Breastfeeding affects the growth and development of infants, and polyunsaturated fatty acids (PUFAs) play a crucial role in this process. To explore the factors influencing the PUFA concentration in breast milk, we conducted research on two aspects: dietary fatty acid patterns and single nucleotide polymorphisms (SNPs) in maternal fatty acid desaturase genes.P < 0.05 as statistically significant.Three hundred seventy Chinese Han lactating mothers were recruited. A dietary semi-quantitative food frequency questionnaire (FFQ) was used to investigate the dietary intake of lactating mothers from 22 to 25 days postpartum for 1 year. Meanwhile, breast milk samples were collected from the participants and tested for the concentrations of 8 PUFAs and 10 SNP genotypes. We sought to determine the effect of dietary PUFA patterns and SNPs on breast milk PUFAs. We used SPSS 24.0 statistical software for data analysis. Statistical tests were all bilateral tests, with Under the same dietary background, PUFA contents in breast milk expressed by most major allele homozygote mothers tended to be higher than that expressed by their counterparts who carried minor allele genes. Moreover, under the same gene background, PUFA contents in breast milk expressed by the mother’s intake of essential PUFA pattern tended to be higher than that expressed by their counterparts who took the other two kinds of dietary.Our study suggests that different genotypes and dietary PUFA patterns affect PUFA levels in breast milk. We recommend that lactating mothers consume enough essential fatty acids to ensure that their infants ingest sufficient PUFAs.The online version contains supplementary material available at 10.1186/s12263-023-00735-0. Breastfeeding affects infant growth and development . For infAccumulating evidence indicates that polyunsaturated fatty acids (PUFAs) play a major role in brain development during the first year of life . In vivoFADS1) and 2 (FADS2) genes. Several studies have demonstrated an association between the FADS enzymes and fatty acid levels [Breast milk is the only source of PUFAs for infants who are exclusively breastfed. The PUFA content depends on the intake of fatty acid precursors that cannot be produced in vivo . PUFAs ad levels –10. The d levels .Examining dietary patterns has emerged as a holistic approach to capturing complex interactions between nutrients and foods . One recAll subjects of this study were healthy Chinese Han lactating mothers who received nursing care in a postpartum nursing center in Changchun from March 2012 to December 2014. The study was approved by the medical ethics committee of the School of Public Health of Jilin University, and all subjects signed informed consent. The inclusion criteria required that the nursing mother had no special eating habits, full-term delivery (37 ~ 42 weeks of pregnancy), infant Apgar score ≥ 8, and voluntary participation in this study. The exclusion criteria were pregnancy-induced hypertension, diabetes, and other metabolic diseases, including infectious diseases such as AIDS, tuberculosis, and other related drugs that affect the metabolism of nutrients. Babies suffering from breast-feeding contraindications were also excluded from the study. In total, 370 pregnant women were included as the research subjects.The sociodemographic information on the subjects was collected through questionnaires, including lactating mothers’ age, height, pre-pregnancy weight, postpartum weight, education level, and monthly household income. The type, quantity, and frequency of foods consumed by pregnant women in the past year were investigated by means of a semi-quantitative food frequency questionnaire (FFQ). In order to ensure the quality of the dietary survey, the special food pictures for the nutritional dietary survey, common food estimation tables, and quantitatively designed standard molds were used to verify the food intake, and the investigators were trained by a professional dietitian to conduct investigations and contact lactating mothers for clarification (if necessary). The questionnaire adopted a dual-entry method to avoid errors. The intake of dietary fatty acids was calculated according to the China Food Composition Tables (Sixth Edition) .FADS1, FADS2, and FADS3 were identified using the International HapMap Project SNP database and the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/snp/). Selected SNPs from the FADS1 gene cluster , FADS2 gene cluster  and FADS3 gene cluster  were genotyped using Sequenom Mass Array system  with validated primers. Each SNP had a minor allele frequency (MAF) above 10% in the Asian population according to the SNP database of the NCBI. The milk samples were thawed at 4℃, and genomic DNA was extracted from 300 μl of the cellular layer using the DNA kit  following the manufacturer’s instructions. The primer sequences of the ten SNPs in this study are shown in Supplementary Table Breast milk was collected between 8:00 a.m. and 11:00 a.m. on any day of the 22–25 days postpartum period. The entire milk from one breast was applied using an electric breast pump, and all samples were delivered to the laboratory by cryogenic transport and quickly transferred to a − 80℃ refrigerator for later testing. The detection of milk PUFAs is the same as the previous study of the research group . The SNPP value of Bartlett test should be less than 0.05. Chi-square test was used to compare the count data between groups. For the measurement data meeting the normal distribution, t test or ANOVA was used for the comparison between multiple groups, and the LSD method was used for the subsequent pairwise comparison; Kruskal–Wallis H test was used for multi-group comparison of skewed distribution measurement data, and Bonferroni method was used for subsequent pairwise comparison. Statistical tests were all bilateral tests, with P < 0.05 as the difference, which was statistically significant.Data analyses were performed with SPSS 24.0 statistical software. Kolmogorov Smirnov test was used to judge whether the data meets the normal distribution. The measurement data meeting the normal distribution was represented by mean ± SD, and the measurement data of skewed distribution was represented by median (IQR); the count data was represented by component ratio. The principal component analysis (PCA) method was used to obtain the dietary fatty acid model. The rotation method used is the maximum variance method. The KMO test coefficient should be greater than 0.6 and the A total of 370 healthy lactating mothers aged 30 years participated in this study. Their monthly family income was divided into 2000 ~ 5000 yuan, 5000 ~ 10,000 yuan, and above 10,000 yuan, accounting for 15.5%, 53.5%, and 31.0%, respectively. Regarding education, 43.8% of the participants had a high school education or below, 46.3% had a bachelor’s degree, and 9.9% had a graduate degree or above. Lactating mothers with normal BMI before pregnancy accounted for 61.3%, gaining 18.5 kg during pregnancy and 39.1 weeks of gestation. The birth weight of the infant was 3.4 kg, and their body length was 50.0 cm. Details are shown in Table Supplementary Table χ2 = 10,414.pc098, P < 0.001), suggesting that the PCA method can be used.In this study, the dietary fatty acid patterns of 370 subjects were analyzed by PCA. There was a strong linear correlation between the research variables and the data structure was reasonable .Ten SNPs were identified in the present study. Table FADS1 rs174547 gene (P = 0.019), whereas the concentration of ALA (P = 0.006) in breast milk was lower in MM than in Mm/mm for the FADS2 rs174575 gene. In the n-6 PUFAs major pattern, the concentration of AA (P = 0.003), DTA (P = 0.019), and DHA (P = 0.032) in breast milk was higher in MM than in Mm/mm with the FADS1 rs174547 gene; the concentration of AA (P = 0.005) and DTA (P = 0.026) in breast milk was higher in MM than in Mm/mm with the FADS1 rs174553 gene; the concentration of LA (P = 0.040), GLA (P = 0.042), AA (P = 0.002), DTA (P = 0.009), and DHA (P = 0.021) in breast milk was higher in MM than in Mm/mm with the FADS2 rs3834458 gene; the concentration of GLA (P = 0.047), AA (P = 0.004), and DTA (P = 0.022) in breast milk was higher in MM than in Mm/mm with the FADS2 rs1535 gene; and the concentration of AA (P = 0.045) in breast milk was higher in MM than in Mm/mm with the FADS2 rs174602 gene. In the essential PUFAs pattern, the concentration of AA in breast milk for the FADS2 rs174602 (P = 0.040) gene was higher in MM than in Mm/mm, and DTA was higher in MM than in Mm/mm for the FADS2 rs174575 gene (P = 0.023), but the concentration of LA in breast milk was lower in MM than in Mm/mm for the FADS3 rs1000778 (P = 0.023) gene. In addition, within the same dietary pattern, the concentration of PUFAs in breast milk did not differ between MM and Mm/mm of FADS2 rs498793, FADS3 rs174450, and FADS3 rs7115739.As shown in Fig. FADS1 rs174547, FADS1 rs174553, FADS2 rs3834458, and FADS2 rs1535 genes had significantly higher concentrations of LA, DGLA, AA, DTA, ALA, and DHA in their breast milk than mothers who were homozygous for the major allele (MM). Further analysis showed that the levels of PUFAs in the breast milk of mothers carrying the minor allele (Mm/mm) did not differ significantly across dietary patterns. In addition, regardless of the lactating mother’s adoption of any of the three dietary patterns, compared with Mm/mm, the milk of mothers homozygous for the major allele (MM) of the FADS2 rs174575 and FADS3 rs7115739 genes was significantly different for DGLA, AA, DTA, EPA, and DHA, which were significantly higher in the breast milk of their counterparts. Similarly, the concentrations of PUFAs in the breast milk of mothers homozygous for the major allele (MM) did not differ significantly across dietary patterns.Overall, as shown in Fig. FADS2 rs174602 and FADS3 rs1000778 minor alleles (Mm/mm) and ingesting the essential PUFAs pattern, the concentration of LA in the breast milk was significantly higher than that in mothers carrying the same genotype and ingesting the other two dietary patterns. However, the concentrations of DTA, EPA, and DHA in breast milk with the essential PUFA pattern were the highest among the three patterns homozygous for the major allele (MM). Mothers who consumed the essential PUFAs pattern and were homozygous for the FADS2 rs498793 major allele (MM) had significantly higher concentrations of DGLA and DHA in their breast milk than mothers of the same genotype who chose the other two dietary patterns. Mothers who consumed the essential PUFA pattern and carried the FADS2 rs498793 minor allele (Mm/mm) had significantly higher concentrations of DTA in their breast milk than mothers of the same genotype who chose the other two dietary patterns. In addition, mothers who consumed the n-3 PUFAs dietary pattern and carried the FADS2 rs498793 allele had significantly lower EPA concentrations in their breast milk than mothers with the same genotype who chose the n-6 PUFAs dietary pattern. Among the Mm/mm of the three patterns, mothers who consumed the essential PUFA pattern and carried the FADS3 rs174450 minor allele (Mm/mm) had significantly higher concentrations of LA, AA, and DTA in their breast milk than those who consumed the n-6 PUFA major pattern with the same genotype; in contrast, mothers who selected the essential PUFA pattern carrying the MM had significantly higher concentrations of DHA in their breast milk than those who consumed the other two dietary patterns with the same genotype. No differences were observed in other PUFAs in the breast milk of mothers homozygous for both MM and Mm/mm under different dietary patterns. The details are shown in Fig. For mothers carrying the FADS1 [The World Health Organization recommends that mothers worldwide breastfeed their babies during the first 6 months of life for optimal growth, development, and health . PUFAs aFADS1 ; howeverFADS1 rs174547, FADS1 rs174553, FADS2 rs3834458, FADS2 rs1535, and FADS2 rs174602 MM was significantly higher than that of Mm/mm, and the content of DTA in breast milk of FADS2 rs174575 MM was significantly higher than that of Mm/mm, indicating that the genotype would affect the content of fatty acids in breast milk. Compared with MM, the concentration of PUFAs secreted in the breast milk of Mm/mm was less; therefore, additional supplementation is needed for infants to have enough fatty acids to ensure adequate nutrition. Studies have found that the proportion of DHA in the breast milk of Mm/mm of FADS1 rs174561, FADS2 rs174575, and FADS2 rs3834458 is lower than that in MM, which are findings similar to our study [FADS1 rs174547, FADS1 rs174553, FADS2 rs3834458, and FADS2 rs1535 genotypes, which was consistent with linkage disequilibrium between them.This study found that in the same dietary model, the concentration of PUFAs in breast milk of FADS2 rs174575 MM was significantly lower than that of Mm/mm, and the concentration of LA in the breast milk of FADS3 rs1000778 MM was significantly lower than that of Mm/mm, indicating that the genotype might affect the concentration of PUFAs in breast milk. ALA and LA are precursors of other PUFAs; therefore, if a mother is a Mm/mm, the activity of the desaturase enzyme is weak and the amount of other PUFAs is low; therefore, the content of ALA and LA in breast milk is abundant. Compared with MM, the content of PUFAs secreted by lactating mothers of Mm/mm was adequate. O’Neill’s et al. reported similar results for plasma [FADS2 rs498793, FADS3 rs174450, and FADS3 rs7115739 were observed, indicating that these three SNPs did not affect the concentration of PUFAs in breast milk. In general, lactating mothers of different genotypes need personalized and accurate nutrition to ensure that they secrete milk with a more balanced nutrition to ensure the healthy growth of infants.The ALA content in the breast milk of r plasma . No signFADS1 rs174547, FADS1 rs174553, FADS2 rs1535, and FADS2 rs383445 MM in the three dietary patterns, but differences in breast milk fatty acids were found among Mm/mm in the three dietary patterns, which further explained that the genotype would affect the content of breast milk fatty acids and that the MM would not be affected by the diet; therefore, ample PUFAs in their breast milk would be present. However, some PUFAs in the breast milk produced by Mm/mm were insufficient, indicating that it is necessary to adjust the diet for additional supplements to ensure that infants have sufficient fatty acid intake. No significant differences in breast milk fatty acids were observed between FADS2 rs174575 and FADS3 rs7115739 Mm/mm in the three dietary patterns; however, differences in breast milk fatty acids between MM in the three dietary patterns, which showed that the genotype affected the content of breast milk fatty acids. Mm/mm was not affected by diet and had adequate PUFAs in their breast milk.This study found no significant differences in breast milk fatty acids among FADS2 rs174602, FADS2 rs498793, FADS3 rs1000778, and FADS3 rs174450 had significantly higher PUFA levels in their breast milk than the other two dietary patterns when consuming an essential PUFA dietary pattern. Therefore, we inferred that the precursor ratio in the diet affected the PUFAs in the breast milk of MM. Among different dietary patterns of the same genotype, fatty acids in most breast milk samples were higher in the essential PUFA pattern. A possible reason is that the essential PUFA pattern is dominated by the essential fatty acids ALA and LA. When the lactating mother is a MM, her desaturase activity is strong, resulting in a higher content of PUFAs such as AA in breast milk. When the lactating mother is a Mm/mm, her desaturase activity is weak, resulting in a higher content of the substrate itself (LA and ALA).Mothers who were MM of The limitations of this study are as follows: (1) only eight PUFAs were detected in breast milk, and 11 PUFAs were used for dietary pattern classification, which is not comprehensive; (2) The PUFAs used in this study for dietary pattern classification did not completely match those detected in breast milk. (3) We conducted a dietary pattern survey using the FFQ without conducting a 3-day and 24-h questionnaire simultaneously, which cannot accurately control the intake of PUFAs in lactating mothers, as in a randomized controlled trial [The main advantages of this study are as follows: (1) Previous studies have primarily focused on the effects of different diets –33 or geOur study suggests that different genotypes and dietary PUFA patterns affect PUFA levels in breast milk. In addition, desaturase activity in the major allele homozygotes is stronger, and more downstream products are metabolized in breast milk, especially in mothers who consume essential fatty acid dietary patterns. Therefore, we recommend that lactating mothers consume enough essential fatty acids to ensure that their infants ingest sufficient PUFAs. In the future, we will conduct a randomized study with greater precision of dietary intake by varying the known quantities of PUFAs in groups of mothers with different genetic variants.Additional file 1: Supplementary Table S1. The primer sequences of ten SNPs in FADS genes (5'-3'). Supplementary Table 2. Dietary polyunsaturated fatty acid intake of lactating mothers. Supplementary Table 3. Effects of rs174547 dominant pattern of FADS1 gene and different dietary patterns on fatty acid concentration in breast. Supplementary Table 4. Effects of rs174553 dominant pattern of FADS1 gene and different dietary patterns on fatty acid concentration in breast. Supplementary Table 5. Effects of rs1535 dominant pattern of FADS2 gene and different dietary patterns on fatty acid concentration in breast milk. Supplementary Table 6. Effects of rs174575 dominant pattern of FADS2 gene and different dietary patterns on fatty acid concentration in breast milk. Supplementary Table 7. Effects of rs174602 dominant pattern of FADS2 gene and different dietary patterns on fatty acid concentration in breast. Supplementary Table 8. Effects of rs3834458 dominant pattern of FADS2 gene and different dietary patterns on fatty acid concentration in breast milk. Supplementary Table 9. Effects of rs498793 dominant pattern of FADS2 gene and different dietary patterns on fatty acid concentration in breast milk. Supplementary Table 10. Effects of rs1000778 dominant pattern of FADS3 gene and different dietary patterns on fatty acid concentration in breast milk. Supplementary Table 11. Effects of rs174450 dominant pattern of FADS3 gene and different dietary patterns on fatty acid concentration in breast milk. Supplementary Table 12. Effects of rs7115739 dominant pattern of FADS3 gene and different dietary patterns on fatty acid concentration in breast milk.


Background and objectivesThe lung function test is a gold standard, guideline-recommended test to detect obstructive airway diseases like asthma and COPD. It is of considerable value in detecting the presence and severity of airflow obstruction in patients with respiratory symptoms. However, the role of spirometry in a routine health checkup is controversial. Spirometry, when used in routine health checkup settings as a case-finding tool for all adults with persistent respiratory symptoms or having a history of exposure to risk factors, is likely to label a relatively large proportion of individuals as diseased with airflow obstruction. Conversely, spirometry is normal in a relatively large percentage of adults who report respiratory symptoms including dyspnea, the respiratory symptom having the greatest impact on quality of life. The objective of this study is to determine the utility of spirometry as a screening test to detect airflow obstruction in otherwise healthy subjects undergoing a routine health checkup.MethodsThis observational study was conducted with 538 health checkup individuals aged 18 and over. A brief history was taken prior to the test. Lung function tests were performed and interpreted as per the Global Initiative for Chronic Obstructive Lung Disease criteria. The anthropometric and spirometric data obtained were compared to other population-based spirometric studies to compare the prevalence of airflow limitation, the risk factors, and smoking history.ResultsOf the total 538 subjects incorporated in the study, 305 (57%) were males and 233 (43%) were females aged between 18 to 80 years with a mean age of 45 years. The male-to-female ratio was 1.3:1 with a mean BMI of 25.9. The overall yield from lung function tests in detecting airflow obstruction was 63 subjects (11.7%), of which 36 (11.8%) were males and 27 (11.5%) were females. Seventy-three subjects (13.5%) were classified as having a small airway obstruction, of which 34 were males (46.6%), and 39 were females (53.4%). The distribution of airflow obstruction by age was with eight subjects (5.4%) in the 18-35 group, 21 subjects (7.8%) in the 36-55 group, and 34 (25%) in the elderly (>55) age group. Although overall smoking history showed no significant association with developing airflow obstruction, significant association with smoking was found in the elderly (>55) age group.Interpretation and conclusionsThe results of the study suggest that lung function tests should be included in routine health checkups in the subset of individuals greater than 35 years of age with or without a history of smoking, in all age groups with a family history of asthma, in individuals with respiratory symptoms and in individuals greater than 55 years of age with a moderate history of smoking. Spirometry is a physiological test that measures how an individual inhales or exhales volumes of air as a function of time. It is invaluable as a screening test for general functional respiratory health. Spirometry is the gold-standard, guideline-recommended test for the diagnosis of obstructive airway diseases (OADs), including asthma and chronic obstructive pulmonary disease (COPD) . It also1), which is the volume delivered in the first second of the FVC maneuver. Spirometry is accepted as the diagnostic test to assess airflow obstruction and classify the severity of disease, based on specific cut points: FEV1/FVC ratio less than 0.7 and FEV1   / e2], where: z = 1.96 for a confidence level (α) of 95%, p = proportion , and e = margin of error.The sample size (n) was calculated according to the formula: n = [zAll the participants were asked to fill up a questionnaire prior to the test (Appendix 1). Age, sex, weight, height, smoking history, occupation, respiratory symptoms, and family history of respiratory illnesses were recorded.1), FEV1/FVC ratio, and forced expiratory flow in mid-expiration (FEF25% - 75%) were acquired. Each test was repeated a minimum of three times and a maximum of five times with adequate rest after each maneuver for best results. All the staff involved in lung function tests are qualified professionals who have received special training.Spirometry was performed using a spirometer device in strict accordance with the American Thoracic Society (ATS) quality control standards. Values like forced vital capacity (FVC), forced expiratory volume in the first second  below 60% of predicted in the presence of a normal FEV1/FVC. Restrictive airway disease was interpreted as an FEV1/FVC ratio greater than 70%, with FVC of less than 70% of predicted.Small airway disease was diagnosed according to maximal mid-expiratory flow  were used to find the significance of study parameters on a continuous scale between two groups. An analysis of variance (ANOVA) was used to find the significance of study parameters between the groups . The statistical software IBM SPSS Statistics 20.0  was used for the analyses of the data, and Microsoft Word and Excel were used to generate graphs, tables, and so on.1 across each age group and sex.A total of 538 subjects aged between 18 and 80 based on the inclusion criteria were incorporated into the study. There were 305 males and 233 females with a male-to-female ratio of 1.3:1. Table Spirometry results were interpreted as per GOLD guidelines. Overall, 347 subjects (64.4%) had normal spirometry, 63 subjects 11.7%) had obstructive defects, 73 subjects (13.5%) were classified as having small airway disease, and 55 individuals (10.2%) demonstrated restrictive defects  had a mild degree of obstructive defect, 36 (57.2%) subjects had a moderate obstructive defect, 10 (15.9%) had a severe obstructive defect, and only one (1.5%) spirometry demonstrated very severe obstructive defect , 21 subjects in those between 36-55 years (7.8%), and 34 in those >55 years (25%) . Sixteen subjects (25.4%) had a mild degree of obstructive defect, 36 (57.2%) subjects had a moderate obstructive defect, 10 (15.9%) had a severe obstructive defect, and only one (1.5%) spirometry demonstrated a very severe obstructive defect. Fifty-five subjects (10.2%) were in the restrictive spirometry group. Seventy-three subjects (13.5%) were classified as small airway disease. The detection rates of obstructive airway disease and small airway disease were similar in both males and females. The average pack years was 9.7 pack years. Although overall smoking history showed no significant association with developing airflow obstruction, significant association with smoking was found in the elderly (>55) age group.1/FVC ratio ≤70% in a routine health checkup population in Mumbai, India. This study also sought to determine whether spirometry as a screening test should be used in a general population or specific groups with certain risk factors like age, BMI, gender, family history of asthma, and smoking. As mentioned earlier, individuals with previously spirometric-diagnosed restrictive or obstructive airway diseases were excluded from the study so that they do not influence the results of these studies.This study sought to investigate the efficacy of spirometry in determining the prevalence of airflow obstruction as per FEVThe prevalence of airflow obstruction in this study was 11.7%. A study by Chakrabarti et al.  in West 1/FVC<70%).Compared to these studies, the detection rates of both obstructive pattern and small airway disease in this study are higher. However, a comparison between this study and other studies is likely to be biased owing to the different study populations and different criteria used for assessing airflow limitation. Our study used the GOLD criteria for diagnosis of airflow obstruction (FEVOur study demonstrated increasing age associated with airflow obstruction. Almost 25% of study subjects in the elderly age group (>55 years) showed an obstructive pattern of spirometry. Similar associations between age and airflow obstruction have been described in studies worldwide -10. AginAn epidemiological study in China indicated that low BMI might increase the risk of COPD, especially in the elderly (60 years and over) populations . HoweverMale sex and increasing age are identified as risk factors in most studies from Asia -15. ThisSmokers constituted 6.5% of subjects, with an average of 9.7 pack years. The average pack years in the elderly age group was 19.8. However, there is a possibility that some subjects may have confounded their smoking history. In elderly subjects, smoking was found to be significantly associated with airflow obstruction. However, in the young and middle-aged groups, smoking history did not attain any statistical significance. This is likely because obstructive airway diseases like COPD predominantly require a cumulative smoking history of approximately 20 pack years or more. It is very unlikely for an individual aged less than 35 years to accumulate a 20-pack-year smoking history. This supports the fact that the duration of tobacco smoking and the number of cigarettes smoked have a cumulative effect on developing airflow obstruction. Therefore, as a global joint effort, effective measures should be taken to lower the smoking rate, eliminate the hazards of tobacco, and maintain and promote human health.In our study, family history was found to be significantly associated with the occurrence of airflow obstruction. Other studies have also identified that family history of asthma in one or more first-degree relatives as a risk factor for asthma . Asthma Dyspnea and cough were the most common symptoms reported by the subjects diagnosed with airflow obstruction (11%). Airflow obstruction was found to be associated with respiratory symptoms like dyspnea, cough, and wheezing. Medbo et al.  emphasizThe study was conducted in Mumbai, a major urban city with significant air pollution because of vehicular emissions, commercial, and industrial activities. This can lead to both short-term, acute, and long-term chronic respiratory problems. The long-term effects of this air pollution can cumulatively lead to chronic irritation of the airways, which initially can cause small airway disease and eventually also lead to chronic obstructive pulmonary disease. These effects happen over time and may demonstrate a spirometry-measurable disease only after significant cumulative years of exposure. This likely contributes to the increasing incidence of obstructive airway diseases in the population above 35 years of age, irrespective of smoking. As we know, the burden of air pollution in India is ever-increasing, and, therefore, the findings of the study can be extrapolated to such age groups across India. This study demonstrates that 25% of the individuals above 55 years of age had obstructive spirometry. This is significant and therefore advocates for screening spirometry in this age group. We will include this clarification in the study. In our current study, the incidence of small airway disease reached 13.5%. Small airway disease may progress to COPD if the modifiable risk factors are not interrupted timely. Therefore, timely identification of these individuals and early intervention are particularly valuable.In this study, 55 individuals (10%) were found to have restrictive airway disease. Restrictive lung diseases may be present in various conditions ranging from primary pulmonary causes like lung parenchymal diseases, interstitial lung disease, pneumoconiosis, sarcoidosis, etc. It can also result because of various extrathoracic causes like obesity, kyphoscoliosis, neuromuscular diseases affecting the diaphragm, ascites, etc. This list of causes of restrictive lung disease is extensive and can be multifactorial. This needs a separate dedicated restrictive lung disease screening which would include chest X-ray, blood tests, CT chest imaging, advanced spirometric studies like diffusion testing, lung volumes, and workup for extrathoracic causes.LimitationsIn this study, there was no statistically significant association found between smoking and obstructive airway disease in the young and middle-aged groups. However, it is possible that some individuals may have confounded their smoking history. Therefore, the association between smoking and obstructive airway disease in this study may be underestimated. Most of the study subjects belonged to the young or the middle-aged age groups. The health checkup population are often composed of both healthy people and people with well-controlled chronic diseases like diabetes, hypertension, ischemic heart disease, hypothyroidism, chronic kidney disease, etc. In comparison to other studies, our study was conducted in the urban population and, hence, had good economic status and lower chances of being exposed to biomass fuel and childhood respiratory infections. In view of all these factors, it is difficult to extrapolate the results of this study to the general Indian population. Therefore, we recommend the need for similar spirometric studies with larger population groups across different parts of the country. Although this study sought to estimate the prevalence of both obstructive and restrictive airway disease, the focus of this study was more on obstructive airway disease. This is because obstructive airway disease is largely because of pulmonary causes whereas restrictive airway diseases may be because of pulmonary and extrapulmonary causes. Therefore, restrictive airway defects found on spirometry may trigger further systemic laboratory and radiological workup based on the history and symptoms. Hence, we recommend the need for a separate restrictive airway disease study to analyze the factors influencing restrictive airway defect and to characterize the target population who should receive screening spirometry.In conclusion, increasing age, respiratory symptoms, smoking history in the elderly, and family history of asthma are factors associated with airflow obstruction. There was no association between gender and airflow obstruction. Also, the association between BMI and airflow obstruction was not significant in this study. This study advocates the use of screening spirometry in subjects of both sexes above 35 years of age with or without any smoking history, people with family history of asthma, people with respiratory symptoms, and elderly people with moderate smoking history for early detection of airflow obstruction in routine health checkup population.


Arachis hypogaea L.) are an allotetraploid grain legume mainly cultivated by poor farmers in Africa, in degraded soil and with low input systems. Further understanding nodulation genetic mechanisms could be a relevant option to facilitate the improvement of yield and lift up soil without synthetic fertilizers. We used a subset of 83 chromosome segment substitution lines (CSSLs) derived from the cross between a wild synthetic tetraploid AiAd (Arachis ipaensis × Arachis duranensis)4× and the cultivated variety Fleur11, and evaluated them for traits related to BNF under shade-house conditions. Three treatments were tested: without nitrogen; with nitrogen; and without nitrogen, but with added0 Bradyrhizobium vignae strain ISRA400. The leaf chlorophyll content and total biomass were used as surrogate traits for BNF. We found significant variations for both traits specially linked to BNF, and four QTLs (quantitative trait loci) were consistently mapped. At all QTLs, the wild alleles decreased the value of the trait, indicating a negative effect on BNF. A detailed characterization of the lines carrying those QTLs in controlled conditions showed that the QTLs affected the nitrogen fixation efficiency, nodule colonization, and development. Our results provide new insights into peanut nodulation mechanisms and could be used to target BNF traits in peanut breeding programs.Peanuts ( Species of the legume family can associate with beneficial soil bacteria called “rhizobia”, which reduce atmospheric nitrogen (Nf carbon . A legumf carbon . DeciphePisum sativum) and broad beans (Vicia faba L.) that cultivated species are less promiscuous in their interaction with indigenous rhizobia compared to wild species. A reduction in host range was also reported between cultivated and wild species for chickpeas (Cicer arietinum L.) [Glycine max L.) [Medicago sativa L.), and common beans (Phaseolus vulgaris L.), the wild species and landraces exceed the improved varieties in nitrogen fixation efficiency [Domestication is the selection, modification, and adoption of wild plant species with traits of interest to humans . Over 12inum L.)  and soyb max L.) , suggestficiency . These rficiency ,15. In cficiency  reportedA. hypogaea L.) are an oilseed legume crop that can meet up to 68% of its nitrogen needs via BNF [Bradyrhizobium [Arachis sp. [Cultivated peanuts  and A. ipaensis (genome B), followed by a duplication of the chromosomes [Peanuts appeared about 9400 years ago from an interspecific hybridization between two wild diploid species, omosomes . The comomosomes ,36. Inteomosomes ,38,39,40In this study, we used a CSSL population derived from the cross between a cultivated peanut variety and a synthetic tetraploid, created by gathering the genomes of the most probable wild diploid ancestors of the cultivated species, to target traits related to BNF. Genotype–phenotype associations allowed for the identification of QTLs for BNF variation. Our results provide new insights into the genetics of peanut nodulation and useful information for pre-breeding programs.4F3) developed at CERAAS . These CSSLs were derived from an interspecific cross between a cultivated Fleur11 variety, used as the female parent, and the synthetic tetraploid AiAd, which is a combination of the genomes of (A. ipaensis KG30076 (BB genome) × A. duranensis V14167 (AA genome))4×, used as the male parent [A subset of 83 CSSLs was selected from a population of 122 CSSLs (BCe parent . Fleur11e parent .B. vignae strain ISRA400 (−N + ISRA400) was added to each pot. ISRA400 is an efficient bacterial strain that was isolated from the nodules of peanut plants grown in Senegal [The 83 CSSLs and the recurrent parent Fleur11, used as a control, were evaluated for their BNF capacity under shade-house conditions in December 2017 and March 2018 in Senegal. Seeds were sterilized with calcium hypochlorite and germinated in Petri dishes, and seedlings were transferred manually into pots containing a leached and sterilized substrate with a low nitrogen content, as described in [ Senegal  in the dFor each treatment, the leaf chlorophyll content and total dry biomass were evaluated. The leaf chlorophyll content was indirectly estimated weekly on the third youngest leaf using SPAD . After nine weeks, the plants were harvested and dried at 65 °C for 72 h before weighing to obtain the total dry biomass.ijky = observed value of genotype i in replication j and block k; µ = mean value of the population; iG = the effect of genotype i; j =r the effect of replication j; jkb = the effect of block k in replication j; and ijkɛ = residual error.Descriptive statistics (mean and standard deviation) were calculated for each trait and each treatment in both experiments. The normality and homogeneity of variance were checked with the Kolmogorov–Smirnov and Levene tests, respectively. An analysis of variance (ANOVA) was performed for each experiment to estimate the genotypes and replication effects on each trait using a standard linear model:Estimates of broad-sense heritability were calculated for each treatment in each environment using the formula:e effect .p < 0.05 using the package multcomp of the R software [When a significant genotype effect was found for a given trait, each CSSL was compared to the recurrent parent Fleur11 with Dunnett’s multiple comparisons test at software , as descsoftware . When a The QTL effect for a given trait was calculated according to the formula below :QTL effevol/vol) and observed using an LSM 700 confocal laser scanning microscope . SYTO9 and propidium iodide were excited to 488 and 555 nm for an emission range of 490–522 nm and 555–700 nm, respectively. For calcofluor, the excitation wavelength was 405 nm and the emission range was 405–470 nm. Nodule sections were also observed under an AZ differential interference microscope  equipped with a DS-Ri2 camera to describe the nodule structure.A cytological analysis was performed to provide information about the QTL effect on the shape, structure, and functioning of nodules. Fresh nodules obtained in controlled conditions were harvested, embedded in 4% agar, and sliced into 80 µm sections using a VT1000S vibratome . The sections were successively incubated in live/dead staining solutions (5 μM SYTO 9 and 30 μM propidium iodide in 50 mM Tris (pH 7.0) buffer; Live/Dead BacLight, Invit-rogen, Carlsbad, CA, USA) for 15 min, and calcofluor white M2R  for 15 min in the dark . The staB. vignae ISRA400), and two traits related to BNF, the leaf chlorophyll content and the biomass, were recorded. The experiment was conducted in two consecutive environments (December 2017 and March 2018). We observed a gradual decrease in the average leaf chlorophyll content in the negative control treatment (−N), while the chlorophyll content remained almost constant in the positive control treatment (+N)  . In the ent (+N) . The ferent (+N) . The resent (+N) . The broent (+N) .To search for significant line × trait associations in each environment, the CSSLs were compared to Fleur11. We considered a significant line × trait association to be specifically linked to biological nitrogen fixation when it was not observed in the control treatments.In the 2017 environment, the Dunnett’s test showed that six lines had a significantly lower leaf chlorophyll content compared to Fleur11 in the inoculated (−N+ISRA400) treatment, while in the negative (−N) and positive (+N) control treatments, no significant difference was observed . The relThe comparison of the total biomass between the CSSLs and Fleur11 in 2017 showed that six lines had a significantly lower biomass than Fleur11 in the −N treatment, and 26 lines had a significantly lower biomass in the inoculated treatment . HoweverIn summary, 15 QTLs were identified for the leaf chlorophyll content and 25 QTLs were identified for the total biomass in 2017 and 2018 . Among tFive CSSLs that showed contrasting phenotypes compared to Fleur11 were selected to validate the QTLs. Among these lines, four  contained less chlorophyll and produced less biomass in the inoculated treatment, and one line (12CS_114) showed higher values for these traits, although these differences were not statistically significant .Differences in the plant growth between Fleur11 and the CSSLs were observed thirty-five days post-inoculation. For the leaf chlorophyll content, no difference was found between Fleur11 and the CSSLs under the −N treatment, while in the inoculated treatment, we observed a significant decrease in the chlorophyll content (~53–75%) for the lines 12CS_004, 12CS_044, 12CS_051, and 12CS_084 in the first and second experiments .B. vignae ISRA400.Regarding the total biomass, except for line 12CS_004 in the first experiment, no difference was found between Fleur11 and the CSSLs under the −N treatment . In the To further investigate the phenotypes of these five lines, we evaluated the traits directly related to nodulation as indicators of symbiotic performance: the nitrogenase activity using an acetylene reduction assay, the number of nodules, the nodule dry weight, and the ratio between the nodule dry weight and the nodule number . The corTwo independent experiments performed under controlled conditions showed that the line 12CS_004 exhibited a significant decrease in all the nodulation traits compared to Fleur11 . SimilarDuring legume–rhizobia interactions, the symbiotic efficiency may depend on the nodule colonization by the rhizobia, and also on the development and functioning of the nodule. To better investigate the effect of QTLs, we examined histological sections of nodules at 35 days post-inoculation. In Fleur11, we observed spherical and determinate nodules of the aeschynomenoid type that exhibited central infected tissue corresponding to the fixation zone, surrounded by uninfected cells corresponding to the inner cortex or nodule parenchyma, the vascular bundles, and the outer cortex a. Similahttps://peanutbase.org/gbrowse_peanut1.0 (accessed on 30 January 2023)” and Orthofinder “https://davidemms.github.io/ (accessed on 23 April 2021), and found 17 orthologs of the nodulation genes previously described in model legumes  is a valuable component of sustainable agriculture. Understanding the genetics of the interaction between plants and rhizobia can facilitate the enhancement of BNF in grain legumes. In this study, 83 chromosome segment substitution lines (CSSLs) were used to map the QTLs of the leaf chlorophyll content and the total biomass under three treatments , and to explore the contribution of wild species to the improvement of BNF in cultivated peanuts.B. vignae ISRA400). Sahrawat et al. [Genetic and phenotypic variations were found for the chlorophyll content and/or the total biomass in the CSSL population . We hypot et al.  showed tt et al. ,49,50,51We observed that some lines, such as 12CS_055 and 12CS_106, presented a total biomass that was significantly lower compared to Fleur11 in the −N treatment; however, in the treatment −N+ISRA400, these lines exhibited a total biomass similar to Fleur11 . These f2 peanut populations have allowed for the identification of syntenic regions, including a pair of homoeologous genes coding for GRAS transcription factors involved in nodulation and nodulation signaling pathway 2 (NSP2) in the chromosomes A08 and B07 [In the 2017 and 2018 environments, the genotype × phenotype associations allowed for the identification of 15 and 25 QTLs for the leaf chlorophyll content and the total biomass, respectively . The QTL and B07 . Due to  and B07  and in e and B07 . The int and B07 .Aeschynomene indica [A. indica [Vigna unguiculata [Aeschynomene, and they are associated with failed rhizobia differentiation.The experiments under controlled conditions allowed for the confirmation of the shade-house results  and poine indica . Interese indica ,70,71. F. indica  and in Vuiculata . These suiculata . Gully euiculata  showed t2+/calmodulin-dependent protein kinase (CCaMK) reduces nitrogenase activity by ~90% and affects rhizobial dissemination during nodule organogenesis. Likewise, the silencing of cytokinin receptor histidine kinase1 (HK1) led to a drastic reduction in the nodule number, nodule size, and nitrogenase activity [Some QTL actions described here are close to those previously observed in functional studies performed using RNA interference to downregulate the orthologs of well-known nodulation genes in peanuts. Sinharoy and DasGupta  showed tactivity . In our A. ipaensis and A. duranensis are not valuable sources of new alleles for increasing BNF. These results suggest either a potential positive impact of domestication and/or the breeding process in the improvement of peanut BNF or a disruption of the symbiotic mechanisms between the wild species and a foreign rhizobia strain as the result of peanut introduction and adaptation in Senegal. Bouznif et al. [B. vignae ISRA400 [Medicago lupulina [GmCRP gene encoding a rhizobial type III effector [A. duranensis and A. ipaensis at the QTL region in the cultivated background of Fleur11 has partially disrupted the symbiotic association with ISRA400 in the absence of coevolution or coadaptation between the Senegalese strain and the wild species. To provide more insights into this assertion, it would be interesting to explore the response of the CSSL population when inoculated with strains coming from South America, where peanuts are native.In our study, at all the QTLs, the wild alleles had a depressive effect on the plant growth and nodulation traits, indicating that when the efficient Senegalese strain ISRA400 is used as a symbiotic partner, f et al.  suggeste ISRA400 . BNF is  ISRA400 . In comm ISRA400 , and spe ISRA400 . Howeverlupulina . In soyblupulina . Recentleffector . We hypoIn this study, an interspecific population of 83 chromosome substitution segment lines was used to map genomic regions controlling biological nitrogen fixation traits in peanuts. We found four QTLs that were consistently associated with peanut nodulation. At these QTLs, the wild alleles had a negative effect on biological nitrogen traits. Then, screening these genomic regions allowed for the identification of 17 orthologs of the nodulation genes. However, more studies are needed to identify the underlying gene(s). Our results provide additional insights into the genetic aspects of peanut nodulation and will contribute to a better knowledge of the potential impact of domestication on the enhancement of the nitrogen fixation capacity of peanuts.


To investigate the effect and safety of recanalization surgery for non-acute occlusion of large intracranial arteries and factors affecting clincial outcomes. Patients with non-acute occlusion of internal carotid artery (ICA), middle cerebral artery (MCA), and vertebrobasilar artery (VBA) treated with recanalization were retrospectively enrolled. The clinical and angiographic data were analyzed. 177 patients were enrolled, including 67 patients with intracranial ICA occlusion, 52 with MCA occlusion, and 58 with VBA occlusion. Successful recanalization was achieved in 152 (85.9%) patients. Complications occurred in 15 patients (8.5%). Followed up for 3–7 months, the 90 day mRS was significantly improved compared with that before the procedure. Among 152 patients with successful recanalization, eight patients experienced reocclusion (5.3%), and 11 patients experienced restenosis (7.2%). Successful recanalization was significantly (P < 0.05) associated with occlusion duration, calcification or angulation of the occluded segment. Complications were significantly (P < 0.05) associated with location of occlusion, hyperlipidemia, and patients’ height. Restentosis or reocclusion at follow-up was significantly (P < 0.05) associated with complications and mRS at 90 days. The significant (P < 0.05) independent risk factors were angulation and calcification for successful recanalization, hyperlipidemia for complications, and mRS at 90 days for restenosis or reocclusion at follow-up. Recanalization surgery may be a safe and effective approach for patients with non-acute symptomatic occlusion of large intracranial arteries, and factors significantly independently associated with successful recanalization, periprocedural complications and restenosis or reocclusion after surgery have been identified for future reference to improve clinical outcomes. Non-acute symptomatic atherosclerotic occlusion of large intracranial arteries beyond 24 h from onset is common with a high rate of recurrent stroke of approximately 7.27% per year in China because of hemodynamic compromise and poor collateral circulation and constitutes a treatment dilemma4. It may affect cognitive function and cause decreased quality of life, thus necessitating timely treatment6. Patients with acute intracranial large artery occlusion may survive the acute stage but will still experience recurrent transient ischemic attack (TIA) or stroke, with a yearly incidence of 6–14%8. Some patients can benefit from surgical treatment if they still have recurrent stroke or progressive aggravation of symptoms after intensive drug treatment11. In earlier studies, low flow extracranial-intracranial artery bypass did not show advantages over drug treatment, nor did it reduce the risk of ipsilateral ischemic stroke13. Recently, some small-sample studies have been performed on the effect of endovascular recanalization on symptomatic non-acute occlusion of intracranial internal carotid artery (ICA)16, middle cerebral artery (MCA)19, and vertebrobasilar artery (VBA)21. For non-acute occlusion of the initial segment of the ICA, hybrid surgery involving endarterectomy and endovascular stenting has achieved a high success rate of 97%15, and for non-acute occlusion of intracranial MCA and VBA, endovascular recanalization is the primary approach which may result in heterogeneous outcomes. Currently, the effect and safety of endovascular recanalization for non-acute occlusion of large intracranial arteries are controversial, and significant factors affecting the clinical outcome, periprocedural complications and restenosis or reocclusion after recanalization are yet to be decided. It was hypothesized that endovascular recanalization would be effective and safe for non-acute occlusion of large intracranial arteries of ICA, MCA and VBA. This study was consequently carried out to investigate this hypothesis and significant factors affecting successful recanalization, periprocedural complications, and restenosis or reocclusion after successful recanalization so as to improve the clinical outcome.Atherosclerotic steno-occlusive disease of large intracranial arteries is a common cause of stroke across the world which is associated with a high risk of recurrent stroke and poor outcomes22. Exclusion criteria were patients with severe diseases precluding surgery or anesthesia, heart diseases , hemorrhagic disease, being allergic or contraindicated to aspirin, clopidogrel, heparin or iodine contrast medium, and elderly patients with advanced diseases or neurological diseases. Standardized medication was applied for initial cerebral infarction. Patients with cerebral infarction were initially treated with standardized medication, including dual antiplatelet therapy and medication for hypertension, hyperlipidemia and diabetes mellitus within 1 month. One month later, single antiplatelet medication and measures for hypertension, hyperlipidemia or diabetes were applied for long-term prevention. If a new infarction occurred within the blood supply area of occluded vessels during medication, it was defined as failure of medical treatment. All patients in this cohort underwent head MRI after occurrence of symptoms to determine the intracranial condition . Then, non-invasive cerebral angiography (MR angiography or CT angiography) and cerebral perfusion examination (MR perfusion or CT perfusion) were performed to determine the patentcy of intracranial vessels and cerebral blood perfusion of the patient. These examinations were completed within 1 week after onset of the disease. If the patient was found to have intracranial arterial occlusion, cerebral digital subtraction angiography was performed to confirm the diagnosis and for possible treatment. The flow chart of enrollment of patients is shown in Fig. This retrospective case-series one-center study performed in a tertiary hospital was approved by the ethics committee of our hospital, and all patients had provided the written informed consent to participate. All methods were performed in accordance with the relevant guidelines and regulations. Between January 2019 and December 2020, patients with symptomactic non-acute occlusion of intracranial ICA, MCA or VBA treated with endovascular recanalization were enrolled. Non-acute occlusion was defined as occlusion of large intracranial arteries beyond 3 weeks after imaging confirmation of the occlusion. Inclusion criteria were patients with symptomactic non-acute occlusion of intracranial ICA, MCA or VBA confirmed by medical imaging, symptoms of ipsilateral ischemia despite standardized medication, no new ischemic cerebral infarction (within 2 weeks), normal arteries distal to the occlusion without Moyamoya-like vessels, cerebral hemodynamic insufficiency or phase II hemodynamic failure with the mean transit time (MTT) > 4 s, decreased cerebral blood flow (CBF) (symptomatic side/asymptomatic side < 0.95) and increased oxygen extraction fraction (OEF) ratio  before endovascular recanalization on computed tomography perfusion (CTP), perfusion weighted imaging (PWI) of magnetic resonance imaging (MRI) or positron emission tomography (PET)Digital subtraction angiography and CT angiography images were collected and evaluated by two senior neurointerventional doctors (deputy director or chief physician with experience of over 5 years). The length of occluded vessels, occluded sites, collateral circulation, hypoperfusion variables and neurological symtpoms were collected and analyzed. The measurement of vascular occlusion length was performed using three-dimensional 3D) angiography images from the proximal end to the distal end of occlusion without interference by potential curves of blood vessels. Cerebral hemodynamics and perfusion-diffusion mismatch were evaluated using MRI PWI Figs.  and 3, CD angiogr23. If there was no or little blood return or endovascular devices could not be navigated through the occluded segment, the procedure was also defined as a failure.The endovascular procedure was performed under general anesthesia, and femoral artery puncture was performed for cerebral angiography to observe the patency and anatomical structure of cerebral vessels. Meanwhile, heparin was injected intravenously to maintain the activated coagulation time of 200–250 ms. After an 8F or 6F guiding catheter was inserted into the common carotid artery, ICA or vertebral artery, an Echelon-10 combined 0.014 in. Transend micro-guidewire  or a Pilot micro-guidewire  combined with a microcatheter  was used to explore the occluded blood vessels. If patients with occlusion of the initial part of the internal carotid artery which presented some difficulties in the procedure of endovascular recanalization, a hybrid approach combining both endarterectomy and endovascular intervention was utilized. When the arterial segment distal to the occlusion was reached, a balloon catheter  was used to expand the occluded segment before stent deployment with a balloon- or self-expandable stent. Balloon dilatation was performed if residual stenoses exceeded 50%, and if the residual stenosis did not respond to balloon dilatation after balloon dilatation had been tried for more than 30 min, the operation was terminated as a procedure failure. If the residual stenosis was less than 20% and the antegrade blood flow of cerebral infarction thrombolysis (TICI) grade IIb or above was established, the recanalization surgery was considered successfulCT scan was performed after surgery to exclude intracranial hemorrhage or new ischemic lesions. Blood pressure was controlled at < 140/90 mmHg for 3 days for patients with hypertension or < 120 mmHg for patients without hypertension. Patients with stent deployment took aspirin (100 mg/day), clopidogrel (75 mg/day) and atorvastatin calcium (20 mg/day) tablets for at least 3 months, and then aspirin (100 mg/day) or clopidogrel (75 mg/day) and atorvastatin calcium from 6 months to 1 year based on clinical symptoms and conditions of the treated artery on cerebral angiography. CT angiography, CTP, MRI PWI, or digital subtraction angiography was carried out 3–6 months after surgery for angiographic follow-up. Recurrent ischemic symptoms, vascular reocclusion rate, and modified Rankin scale score (mRS) were recorded during follow-up.2), clinical symptoms , hypertension (antihypertensive treatment or diastolic pressure > 90 mm Hg or systolic pressure > 140 mmHg), hyperlipidemia (low-density lipoprotein cholesterol > 1 g/L or on lipid-lowering therapy), diabetes mellitus , alcohol abuse , smoking history, occlusion of the artery, occlusion duration, length, arterial calcification and angulation were recorded and analyzed. Calcification at the occlusion site was defined as any form of calcification found at the occluded arterial segment by head and neck CT angiography. On CT imaging, calcification was hyperdense foci along the artery with a peak density greater than 130 Hounsfield units24. Arterial angulation at the occluded segment was defined as the angle formed between the axial extending line of the proximal segment and that of the distal segment . Categorical data were presented as numbers and percentages and tested with the Chi square test, and measurement data were presented as mean ± standard deviation if in normal distribution and tested with the student t test. If in skew distribution, the measurement data were presented as median and interquartile range and tested with the Mann–Whitney U test. Factors significantly affecting successful recanalization, periprocedural complications and restenosis or reocclusion were analyzed using the univariate and multivariate logistic regression analysis. Receiver operating characteristic (ROC) curve analysis was performed for the risk factors for successful recanalization and restenosis or reocclusion. The statistically significant P value was set as < 0.05.This study was approved by the Ethics Committee of Henan Provincial People’s Hospital, and all patients had provided written informed consent to participate.Human ethics guidelines were obeyed in doing this study.A total of 177 patients were enrolled, including 67 patients with ICA occlusion, 52 with MCA occlusion, and 58 with VBA occlusion, with a female to male ratio of 51/126 and an age range of 38–80 (mean 58.6 ± 9.3) years  patients with ICA occlusion, six (9.0%) patients underwent carotid endarterectomy of ICA prxomal orifice occlusion and additional stent deployment for recanalization of intracranial occlusion Table . Sixty-o7.9% patiIn recanalizing ICA occlusion, three (4.5%) patients experienced periprocedural complications within 7 days after the procedure, including one (1.5%) case with cerebral infarction, one (1.5%) with cerebral hemorrhage, and one (1.5%) with internal carotid cavernous fistula. In patients with MCA occlusion, periprocedural complications occurred in five (9.6%) cases within 7 days, with cerebral infarction in two (3.8%) patients, cerebral hemorrhage in one (1.9%), subarachnoid hemorrhage in one (1.9%), and cardiac arrest in one (1.9%). In recanalizing VBA occlusion, three 5.2%) patients experienced complications within 7 days after the procedure, including cerebral infarction in two (3.5%) patients and cerebral hemorrhage in one (1.7%). Among 25 patients who failed the recanalization procedure, four (16%) patients with MCA occlusion experienced complications within 7 days after the procedure, including cerebral infarction in two patients and subarachnoid hemorrhage in the other two. The total complication rate among all patients was 8.5% (15/177), with a complication rate of 4.5% in patients with ICA occlusion, 17.3% in MCA occlusion, and 5.2% in VBA occlusion, with the complication rate in recanlization of MCA occlusion significantly (P < 0.05) greater than that in the other two groups  months after the procedure Table , and theAmong 25 (13.8%) patients without successful recanalization who were treated with antiplatelet therapy, the mRS score was aggravated in six (24.0%) patients including two patients with ICA occlusion, one with MCA occlusion, and three with vertebrobasilar artery occlusion.Among all patients, successful recanalization was significantly associated with occlusion duration P = 0.039), calcification (P < 0.0001) or angulation (P = 0.0002) of the occluded segment, with longer occlusion duration, calcification and greater angulation of the occluded segment significantly decreasing the recanalization rate  associated with location of occlusion, hyperlipidemia, and patients’ height in univariate logistic regression analysis, but only independently significantly (P = 0.002) associated with hyperlipidemia  in multivariate logistic analysis (Table Occurrence of restentosis or reocclusion was significantly (P < 0.05) associated with complications and mRS at 90 days in univariate logistic regression analyses Table . Multiva34, a scoring system for successful recanalization of intracranial arterial occlusion was estalibshed  associated with calcification or angulation of the occluded segment, MCA recanalization with angulation, occlusion length and right side occlusion, and VBA recanalization with occlusion length, angulation, calcification and age for VBA occlusion. The significant (P < 0.05) independent risk factors were angulation and calcification for successful recanalization, hyperlipidemia for complications, and mRS at 90 days for restenosis or reocclusion at follow-up.35. Clinically, atherosclerotic stenosis and occlusion of the ICA are common, with frequent local calcification. Success recanalization of ICA occlusion has been reported to range 70–90% with a periprocedural complication rate of 10–20%33. In our study, the successful recanalization rate of ICA occlusion was 87.3% with a periprocedural complication rate of 6.5%. The relatively high successful recanalization rate of ICA occlusion with a low complication rate results firstly from the straightness of the occluded segment, and if the ICA opening is involved, hybrid surgery can be performed using both endarterectomy and endovascular recanalization. The ICA has fewer branching arteries, and dissection caused by a micro-guidewire may not result in severe consequences. At the ICA intracranial clinoid segment, because the anterior choroidal artery and posterior communicating artery are consistent with the axial direction of ICA, a microcatheter or micro-guide wire may easily enter these arteries and cause arterial occlusion or bleeding if operated carelessly and roughly. To prevent possible complications, angiography with multiple projections for monitoring and timely reshaping of the tip of the micro-guide wire are the key. The superior pituitary artery from the inferior wall should not be ignored, and cares should be taken when exploring the occluded segment beyond the lower edge of the ICA contour. When exploring the ophthalmic segment which is bent, the guide wire can easily exert force on the local anterior upper wall and cause dissection. It is recommended to use a microcatheter with the tip shaped to 45°. It may be more reasonable for the guide wire to deviate to the lower wall in the proximal segment and to the upper wall in the distal segment when exploring the occluded segment. If a dissection in the ICA is too long and the micro-guide wrie cannot enter the true cavity in time, it is wise to terminate the operation because repeated operation may damage the opening of the posterior communicating artery and destroy formed collateral compensation.Anatomically, from the opening to the bifurcation of the ICA, the ICA is relatively long and divided into 7 segments according to the Bouthillier classification approach29. The course of the occluded M1 segment can refer to that of the other side, with the course being mostly straight towards the out rear side, and the M1 segment measures 15–20 mm in length and 2.0–2.5 mm in diameter. The posterior superior wall sends out slender lenticular arteries while the anterior inferior wall sends out the temporal branches34. In our study, the successful recanalization rate for MCA occlusion was 88.5%, with the highest periprocedural complication rate of 17.3% among all patients and a reocclusion rate of 4.3%. There are many perforating arteries in the M1 segment of the MCA, and serious infarction of the conducting fibers in the basal ganglia and radial crown may lead to severe disability and poor prognosis if these perforating arteries are injuried during endovascular operation for recanalization. Moreover, the tip of the micro-guide wire can enter the slender insular arterial branches of the M2 segment and injure them if maneuvered roughly, resulting in intracerebral hemorrhage and a higher periprocedural complication rate. In order to reduce the perioperative complication rate, suboptimal recanalization of the MCA occlusion is often performed with greater residual stenosis, which may lead to poor follow-up outcomes. In patients with better effects of MCA recanalization, the imaging manifestations are mostly watershed infarction and large-scale cortical hypoperfusion, which is caused by short occlusion of M1 segment with the original stenosis locating in the front and lower part of M1 without occluding the perforator artery of the MCA. The potential lacuna and thrombus-occluded segment are located in the rear and upper part of M1 segment, which is right in the direction of exploration of the micro-guide wire.Recanalization of MCA occlusion is mainly at the M1 segment. Because the blood vessels at the M2 segment and beyond are small, recanalization of these vessels is highly risky and rarely performed26. If occlusion of the intracranial segment involves the posterior inferior cerebellar artery, it can cause a large area of cerebral infarction in the lower part of the cerebellar hemisphere. When the infarction exceeds 1/3 of the cerebellar hemisphere, cerebellar tonsillar hernia may take place. If the anterior spinal artery or perforator artery of medulla oblongata is occluded, respiratory and swallowing functions will be affected, leading to poor consciousness and severe clinical symptoms. The normal basilar artery courses straight and measures on average 3.2 mm in diameter and 2.5 mm–3.0 cm in length. The dorsolateral perforating branches are abundant and supply blood to the midbrain and brainstem. Occlusion of longer segments often leads to serious disability, with little significance for recanalization in non-acute stage. In patients with short occlusion and poor collateral compensation, the symptoms may be repeated with delayed progression, and recanalization may benefit these patients at the non-acute stage32. In our study, the occlusion was located in the intracranial VBA which was recanalized in 82.8% patients with a periprocedural complication rate of 5.2%.The intracranial segment of the vertebral artery runs straight, and its branches are mainly the posterior inferior cerebellar artery, anterior spinal artery and some perforating arteries of the medulla oblongataFor patients with eggshell-like and long-segment calcification occlusion on imaging presentation, we should retreat from trying to recanalizing the occlusion. Even if the occlusion is recanalized, severe perioperative complications and poor long-term prognosis may be resulted because calcification is significantly associated with successful recanalization as found in our study. If arterial dissection is mistakenly created, the operation should be terminated immediately to prevent further damage to the vessel, especially in the intracranial segment of vertebral artery. This segment has small blood vessels supplying the medulla oblongata, which is closely related to the respiratory center, and injury to these vessels may lead to acute or subacute respiratory disorders. In many cases of injury, there are no premonitory symptoms, and special care should be taken in clinical postoperative management to prevent apnea or asphyxia. To prevent and reduce periprocedural complications, medical imaging before recanalization should be carefully evaluated for the occlusion length and vessels distal to the occlusion. High-resolution MRI is helpful to distinguish the plaque orientation and true segments of occluion. In some patients, angiography through a micro-catheter proximal to the occlusion can be used to assess the occlusion.37. Thus, endovascular treatment was selected at around 3 weeks after imaging-confirmed occlusion of intracranial arteries based on studies in the literature37.In our study, over 80% of the patients achieved an mRS score of 0 at 90 days post recanalization. The prognoses were indeed very good. Possible reasons were described as follows. Most patients enrolled had only mild symptoms with the mRS ≤ 2 in 91.2% and mRS > 2 in the rest 8.8% (n = 16). Patients with mRS > 2 had definitive hypoperfusion which would result in good prognosis after successful recanalization and restoration of perfusion. Moreover, a considerable number of patients were in the recovery stage, and the effect of natural functional recovery plus endovascular reconstruction of blood flow resulted in good prognosis. Endovascular recanalization and restoration of blood flow improved the hypoperfusion in some brain tissues and restored the function of some brain dormant parts at hypoperfusion. On the other hand, our study suggests that patients with mild disability and insufficient blood perfusion are one of the potential indications for recanalization in the non-acute phase of cerebral artery occlusion. In this study, all enrolled patients had survived the initial acute stage of arterial occlusion. Patients requiring emergency surgery, with severe disability or insignificant hypoperfusion were not included. For patients with short segment of occlusivon and a small amount of compensation in the distal segment, neurological symptoms will likely recur due to insufficient compensation, and for these patients, endovascular recanalization is beneficial. However, the timing of recanalization is difficult to decide. Early recanalization is conducive to saving the brain tissue at risk of poor perfusion and reducing the recurrence and progression of cerebran infarction. Nonetheless, early recanalization may involve fresh thrombus and new infarction, leading to increased risk of perioperative thrombus shedding, bleeding transformation and stent reocclusion38. In atherosclerotic diseases, plaque calcification often develops through an inflammation-dependent mechanism of atherosclerotic progression39. Moreover, atherosclerotic calcification is frequently coexistent with arterial tortuosity or angulation, whereas angulation is an independent factor significantly affecting successful recanalization and restenosis or reocclusion of in stent recanalization of intracranial arterial occlusion as revealed by our study. Both calcification and arterial angulation result in poor compliance of arteries to balloon angioplasty and necessitate application of a greater balloon inflation pressure38, which may cause subsequent arterial dissection or rupture, increasing the periprocedural complication rate. When the calficied or angulated artery of occlusion was not sufficiently dilated, a stent might not be able to apposite closely to the wall, leading to restenosis or reocclusion. Restenosis or reocclusion occurred more often in the ICA ophthalmic segment followed by ICA cavernous segment, VBA, and MCA. The ophthalmic and cavernous segments of the ICA are more curved with thicker arterial wall which are not readily responsive to balloon angioplasty or compliant to stent deployment as the other vessels do. Periprocedural complications like arterial dissection, injury and thrombus formation may cause greater restenosis or reocclusion of stented artery, and with greater follow-up durations, more instent intimal hyperplasia will develop, resulting in more restenosis or reocclusion. Occlusion duration significantly affected successful recanalization because longer time would cause longer and stronger occlusion by intimal hyperplasia. Periprocedural complications occurred more often in recanalization of MCA occlusion than in VBA or ICA as analyzed above, and hyperlipidemia may also significantly affect the complications as revealed by our study. The risk factors for intracranial atehrosclerosis include diabetes mellitus, hypertension, hyperlipidemia and cigarette smoking40, however, only hyperlipidemia was identified as an independent significant factor affecting complications, which needs to be confirmed. Moreover, patients’ height was also associated with the presence of complications, which also needs further study for confirmation.Our study identified calcification as one independent factor significantly associated with restenosis or reocclusion. This factor had also been confirmed to significantly link to instent restenosis when using stent angioplasty for intracranial atherosclerotic stenosisFor successful recanalization of ICA and VBA occlusion, angulation and calcification were two independent risk factors. For MCA recanalization, right side occlusion, occlusion length and angulation were three independent risk factors, whereas for VBA recanalization, occlusion length and older age were independent risk factors in addition to angulation and calcification. For restenosis or reocclusion, only mRs at 90 days was an indepent risk factor. For the continuous variable of angulation as a significant independent risk factor for recanalization, the cutoff value was 76.9° for ICA recanalization, 16.0° for MCA recanalization, and 29.4° for VBA recanalization, which indicates that an angulation greater than the above cutoff values will result in an increased successful recanalization rate. The cutoff value of the independe risk factor of occlusion length was 7.3 mm for MCA recanalization and 25.2 mm for VBA recanalization, suggesting an occlusion length greater than the above cutoff values will lead to a decreased recanalization rate.Based on our outcomes, clinical experience and studies published in the literature, we have coined a risk classification for recanalizing occlusion of large intracranial arteries Table , which nThis study had some limitations, including the retrospective and one-center study, a small cohort of patient, insufficient quantitative perfusion data, patient’s heterogeneity, Chinese patients enrolled only, short-term follow-up, and no randomization, which may all produce some bias to affect the generalization of the study. Explanation of the outcomes should be cautious. Future studies will have to resolve all these issues to improve and confirm the outcome.In concsluion, recanalization surgery may be a safe and effective approach for patients with non-acute symptomatic occlusion of large intracranial arteries which do not respond to medications, and factors significantly independently associated with successful recanalization, periprocedural complications and restenosis or reocclusion after recanalization have been identified to improve the outcome in future practice.


However, animal studies remain mandatory in the upscaling before clinical studies. Orthotopic and syngeneic grafting is a robust model to test a drug efficiency in a tumor and its microenvironment.Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with increasing incidence. Even if progress have been made, the five-year overall survival remains lower than 10%. There is a desperate need in therapeutic improvements. In the last two decades, new We described a method for orthotopic and syngeneic graft of KRAS mutated, p53 wildtype, 8305 cells in a C57BL/6J mouse model.With this microsurgical method, 30 mice were grafted, 24 by a junior and six by a senior, resulting in 95,8 and 100% of  successful tumoral implantation, respectively. Twenty mice underwent ultrasound follow-up. It was an efficient method for the tumoral growth evaluation. At day 16 after grafting, 85% of the tumors were detectable by ultrasound, and at day 22 all tumors were detected.The presented method appears to be a robust and reliable method for pre-clinical studies. A junior master student can provide positive results using this technique, which can be improved with training. Adding to historically two-dimensional (2D) culture of tumor cells, three-dimensional (3D) cultures, including co-cultures in spheroids and organoids, have been developedHere we aimed to describe an easy and reproducible method for orthotopic and syngeneic pancreatic cancer cell graft in mice. To illustrate the feasibility of this method, we analyzed the results of the first 24 orthotopic pancreatic cancer cell graft performed by our mastership student and six by an experienced team.All experiments were in accordance with institutional guidelines and European animal protection law and approved by the responsible government agency (agreement number 2019061900061441).G12D (p53 wild type) and generously provided by Dr. D. Saur . Mouse tumor cells were maintained in Dubelcco’s Modified Eagle Medium (DMEM) from Sigma Aldrich (ref D6429) containing 10% foetal bovine serum (FBS) (Life Technologies) supplemented with L-Glutamine , penicillin and streptomycin (P/S). The day of the procedure, the correct number of cells  was centrifuged at 1,200 rotation per minute (RPM) for 3 minutes, washed with phosphate buffer saline (PBS) and re-suspended in 20 uL of Dubelcco’s PBS from Sigma Aldrich (ref. 8537). Cells were kept on ice before injection for a maximum of 3 hours.Mouse pancreatic tumor cells are tumor cells isolated from p48-Cre LSL-Krasad libitum in each cage. Mice were monitored for a week in our facility, and, one day before the procedure, mice were shaved (left flank) with an electrical instrument.Eight-week-old C57BL/6J female mice were dispatched after reception in cages up to five animals per cage. Room temperature was kept between 19 and 22 °C and humidity between 55 and 65%. Food and water were available Mice were treated  with the analgesic buprenorphine  at least 30 minutes before the procedure.Mice were put under general anesthesia using isoflurane 2,5% by inhalation with a flow of 1 L/min. Maintain of the anesthesia was performed with a facial mask with a flow of 0,4 L/min. The whole procedure was performed on mice kept on a heating pad (37 °C). General anesthesia was considered adequate in the absence of any reaction/reflex after toe pinch. A droplet of an eye gel  was added in both eyes of anesthetized mouse to avoid eye dehydration.Mice were placed on the right side, exposing the left flank to the operator. At this time, the correct positioning of the facial mask was verified . The skiA short incision (< 1 cm), 1 cm from the grill cost on a line between the grill cost and the start of the tail, was done using straight scissors. Incision was performed in the coronal plan in a head-to-toe direction between two clamps –1d. A suA total of 20,000 cells, kept on ice during the process and diluted in 20 μL of PBS, was injected in the pancreas using a needle  . The appThe peritoneum was closed with absorbable 5/0 suture (Novosyn) . Skin waA second intra-peritoneal (IP) injection of buprenorphine  was performed 8 hours after the first one.Mice behavior was verified in the evening of the procedure, in the next morning  and then every day. Staples were removed at post operative day (POD) 8.· π* a/2 · b/2 · c/2; a, b and c being the diameters measured).Mice were weighted twice a week. At POD 16, the presence of tumor within the pancreas was evaluated by ultrasound using Aixplorer (Supersonic) echograph and SL-22 transducer. The tumor volume follow-up was performed twice a week by ultrasound, and tumor volume was calculated using an ellipsoid model .Total success was defined by the presence of a pancreatic tumor without subcutaneous tumor. Partial success was defined by the presence of a pancreatic tumor associated with a subcutaneous tumor. Failure was defined by the absence of any pancreatic tumor. End points were defined by either a weight loss greater than 20%, or a total tumor burden (subcutaneous tumor + pancreatic tumor) reaching more than 700 mmAt the day of sacrifice, mice were put under general anesthesia, intracardiac punction was carried out to collect the mice blood, and a cervical dislocation was performed. The intracardiac punction was performed using a needle (#300600) inserted just above sternum  of the animal with an inclination of 45° in the direction of the head. The presence of blood within the needle (even before any aspiration) allows the identification of the right position within the cardiac cavity. The punction allows to collect up to 900*μL of blood.At sacrifice, we systematically searched for any sign of peritoneal carcinomatosis or solid-organ metastasis. Liver, spleen, lungs, and pancreas were extracted. The pancreas (pancreatic parenchyma plus tumor) was then weighed after being separated for the spleen. Organ samples were kept in formalin for 24 hours and then stored in 70% ethanol before paraffin inclusion.Twenty-four mice were grafted by a junior (a master 2 student) and six by a senior operator (experienced member of the Centre de Recherche en Cancérologie de Toulouse team in Toulouse) .3, and 10 mice were sacrificed at intermediate endpoint–five were sacrificed when the tumor volume reached 200 mm3 and five at POD 31 independently of the tumor volume.Over the 24 mice grafted by the junior operator, ten were sacrificed at POD 32 without ultrasound follow-up, four mice were sacrificed when the tumor reached 500 mm3 and subcutaneous tumor was 532 mm3). Eleven mice had subcutaneous tumors (42%). Over the 23 mice sacrificed after day 31, three (13%) had macroscopic liver metastasis. No macroscopic pulmonary or peritoneal carcinomatosis metastasis was found.Over the twenty-four mice, twenty-tree  mice developed a pancreatic tumor. No mice were sacrificed because of clinical worrisome feature or important weight lost. Weight was stable during the follow-up in each group . Grafts All subcutaneous tumors seen at sacrifice were localized at incision site and had been identified during ultrasound examination. Over the 14 mice for which tumor growth was followed by ultrasound, 85,7% of them presented a tumor at day 16 after grafting, and 100% at day 22. Mean tumor volume evolution for each group is represented in Regarding the six mice grafted by the senior operator, all of them developed a tumor, and two had a subcutaneous graft (33%), which resulted in 67% successful and 33% partially successful grafts.Although there was some heterogeneity in tumor growth, both junior and experienced team tumor growths followed an exponential form  and 2c. Here we described our method for orthotopic pancreatic cancer graft and follow-up in a syngeneic and immunocompetent murine model. No previous experience in small animals’ surgery was required as shown by our results obtained from an inexperienced master student . Although most of the animals showed a subcutaneous tumor, only one was sacrificed because of this ectopic tumor burden. It was considered as a graft failure in our definition as it constituted a reason for early sacrifice. Our master student had a 95,8% of graft intake with the presence of subcutaneous tumors in 42% of the animals, while experienced operator reported 100% success with 30% subcutaneous tumors.3 emphasizing the importance of this endpoint volume. Larger volume end points might have led to unnecessary animal discomfort.Mice’s weights were stable during the follow-up, showing good tolerance of the procedure. Interestingly, we report one mouse with an important weight lost which coincided with a tumor volume reaching almost 500 mm-,In our study, the tumoral growth was following an exponential model which is consistent with previously reported studiesSome authors rather use heterotopic grafting (subcutaneous) as tumoral growth can be directly followed up by palpation and measured with a caliper,Moreover, while heterotopic graft is methodically easier, it was demonstrated to be less robust for studying drug efficacy. Treatment efficiency differed due to the difference in vascularization systemDifferent models for therapeutic screening are available; every model has its own advantages and limitations. Combination of these models is useful: for example, spheroid and organoid models are used for drug testing as they can reduce the number of animals needed necessary for drug validationTherapeutic innovations are needed to improve pancreatic cancer treatment. Murine models remain a good tool to recreate, in pre-clinical studies, the complexity of pancreatic cancer environment. In this study, we described a robust and reproducible method for grafting pancreatic cancer cells in an immunocompetent mouse model. We also described a useful method of ultrasound for an efficient tumor growth evaluation. This method is reliable and can be performed by a junior student after a short training.