of the end plates; and (3) invasive challenge discography results . FurtherKlessinger et al. reported a short-term repeat surgery rate as high as 18.7% for LNP, making pre-operative assessment crucial for preventing poor outcomes . HoweverSix ML algorithms were used to develop the models, including SVM, LGBM, XGB, XGBRF, CatBoost, and iRF, and their performance was compared. As a result of comparing overall performance, including accuracy, sensitivity, specificity, F1 score, and AUC , the iRFML algorithms analyze data automatically and generate rules for predicting unknown data . Deep leAccording to the results of feature importance based on these influential factors , patientIn this study, the iRF ML model achieved the highest accuracy for predicting pain improvements in patients with LDDD following LNP. It can contribute to LDDD prognostic prediction and provide a reference for LNP decision-making. However, this retrospective study has several limitations. (i) The case number was small, resulting in the unsuccessful fitting of DL models. (ii) Uniformity of images was difficult to achieve due to differences in the MRI machines used to obtain images or in the image format. (iii) No indicators of LDDD severity were available beyond the VAS score. (iv) Individual differences in IDTs were not examined in this study. (v) Missing data might also have affected the study outcomes. It is essential to compare prospective differences between iRF ML model predictions and real-world results. Therefore, further work on model validation based on a comparison of results is required.


Univariate and multivariate logistic regression analyses were used to determine independent risk factors for esophageal stricture after ESD and establish a nomogram model for the development group. The prediction performance of the nomogram model is internally and externally verified by calculating C-Index and plotting the receiver operating characteristic curve (ROC) and calibration curve, respectively. The results showed that Age, drinking water temperature, neutrophil–lymphocyte ratio, the extent of esophageal mucosal defect, longitudinal diameter of resected mucosa, and depth of tissue invasion (P < 0.05) were independent risk factors for esophageal stricture after ESD. The C-Index of the development group and validation group was 0.925 and 0.861, respectively. The ROC curve and area under the curve (AUC) of the two groups suggested that the discrimination and prediction performance of the model were good. The two groups of calibration curves are consistent and almost overlap with the ideal calibration curve, indicating that the predicted results of this model are in good agreement with the actual observed results. In conclusion, this nomogram model has a high accuracy for predicting the risk of esophageal stricture after ESD, providing a theoretical basis for reducing or avoiding esophageal stricture and guiding clinical practice.Esophageal stricture is a common complication after endoscopic submucosal dissection (ESD) for superficial esophageal cancer and precancerous lesions, we intend to investigate the independent risk factors of esophageal stricture after ESD by adding the data of included living habits, established a nomogram model to predict the risk of esophageal stricture, and verified it by external data. The clinical data and living habits of patients with early esophageal cancer and precancerous lesions who underwent ESD in the Affiliated Hospital of North Sichuan Medical College and Langzhong People's Hospital from March 2017 to August 2021 were retrospectively collected. The data collected from the two hospitals were used as the development group (The online version contains supplementary material available at 10.1186/s12876-023-02855-8. Esophageal cancer (EC) is the seventh most common cancer and the sixth most common cause of cancer death in the world, and new cases and deaths in China account for 53.7% and 55.7% of the global total, respectively . EsophagAt present, with the development of endoscopic techniques, endoscopic submucosal dissection (ESD) has gradually become the treatment of choice for early esophageal cancer and precancerous lesions . EsophagThe nomogram model plays an important role in predicting the individualized assessment of patients, the occurrence of diseases and survival prognosis –12. HoweIn this paper, we retrospectively collected patients with superficial esophageal squamous cell carcinoma and precancerous lesions who underwent esophageal ESD in two hospitals: North Affiliated Hospital of Sichuan Medical College and Langzhong People's Hospital from March 2017 to August 2021, and collected the clinical data and living habits of these patients. The date of first patient data access for this study was January 20, 2022. After strict inclusion criteria and exclusion criteria, 256 patients in the Affiliated Hospital of North Sichuan Medical College and 105 patients in Langzhong People's Hospital were included, and relevant data were collected as the development dataset and validation dataset, respectively. In this study, we routinely used oral steroids in patients with more than 3/4 of the circumferential percentage of mucosal defects, who started oral prednisone acetate at a dose of 30 mg/day on the third postoperative day, which was reduced by 5 mg every 2 weeks. When the dose was reduced to 20 mg/day, 5 mg was reduced every week. Steroids were administered for a total of 8 weeks. In the development group, 67 patients received steroids; in the validation group, 23 patients received steroids. Postoperative re-examinations at endoscopes were performed every 3 months during the first year after ESD to determine whether esophageal strictures developed. This study is a retrospective design and we applied to the ethics committee for a waiver of informed consent, which waived the need for participant consent. The ethical study was performed at The Affiliated Hospital of North Sichuan Medical College under the Declaration of Helsinki and the protocol was approved by the ethics committee of the Affiliated Hospital of North Sichuan Medical College (batch number: 2022ER018-1).The inclusion criteria included: (1) preoperative comprehensive imaging examination to rule out peripheral lymph nodes and distant metastasis; (2) preoperative endoscopic biopsy findings suggestive of early ESCC or precancerous lesions; (3) endoscopic ultrasonography was performed to rule out cases with tumor invasion depth exceeding the mucosal layer; (4) none of the patients had serious underlying diseases.Exclusion criteria included: (1) incomplete, blurred, or lost patient-relevant data; (2) Postoperative pathology after ESD revealed esophageal adenocarcinoma, papilloma, spindle cell tumor, etc., with the depth of tissue invasion exceeding 200 μm in the submucosa; (3) loss to follow-up or patient death; (4) patient with a history of other tumors.Pathological diagnostic criteria: According to the criteria of the World Health Organization , patholo(1) clinical data: Age, gender, preoperative high neutrophil–lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lympho-monocyte ratio (LMR), the longitudinal length of the resected mucosa, lesion location, en bloc resection, percentage of the circumferential mucosal defect, postoperative pathological type, positive margin, and depth of Invasion.(2) living habits data: Long-term smoking, long-term drinking, high protein diet, fruit and vegetable intake, eating speed, pickled food, fried food, and high-temperature water intake.In both the development and validation groups, we collected data on the clinical and lifestyle habits of patients with early esophageal cancer and precancerous lesions treated with ESD.NLR, PLR, and LMR: Preoperative blood routine was collected from patients, including neutrophil, lymphocyte, and monocyte counts. The neutrophil–lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and Lympho-monocyte ratio (LMR) were calculated for each patient, and then the median of the above parameters was found. Those above the median were in the high-value group, and those below the median were in the low-value group.Sites of esophageal lesions: The sites of esophageal lesions were classified according to the latest segmented criteria : 1) cervDepth of tissue invasion: All of them were based on the Paris classification criteria for gastrointestinal tumors in 2002 . The depAssessment of living habits: Referring to the questionnaire survey on living habits by Liu et al. . If the Esophageal stricture: Esophageal stricture was defined if the patient had symptoms of dysphagia or if standard endoscopy (9.8 mm in diameter) could not pass through the esophagus .Data were statistically analyzed using SPSS 25.0 software and R 4.2.0 software. Enumeration data were compared by chi-square test, measurement data were expressed as mean ± standard deviation P < 0.05) to obtain independent risk factors for esophageal stricture after ESD. Based on the results of multivariate logistic stepwise regression analysis, a nomogram model for predicting esophageal stricture was successfully developed using R 4.2.0 software, and the model fit was assessed by the Hosmer–Lemeshow test.SPSS 25.0 software was used for univariate analysis of variables in the developmental group, and multivariate logistic stepwise regression analysis was performed for statistically significant variables  and a validation group (n = 105), with a ratio of 7:3. In the development group, there were 147 males and 109 females, with an average age of 63.01 ± 8.397 (years); in the validation group, there were 53 males and 52 females, with an average age of 63.10 ± 7.252 (years). All patients were followed up, and postoperative endoscopic reexamination was performed every 3 months in the first year, if there were no significant abnormalities, subsequent reexamination was performed once a year. In the development group, 38 patients developed postoperative stenosis, with an incidence rate of esophageal stenosis of 14.8%; in the validation group, 20 patients developed postoperative stenosis, with an incidence rate of esophageal stenosis of 19.05%. The basic characteristics and living habits of the development group and validation group are shown in Table A total of 361 patients after esophageal ESD were included in this study, consisting of a development group (P < 0.05). In order to control for confounding factors, we performed multivariate logistic stepwise regression for the above statistically significant factors. The results showed that age of more than 60 years, long-term high-temperature water intake, preoperative high NLR, esophageal mucosal defect of more than 3/4, longitudinal diameter of resected mucosa, and depth of tissue invasion of more than M2 were independent risk factors for the esophageal stenosis after ESD. We found a cutoff value of 4 cm for the longitudinal length of the resected mucosa by SPSS software, with a Youden index of 0.603, corresponding to a sensitivity of 84.2% and a specificity of 76.1%.Table Collinearity analysis of age, drinking temperature, NLR, longitudinal diameter of resected mucosa, circumferential range of mucosal defects, and depth of tissue invasion resulted in variance inflation factors (VIFs) of 1.023, 1.034, 1.010, 1.466, 1.496, and 1.094, respectively, which indicated that there was no multicollinearity among the six independent risk factors. Based on the results of logistic regression analysis, we developed an individualized prediction model for esophageal stricture after ESD using R, the nomogram model . The performance of the nomogram model was verified by calculating the C-Index and plotting the ROC curve and the calibration curve. First, we first internally validate the model. We calculated the C-Index of the development group to be 0.925, plotted the ROC curve . Previous studies have shown that the extent of esophageal mucosal defects, the depth of tissue invasion, and the length of longitudinal mucosal defects are reliable independent risk factors for esophageal stricture after ESD [This study found that age of more than 60 years, long-term high-temperature water intake, preoperative high NLR, esophageal mucosal defect of more than 3/4, longitudinal diameter of resected mucosa, and depth of tissue invasion of more than M2 were independent risk factors for esophageal stenosis after ESD . In fact, the formation of esophageal stricture itself requires the participation of inflammatory mediators, which play an important role in scar repair [In previous studies, inflammatory markers such as NLR, PLR, and LMR are often used to predict the stage, differentiation, invasion, and prognosis of tumors, and have good predictive value , 24. Ther repair . TherefoP = 0.003, OR = 11.229, 95% CI: 2.315—54.466). Funakawa et al. found that there was no significant difference in age, gender, alcohol consumption and smoking, and tumor location between the esophageal stenosis group and the non-stenosis group after ESD [The incidence of cancer increases with age, which is a risk factor for esophageal cancer and is commonly used to predict the occurrence and prognosis of cancer , 27. Thefter ESD . HoweverP < 0.001, OR = 13.378, 95% CI: 3.987—44.882), but not with high-protein diet, fruit and vegetable intake, pickled food, fried food, eating speed, long-term smoking and alcohol consumption (P > 0.05). High temperature have been reported to lead to esophageal mucosal edema and inflammation formation, long-term will lead to surrounding fibrous hyperplasia, collagen accumulation, scar contracture, and ultimately esophageal scar stenosis [Liu et al. established an age-stratified risk prediction model for esophageal squamous cell carcinoma and its precancerous lesions on the basis of a population-based large endoscopic screening trial, which included predictors of lifestyle habits and dietary status . In recestenosis . TherefoIn this study, nomogram models were established to predict the risk of esophageal stricture after ESD, and the models were internally and externally validated by calculating C-Index, plotting ROC curves, and calibration curves. The c-index of the development group and the validation group were 0.925 and 0.861, respectively, and the ROC curves of the two groups suggested that the discrimination and prediction performance of the model were good. The two groups of calibration curves were basically consistent and almost overlapped with the ideal calibration curve, indicating that the predictions of the nomogram model were in good agreement with the observations. Since the data sources between the development group and validation group are different, there are some differences in the patient's living habits, physician technical level, etc., but this does not significantly affect the discrimination and calibration of this prediction model. External validation showed that the model had good performance in predicting esophageal stricture after ESD, indicating that the model can be well-suitable for different populations and medical centers, which is important to reduce or avoid the occurrence of esophageal stricture.However, this study still has some limitations. This study was a multi-center, retrospective, small-sample design, so the clinical effectiveness needed to be verified in a large-sample, prospective study; secondly, the evaluation of the patient's living habits and other indicators had certain subjectivity, and the relevant factors should be quantified by the standard for accurate analysis; lastly, due to the limited factors included in this study, such as the technical level of endoscopists, patient's work, living environment, and other factors may also affect the study outcome, it needs to be considered in the future relevant studies.In this study, a nomogram model was established to predict the risk of esophageal stenosis after ESD. Both internal and external validations showed that the model has good predictive performance and can be well adapted to different populations and medical centers. It is a free tool beneficial to clinical risk assessment, which has a positive significance for preventing esophageal stenosis and guiding clinical practice. However, more prospective validation is needed in the future.Additional file 1.Additional file 2.