Accumulating evidence supports the efficacy of administering natalizumab (NZ) with extended-interval dosing (EID) in patients with relapsing-remitting multiple sclerosis (RRMS).We switched NZ dosing from 4-week to 6-week intervals in patients with RRMS, and investigated the effect on serum neurofilament light chain (sNfL) concentrations.N = 45). The other cohort received EID  both at baseline and during follow-up . Serum samples were collected in the EID4–6 cohort at every NZ infusion, for 12 months. The primary outcome was the change in sNfL concentrations after switching to EID.We included two cohorts of patients with RRMS treated with NZ: one received the standard-interval dosing (4 weeks) at baseline, and were switched to 6-week intervals  = 6.1), and it remained unchanged at 12 months. Moreover, individual sNfL concentrations did not change significantly after extending the NZ dosing intervals. In addition, the EID4–6 and EID5/6 cohorts had similar baseline sNfL concentrations.We concluded that extending the NZ dosing interval did not increase axonal damage, as determined with sNfL, in patients with RRMS. When NZ binds to α4 integrin on the surface of leukocytes, it prevents leukocyte migration from the blood into the central nervous system (CNS). NZ effectively reduces disease activity in RRMS, and it is well tolerated with a few adverse effects.2 The main drawback of NZ is that it increases the risk of progressive multifocal leukoencephalopathy (PML),3 a John Cunningham (JC virus) virus infection that often gives rise to severe impairment and is lethal in 24% of patients treated with NZ.4 In non-randomized observational studies, extended-interval dosing (EID) was associated with a significantly lower risk of developing PML, compared to SID,5 but it had similar therapeutic efficacy.69 Very recently, therapeutic efficacy has also been demonstrated to be maintained in EID with NZ in a randomized controlled study.10Natalizumab (NZ) is a monoclonal antibody used for treating patients with relapsing-remitting multiple sclerosis (RRMS).11 However, current evidence has suggested that signs of inflammatory activity and neurodegeneration may escape detection with conventional monitoring.1214Previously, when switching NZ dosing from SID to EID, disease activity and progression was generally monitored with conventional cerebral magnetic resonance imaging (MRI) and clinical evaluations.15 a marker of axonal damage that can be determined in cerebrospinal fluid (CSF)16 as well as in blood.18 There is accumulating evidence that NfL is a reliable biomarker of disease activity in RRMS,20 that may also reflect therapeutic efficacy.21 Consequently, NfL has served as an additional outcome measure in clinical trials.22The most promising soluble biomarker in MS is neurofilament light (NfL),This study aimed to determine whether switching NZ treatment intervals from SID to EID might affect serum neurofilament light chain (sNfL) concentrations in patients with RRMS. To reduce potential effects of other factors on sNfL levels, we selected patients who lacked signs of disease activity in clinical and MRI examinations.23 and had been receiving 300 mg NZ  intravenously, every 4, 5, or 6 weeks, for at least 1 year. They should not have any relapse or new or enlarging lesions on MRI within 6 months prior to baseline. After signing informed consent forms, patients were consecutively enrolled in the study. The first patients were included on the 1st of Oct 2019 and the last follow-up visit was on the 1st of June 2021. The inclusion process is illustrated in This prospective observational single-center study was conducted for 12 months at the MS center, Sahlgrenska University Hospital in Gothenburg, Sweden. Eligible patients had RRMS, fulfilled the 2017 McDonald criteria,The study participants were divided into two cohorts, based on the NZ infusion interval; one cohort had received NZ at 4-week intervals prior to baseline, and they were switched to receive EID at 6-week intervals (EID4–6). The other group had received EID at 5- or 6-week intervals at baseline, and continued extended dosing (EID5/6). Except for one patient, the patients in EID5/6 who received EID at 5-week intervals at baseline switched to 6-week intervals.24 scoring were performed at baseline and at 48 weeks , and then every 6 weeks, up to 48 weeks. In the EID5/6 cohort, blood was drawn at baseline (week zero), at 5/6 weeks, and at 12 weeks. In both study cohorts, MRI scan was performed at baseline, at 24 weeks, and at 48 weeks, and clinical examination with Expanded Disability Status Scale (EDSS)48 weeks .http://www.msreg.net) and from electronic patient records. A relapse was defined as the appearance of new or worsening neurological symptoms compatible with MS that lasted more than 24 hours in the absence of any factor that could cause worsening of symptoms (i.e. a pseudo relapse). Disability was assessed at clinical visits or via telephone due to the COVID-19 pandemic, and scored with EDSS or telephone-EDSS.25 Significant disability progression was defined as an increase of 1.5 points from a baseline EDSS score of 0, an increase of 1 point from a baseline EDSS score of 1–5.5, and an increase of 0.5 point from a baseline EDSS score >5.5. The MRI protocol included the brain and cervical spinal cord with T1-weighted images, T1-weighted images with gadolinium contrast, T2-weighted images, fluid-attenuated inversion recovery (FLAIR) images, and diffusion-weighted imaging (DWI), performed according to the Swedish guidelines.26 No evidence of disease activity (NEDA-3) was defined as a lack of relapse, no new or enlarging lesions detected on MRIs, and no significant progression during the study period.27Baseline demographic and clinical characteristics were retrieved from the Swedish MS Registry . Briefly, the samples, including internal quality control samples, and calibrator stock were removed from storage and allowed to thaw at room temperature. The RGP reagent was shaken for 30  minutes at 800  r/min and heated to 30°C. The calibrators, samples, and QCs were vortexed for 30  seconds at 2000  r/min. The internal calibrators, samples, and QCs were additionally centrifuged for 10  minutes at 4000g. Calibrators, samples, and QCs were added to the plate and covered with sealing tape. Reagents, samples, and calibrators were run in the HD-1 Analyzer using a 4× dilution. The intra-assay and inter-assay coefficients of variation were 10%.All NfL analyses were performed by board-certified laboratory technicians who were blinded to clinical data. To minimize variation, baseline and follow-up samples were analyzed side-by-side on each assay plate using one batch of reagents. In addition, samples from healthy controls were randomly analyzed in each assay plate. All analyses were performed at room temperature. Serum NfL concentration was measured using the SimoaThis study was conducted in accordance with the Declaration of Helsinki and the International Good Clinical Practice guideline. The study was approved by the Regional Committee for Medical Research Ethics, Gothenburg (EPN-460-13) and the Swedish Ethical Review Agency (DNR 2020-04900). Written informed consent was obtained from all participating patients.28 For matched pairs, comparisons within groups were performed with Fischer’s Non-Parametric Permutation test. Analysis of covariance (ANCOVA) was performed to adjust for age. The Wilcoxon signed-rank test was performed to evaluate differences between sNfL and plasma neurofilament light chain (pNfL) and changes in EDSS from baseline to 12 months. Pearson’s correlation coefficients were used to evaluate correlations between sNfL and age or body mass index (BMI). Pearson’s correlation analysis and the Shrout-Fleiss reliability random test29 were performed to compare sNfL and pNfL. The Mersenne Twister was used for random number generation.Statistical analyses were performed with SAS 9.4 , SPSS version 23 , and GraphPad Prism 9.3 . For continuous variables, comparisons between groups were performed with Fisher’s Non-Parametric Permutation Test.The study included 73 patients with RRMS; 48 had been treated with SID and 25 had been treated with EID. Three patients in the EID4–6 cohort were excluded due to concomitant conditions with neurological injuries . The demp = 0.68). Although three patients converted from RRMS to secondary progressive MS during follow-up, only one showed a significant increase in the EDSS. Of the other two patients, one showed an increase of 0.5 points in the EDSS, and the other showed no change in the EDSS, but experienced a progressive reduction in walking distance. Accordingly, overall, NEDA-3 was achieved in 66/70 patients (94%).One patient in the EID5/6 cohort experienced a relapse and a new non-enhancing lesion was detected on MRI. However, none of the other patients exhibited clinical or MRI signs of disease activity. In the EID4–6 cohort, no significant change was observed between the mean EDSS values at baseline and at 48 weeks  was 10.5 ng/L (standard deviation (SD) = 6.1) . We compared changes in mean sNfL concentrations between all samples, but also between baseline and the early period (weeks 6–18), and between baseline and the entire study period (weeks 6–48). Serum NfL concentrations remained stable throughout the 48-week study period with no significant change in the mean sNfL at any time-point .In the EID5/6 cohort, the mean sNfL concentration at baseline was 10.8 ng/L (SD = 5.3) and for all samples 10.3 ng/L (SD = 4.2), which was of similar level as the mean sNfL in the EID4–6 cohort  before extending the NZ dosing interval (baseline). There was no statistically significant difference in the baseline mean sNfL between EID4–6 and EID5/6  = −2.97–2.70). Hence, we found no significant increase of sNfL in NZ-treated patients with EID compared with SID. Furthermore, the sNfL concentrations did not change significantly in the patient who experienced a relapse and a non-enhancing new lesion or in patients who converted to secondary progressive MS.R = 0.48, p < 0.001), but not with BMI , EDSS (p = 0.243) or gender (p = 0.979). After adjusting for age, the mean sNfL  was 11 ng/L (95% CI = 9.6–12.3).In the EID4-6 cohort, we investigated the inter- and intra-individual variation of sNfL in order to evaluate sNfL as a biomarker for individual patients. The mean sNfL  was 10.8 ng/L (SD 5.9), and the median sNfL 8.9 ng/L (range = 4.5–39.4). We found that sNfL concentrations varied significantly with age .We set the mean + 1 SD (16.7 ng/L) and the mean + 2 SD (22.6 ng/L) as cut offs for abnormality. We found one or more samples above this limit in six patients  and three patients , respectively. Furthermore, in three patients, all samples were above 1 SD, and in one patient, all samples were above 2 SD. None of these patients had clinical or MRI signs of disease activity or disease progression, but all were within the oldest age quartile (49–73 years) of the EID4–6 group. All patients with sNfL concentrations under or equal to 16.7 ng/L (i.e. 1 SD above the mean) were 48 years or younger. In contrast, 6/13 (46%) older patients had one or more samples with sNfL concentrations above 16.7 ng/L . The levels of sNfL and pNfL were highly correlated . p < 0.001).Both sNfL and pNfL were collected from a randomly selected subgroup of the EID4–6 cohort  have been associated with chronic or smoldering lesions.13 Recently, increased sNfL levels were found in RRMS and progressive MS without recent disease activity but with two or more PRLs.37 Thus, although smoldering MS is considered a slow process, it may give rise to elevations in sNfL levels. In the same study, PRLs were associated with disease severity. In contrast, this study population included only patients with RRMS at baseline and with a few exceptions they did not progress at follow-up. Besides, the observational period was limited to 12 months which probably was too short to detect increases of sNfL due to smoldering MS. However, we cannot rule out that EID with NZ can impact such disease activity.Even though smoldering MS activity may escape detection by conventional monitoring of RRMS,8 In another study, sNfL remained stable for up to 8 weeks after discontinuing NZ, due to JC virus antibody positivity, but increased sNfL levels were detected at follow-up.38 In a group of 60 stable MS patients on SID NZ treatment, no change in sNfL was seen at 6 months after switch to ⩾35 days dosing interval.39 However, our study was the first to monitor sNfL at a relatively high frequency (every 6 weeks) for an extended period of time (48 weeks).We monitored sNfL levels to detect new disease activity in patients treated with NZ that switched from SID to EID. Previous studies have used sNfL to evaluate potential disease recurrences in patients who switched from SID to EID. In one previous study, 34 patients with RRMS switched from SID to EID , and sNfL was essentially unchanged from baseline, after 12 months of NZ treatment.40 When we determined sNfL concentrations every 6 weeks, we found that most NZ-treated patients had only minor fluctuations of sNfL. We also found that sNfL concentrations were slightly, but significantly higher than pNfL concentrations, consistent with findings from a previous study.41 With a few exceptions, included patients had essentially no disease activity or disability progression and 94% achieved NEDA-3. Although this selected cohort had apparently very low disease activity prior baseline and during the 48-week study period, approximately 11% and 4% of samples had sNfL concentrations greater than 1 SD and 2 SD, respectively, above the mean sNfL. However, most of those samples were obtained from a few patients who had relatively high sNfL levels during the entire study period. In these patients, the elevated sNfL levels were not associated with disease activity or progression. Among the six patients with sNfL values above 1 SD, two had diabetes, which is known to influence sNfL levels,42 and all six patients were within the oldest quartile of the EID4–6 group. Similarly, in a previous study, the sNfL variance was higher among individuals of older age, in a population of patients with RRMS who had been followed with repeated serum sampling.43 The sNfL inter-individual variability seemed larger than the intra-individual variability, suggesting that the best utility of sNfL measurements is for individual longitudinal follow-up of younger adult age. Nevertheless, in our cohorts, some patients displayed unexplained variations in sNfL levels, which suggested the possibility that subtle disease activity could have occurred, but was not detected with conventional monitoring.12Repeated serum sampling provided the means to investigate treatment effects at both the individual and group levels over time. Due to the temporal change of NfL after a relapse or CNS injury, the interval between testing sNfL should not exceed 3–6 months.43 there is an unmet need for data on sNfL variability in clinically meaningful subgroups of patients with MS. Perhaps the most important such cohort would be patients with RRMS that receive disease-modifying treatment, particularly those with stable disease monitored with conventional clinical and MRI measures. Our results from repeated sNfL determinations, in most cases, confirmed the stability of clinical and MRI measurements, but individual deviations could occur. A second limitation was that, in contrast to previous studies,44 we identified new disease activity in only one patient. Thus, we could not investigate the utility of sNfL testing for detecting disease activity in our patient cohort. Another limitation was the lack of healthy control subjects to serve as a reference for comparing sNfL levels to those found in patients with stable disease under NZ treatment. However, the sNfL levels of this study population were similar to those observed in healthy control subjects in a previous study of ours, using similar Simoa assay,17 but slightly higher than those reported in other studies.43This study had some limitations. One limitation was the relatively small number of patients included, preventing us from establishing a valid age- and BMI-adjusted reference sNfL concentration in stable RRMS. Although some population-based surveys of healthy subjects have provided age- and BMI-adjusted sNfL concentrations,In conclusion, based on repeated determinations of sNfL measurements over 48 weeks, we did not find any signs of increased axonal damage in patients who received NZ and switched from SID to EID. Our data supported the notion that sNfL monitoring was most reliable for monitoring younger adult patients with RRMS, while increased sNfL concentrations may still occur in stable RRMS where confounding factors such as comorbidities are more common.Click here for additional data file.Supplemental material, sj-tif-1-msj-10.1177_13524585221108080 for No increase of serum neurofilament light in relapsing-remitting multiple sclerosis patients switching from standard to extended-interval dosing of natalizumab by Magnus Johnsson, Helen H Farman, Kaj Blennow, Henrik Zetterberg, Clas Malmeström, Markus Axelsson and Jan Lycke in Multiple Sclerosis Journal


Glucose-dependent insulinotropic peptide (GIP) and meal ingestion increase subcutaneous adipose tissue (SAT) perfusion in healthy individuals. The effects of GIP and a meal on visceral adipose tissue (VAT) perfusion are unclear.Our aim was to investigate the effects of meal and GIP on VAT and SAT perfusion in obese individuals with type 2 diabetes mellitus (T2DM) before and after bariatric surgery.15O-H2O positron emission tomography–magnetic resonance imaging at 3 time points: baseline, 20 minutes, and 50 minutes after the start of stimulation. Obese individuals were studied before and after bariatric surgery.We recruited 10 obese individuals with T2DM scheduled for bariatric surgery and 10 control individuals. Participants were studied under 2 stimulations: meal ingestion and GIP infusion. SAT and VAT perfusion was measured using P = .04) and GIP-infusion (P = .002) were blunted in the obese participants compared to controls. VAT perfusion response did not differ between obese and control individuals after a meal or GIP infusion. After bariatric surgery SAT perfusion response to a meal was similar to that of controls. SAT perfusion response to GIP administration remained lower in the operated-on than control participants. There was no change in VAT perfusion response after bariatric surgery.Before bariatric surgery the responses of SAT perfusion to meal (The vasodilating effects of GIP and meal are blunted in SAT but not in VAT in obese individuals with T2DM. Bariatric surgery improves the effects of a meal on SAT perfusion, but not the effects of GIP. Postprandial increase in SAT perfusion after bariatric surgery seems to be regulated in a GIP-independent manner. The responses of adipose tissue (AT) to natural cues or challenges, such as eating, and related gastrointestinal hormones are not thoroughly understood in obesity. The normal physiological response of AT to meal ingestion is the uptake of nutrients and storing of triglycerides (TGs) in intracellular lipid droplet(s). In obesity, insulin resistance of AT is one major factor contributing to unbalanced insulin response, and furthermore in type 2 diabetes mellitus (T2DM), AT becomes a sink of glucose: Increasing fat mass can compensate for reduced glucose uptake per mass of AT .Bariatric surgery is an effective way to achieve rapid and sustained weight loss . In addiGlucose-dependent insulinotropic peptide (GIP) is an incretin hormone produced by K cells in the proximal small intestine, and it is released in response to the ingestion of a meal containing either glucose or fat . FurtherGIP increases subcutaneous adipose tissue (SAT) blood flow in the presence of insulin . This re15O-H2O PET radiotracer. After a baseline 15O-H2O PET scan of the abdominal region, the scan was repeated 20 and 50 minutes after meal ingestion and in another scanning session during GIP infusion. During the scanning sessions plasma levels of glucose, insulin, GIP, and GLP-1 were measured at time points 0, 15, 30, 45, 60, and 90 minutes. The morbidly obese participants were scanned before bariatric surgery and the same protocol was repeated approximately 2 months  after surgery.This is part of the GIP-PET study  described previously . BrieflyPET scans were performed using a Philips Ingenuity combined PET/MRI scanner .15O-H2O (radiowater), and a dynamic emission scan of the abdominal region was performed. Radiowater was produced using the Hidex Radiowater Generator (Hidex Oy). Tissue-specific perfusion was calculated using the 1-tissue compartment model, a method based on the principle of exchange of inert gas between blood and tissues. Input functions were derived from the images by drawing a volume of interest in the abdominal aorta. Analysis of PET images was conducted using Carimas software (Turku PET Centre) (Tissue-specific perfusion was quantified with an intravenous injection of  Centre) , 30.The study protocol was reviewed by the local ethical committee of the Hospital District of Southwest Finland, and the study was carried out according to the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. Written informed consent was signed by all study participants before any study procedures and inclusion in the study.RRID:AB 2884907) as was plasma GIP . Electrochemiluminescence immunoassays were used to measure plasma insulin  and C-peptide concentrations .Plasma GLP-1 was measured by an enzyme-linked immunosorbent assay  were reported by Asmar et al (Comparison of means was performed with 2-way Insulin sensitivity is expressed as homeostatic model assessment of insulin resistance (HOMA-IR) and 2-hour oral glucose sensitivity index .AT volumes were calculated by measuring SAT and VAT from a single MRI slice at the level of L3, and calculating total SAT and VAT mass using predictive equations by Schweitzer et al . AT voluGeneral characteristics of the study individuals are provided in P = .62) and VAT (12.12 ± 6.24 vs 10.45 ± 5.78 mL/100 g/min) were similar between control and obese participants , but in control individuals there was a significant increase from baseline (P < .05) (P < .01) as well as controls (P < .05), suggesting a delayed response in obesity  . After 5 obesity .P < .05, interaction time × group). Interestingly, blood flow over time of VAT after the mixed meal was similar between obese and control participants  and obese (P < .01) participants before surgery (P < .001) and obese individuals  and in obese participants .GIP infusion had increased SAT blood flow from baseline after 20 minutes in control   .P < .01) and insulin sensitivity and glucose homeostasis had improved . Bariatric surgery reduced SAT mass by 7.62 ± 3.02 kg, and VAT mass was reduced by 1.56 ± 0.86 kg. SAT and VAT mass both remained higher in obese individuals after bariatric surgery compared to controls  as was VAT blood flow . Likewise, there was no statistically significant change in unstimulated SAT  or VAT  blood flow after bariatric surgery compared with measurements before surgery.After bariatric surgery baseline (0 minutes) SAT blood flow was not significantly different between control and obese individuals  (see P < .01), along with an increase in VAT blood flow (P < .01) (P < .05) and VAT (P < .01) blood flow were elevated compared to baseline.In obese patients after bariatric surgery, SAT blood flow response to a meal resembled the response of controls (ols) see . VAT resP < .01) . SimilarP < .01) and at 50 minutes (P < .05) (see P < .01) and at 50 minutes (P < .01)  see . After s.01) see  in obeseP < .05, interaction time × group)   (see SAT blood flow over time after GIP infusion was higher in controls compared with obese patients after surgery (oup) see . Change oup) see  or GIP ioup) see .There was no difference is SAT or VAT blood between the bariatric surgery groups  after a mixed meal or GIP infusion for time × group interaction.We measured changes in plasma glucose, insulin, and incretin (GIP and GLP-1) levels during scanning. These results of glucose, insulin, GIP, and GLP-1 concentrations have been previously reported , 26 and After meal stimulation, control and presurgery participants showed an increase of plasma glucose at the 45-minute sample time, whereas postsurgery patients’ more robust plasma glucose response was seen already 15 minutes after the meal . SimilarDuring GIP infusion plasma glucose levels of obese individuals before surgery decreased from baseline, whereas no change was seen in controls or obese participants after surgery . Plasma P < .01) and a positive association between SAT blood flow AUC and plasma glucose AUC  during GIP stimulation, as shown in Supplementary Fig. 1 (Before surgery we found a strong negative correlation during GIP infusion with SAT blood flow area under the curve (AUC) and plasma insulin AUC  (P < .01) during GIP infusion. During mixed-meal stimulation a strong association between SAT blood flow AUC and plasma GIP AUC  was found  . There was found .P < .01) during mixed-meal testing.In control individuals VAT blood flow AUC correlated with plasma GLP-1 AUC . It has been shown that SAT is reduced more by volume after bariatric surgery, but a larger portion of VAT is reduced, and the reductions are mutually associated (VAT is a more metabolically active adipose depot, and it has been more strongly associated with obesity-related complications than SAT , 17, 18.sociated . It has sociated , 38, 39.sociated , VAT wassociated , 41. Thesociated , 26, 42.sociated , 26. ThiA strength of this study is the ability to measure blood flow in SAT and VAT under nonstimulated and stimulated conditions both in lean and obese individuals—as well as before and after bariatric surgery–induced weight loss. However, this study has some limitations. The follow-up time of this study was relatively short: The mixed-meal test was repeated on average 69 days and the GIP infusion test 80 days after bariatric surgery. Since the obese participants were still in the process of rapid weight loss, it is possible that more pronounced changes in AT blood flow could be found later after surgery. We also observed no difference in VAT blood flow between controls and obese individuals, as well as no difference between SAT or VAT blood flow at baseline between controls and obese participants, which could be due to high interindividual variation. Whether the changes are more dependent on changes in insulin sensitivity and glucose homeostasis or weight loss is not obvious. It should be noted that while AT blood flow is linked to glucose uptake and influx and outflux of fatty acids in AT, blood flow does not directly measure the substrate metabolism of AT.In conclusion, obese individuals with T2DM have a blunted response to mixed-meal ingestion and GIP infusion in SAT. After bariatric surgery SAT blood flow response to a meal improves and resembles the response of healthy controls; however, no improvement was seen in response to GIP infusion. Interestingly these same changes are not seen in VAT: There was no difference between controls and obese participants either after meal ingestion or GIP infusion. After bariatric surgery VAT blood flow increased more rapidly after mixed-meal ingestion, but overall remained the same as before surgery. This emphasizes the higher metabolic activity of VAT, as blood flow remains more sensitive to stimulation by a meal or GIP even in obesity compared with SAT. It seems that the vasodilative effects of GIP are blunted in SAT but not VAT, and that the improved meal response seen in SAT could be mediated via regulators other than GIP.


Ghi_A09G08736, which is homologous to SAUR46 in Arabidopsis thaliana, was detected in our study. In addition, transcriptomic evidence was provided to show that gravity and light were implicated in the FBA formation. This study provides new insights into the genetic architecture of FBA that informs architecture breeding in cotton.Fruit branch angle (FBA), a pivotal component of cotton plant architecture, is vital for field and mechanical harvesting. However, the molecular mechanism of FBA formation is poorly understood in cotton. To uncover the genetic basis for FBA formation in cotton, we performed a genome-wide association study (GWAS) of 163 cotton accessions with re-sequencing data. A total of 55 SNPs and 18 candidate genes were significantly associated with FBA trait. By combining GWAS and transcriptome analysis, four genes underlying FBA were identified. An FBA-associated candidate gene  Gossypium spp.) is an important economic crop cultivated worldwide and the largest source of textile fiber  and amyloplasts , which is the functional ortholog of the SGR5 gene in Arabidopsis, negatively regulates leaf angle by controlling the adaxial growth of tiller node and lamina joint in rice  angle is complex, and many environmental signals including light and gravity play an essential role in branch angle formation. To date, many gravity-related genes associated with branch angle have been identified. For instance, shoot gravitropism (SGR) family change the branch angle by attenuating gravity perception in bidopsis . SGR9 atloplasts . SGR5 isal cells , and Loo in rice . Many st sensing . In riceectively . CsLAZY1a plants . Light ichrome B . In the chrome B , and EIDchrome B .BnaA3.IAA7, which encodes an Aux/IAA protein, reduced branch angle in rapeseed (SMALL AUXIN UPREGULATED RNA 10 (SAUR10) is specifically expressed at the abaxial side of the branches and this localized activity is influenced by hormones, light conditions and the Arabidopsis MADS-domain factor FRUITFULL, which also has an effect on branch angle formation  has been gradually applied to the study of complex traits in crops. GWAS has been performed on tiller angle in rice , leaf anbreeding . The appGossypium hirsutum cultivars with wide variations were collected in Korla and Shihezi, Xinjiang in this study. Through GWAS, genetic loci and key candidate genes related to FBA were explored. Moreover, we performed transcriptome sequencing of two G. hirsutum cultivars with significant FBA difference, and identified key regulatory genes and pathways, which laid a foundation for gene function verification, molecular mechanism study of FBA formation and cotton genetic improvement in the future.The proper fruit branch angle (FBA) can be helpful for adaptation to mechanistic harvesting and yield improvement. Although some QTLs or genes regulating FBA in cotton have been previously identified through parental mapping populations and natural populations , the effThe 163 upland cotton germplasm resources, which were composed of 152 white-fiber cultivars  and 11 bIn squaring stage, we connected the main stem from the 3rd to 5th fruit branches, which were divided into upper side and lower side for anatomical observation. The plant samples were immersed in FAA stationary solution . Samples were rehydrated in two changes of xylenes for 20 min and 75% alcohol for 5 min, rinse with running water. Subsequently put samples into safranin O staining solution for 15–30 s and dehydrated in a gradient alcohol series , after using plant solid green staining solution staining 6–20 s. Finally, put samples into three cylinders of xylene for 5 min each time, mount the tissue section with neutral balsam. A panoramic MIDI automatic digital slide scanner  was used for image processing and quantification.In the field, FBA was measured as the angle between the cotton stem and fruit branch and was recorded for the third to fourth fruit branch from the top to bottom and 10 typical plants were selected in 4 environments. The average value from 10 plants represents the phenotypic data of each accession and FBA across two replicates within 1 year was used for GWAS. A digital protractor was employed to measure the angle between the main stem and fruit branch of cotton.Spss25.0 was used for the statistical analysis of FBA. Correlation analysis of FBA for the association panel across different environments was performed in R software. QTL IciMapping 4.2 was used for analysis of variance (ANOVA) and broad-sense heritability. The broad-sense heritability was estimated according to the following equation: G. hirsutum reference genome TM-1 (WHU_updated v1) . Clean reads derived from 163 accessions were aligned against the ated v1)  using BWated v1) . After aated v1) . Subsequdmixture . The numdmixture . The phyN) . The best linear unbiased prediction (BLUP) and individual environment value was used as the phenotype for association analysis. Genome-wide association analysis was performed between SNPs and trait using the genome-wide efficient mixed-model association (GEMMA) (Q) and kinship (K) were employed to correct stratification.The association panel containing 163 samples, has a total of 2,499,987 SNPs (MAF ≥ 0.05). We used the sliding window method with default settings for 50 bp window size and 10 bp step size to view the SNPs across the whole genome. The threshold line of association analysis was set at −lg (1/ (GEMMA)  with a mQ ≤ 20) reads with more than 50% bases were removed  . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . The libraries were sequenced by Novogene  with an Illumina HiSeq  system. To gain more reliable data, reads from each data set with more than 10% N bases and low quality ( removed . Eventuaated v1)  by Hisatp-value by using the Benjamini and Hochberg’s method. Genes with a minimum 2-fold difference in expression, |log2FoldChange| > 1 and FDR < 0.05 were considered as DEGs.DESeq2 R package (1.20.0)  was usedp-value of the GO enrichment analyses were adjusted using the Benjamini and Hochberg’s method, and FDR < 0.05 was chosen as the threshold value for determining significantly enriched GO terms. Based on all expressed genes, PCA was carried out to explain the relationship among all samples.Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package. The −ΔΔCt) values  values . The reaWe constructed a high-density map with genetic variation based on re-sequencing data of 163 accessions with an average of 12.12-fold sequencing depth . In totaH2) of FBA was 68.70%  was used to determine correlation of phenotypic data between two environments. The minimum r was 0.24 (E2 and E3) and the maximum r was 0.46 . SNPs with p-value <5.31 × 10−6 were considered as significant SNP-trait associations. GWAS for each environment and BLUP phenotypes were performed. It is found that a total of 55 SNPs with significant association to FBA were identified, with 31 and 24 SNPs on chromosome A09 in two environments and D11 in single environment, respectively , which are significantly different in the FBA, were selected from the population. The average FBA of Xinluzhong 45 and Xinluzao 2 is 58.80° and 44.40°, respectively (p < 0.001) and between the upper side and lower side of per cultivar (p < 0.05)  ,D.To explain the differences in FBA of the fruit branch, the axillary buds (A), upper side (U) and lower side (L) of stem tissue were collected. Then, RNA sequencing was performed on these tissues at different stages of Xinluzhong 45 (Z45) and Xinluzao 2 (Z2) with three biological replicates. A total of 111.77 Gb of clean data were obtained by RNA sequencing. The Q30 reached 94.15%, multiply mapped reads and uniquely mapped reads accounted for 3.58–4.01% and 90.77–94.17% of these reads, respectively . To asseTo uncover the functional implications of 12,686 DEGs, the GO enrichment analysis was performed. The DEGs of all tissues were clustered into 5 categories according to expression patterns. GO enrichment analysis indicated that these five types of DEGs were mainly concentrated in ‘response to gravity’ (GO: 0009629), ‘regulation of actin filament length’ (GO:0030832), ‘response to red or far-red light’ (GO:0009639), ‘flavonoid metabolic process’ (GO:0009812), ‘gibberellic acid mediated’ (GO:0009937) and ‘abscisic acid-activated signaling pathway’  were identified from the 12,686 DEGs  of the large-FBA  was an important gene controlling cotton plant architecture, through its effects on hormones signal pathways, and over-expression of GhDREB1B confers a significant reduction in FBA  in stem and inflorescence branches. SAUR10 has an effect on branch angle as a function of auxin, brassinolide, light conditions and FUL (BnaC03g14890D (SAUR30) and BnaC03g16420D (SAUR55), were considered as key candidate genes controlling branch angle formation in B. napus  of the large-FBA  repository, accession number PRJNA874737.XN and MW designed and supervised the research. YW, XN, LL and MW revised the manuscript. ZP, YY, CY, QY, and XZ guided the content of the article. PS, NA, GS, CG, LC, LL, and XT investigated phenotypic of FBA in Korla and Shihezi. PS and QA collected the sample for RNA-seq. PS, JW, and YP performed the data analysis. PS and YP finished the writing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Fund for National Natural Science Foundation of China (no. 3206150567), the Innovation Leadership Program in Sciences and Technologies for Young and Middle-aged Scientists of Xinjiang production and Construction Corps, China (no. 2021CB028), Corps science and technology innovation talent plan-Science and Technology Commissioner (no. S2020CB1877), and the Key Programs for Science and Technology Development of Shihezi city, Xinjiang production and Construction Crops, China (no. 2022NY01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the study, we determined the influence of age, body mass index (BMI), and waist-to-hip ratio (WHR) on insulin response to oral glucose load (OGTT) and on insulin sensitivity (Si) and ß-cell function in young women with PCOS. One hundred fourteen patients with PCOS and 41 controls with normal basal plasma glucose were studied. A 75-g OGTT was performed to determine glucose tolerance and insulin response. Insulin sensitivity and ß-cell function were studied using a modified frequently sampled IV glucose tolerance test (FISGTT) to determine the acute insulin response (AIRG), as well as Si by minimal model analysis. Si was decreased in PCOS women , but no difference in AIRG existed between the PCOS and control group . BMI and WHR correlated negatively with Si , but not with AIRG . Increasing age correlated negatively with AIRG . There was a significant interaction between disease (PCOS), BMI, and WHR on Si as well as between age and PCOS on AIRG. Thus, patients below the age of 25 with PCOS showed enhanced AIRG  and decreased Si  compared to age-matched controls. In conclusion, these data suggest that not all patients with PCOS have basal and stimulated hyperinsulinemia, insulin resistance, and impaired glucose tolerance. Based on these data in young PCOS subjects, the development of insulin resistance and T2DM may be prevented with appropriate treatment strategies.Insulin resistance is believed to be an integral component of the polycystic ovary syndrome (PCOS). Beta ( Knockout experiments confirm that type 2 diabetes is a “2-hit” disease, in which insulin resistance is necessarily accompanied by a ß-defect, preventing the compensatory up-regulation of insulin secretion [Polycystic ovary syndrome (PCOS) is one of the most frequent endocrinopathies in women of reproductive age . The diaecretion . The preecretion . The cliecretion . A previecretion  suggesteecretion .i), and acute insulin response (AIRG) during a frequently sampled glucose tolerance test (FSIGTT) as well as the interactions of age, body mass index (BMI), and waist-to-hip ratio (WHR) with the PCOS disorder in a relatively young group of women with PCOS.Thus, the aim of the present study was to investigate the oral glucose tolerance test (OGTT), insulin sensitivity (S2), and WHR was determined by measuring the waist in centimeters at its smallest circumference and the hip in centimeters at the largest circumference. A BMI of 25 kg/m2 was determined as the borderline between overweight and nonoverweight subjects. All the investigated subjects had normal fasting plasma glucose (≤5.6 mmol/l) except one PCOS patient who had fasting plasma glucose of 5.8 mmol/L. All controls had normal glucose tolerance based on 2-hr plasma glucose levels during OGTT [114 women with PCOS and 41 years of age and BMI-matched healthy women were referred consecutively to the outpatient clinic of the Clinic for Endocrinology, Diabetes, and Diseases of Metabolism for clinical hyperandrogenism and/or menstrual dysfunction. PCOS was diagnosed according to the Rotterdam workshop criteria, i.e., the presence of at least two among the three following features: clinical and/or biochemical hyperandrogenism, chronic oligoanovulation, and polycystic ovary morphology (PCOM), after exclusion of secondary causes . Appropring OGTT . Women wOral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) with frequent blood sampling (FSIGTT) were conducted on separate occasions in all subjects. Tests were performed after 3 days on a 300 g per day carbohydrate diet and after an overweight fast of 10 hr. Blood samples for plasma glucose and plasma insulin were drawn at baseline and every 30 min for 2 hr, after a 75-g glucose load. The modified IVGTT (FISGTT) was also performed after overnight fasting, according to the previously published procedures , 12. Afti) was calculated by a minimal model analysis using the MINMOD computer program [G) was calculated as the mean increase in insulin levels calculated from 2 to 10 min of IVGTT. The disposition index was calculated according to the following formula: DI = Si × AIR. The glucose tolerance to IV glucose load (Kg) was calculated according to the standard procedures [T-test, and age and BMI multivariate probability distribution was compared using the peacock test. Multivariate probability distributions were not different between the groups (p=0.305).The area under the glucose (AUCG) and insulin (AUCI) response curves during OGTT was calculated by the standard trapezoidal rule. The insulin sensitivity index . All analysis was done for the whole group, but some data are presented for obese and nonobese separately. All analysis was performed using SPSS and the R software package.p < 0.001) in PCOS than in control women (3.73 ± 0.16 vs. 1.89 ± 0.10 nmol/L). As regards PCOS clinical phenotypes, 109 (95.62%) women had hyperandrogenism and polycystic ovary morphology (PCOM) and 4.38% (5 women) had hyperandrogenism and oligoanovulation.The clinical characteristics of PCOS women and controls are presented in p > 0.05) (p < 0.05). Plasma glucose at the 2 hr point of OGTT was higher in the PCOS group than in controls (p < 0.05)  . There w < 0.05) , Table 2 < 0.05) . BMI (Fi < 0.05)  and WHR  < 0.05)  were pos < 0.05) . AUCG wap < 0.05)  (Tables p < 0.05)    Tables . BMI and Figures . Signifi Figures , as well Figures .i was decreased in patients with PCOS compared to controls (p < 0.05) (i (p < 0.001)  (G decreased in both groups. Interaction between age and PCOS on this parameter shows that AIRG declines more with aging in the PCOS group (p < 0.001) (i × AIR) was decreased in the PCOS group but not significantly compared to controls (p > 0.05) (G was higher in the PCOS subgroup A than in age-matched controls (p < 0.05) (i was decreased in the PCOS group (p < 0.05) and DI was not significantly decreased in the PCOS subgroup A compared to the control subgroup A (p=0.061) . A positive relationship between BMI and WHR with PCOS was also confirmed (p < 0.05). The best predictors of BMI as well as WHR were presented in S < 0.05) . There w Figures . Results > 0.05) . With in< 0.001) . Disposi > 0.05) . When PC < 0.05) . Si was  Figures   and diabetes increased in PCOS , 15. Ourcontrols . Furthercontrols . Furthercontrols .Android obesity, clinically confirmed as increased WHR, is an especially strong risk for insulin resistance and other factors that predispose to premature cardiovascular disease . AlthougG) in healthy subjects is a constant value that has been termed the disposition index (DI) [G will be higher as a compensatory mechanism in the state of insulin resistance [The product of insulin sensitivity (Si) and insulin response (AIRdex (DI) . It is esistance . On the sistance . In our G between PCOS and controls. Results from this study clearly show a significant negative correlation between AIRG and increasing age. With an increase in age, AIRG falls faster in patients with PCOS. Interestingly, our data did not confirm the significant influence of obesity on this parameter [Interestingly, the data show that there is no difference in AIRarameter .ß-cell function could be an important determinant of the development of T2DM in many of these women [ß-cell dysfunction seems to be an integral characteristic of this syndrome [While insulin resistance is a factor in the development of PCOS, the associated failure of pancreatic se women –28. Althse women , 30. Thusyndrome . A recensyndrome , 31.ß-cell secretory defects may contribute to increased carbohydrate intake and subsequently obesity and insulin resistance. Current data support the notion that glucose intolerance and frank T2DM are found in combination with the sign of ß-cell exhaustion in a significant portion of young women with PCOS. These subjects with PCOS may manifest later stage carbohydrate intolerance and T2DM after long-standing insulin resistance in susceptible women [According to a previously published report , ß-cell le women . In thisG in comparison with age-matched controls as well as compared to older PCOS women (subgroup B). The exaggerated AIRG in our young patients with PCOS could be a compensatory response to underlying insulin resistance. Despite the compensatory response, this group failed to achieve a DI observed in controls.In the youngest PCOS women (subgroup A), we demonstrated exaggerated AIRG was enhanced, the DI was still decreased compared to age-matched controls due to significantly impaired Si, placing these patients at heightened risk for T2DM. Studies on young adolescent girls may provide clues about the pathophysiology of PCOS since this is an age when early clinical signs are manifested. Thus, this may be the time to initiate hygienic and medical interventions to retard the development of impaired glucose intolerance and T2DM [Current data are in concert with prior reports that significant abnormalities in insulin secretion are already present in patients with PCOS <25 years , 32. Altand T2DM –31, 33. and T2DM , 34–49.The major strength of this study is the large number of PCOS women investigated using minimal model analyses to evaluate acute insulin response and assess insulin secretion as well as insulin sensitivity. However, there are also limitations in the study. This is an observational study, and cause-effect relationships cannot be firmly established. In addition, as most women in this cohort had a hyperandrogenic PCOS phenotype, the results of the study may be more applicable to these subjects.ß-cell secretion before T2DM develops.In conclusion, current observations underlie the importance of interactions between PCOS, BMI, age, and WHR. This investigation has also shown that not all patients with PCOS demonstrate basal and stimulated hyperinsulinemia, insulin resistance, and impaired glucose tolerance, particularly early in the evolution of PCOS as a clinical entity. Our data concerning subjects younger than 25 years underscores the importance of establishing the diagnosis of PCOS in adolescence, and the institution of appropriate therapy targeting insulin resistance and


Analyses of the calibration charts show that nomograms have good performance and clinical applicability. In our study, renal color ultrasound parameters correlated well with TA/IF in IgAN. By establishing a conventional color ultrasound prediction model, we can accurately gauge the extent of TA/IF in patients with IgAN for clinical applications.In this study, we aimed to explore the clinical value of routine color ultrasound parameters in the evaluation of tubular atrophy and interstitial fibrosis (TA/IF) in IgA nephropathy (IgAN). We enrolled 725 patients with IgAN who underwent renal biopsy at the First Affiliated Hospital of Anhui Medical University between January 2019 and May 2022. Examinations were performed to measure the routine ultrasound renal parameters and renal biopsy was done within next three days. Univariate and multivariate analyses were used to determine the correlates and the independent predictors of TA/IF. Simultaneously, a nomogram based on risk indicators was created to predict TA/IF. Univariate and multivariate analyses showed that sex ( IgA nephropathy (IgAN) is the most common primary glomerulonephritis in China and is the main cause of end-stage renal disease ,2. ReporTherefore, a noninvasive and accurate technique is needed to detect and monitor TA/IF. Complex and practically difficult techniques of functional imaging have been tried in some studies without getting ideal results . ClinicaThis study was approved by the institutional review committee of the First Affiliated Hospital of Anhui Medical University (PJ2022-11-29). We enrolled 725 renal biopsy-proven IgAN patients at the First Affiliated Hospital of Anhui Medical University from January 2019 to May 2022.The inclusion criteria were as follows: (1) renal biopsy-proven IgAN; (2) at least 10 glomeruli in the specimen visible under light microscope; (3) age of the patient more than 18 years.The exclusion criteria were: (1) acute renal damage, heart valve disease, and heart failure; (2) renal artery stenosis or urinary tract obstruction; (3) cysts or tumors in the kidneys; (4) Doppler patterns suggestive of renal artery stenosis .Four radiologists with a minimum of 5 years’ experience in routine ultrasound performed the examinations and acquired images .The ultrasound instruments used to acquire the images in this study included: Mindray Resona 7 ; GE Vivid E9 ; Aplia 500 ; a 2–5 MHz frequency convex array transducer (C5-2) was used as a probe.The patient fasted for more than 8 h. Patient was made to lie in the supine position. The following parameters were measured by ultrasound (during end-inspiratory breath-hold): kidney size  and renal echo . Images were obtained in Doppler and pulse Doppler modes. The ultrasonic probe is gently positioned in the right abdomen in the form of oblique projection, visualizing the kidney as a longitudinal image, placing the Doppler beam on the trunk of the right renal artery, measuring the peak systolic velocity (PSV) of the main renal artery (MRA), and measuring the PSV and end diastolic velocity (EDV) for an average of 2–3 times to calculate the resistance index (RI)=(PSV – EDV)/PSV . IntrareThe indicators of a renal biopsy are as follows: (1) acute nephritis syndrome ; (2) primary nephrotic syndrome ; (3) clinical diagnosis of denatured red blood cell hematuria is unclear; (4) proteinuria continues to be greater than 1 g/d, and the diagnosis is unclear.Renal biopsy from right kidneys was performed by two experienced nephrologists within three days of renal ultrasound measurements. Light, electron, and immunofluorescence microscopies were used to examine biopsied tissues. All biopsy samples to be valid, needed to have at least 10 glomeruli each on light microscopy. MEST-C criteria put forward in Oxford classification of IgAN were used to score the following histopathologic findings: mesangial cell proliferation (M0/M1), endocapillary hypercellularity (E0/E1), segmental glomerulosclerosis (S0/S1), tubular atrophy/interstitial fibrosis , and crescents (C) . The perp < 0.1) thus discovered were analyzed by multifactor logistic regression analysis to determine the independent predictors significantly related to TA/IF. Nomograms were constructed using the variables in the final multiple logistic regression model to evaluate the indicators of TA/IF. The discriminative ability of predictive nomograms was evaluated by Harrell’s concordance index (C-index) [C-index and area under curve have similar meanings. The highest value of C-index is 1, which means a perfect distinction and the minimum value is 0.5, representing random distinction. Calibration is used to compare the probability predicted by our nomogram with the actual probability of TA/IF. p < 0.05 (two-tailed) indicated significance. IBM SPSS Statistics 26 statistical software package , R language software and RMS software package  were used.Due to limited number of samples, TA/IF1 and TA/IF2 cases were combined into one group. Single factor logistic regression analysis was used to analyze the correlation between ultrasonic parameters and TA/IF. The related factors (C-index) . C-indexA total of 725 patients with IgAN  were included in this study with 346 male (47.7%) and 379 female (52.3%), and an average age of 39.5 ± 12.5 years. Among the cases, 541 cases were TA/IF0, while 184 were TA/IF1 and TA/IF combined. The clinical and pathological characteristics of patients are listed in p = 0.005), sex (p < 0.001), renal length (p < 0.001), PSV of MRA (p < 0.001), RI of MRA (p = 0.005), PSV of SRA (p < 0.001), cortex echo (p < 0.001), and the thickness of renal cortex (p = 0.076) were associated with TA/IF. The above factors with p < 0.1 were further included in the multivariate logistic regression. The results showed that sex , renal length , RI of MRA , PSV of SRA , and cortex echo  are independent predictors of TA/IF (Routine ultrasonic characteristics of IgAN patients are shown in of TA/IF . RI of MC-index of 0.765 (95%CI = 0.727–0.803)  . The cal7–0.803) .C-index of 0.765. With this model, we can estimate TA/IF in patients with IgAN. To the best of our knowledge, this is the first large sample-size study to use color ultrasound to predict IgAN TA/IF. Irreversible damage to the nephrons, due to chronic glomerular or tubular injury, lead to TA/IF around the damaged nephrons [p = 0.01). Based on our experience, the Oxford classification predicts progressive kidney disease, but the degree of TA/IF is the only independent predictor [Based on univariate and multivariate analyses of large number of renal biopsy samples and certain ultrasound parameters in IgAN patients, this study systematically analyzed the relationship between color ultrasound parameters and TA/IF. Our results show that sex, renal length, RI of MRA, PSV of SRA, and cortex echo are the five independent noninvasive predictors of TA/IF. Moreover, we developed nomograms based on the identified indicators because nomograms can integrate all these variables and estimate the TA/IF degree of each patient separately. The nomogram model based on color ultrasound indicators showed good discrimination ability, with a redictor . Yu et aredictor . Coppo eredictor . The degredictor . The evaMale is independently associated with poor kidney outcomes . Huang eSimplicity and convenience of color ultrasound makes it the preferred method for renal morphology and blood flow evaluation by nephrologists. Longitudinal renal length is considered to be a key indicator of chronic kidney disease because it decreases with the gradual decline of eGFR . In IgANThe limitations of our study are as follows: (1) the use of time-varying covariates in proportional hazard models may produce time-varying confounding limitations in a single-race single-center design; (2) only adults are included in our analyses, so the prediction model may not be applicable to children; (3) in this study, TA/IF1 and TA/IF2 were merged, the study had limited TA/IF2 samples, which can be further studied in future research.In conclusion, the results of our study bring out the correlation between color US parameters and TA/IF in IgAN. By establishing a US prediction model, we can accurately gauge the degree of TA/IF in patients with IgAN. This model is convenient and noninvasive and hence applicable in daily clinical practice after validation from future studies.


I2 = 63.735%). We conducted a meta-analysis of the factors affecting patency after microsurgical IVE, finding that the presence of motile sperms in epididymal fluid , anastomosing bilaterally  and distally  lead to higher patency rates. IVE is an effective treatment for EOA. The presence of motile sperms found in the epididymal fluid, anastomosing bilaterally and distally are significantly correlated with higher patency rates.A systematic review and meta-analysis of microsurgical vasoepididymostomy (MVE) for treating epididymal obstructive azoospermia (EOA) with different intussusception techniques. We conducted a comprehensive literature search using PubMed, Embase, and the Cochrane Central Register of Controlled Trials, retained literature related to obstructive azoospermia or male infertility and vasoepididymostomy, proactively reviewed other relevant literature, supplemented valuable references, and excluded studies that did not use intussusception and where valuable statistical data were difficult to obtain. Event rate and risk ratio (RR) were estimated. Patency rates were investigated. The influence of motile sperms found in the epididymal fluid, anastomotic sides and sites on patency was evaluated. 273 articles were comprised in this analysis, and 25 observational studies were eventually included, with a total patient sample of 1400. The overall mean patency rate was 69.3% (95% confidence interval [CI] 64.6–73.6%;  Microsurgical longitudinal intussusception vasoepididymostomy (LIVE) has been the preferred operation for EOA treatment since 20043, and this procedure requires superior surgical skills and meticulous surgical technique4.Microsurgical vasoepididymostomy (MVE) is an effective technique for epididymal obstructive azoospermia (EOA) to improve male fertility. Since the new century, intussusception vasoepididymostomy (IVE) has become the mainstream mode of MVE, which has a higher surgical patency rateEvaluating the differences in surgical results with different techniques and finding out the factors affecting surgical results will provide the basis for the decision-making of surgical methods and obtain better surgical results.This analysis aims to systematically review the evidence for IVE treatment of EOA, explore differences in outcomes among different IVE techniques, and provide a meta-analysis of their effectiveness.This systematic review and meta-analysis were reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and MOOSE Guidelines for Meta-analyses and Systematic Reviews of Observational studies.I2 values.Databases searched included PubMed, Embase, and the Cochrane Central Register of Controlled Trials from inception to March 20, 2022. Exhaustive electronic search was conducted by two independent authors (Wang S. and Fang Y.) using the subject terms ((“azoospermia” [All Fields]) OR  OR  OR (“epididymis obstruction” [All Fields]) OR  OR (“infertility” [All Fields]) OR (“fertility” [All Fields])) AND ((“vasoepididymostomy” [All Fields]) OR (“epididymovasostomy” [All Fields])). The two independent authors evaluated the searching results. The senior author (Hong K.) reviewed these articles to ensure their relevance to our study and determined the articles included. The search strategy was initially developed for PubMed and subsequently adapted for the other databases. Event rate and risk ratio (RR) were estimated using a random‑effects model. Heterogeneity was investigated using the Q statistic and During the screening, we included articles evaluating the effect of vasoepididymostomy on EOA patency rate and those comparing the effect of different factors on patency. We excluded review articles, animal studies, < 10 case series, and a previous meta-analysis. We excluded articles in which IVE was not carried out in treating EOA. There are no language or publication date restrictions for this analysis.We extracted the following data from the retrieved studies: author names and years of publication, methods of study design, surgical techniques, sample sizes, patients’ and partners’ ages, patency rates, pregnancy rates and follow-up. We also extracted intraoperative information, including the presence or absence of motile sperms in epididymal fluid, anastomotic sides and sites of epididymal.https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools, was used for uncontrolled before-after studies. This tool is composed of 12 items that could be answered as “Yes”, “No”, “Not Reported” or “Not applicable”, and the overall quality of each study could be classified as “Good”, “Fair” or “Poor”. In this analysis, however, the 12th item should always be answered as “Not applicable”, thus the highest quality score theoretically is 11.The NIH “Quality Assessment Tool for Before-After (Pre-Post) Studies with No Control Group”, which can be achieved at P values are two-tailed, and P < 0.05 was considered statistically significant.RevMan 5.3 (Cochrane Community) and Comprehensive Meta-Analysis V3  were used for the meta-analysis. SPSS 24.0 (IBM) was used for statistical analysis of quality assessment. Event rates or RR and 95% confidence interval (CI) for dichotomous variables were investigated. Due to the heterogeneity of the included studies, the Mantel–Haenszel random-effects model was used. All P value is < 0.10.Q statistics were used to test homogeneity between studies. Homogeneity is rejected when the Q statistic The effects of IVE on patency, in total and in different techniques respectively, were shown by the forest plots, which contain a pooled estimate of the effect (event rate or RR) as a dashed vertical line with a diamond at the bottom representing the 95% CI. Individual studies are represented as squares with their CIs, and the weight of each study is represented by the proportion of its corresponding square.28. A PRISM flow chart describing the results of study identification and selection is shown in Fig. Among 273 retrieved articles, 25 met the eligibility criteria and were included in the system reviewAmong these 25 studies (9 prospective cohort studies and 16 retrospective cohort studies), 1400 patients in the meta-analysis were aggregated qualitatively and quantitatively .The publication bias in this analysis was shown in funnel plots Fig. . There w4 sperms/ml (8) and > 106 sperms/ml (1). In other studies, intact sperm (2), motile sperm (1), or any sperm (9) seen in the ejaculates were considered patency. The definition of patency was not clear in the remaining 4 studies. Natural pregnancy observed is always considered patency.The definition of patency after IVE varied among all the 25 studies. 9 studies defined patency in different sperm concentrations: > 10I2 = 63.735%), 3-suture double-armed triangulation and 2-suture double-armed transverse IVE mean patency rate was 77.2%  (75.9 and 80.0% respectively). LIVE mean patency rate was 67.6% .Patency was analyzed according to different surgical techniques in all the 25 studies (27 sequences) Figs. , 4 and 5Although “pregnancy rates” were reported in 20 studies Table , the defFactors affecting patency after IVE were analyzed and presented by forest plots Fig. .Figure 6P = 0.001).In 10 studies involving 626 patients, those with the presence of motile sperms found in epididymal fluid showed higher patency rate .13 studies involving 839 patients analyzed the patency rate according to anastomosis sides . Compared with the unilateral group, patients in the bilateral group exhibited higher patency rate .The effect of anastomotic sites on patency rate was analyzed in 11 studies involving 796 patients. Distal anastomosis leads to a higher patency rate than proximal one  involving 1400 patients, the efficacy of IVE was analyzed, and the total patency rate was 69.3%, which confirmed that this surgical technique had good efficacy in treating patients with EOA, and was proved to have a higher patency rate than previous MVE surgical techniques1.Several systematic evaluations have preliminarily confirmed the safety and efficacy of MVE in the treatment of EOA3.Contrary to common belief, we found that other IVE techniques (3-suture double-armed triangulation and 2-suture double-armed transverse IVE) can achieve a higher patency rate than LIVE (77.2 vs. 67.3%). We tried to explain this result as follows. The triangulation technique, which is not a mainstream technique nowadays, was reported in earlier studies. As was described in the earlier studies, in the cases where the epididymal tubule was too small to accommodate three micro sutures, the 2-suture technique, especially the longitudinal technique, should be used. Therefore, the triangulation technique showed a higher patency rate because of the bias of its patient population with a relatively wider epididymal tubule. It is similar to the 2-suture transverse technique.Currently, LIVE is considered to be the preferred operation for treating EOA30.Several studies have suggested that motile sperm detected in epididymal fluid, bilateral, and distal epididymal anastomosis can improve the patency rate. In this analysis, we confirmed that motile sperms found in epididymal fluid (80.8% [291/360] vs. 56.8% [151/266]), bilateral anastomosis (74.3% [746/641] vs. 53.55% [106/198]) and distal anastomosis (73.9% [465/629] vs. 48.5% [81/167]) lead to obvious higher patency rate. According to this conclusion, urologists can make more appropriate surgical decisions under certain objective conditions, to improve the patency rate and promote the rehabilitation of male fertility. Bilateral IVE should be considered in all circumstances that are possible in EOA. Moreover, IVE may lead to natural pregnancy, avoiding the risks of assisted reproductive technology (ART) to both female partners and fetuses21; Zhao, L. et al.18) which may cause unpredictability bias. Increasing the sample size can further eliminate the influence of bias. Combined with the result that “different IVE techniques obtain different patency rates” found in this analysis, which is contrary to popular belief, it is feasible to design randomized controlled trial (RCT) studies of different surgical techniques on the patency rate of IVE. Finally, other factors which may affect patency rates were not systematically reviewed and analyzed limited by the number of studies.At the same time, we recognize several limitations of this analysis. First of all, we found differences or ambiguities in the definitions of “patency rate” and “pregnancy rate” in previous studies. Especially for “pregnancy rate”, the same name means different parameters indeed. Then, this analysis only included observational studies and most of the studies (16) were retrospective. In addition, several included studies were performed in same centers (Peng, J. et al.Microsurgical vasoepididymostomy using intussusception technique proved to have good patency rates. Thus, IVE is effective in improving the fertility of men from EOA. The presence of motile sperms in perioperative epididymal fluid, and anastomosing bilaterally and distally are significantly correlated with higher patency rates in IVE.


To describe the use of warfarin and direct oral anticoagulants (DOACs) in patients with atrial fibrillation (AF) and chronic kidney disease (CKD), to evaluate changes in renal function over time and predictors of rapid decline, and to describe time in therapeutic range (TTR) and predictors of poor TTR among patients on warfarin.2), mild CKD (60–89 mL/min/1.73 m2), moderate CKD (30–59 mL/min/1.73 m2), severe CKD (15–29 mL/min/1.73 m2) and end-stage CKD (<15 mL/min/1.73 m2)/dialysis. TTR was estimated using international normalised ratio (INR) measurements. Predictors of eGFR decline over time and of poor TTR were estimated using regression analysis. Between 2013 and 2018, use of DOAC increased from 9.2% to 89.3%, with a corresponding decline in warfarin. A similar trend was observed in patients with mild to moderate CKD, while DOAC over warfarin increased slower among patients with severe to end-stage CKD/dialysis. In patients treated with warfarin, the median TTR was 77.1%. Worse TTR was observed among patients with severe CKD (70.0%) and end-stage CKD/dialysis (67.5%). A gradual annual decline in eGFR was observed (−1.1 mL/min/1.73 m2), with a more rapid decline among patients with older age, female sex, diabetes mellitus and/or heart failure.Using data from AuriculA, the Swedish oral anticoagulation registry, patients with AF on warfarin or DOAC were identified between 2013 and 2018 (N=6567). Estimated glomerular filtration rate (eGFR) was calculated and categorised into normal (≥90 mL/min/1.73 mIn patients with AF, use of DOAC has steadily increased across different CKD stages, but not in patients with severe to end-stage CKD/dialysis despite these patients having poor INR control. Patients with AF have a gradual decline in renal function, with a more rapid decline among a subgroup of patients. There are conflicting data on whether warfarin treatment is safe and effective in patients with chronic kidney disease (CKD), particularly in those with end-stage CKD.The absolute change in renal function over time and the predictors of rapid decline in renal function among patients treated with warfarin or direct oral anticoagulants (DOACs) are also unknown.Treatment with DOAC has increased drastically between 2013 and 2018, with a corresponding decrease in use of warfarin in patients with atrial fibrillation (AF).Similar trends were seen in patients with mild to moderate CKD, while use of DOAC in preference to warfarin increased considerably slower among patients with severe CKD and end-stage CKD/dialysis.In patients treated with warfarin, poor warfarin control with low time in therapeutic range was observed to a higher extent among those with severe CKD and end-stage CKD/dialysis.Also, there was a gradual decline in renal function over time in patients with AF, with a more rapid reduction among patients with older age, female sex, diabetes mellitus and/or heart failure.This study underlines the importance of close follow-up of renal function in selected patients at risk to allow timely adjustment of oral anticoagulant treatment in terms of drug of choice and correct dosage.For patients on warfarin with severe CKD and end-stage CKD/dialysis, close follow-up of international normalised ratio or change in oral anticoagulant strategy might be warranted.Clinical practice guidelines recommend direct oral anticoagulants (DOACs) over warfarin for stroke prevention in patients with atrial fibrillation (AF) having risk factors for ischaemic stroke.8All DOACs are dependent on renal clearance to various degrees, causing a potential risk of excessive drug exposure and consequently a higher risk of associated bleeding in patients with CKD.12 142.2.1Despite reports suggesting favourable benefit to risk ratios with DOAC versus warfarin in patients with worsening renal function, the Swedish Medical Product Agency recommends warfarin over DOAC in patients with CKD and with estimated glomerular filtration rate (eGFR) below 25–30 mL/min/1.73 mCurrently, there are ongoing RCTs evaluating DOACs versus warfarin in patients with severe to end-stage CKD. While awaiting the results, it is important to understand how healthcare professionals weigh the benefits and risks and interpret the data in relation to guidelines when deciding which OAC to prescribe to patients with AF and CKD. Thus, the aims of the present real-world study were to illustrate (1) treatment patterns with warfarin and DOACs in patients with AF and CKD, (2) TTR in patients with AF and CKD treated with warfarin, (3) predictors of poor TTR in patients treated with warfarin, (4) changes in eGFR over time in patients treated with warfarin or DOACs, and (5) predictors of worsening eGFR in patients treated with warfarin or DOACs.The study cohort was identified in the Swedish national quality register for AF and oral anticoagulation (AuriculA). AuriculA has over 150 000 active patients and is estimated to include approximately 50% of all patients with AF in Sweden.10.1136/openhrt-2022-002043.supp1Supplementary data2), mildly decreased (eGFR 60–89 mL/min/1.73 m2), moderately decreased (eGFR 30–59 mL/min/1.73 m2), severely decreased (eGFR 15–29 mL/min/1.73 m2) and end-stage CKD (eGFR <15 mL/min/1.73 m2)/dialysis.22Information about medical treatment with warfarin or DOAC and dosages was obtained from AuriculA. Information about eGFR was calculated based on serum creatinine concentrations using the Chronic Kidney Disease Epidemiology Collaboration equation.2 for categorical variables. Treatment strategies with warfarin and DOACs over years were illustrated using line graphs with proportional percentages. Multiple regression analysis was used to assess the association between CKD stage and TTR, with TTR being the dependent variable and baseline characteristics being the independent variables , prior myocardial infarction, prior revascularisation (percutaneous coronary intervention or coronary artery bypass graft surgery), heart failure, peripheral vascular disease, chronic obstructive pulmonary disease (COPD), cancer, prior major bleeding, and concomitant antiplatelet therapy (aspirin or P2Y12 inhibitor)). This analysis was performed on patients treated with warfarin and in whom serial INR measurements were available . Temporal changes in eGFR from index date and onwards were illustrated using line graphs indicating median values with CIs. The association between baseline characteristics and the annual change in eGFR was examined using logistic regression analysis, where an annual drop in eGFR ≥20% was the response variable, with baseline characteristics being the independent variables . This analysis was performed in patients with available serial measurements of eGFR . All statistical analyses were performed using R V.4.1.1 (The R Foundation). Two-sided p value <0.05 was considered statistically significant.Patient characteristics, risk factors, comorbidities and medical treatment were reported in a tabular format with median and IQR for continuous variables and with frequencies and percentages for categorical variables across the prespecified CKD strata. Statistical differences were reported using Kruskal-Wallis for continuous variables and using χ2DS2-VASc score   in and sex) .Between 2013 and 2018, the proportion of patients treated with warfarin decreased from 90.8% to 10.7%, while the proportion treated with DOAC increased from 9.2% to 89.3% . When caIn total, 190 814 INR measurements (median of 49 measurements per patient) were available during a median follow-up of 1.7 years. The median TTR was 77.1% in patients treated with warfarin, with marginally poorer TTR observed among patients with worsening CKD stage . In patiClinical predictors associated with poor TTR are presented in 2, and a total of 1415 (34.9%) patients had a decline in eGFR ≥20% from baseline (2 and −1.1 (−4.7 to 2.1) mL/min/1.73 m2, and a total of 955 (36.6%) and 460 (31.9%) patients had a decline in eGFR ≥20% from baseline, respectively  were available during follow-up. Serial eGFR measurements with at least two measurements over a 1-year period were available in 4055 (61.7%) patients included in this study. Among the subgroup of patients with serial measurements of eGFR, the median annual decline in eGFR was −1.1 (25th–75th percentile: −4.4 to 1.3) mL/min/1.73 mbaseline . In patiectively . In addiectively .Worsening eGFR with an annual decline ≥20% from baseline was associated with several patient characteristics and comorbidities in the adjusted analysis, including age, female sex, diabetes mellitus and heart failure .We showed in this real-world evidence study that use of DOAC has increased between 2013 and 2018, with a corresponding decrease in warfarin in patients with AF. A similar trend was seen for patients with mild to moderate CKD, while use of DOAC in preference to warfarin increased considerably slower among patients with severe CKD and end-stage CKD/dialysis. In warfarin-treated patients, poor TTR (<70%) was observed to a higher degree among patients with severe CKD and end-stage CKD/dialysis. In addition, this study verified that there is a gradual decline in renal function over time in patients with AF, with a more rapid reduction among patients with older age, female sex, diabetes mellitus and heart failure.CKD is a common finding among patients with AF and it is believed that CKD might contribute to the development and progression of AF.292.In the present study, we found that treatment with DOACs steadily increased with time since their introduction, with a concurrent decline in warfarin. This trend was observed in patients with mild to moderate CKD, but not in patients with severe CKD or end-stage CKD/dialysis. This is in accordance with current recommendations provided in guidelines and is similar to changes seen across other European countries.2, with a more pronounced decline observed among patients with older age, female sex, diabetes mellitus and heart failure. Thus, in patients with normal renal function and with no risk factors, there seems to be only a modest need for close monitoring of renal function. However, for patients with poor renal function at treatment initiation or for patients with the above risk factors for rapid decline in renal function, more frequent monitoring of renal function might be advisable. For patients treated with DOACs, which are all partially eliminated by the kidney, frequent monitoring of renal function might be justified in those with risk factors for rapid decline in renal function or for patients where dose reduction or treatment discontinuation might be warranted due to renal limitations. As an example, the results showed that a quarter of the patients with an eGFR above 50 mL/min/1.73 m2 had an eGFR below 50 mL/min/1.73 m2 after a median follow-up period of 1.7 years, a cut-off point where most of the DOACs need to be dose-adjusted.Another important finding in this study is that patients with AF have a gradual decline in renal function over time. These findings are in accordance with prior observations made in patients with AF.The present study has several strengths and some limitations that merit acknowledgement. The key strength is the inclusion of consecutive real-life patients with AF with no loss to follow-up. Furthermore, the good availability of measurements of creatinine-based eGFR and INR and, in many patients, serial measurements of eGFR and INR are also strengths. However, for serial measurements of eGFR, only 61.7% of patients had two or more measurements available over a 1-year period, which is a limitation to parts of this study. The inclusion of patients with complete information on baseline characteristics and antithrombotic treatment provided opportunities to study the associations between renal function and TTR and time-dependent decline in renal function over time. A limitation is that we only had access to renal function covering patients in one region of Sweden, which might not be representative of nationwide estimates. Another limitation is that we did not have access to the INR measurements made using home monitoring devices. However, the study population had a median of 44 INR measurements per patient during the entire follow-up and the INR control in this cohort is likely to be closer to daily clinical practice than in clinical trials. Finally, we only accounted for comorbidities and medications available at baseline when assessing the associations between renal function, TTR and decline in renal function over time and thus residual confounders might be present.In real-life patients with AF, treatment with DOAC over warfarin has dramatically increased during the last years across normal and moderate levels of CKD. However, this trend was not observed among patients with severe to end-stage CKD/dialysis despite these patients having poor INR control. A gradual decline in renal function was observed among patients with AF over time, with a more rapid decline observed among those with older age, female sex, diabetes mellitus and/or heart failure. A close follow-up of patients at risk might be crucial to timely adjust OAC treatment in terms of drug of choice and correct dosage.


Armeria maritima subsp. elongata. Three accessions  from relatively dry sandy soil habitats in the Baltic Sea region were selected and compared using both in vitro cultivated shoot explants and long-term soil-cultivated plants at flowering stage. Growth of root non-forming explants treated with increasing concentrations of NaCl was significantly inhibited starting from 110 mmol L−1, and the rate of shoot formation was even more sensitive. Significant differences in morphology and responses to salinity were found between different accessions. For soil-grown plants, biomass accumulation in above-ground parts was relatively little affected by salinity in AM1 and AM2 in comparison to that in AM3. Differences in ion accumulation were evident between the accessions as well as in respect to cultivation system used. Maximum accumulation capacity for Na+ was up to 2.5 mol kg−1 both in shoot explant tissues and in old leaves of soil-grown plants treated with NaCl, but that for K+ reached 4.0 mol kg−1 in old leaves of soil-grown plants treated with KCl. Non-ionic component of osmotic value was relatively high in old leaves and significantly increased under NaCl treatment, especially for AM2 and AM3 plants at moderate salinity, but in AM1 only at high salinity. In contrast, it significantly decreased in old leaves of AM2 plants treated with increasing concentration of KCl. It can be concluded that a wide salinity tolerance exists within A. maritima accessions from dry sandy soil habitats, associated with the ability to accumulate surplus ions both in salt glands and old leaves.The aim of the present study was to compare tolerance to salinity and ion accumulation potential of  Increasing soil salinity is one of the major threats to agricultural production, especially, in a light of global climate change-dependent increase in environmental heterogeneity ,2,3. The+ toxicity in the form of NaCl. In the context of salinity, K+ has been analyzed mostly in a view of necessity to maintain high cytoplasmic K+/Na+ concentration ratio [+ excluders, regulating tissue electrical conductivity (EC) by changes in K+ concentration, K+ excluders, regulating EC by changes in Na+ concentration, or tight EC regulators [+ in substrate will have the same negative effects than Na+ [– [In contrast to salt exclusion approach used by salt tolerant glycophytes, tight control of compartmentation of salinity-related ions is one of the adaptive mechanisms used by plants native to saline habitats . As ionion ratio ,7. It wagulators . There ithan Na+ . It was n Na+ [– .Trifolium fragiferum and concluded that high intraspecies variability in morphological and physiological responses to salinity exist between geographically isolated populations [Halophyte species, native to salt-affected habitats, represent a valuable resource for studies of physiological mechanisms related to salinity tolerance ,12. Usuaulations .Atriplex halimus [Sesuvium portulacastrum during high salinity [Salicornia europaea [Salicornia brachiata [Limoniastrum monopetalum [Crithmum maritimum [Tissue culture has been often used as a tool for screening crop plant genotypes for their salinity tolerance ,21,22. I halimus . Axillarsalinity . Shoot ceuropaea , Salicorrachiata  and Limoopetalum  were usearitimum . Severalaritimum ,31. Comparitimum .Armeria maritima (Mill.) Willd. (Plumbaginaceae) is characteristically found in temperate open habitats with dry, saline, sandy or metal-rich soil [A. maritima plants from eight populations representing five different habitats in Britain  it was found that each population showed different ecological responses viewed as a result of local adaptation [A. maritima exhibit phenotypic plasticity [A. maritima in salt-affected habitats raises the question of salt tolerance of the species and its mechanisms. Previously, all ecotypes of A. maritima from different habitats have shown relatively high salinity tolerance, similar to that of species from brackish conditions of upper salt marsh, and even shoot growth stimulation at low salinity [A. maritima plants from different populations is related to phenotypic plasticity or local genetic adaptation. In this respect, it is reasonable to ask whether there are differences between A. maritima accessions growing in habitats with relatively similar conditions but being geographically isolated, in respect to their responses to salinity. Recently we performed a comparative study of heavy metal tolerance and metal accumulation potential of three geographically isolated A. maritima accessions from a dry coastal meadow, and showed significant species-wide metal tolerance and extremely high accumulation potential of A. maritima but with some accession-specific differences [A. maritima subsp. elongata accessions from a dry coastal meadow in controlled conditions. A special attention was paid to possible different growth responses and ion accumulation potential to variable treatment with NaCl or KCl. It was hypothesized that similar effect of Na+ and K+ on growth of A. maritima plants will be found.Rosette-forming evergreen perennial species ich soil . In a staptation . Howeverasticity . Presencsalinity . Howeverferences . TherefoA. maritima subsp. elongata from three geographically isolated micropopulations growing on sandy soils in water reservoir-associated meadows were used as propagation material  was measured in natural habitats using HH2 m equipped with WET-2 sensor  at five spots at least 5 m apart. For all micropopulations, soil was non-saline or slightly saline .−1 sucrose and 6 g L−1 agar was used for initial culture, followed by multiplication on Murashige and Skoog medium containing sucrose and agar supplemented with 0.1 mg L−1 1-naphthaleneacetic acid and 1 mg L−1 6-benzylaminopurine. Rooting was performed on the same medium with 0.2 mg L−1 1-naphthaleneacetic acid. Rooted explants were acclimatized ex vitro in a peat substrate for two weeks and further used for in planta experiment.Seeds of AM1 and AM2 were used as explants for establishment of tissue culture. All details on culture initiation and multiplication were as described previously . Briefly−2 s−1 of photosynthetically active radiation at 25 °C. After 4 weeks, the experiment was terminated. Multiplication rate was evaluated by counting number of shoots per explant. Fresh and dry mass (after drying at 60 °C for 72 h) of tissues were measured. Tissue water content was expressed as g of H2O per g dry mass.Shoot explants at multiplication stage were used for treatment with NaCl. Necessary concentration of NaCl  was addev/v), and placed in an experimental automated greenhouse  with supplemented light from Master SON-TPIA Green Power CG T 400 W  and Powerstar HQI-BT 400 W/D PRO  lamps , 16 h photoperiod, day/night temperature 24/16 °C, relative air humidity 60 to 70%. Salinity treatment . After change of temperature regime to day/night temperature 15/20 °C for two additional weeks, seedlings were transplanted to 0.25 L plastic containers with a mixture of quartz sand  and garden soil  1:3 (v/v) and acclimatized to greenhouse conditions. Final transplantation to 0.5 L plastic containers with the same substrate was performed after two weeks. Fully developed 2-month-old plants in a vegetative stage were assigned to one of 12 treatments  and working solution contained 25 mL of each per 10 L deionized water, used with a rate 100 mL per container.During cultivation, individual containers were randomly redistributed weekly on a greenhouse bench. Substrate water content was monitored with HH2 moisture meter equipped with WET-2 sensor  and kept at 50 to 60%. Every third week plants were fertilized with Yara Tera Kristalon Red and Yara Tera Calcinit fertilizers . A stock solution was prepared for each fertilizer , leaves of different age). In experiment with AM1 and AM2, older leaves did not fully decay, as only part of the particular leaf was becoming brown and dry. Therefore, instead of designating these leaves as “decayed”, all leaves were sorted either as “old” or “young” according to their position and general morphological appearance. In experiment with AM3, individual older leaves decayed fully, therefore, all leaves were sorted either as “decayed” or “living” but for sake of comparison were indicated as “old” or “young”, respectively. Inflorescences were counted, and the length of flower stalks was measured. Plant material was weighed separately before and after drying in an oven at 60 °C for 72 h. Water content was calculated as g H+ concentration and K+ concentration in water extract by respective compact meters and analysis of osmotic activity by a freezing point osmometer. Tissues were ground with mortar and pestle to a fine powder and 10 mL of deionized water was added. The homogenate was stirred with pestle for 1 min. After filtration through nylon mesh cloth (No. 80) homogenate was used for measurement of ion concentration by LAQUAtwin compact meters B-722 (Na+) and B-731 (K+), and electrical conductivity by LAQUAtwin conductivity meter B-771  and measurement of osmotic value. For osmotic value analysis, 50 μL of extract were transferred in a 1.5 mL Eppendorf tube and placed in a freezing point osmometer Osmomat 3000 Basic  and operated according to the manufacturer’s instructions. Using a standard curve for different concentrations of NaCl and KCl, the osmotic value caused by the total concentration of Na+ and K+ was calculated according to the actual Na+ and K+ concentration of each sample extract. For each sample, the difference between the total osmotic value and the osmotic value due to Na+, K+ and Cl– ions was calculated and designated as “non-ionic osmotic value”, which showed the osmotic effect of other osmotically active ions  or non-ionic compounds. At least three analytical replicates were performed for each sample and the average value was calculated.All analyses were performed in triplicate, using representative tissue samples from individual biological replicates. Plant tissues were homogenized by crushing and a sample (0.2 g) was taken for analysis of EC, Nap < 0.05.Results were analyzed by KaleidaGraph . Statistical significance of differences was evaluated by one-way ANOVA using post-hoc analysis with minimum significant difference. Significant differences were indicated at A. maritima cultivated on multiplication medium was negatively affected by increasing concentration of NaCl in the medium (−1 (−1) biomass accumulation was inhibited by 46 and 49%, and proliferation by 56 and 54%, for AM1 and AM2, respectively. Water content in explants showed a tendency to increase at 44 mmol L−1 NaCl, followed by a significant decrease at 110 mmol L−1  in old leaves of plants from accession AM3 treated with 217 mmol L−1 NaCl (+ was 4.03 mol kg−1 (157 g kg−1) in old leaves of plants from accession AM2 treated with 217 mmol L−1 KCl  [+ influx and tissue accumulation or the cytosolic K+/Na+ ratio in the development of toxicity, are currently insufficiently substantiated and require a new, critical approach”.Traditionally, Nad “good” . Of cour. (2013) , “... se+ and K+ in a form of chloride salt on growth of A. maritima plants was nearly identical. However, there were differences in water content in older leaves as well as in osmotic adjustment between the two cations. First, leaves of accession AM2 showed decreasing water content with increasing KCl concentration, reflecting increase of partial dieback of these leaves  are responsible for most of osmotic adjustment both in halophytes as well as glycophytes. In A. maritima, degree of adjustment by inorganic ions vs. organic osmolytes varied between NaCl and KCl treatments and different accessions, as well as between different plant parts, (Salicornia bigelovii, contribution of K+ to osmotic regulation is low in comparison to that of Na+ [According to the generally accepted mechanism of salinity tolerance in halophytes, Naytoplasm . Role ofnditions . Early sreatment . There ireatment . In addibetaines  and evenbetaines . Howeverbetaines . Accordit parts, . Non-iont parts, A. In cont parts, D. It hast of Na+ .+ is stored in vacuoles of mesophyll cells [+ is also stored in vacuoles, both regulating osmotic potential and acting as the main cellular reserve of K+ [A. maritima plants from salt marsh redistributed K+ from shoots to roots with increasing salinity [+ concentration in leaves (In Na-accumulating halophyte species, Nall cells , and theve of K+ . Earliersalinity , but no n leaves .A. maritima accessions AM1 and AM2 was negatively affected by increasing NaCl concentration (+ transfer to shoots. Stimulated development of Casparian strips in roots under salinity can act as a barrier for passive apoplastic flux of Na+, resulting in a large concentration gradient between roots and shoots [+ (+ concentration in cultivated shoot explants (≤1 mol kg−1, It has been a matter of long scientific debate if plant salinity tolerance assessed in conditions of tissue culture fully reflects that in whole plants cultivated in soil-like substrate . For sixntration B, and alntration A in condntration . One of d shoots ,61, simid shoots . Howeverhoots [+  in compahoots [+ , and Na+A. maritima, associated with the ability to accumulate surplus ions both in salt glands and old leaves. Both Na+ and K+ in a form of chloride salts had similar effect on plant growth, but with significant differences in osmotic adjustment by inorganic ions vs. organic osmolytes. Further studies aiming at dissecting physiological and biochemical mechanisms related to salinity tolerance of A. maritima and differences in osmotic and antioxidative protection of internal environment in different accessions due to various salinity types are necessary. Most importantly, functional diversity needs to be related to possible genetic diversity of different ecologically and geographically isolated micropopulations of A. maritima at the Northern range of distribution, to get further insight into abiotic stress adaptation mechanisms of the species.It can be concluded that a species-wide salinity tolerance exists within dry sandy soil accessions of