Objective: To systematically review the effects of different resistance training (RT) protocols on bone mineral density (BMD) in postmenopausal women.Methods: Randomized controlled trials (RCTs) on the resistance training in improving bone mineral density for postmenopausal women were searched in databases including ProQuest, PubMed, Cochrane Library, Embase, and Web of Science. The retrieval time range was from the establishment of the database to May 2022. The included literature was independently screened and relevant data was extracted by two reviewers. The systematic review followed the Joanna Briggs Institute (JBI) methodology for reviews of quantitative evidence. Quality of risk was assessed using the Physical Therapy Evidence Database (PEDro) scale, risk of bias was assessedusing the Cochrane RoB2 tool and a network Meta-analysis was performed on the data using Stata 16.0.Results: A total of 19 studies, which included 919 subjects, were eventually acquired. The results of the network Meta-analysis showed that moderate intensity resistance training was superior in improving lumbar spine bone mineral density (LS BMD) and femoral neck bone mineral density (FN BMD) compared to the control group , with a statistically significant difference (p < 0.05). There was, however, no statistically significant difference between the groups in terms of increasing total hip bone mineral density (TH BMD) and trochanter bone mineral density (Troch BMD), although moderate intensity training tends to increase bone mineral density (p > 0.05). In addition, when training frequency is taken into consideration, 3 days/week of moderate intensity training (3MI) was superior to 2 days/week (2MI) in improving lumbar spine bone mineral density , and moderate intensity training was superior to low and high intensity resistance trainings at training frequency of 3 day/week, with statistically significant differences (p < 0.05). The cumulative probability ranking results indicated that 3MI was the optimal option in improving lumbar spine, femoral neck, total hip and Troch bone mineral density. Subgroup analyses combining interventions time showed that for lumbar spine and femoral neck bone mineral density, 3MI protocol with intervention duration within 1 year (≤48 weeks) had a significant advantage over other interventions, while this advantage was no longer significant with the intervention duration of more than 1 year (>48 weeks).Conclusion: Current evidence shows that moderate intensity resistance training for 3 days/week can be preferred clinically to improve bone mineral density in postmenopausal women, and it is recommended that the duration of the same training should not exceed 1 year. Nevertheless, more high-quality studies are needed to verify the above conclusion. Bone loss is significantly accelerated with the loss of estrogen after menopause in elderly women. Studies have shown that during 1–10 years after menopause, the annual loss rate of human bone mass is 1.5%–2.5% . BMD andIt is proved that exercise training, such as weight bearing, progressive resistance training, strength training, etc., can significantly reduce fall rate to improve function recovery and decrease fracture risk . In addiThe systematic review of this paper was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA); this study has been registered with the International Prospective Registerof Systematic Reviews (PROSPERO) under the registration number: CRD42020212253.Using the PICO  strategy, the studies that meet the following criteria were included in the study. Based on the previous studies , the tra(1) Concurrent drug therapy during resistance training;(2) Mixed combination of exercise interventions apart from resistance training;(3) Had undergone a high intensity physical training in the 6 months prior to the trial;(4) Exercise intervention intensity not marked in detail or intensity expressed in a non-RM way;(5) Liver, kidney and endocrine system diseases that affecting bone metabolism;(6) Abstracts, reviews, conference reports, etc.;(7) Multiple publications of the literature;(8) Non-extractable data;(9) Full text not available;(10) Lack of outcome measuresThe exclusion criteria adopted in the present study are as follows:In this study, the search strategy was developed and implemented according to PRISMA. A systematic search was conducted in databases including ProQuest, PubMed, Cochrane Library, Embase, and Web of Science with a retrieval time range from the establishment of the database to May 2022.The search terms included bone mineral density (BMD), resistance training, strength training, and postmenopausal. The specific search strategy was as follows (PubMed was used as an example):  AND (exercise OR resistance training OR resistance OR weight lift OR strength training OR strength exercise OR weight training) AND .After duplicate data was removed using EndnoteX9, two reviewers independently analyzed the titles and abstracts of all literature retrieved from the database. Then, they browsed through the entire articles, further screened out those that met the inclusion criteria, downloaded the full text, and divided them into three groups-included, possibly included, and excluded. For any disagreement, articles were reevaluated. If disagreements persisted, group discussions were held to resolve it.The following references were extracted from each study by two reviewers using pre-assigned tables, including the first author, year of publication, country, intervention time, sample size (enrollment/disenrollment), mean age of participants, exercise intervention plan  and outcome measures, the main result and complication.Methodological quality and risk of bias of the included studies were assessed using the Joanna Briggs Institute (JBI) Checklists Critical Appraisal Tool for Randomized Controlled Trials (RCTs) , PEDro s2.Data preprocessing and analysis were performed by two reviewers. The raw data was preprocessed using Microsoft Office Excel and results were indicated as the difference value between the endpoint and base values. All results were converted into a mean value and its standard deviation (ΔMean ± SD) in uniform unit of g/cmNetwork Meta-analysis and graphical plotting were performed for relevant data using Stata 16.0 software. The outcome measures were continuous variables and rated by the same scale, so the weighted mean difference (WMD) and 95% confidence interval (CI) were taken as effect sizes. The network evidence diagram of direct comparison between intervention intensity-frequency combinations was plotted; then the consistency of each outcome indicator’s closed loop was evaluated using the loop inconsistency test, where it indicated a good consistency between the direct and indirect evidence if 95% CI of the loop inconsistency factor (IF) contained 0 . The resApart from the above, stability of the study results was verified using subgroup and sensitivity analyses. Subgroup analyses was performed according to the duration of intervention, while sensitivity analyses was performed by excluding studies with a sample size of less than 10.A total of 4,208 studies were retrieved, and based on multiple screenings, 19 studies with a total of 919 patients were finally included. The flow chart of selection process in this study is shown in A total of 19 studies  with 919The JBI  was employed to determine the quality of articles in randomized controlled trials, which revealed the absence of blinding of participants (19/19), treatment providers (19/19), and outcome evaluators (15/19). This may lead to performance or detection bias, especially for subjective outcome measures .Using ROB two to assess methodological quality and bias in the included studies . The majp < 0.05]; 3MI was superior to 2MI , 3LI , and 3HI , which indicated that RT at moderate intensity was better than the other two intensities and that high frequency training at moderate intensity was better than low frequency training. Moreover, the SUCRA results in A total of 16 studies with 722 subjects were included in this part. The network evidence diagram is shown as p < 0.05]; in the pairwise comparison of the other groups, although 3MI had a significant advantage on FN BMD, the difference was not statistically significant (p > 0.05). Besides, the cumulative probability ranking in A total of 19 studies with 767 subjects were included. The network evidence diagram is shown as p > 0.05). Meanwhile, the cumulative probability ranking in A total of eight studies with 282 subjects were included in this part. The network evidence diagram is shown as p > 0.05). Meanwhile, the cumulative probability ranking in A total of eight studies with 393 subjects were included in this part. The network evidence diagram is shown in It can be seen from In this part, sensitivity analyses were also performed by removing studies with sample sizes less than 10 to verify the robustness of our results.In regard to LS BMD, studies including Bemben-2000 and Maddalozzo-2000 were removed. The network evidence diagram and loop inconsistency test are shown in In regard to FN BMD, studies including Bemben-2000, Pruitt1995 and Maddalozzo-2000 were excluded. The network evidence diagram and loop inconsistency test are shown in With respect to TH BMD, studies including Bemben-2000 and Maddalozzo-2000 were removed. The network evidence diagram and loop inconsistency test are shown in Furthermore, considering that differences in intervention time might have an impact on the results of this study, subgroup analyses of LS BMD and FN BMD were performed on the basis of different intervention time. Two subgroups were set up: Intervention time ≤48 weeks group and intervention time>48 weeks group. Whereas, TH BMD, Troch BMD was not available for subgroup analysis due to the number of included studies.p < 0.05), and was also superior to 3LI and 3HI (p < 0.05). For RT at moderate intensity, 3MI was better than 2MI in improving BMD, with a statistically significant difference (p < 0.05). In intervention time>48 weeks group, the network evidence diagram and loop inconsistency test are shown in p > 0.05). Additionally, the cumulative probability ranking results of both subgroups . In contrast, in the subgroup with intervention time>48 weeks, the results of forest plot (p < 0.05). The results of the cumulative probability ranking  was mostly better than 2 days/week (2MI), and this difference was more pronounced in LS BMD. This was because the frequency of loading was the main influence factor that induced a bone adaptive response that could directly affect bone formation. PINHEIRO  demonstrPrevious studies have shown that exercise produces adaptive bone development only when it achieved cumulative time and volume. The bone reconstruction cycle generally takes 3–4 months, and the bone needs 7–9 months to achieve a new stable level of bone volume after alteration ; hence, The results of this meta analysis are helpful to refine the exercise prescription of resistance exercise and provide evidence for improving bone mineral density in postmenopausal women. There are a large number of postmenopausal women in the world and a lot of money is spent on the fight against osteoporosis. The development of resistance movement does not need to invest expensive equipment and large areas, which can save a lot of medical expenses. In addition, resistance exercise has good safety, and participants will feel the pleasure of dopamine secretion during exercise . To sum Although we included all interventions in this network meta-analysis to obtain comprehensive results, the study had certain limitations. Firstly, the population included in the study was not identical in terms of daily exercise habits and exercise sites for RT, and most RCTs did not describe details of the randomized scheme, allocation concealment and blinding of subjects and researchers with regard to article quality assessment, which could lead to potential heterogeneity. Secondly, this analysis included only studies that quantified exercise intensify using 1RM. However, only 19 studies were qualified through screening under the precise classification. The lack of direct evidence may limit the reliability of the results and it’s necessary to treat it with caution. Finally, since the included studies ended up being available only in English, there may be language bias.In conclusion, moderate intensity and high frequency  may be the optimal training protocol to improve LS, FN, TH and Troch BMD in postmenopausal women. Considering the effect of the exercise cycle, it is recommended that the same training protocol should preferably last no longer than 1 year in order to reasonably regulate bone adaptation and produce new strain distribution. However, due to study limitations, the conclusion drawn in this paper need to be further validated by more high-quality studies.


Patients with cutaneous psoriasis (PsC) and psoriatic arthritis (PsA) are reported with increased cancer risk, but the underlying mechanism is less clear, especially the association between the presence of PsA and cancer risk. Motivated by the role of ferroptosis in the progression of cancers as well as inflammation response in psoriasis, this experiment attempts to investigate the relationship between ferroptosis regulators and hub genes in PsA by bioinformatic analysis. The findings revealed an exclusive correlation between CISD1 (ferroptosis regulator) and CLEC2B (hub gene) in PsA group as well as multiple cancer types. Furthermore, CLEC2B was discovered differentially expressed in a variety of cancers and is closely associated with immune cell infiltration as well as immune checkpoints. These results indicate that ferroptosis may act as a bridge between psoriatic arthritis and the onset of certain malignancies. A number of comorbid conditions, such as depression, cardiovascular disease, and particularly cancer, compound the impact of PsD. The incidence of cancer and cancer-related mortality seem to be higher in people with PsD, but the underlying association is much less clear3. To improve the understanding of the underlying mechanisms of this increased risk. Cutaneous psoriasis (PsC) and psoriatic arthritis (PsA) are fall under the umbrella of psoriasis disease5. PsA is a chronic, progressive, inflammatory arthritis associated with PsC that can impact a variety of organ systems6. To date, little is known about the cancer-related genomics in patients with PsD, even less is clear whether presence of PsA is associated with cancer risk in psoriasis patients.Psoriasis disease (PsD) is a polygenic, hyperproliferative chronic inflammatory disease with unknown etiology7. According to research, ferroptosis is likely to inhibit tumor formation and/or progression, which means inducing ferroptosis is a promising anticancer strategy. Activation of lipid ROS and iron metabolism or suppression of antioxidant metabolism is the primary approach of ferroptosis induction based cancer therapy9. Interestingly, ROS occurs systemically and locally in psoriasis10. Many current treatments further increase oxidative stress and ROS production to accelerate the removal of damaged epidermal cells11. The induction of ferroptosis in psoriatic keratinocytes, particularly lipid peroxidation, has been proven to contribute to inflammatory responses12. Therefore, we propose the hypothesis that association analysis between hub genes contributed to PsA development and ferroptosis regulators may provide a new perspective on the pathogenesis of high cancer risk in patients with PsA.Ferroptosis, a new form of regulated cell death that is iron- and reactive oxygen species (ROS)-dependent, has attracted much attention in cancer development and treatment13. Several biomarkers and pathways have been implicated in the development of PsD in previous studies on microarray data15. Moreover, genetic and proteomic studies also confirmed the overlap and differences between PsC and PsA. Specifically, whole blood gene expression analysis suggests that innate immunity may play an important role in the pathogenesis of PsA through TLR signaling, NF-kB, and various chromatin remodeling complexes. NOTCH2NL, HAT1, CXCL10, and SETD2 were found most notably associated with PsA patients16. But still no studies have been conducted on the differential analysis of ferroptosis regulators between PsA and PsC.There is growing interest in microarray platforms as a way to detect genetic alterations and to determine biomarkers for many diseases19, including GSEA/DO enrichment analysis, and PPI network construction to obtain hub genes. Subsequently, association analysis of hub genes with differentially expressed ferroptosis regulators was performed. Co-expression interactions in pan-cancer were analyzed using genes with exclusive correlation in PsA to further narrow down the hub genes. CLEC2B, the final standout, was also discovered to be differently expressed in a variety of malignancies and to have a substantial link to prognosis and immune infiltration in a variety of tumors.In this study, we set up 2 comparisons including PsA-CL  group and PsA-PsC group. Overlapped differentially expressed genes obtained from aforementioned two comparisons were considered as important genes contributed to PsA development, which were used for further analysisAs a result, we propose that CLEC2B, via ferroptosis regulators, may be a bridge gene for disease progression and a high cancer risk in psoriatic arthritis.The flow chart of this study is shown in Fig. http://www.ncbi.nlm.nih.gov/geo)20. The uniformly normalized pan-cancer dataset was downloaded from the UCSC (https://xenabrowser.net/) database21: TCGA TARGET GTEx .Gene expression datasets were collected from the Gene Expression Omnibus database  between each group in the dataset, and a volcano map and heatmap of DEGs was drawn respectively.Raw data were processed and analyzed using R (version 4.0.2)25. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using GAGE26 followed by visualization with the ggplot2 R package27. Annotation of the genes and pathways was provided by the KEGG database (http://www.genome.jp/kegg)28. Disease Ontology (DO) enrichment analysis was performed with an online tool Enrichr (https://maayanlab.cloud/Enrichr/). FDR < 0.05 was considered significant enrichment.Gene Ontology (GO) on DEGs were performed respectively using “clusterProfiler” packagehttps://string-db.org/) to construct PPI networks with a minimum required interaction score of 0.429. For further analysis, Cytoscape software30 was used with the download interaction information. Significant genes were determined by the cytoHubba plugin31 as hub genes.To characterize the crucial DEGs and clusters in skin scar, we used an online tool STRING , the darker the color, the stronger the relation. Asterisks (*) stand for significance levels, ** for p < 0.01, * for p < 0.05.https://starbase.sysu.edu.cn/)33 was utilized to do a co-expression analysis of target hub genes and major ferroptosis regulators that have exclusive linkages in PsA to further characterize the target genes. Target genes were identified based on the number of cancer species with |co-efficient R| ≥ 0.2 and P < 0.05.ENCORI  and analyzed difference significance using unpaired Wilcoxon Rank Sum Tests. Asterisks (*) stand for significance levels, ** for p < 0.01, * for p < 0.05. The results were visualized by ggplot34. Furthermore, we performed log2(x + 0.001) transformation for each expression value, and finally we also excluded the cancers with less than 10 samples in a single cancer species. The connection between the CLEC2B expression and the prognosis of patients, including overall survival (OS) in multiple types of cancer was examined using forest plots and Kaplan–Meier curves. The hazard ratios (HRs) and 95% confidence intervals were calculated using univariate survival analysis.We obtained a high-quality prognostic dataset of TCGA from previous prognostic studies of TCGA published in Cell35 and Timer36 methods of R package IOBR37. We calculated spearman's correlation coefficient between gene and immune cell infiltration scores in each tumor using the corr.test function of R package psych (https://CRAN.R-project.org/package=psych) to determine the significantly correlated immune infiltration scores. Additionally, the expression data of CLEC2B and 60 marker genes of two types of immune checkpoint pathway genes (Inhibitory(24), Stimulatory(36))38 in each sample was also explored, and the spearman correlation between CLEC2B and the marker genes of the five immune pathways was also calculated .The gene expression profile of each tumor was extracted and mapped to GeneSymbol. Further, the infiltration score of each immune cell type in each tumor for each patient was re-evaluated based on gene expression, using the xCellAfter searching in Gene Expression Omnibus database with the inclusion criteria included: (1) patients with psoriatic arthritis or cutaneous psoriasis; 2) whole blood; 52 blood samples from GSE61281 were obtained . We first performed background correction and data normalization after collecting the dataset, and the results are given in box plots | ≥ 1. As shown in the volcano plots, totally 273 DEGs from PsA-CL group and 86 DEGS from PsA-PsC group were obtained . Compared with PsC, the expression levels of ACSL4, CISD1, EMC2 and SAT1 were increased in PsA group, but the level of CARS was decreased . As shown in Fig. After investigating the pan-cancer section of ENCORI, an online tool, CISD1 was discovered to be highly linked to CLEC2B, CLEC12B and CSTA in a variety of cancer types. We also used |Coefficient-R| ≥ 0.2 as well as p < 0.05 to retrieve the cancer kinds and showed the findings in Fig. 39 and STRING29 were used to obtain correlated gene sets of CLEC2B and CISD1  and difference significance analysis using unpaired Wilcoxon Rank Sum and Signed Rank Tests. As shown in Fig. We used the coxph function of the R package to build a Cox proportional hazards regression model to analyze the relationship between CLEC2B expression and prognosis in each tumor, and statistical tests were performed using Logrank test to obtain prognostic significance. We finally observed that upregulated CLEC2B expression was remarkably associated with poor OS in 10 tumor types  and downregulated expression was correlated with poor OS in 2 tumor type (SKCM and ALL). The results of Kaplan–Meier analysis were consistent with the above findings Fig. .Figure 8Among 38 subtypes of immune cells, we found that the CLEC2B expression significantly correlated with most subtypes in different tumor types Fig. . As show41. Contrary to the established nature of the associations between these comorbidities including depression and cardiovascular disease, the underlying mechanism of high cancer risk and psoriasis disease appear to be much less well-established42. Even less is known about whether psoriatic arthritis raises the risk of cancer in psoriasis sufferers. Given the importance of ferroptosis in cancer development as well as inflammatory response in psoriasis43, we attempted to employ bioinformatics analysis to investigate the association between ferroptosis and psoriasis disease, specifically psoriatic arthritis.The risk of cancer developing and incidence in people with psoriasis disease has raised some concern44. Our results similarly demonstrate the relationship between CLEC2B and natural killer cells. Additionally, we discovered that CLEC2B collaborated with the ferroptosis regulator CISD1 to connect iron-sulfer cluster binding and oxidative phosphorylation, further demonstrating the strong connection between CLEC2B and ferroptosis.In this investigation, a total of 10 hub genes Fig. E and 3 d46. Similar to the findings of prior studies, this study found vital functions for CLEC2B in numerous cancers, including changes in expression, correlation with prognosis, infiltration of multiple immune cells and even immune checkpoints in multiple cancer types. Given that, CLEC2B was considered as a key gene which bridges the gap between the development of psoriatic arthritis and cancer through ferroptosis. However, the link between CLEC2B and ferroptosis regulator CISD1 in various malignancies varied in strength and type, which implies that even the same two genes function differently under various regulatory systems in malignant tumors, demonstrating the complexity and unknowable nature of cancer etiology.Based on aforementioned results, we further explored the function of CELC2B in pan-cancer. In previous studies, CLEC2B has been identified as a marker for a variety of cancers, including clear cell renal cell carcinoma, melanoma, and pancreatic adenocarcinomaThis is the first time that CLEC2B has been found to be associated with the development of psoriatic arthritis through natural killer cells. More importantly, as a key gene exclusively linked to ferroptosis regulators in psoriatic arthritis, CLEC2B is differentially expressed in a variety of cancers and is closely associated with immune cell infiltration as well as immune checkpoints. This study may provide new research insights into the high cancer risk in patients with psoriatic psoriasis.


We also analyze cases with deep venous thrombosis (DVT) and/or pulmonary embolism (PE) under treatment with unfractionated heparin. The non-pregnant women (n = 13) and healthy pregnant women (at 9–13 weeks of gestation (n = 13), at 27–30 weeks of gestation (n = 14), at 35–38 weeks of gestation (n = 14)) were cross-sectionally studied, while the normal pregnant women at delivery (n = 14) were sequentially investigated. Blood samples from those patients with PPH (n = 15) and DVT and/or PE (n = 11) were also obtained and compared with those of normal women. Significant changes of clot formation parameters were observed in all parameters and, interestingly, fibrinolytic parameter (LY30) was maintained at a low value even within 120 min after placental delivery  and also in cases with uterine atony . The parameter that indicates the effectiveness of heparin showed strong correlation (R = 0.788) with activated partial thromboplastin time. Thromboelastography may be less sensitive to fibrinolysis in the conditions of uterine atonic bleeding.The aims of this study are to evaluate coagulation and fibrinolytic features using TEG6s Thromboelastography is a simple, rapid and accurate system for analyzing coagulopathy using whole blood at the bedside, and it has been widely used in the critical care of emergent situations all over the world ,2,3. ThiPregnancy, however, is an ambivalent condition where women are occasionally faced with postpartum hemorrhage (PPH), which occurs around the time of placental removal, and an increased risk of deep venous thrombosis (DVT) and/or pulmonary embolism (PE) caused by enhanced production of coagulation factors, vascular dilatation, and restricted venous return by the gravid uterus as a result of maternal physiological changes. ,10. In t® system , which is newly developed, enables four different assays simultaneously from a single blood sample: CK (citrated kaolin), CRT (citrated rapidTEG), CKH (citrated kaolin with heparinase) and CFF . Among them, CKH contains kaolin and heparinase to neutralize the effects of heparin in the blood. The time to initial clot formation, or R time, of CK will be longer than the R time of CKH in a blood sample with heparin [® device has been found to have excellent repeatability within device  [® system will help clinicians to manage PPH patients and to monitor anti-coagulant therapy during pregnancy and postpartum; however, only limited papers on the subject have been published so far [Some studies have reported on the usefulness of thromboelastography as a tool in which to evaluate coagulative conditions and to guide obstetricians toward prompt treatment. The TEG6s heparin . The TEGd so far . ® in normal pregnant courses, in the early postpartum period and in cases with PPH. We also analyze cases with DVT and/or PE under treatment with heparin.The aims of this study are to evaluate coagulation and fibrinolytic features using TEG6sUncomplicated women with a singleton pregnancy who were managed and delivered at Osaka Metropolitan University Hospital were included in this study from October 2020 to February 2022. The study was approved by the institutional review board (Approved Number: 2020-244), and all patients gave their informed written consent before inclusion. Additional venous blood draws were performed at the timing of a routine schedule: 1st trimester; 9–13 weeks of gestation, early 3rd trimester; 27–30 weeks of gestation, and mid-3rd trimester; 35–38 weeks of gestation. Sequential venous blood samples were also obtained at the timing of delivery from patients without PPH (estimated blood loss (EBL) <500 mL; during 1st stage of labor and 15, 30, 60, and 120 min after placental delivery). The blood samples of PPH patients due to uterine atony (EBL of ≥2000 mL) were obtained within 2 h after placental delivery and before the start of any interventions such as transfusion and/or uterine artery embolization. The blood loss was estimated with the weight of the postpartum-pad and gauze. Control samples were also taken from non-pregnant healthy women. All data of the non-pregnant women and pregnant women were collected cross-sectional, while the data of the women at delivery were collected sequential. We also obtained blood samples from those patients with DVT and/or PE and who were treated with unfractionated heparin before and/or after delivery. Once patients were diagnosed with DVT and/or PE, treatment with continuous heparin infusion was initiated with monitoring to ensure that activated partial thromboplastin time (APTT) values were 1.5 to 2.5 times with control values. When APTT values reached targeted values, heparin dosing was determined and then patients were switched to subcutaneous heparin injection at the same dose. ®. Briefly, whole blood (2 mL) was collected in anticoagulation tubes containing 3.2% sodium citrate, and the sodium citrate was mixed thoroughly with the sample before testing. The test cartridges were kept refrigerated and the samples were kept at room temperature until inspection. As a rule, testing was performed within 2 h of blood collection, but in most samples were tested within a few minutes because there were two TEG6s® machines in our delivery rooms. In this case, 300 µg of the collected blood was injected into the test cartridge using the pipette provided. Among the thromboelastographic parameters, reaction time (R) is the time from the start of analysis until the thrombus amplitude reaches 2 mm and reflects the adequacy of coagulation factors. Clot formation time (K) is the time from the thrombus amplitude reaching 2 mm until it reaches 20 mm, and this reflects clot kinetics. The alpha angle (Angle) is the slope of a tangent line from the tracing at the midpoint between R and K and reflects the fibrinogen level. Maximum amplitude (MA) is the absolute thrombus strength and reflects platelet function, and clot lysis at 30 min (LY30) reflects clot lysis [® thromboelastographic parameter trace of a patient who developed DVT at 12 weeks of gestation and started heparin therapy; the red line is CK, the purple line is CRT, the green line is CKH, and the blue line is CFF. The difference between CK and CKH clearly shows the effect of the heparin administration. We defined the difference between R of CK and R of CKH as delta R (delta R = R of CK—R of CKH). For the measurement of APTT, Levohem APTT-SLA  was used as the measuring reagent and Sysmex CN-6000  was used as the coagulation meter.Blood samples were collected by peripheral venipuncture of an upper limb and analyzed by TEG6sot lysis . The CK ot lysis . Figure n (%) and tested using the Kruskal-Wallis test. Statistical comparisons between the two groups were tested with the Mann-Whitney test. Differences in results were considered statistically significant when p < 0.05. Correlations with R values above 0.7 were considered as a strong correlation.Statistical data were processed and analyzed using the software SPSS (version 24.0). All data were expressed as median (range) and n = 4) and gestational diabetes mellitus (n = 2). Finally, blood samples were collected from 13 cases at 9–13 weeks of gestation (9–13 GW group), 14 cases at 27–30 weeks of gestation (27–30 GW group), and 14 cases at 35–38 weeks of gestation (35–38 GW group).We recruited cross-sectional, 13 healthy, non-pregnant women (NP group) and 47 uncomplicated pregnant women, 15 patients with PPH (PPH group), and 11 patients with DVT and/or PE (DVT/PE group). Six uncomplicated pregnant women were excluded because of subsequential revealed hypertensive disorder in pregnancy , 14 women in the 15 min after placental delivery group (15 min group), 11 women in the 30 min after placental delivery group (30 min group), 14 women in the 60 min after placental delivery group (60 min group), and 14 women in the 120 min after placental delivery group (120 min group).® system. Significant changes were observed in all parameters and, interestingly, LY30 was maintained at a low value even within 120 min after placental delivery and also in cases with PPH. In addition, thromboelastographic parameters in CRT, CKH and CFF assay are shown in As shown in ® in normal pregnant women, early postpartum, and PPH cases in Japanese population. We observed the decrease of K, and LY30 and the increased Angle and MA throughout pregnancy before labor onset. In addition, compared with data during pregnancy, clot formation was further strengthened, and clot lysis stayed at low activity during labor and in the early postpartum period. In the PPH group, clot formation was shown to be delayed by the increase of R and K, and by the decrease of Angle, although the strength of the clot was maintained, as shown by unchanged MA and LY30. This observation suggests that the coagulopathy in this setting with atonic bleeding may appear in delayed R and K and decreased Angle caused by shortening of coagulation factors except fibrinogen and/or platelet counts. In the DVT/PE group, delta R showed a significantly strong correlation with the APTT ratio, thus suggesting that delta R well reflects the effectiveness of heparin therapy. As such, we firstly demonstrated coagulation and fibrinolysis specificity using TEG6s® was designed to reduce the technical requirements of pipetting the blood sample, and vulnerability to vibration of TEG5000®, and to offer more reliable data for the healthcare providers at emergent critical care [®, some researchers investigated the hemostatic parameters using TEG5000®  in the 3rd trimester of normal pregnancy, and the enhancement of clot formation was observed in all reports [® system. As to the fibrinolytic parameter, however, there remains some room for verification. Since fibrinolytic steps are expected to change with time after placental delivery and seem to greatly affect the results, the timing of blood sampling should be carefully determined [Using resonance-frequency elasticity measurements and a disposable multichannel microfluidic cartridge, TEG6scal care . Before  reports ,19,20. Ttermined ,22. Accotermined ,19,20. I® parameters by blood loss volume in PPH cases. Based upon their observation that Angle and MA significantly decreased, they reported that impaired hemostasis was observed in cases with a blood loss of more than 2000 mL [® Delta . They demonstrated that women with placental abruption with severe coagulopathy required higher doses of fibrinogen concentrate than women with other causes [® Sigma , a fully automated successor, has also been introduced and reported to correlate with Claus fibrinogen [® guided resuscitation, Hurwich et al. reported a case in which the management protocol of massive hemorrhage in non-obstetrical fields, such as cardiac surgery and trauma, was applied and that the patient was successfully revived after cardiac arrest [Based upon the data at the level of bleeding over 2000 mL, Karlsson et al. examined TEG5000 2000 mL . More rer causes . Furtherr causes . Their obrinogen . As for c arrest ,28,29. Ac arrest ,27,28,29Owing to maternal physiological changes, such as the enhanced production of coagulation factors, vascular dilatation, and restricted venous return by the gravid uterus, venous thromboembolism is a major cause of maternal morbidity and mortality ,14. If a® to other devices , and that we did not assess thromboelastographic parameters in cases with placental abruption or amniotic fluid embolism, where life-threatening coagulopathy rapidly occurs. Furthermore, thromboelastography may be less sensitive to fibrinolysis, except in conditions of excessive hyperfibrinolysis [The limitations of the present study are that the small number of the patients for each group and the data of the pregnant women were collected cross-sectional, and that we did not compare TEG6sinolysis ,34,35. Winolysis . On the inolysis ,38. Thus® in normal pregnant courses, in the early postpartum period and in cases with PPH. Our data of thromboelastographic parameters system will allow us to improve comprehension coagulation and fibrinolytic statuses. We believe that the present study helps to explore the pathology of PPH, and our newly demonstrated data will encourage us to initiate prompt and individualized treatment for coagulopathy. This is the first study to evaluate the coagulation and fibrinolytic feature using TEG6s


To evaluate the duration of cooling after cryotherapy on the skin over stifle joints in dogs after tibial plateau leveling osteotomy (TPLO) surgery using thermography.Seventeen client-owned dogs of various breeds were enrolled in the study. Dogs underwent TPLO surgeries, and on the next day, thermal imaging was performed on the operated stifle prior to cryotherapy for baseline. Orthogonal views were repeated at 30-min intervals until the skin over the stifle had thermally equilibrated. An ice pack was applied for 20 min on the medial and lateral aspects of the stifle. Each stifle was then re-imaged every 15 min for the first 60 min then every 30 min subsequently until the temperature was within 1°C of the pre-cryotherapy temperature.Mean skin temperature of the medial view showed no significance difference compared to baseline value at 45 min after cryotherapy was discontinued and after 60 min for the lateral and cranial views. Mean skin temperature was overall higher in the medial view compared to the lateral and cranial during the rewarming period (except immediately after cold application). Mean skin temperatures of all views combined showed a significant decrease in temperature during cryotherapy application, with a slow increase until a plateau was reached after 45 min of rewarming.Dogs undergoing TPLO for cranial cruciate ligament injury showed quicker rewarming period of superficial tissues compared to previous studies. Cryotherapy is a beneficial modality to reduce superficial tissue temperature in dogs undergoing TPLO, acknowledging that these dogs may require more frequent cryotherapy post-operatively due to more rapid rewarming time compared to dogs without surgery. Cryotherapy, or the use of cold to cool tissues, is a commonly used rehabilitation modality following injury, surgery, and strenuous activity to relieve pain and minimize inflammation. In post-surgical patients, cryotherapy may be used in the immediate recovery period and following exercises in patients undergoing rehabilitation. There are several cryotherapy methods, including conduction , convection (immersion baths), and evaporation (vapocoolant sprays). The benefits of cryotherapy have been well documented, including, vasoconstriction, decreased cellular metabolism, decreased swelling, reduced enzyme-mediated tissue damage, and analgesia –7. AlthoTibial Plateau Leveling Osteotomy (TPLO) is one of the most frequent procedures performed in dogs where post-operative cryotherapy is applied. The significant levels of tissue and bone damage induced by the surgery lead to edema, inflammation, and bruising around the stifle, making dogs undergoing TPLO good candidates for post-operative cryotherapy .via needle thermistors, which are invasive and require sedation or anesthesia in veterinary patients. Furthermore, medications used to induce sedation and anesthesia may cause significant hemodynamic changes in veterinary patients, including vasoconstriction or vasodilatation  surgery for cranial cruciate ligament rupture at the University of Tennessee College of Veterinary Medicine were evaluated between June 2016 to July 2017. The dogs were enrolled after obtaining written client consent and meeting the criteria of being otherwise healthy based on physical examination, orthopedic exam, radiographs, and pre-operative blood work. Dogs were not included if they had any medical history that would prevent them from undergoing anesthesia. Dogs were also not included if there were clinically apparent bilateral cruciate ligament injuries or the data collection was incomplete. All study procedures were reviewed and approved by the University of Tennessee Institutional Animal Care and Use Committee.Dogs underwent routine anesthesia, surgical prepping and TPLO surgery. Surgeries were performed by two ACVS Diplomate surgeons (D.L.M. and J.P.W.). Orthogonal radiographs were obtained immediately after surgery to verify anatomic limb alignment and appropriate implant positioning. Post-operatively, dogs received appropriate analgesic treatment as determined by an anesthesiologist. A sterile adherent Telfa® island dressing  was placed to cover the entire length of the incision (either 2 x 3″ or 3 x 4”), and a modified Robert Jones bandage was placed on the operated leg prior to continuing recovery in the intensive care unit.The day after surgery, patients were relocated to a room with ambient temperature set of 21.0 ± 1.0°C for thermographic evaluation. The modified Robert Jones bandage was removed, but the Telfa® island dressing was left intact. Triplicates of thermographic images using a digital infrared thermal imaging system  were obtained from operated stifles. The camera was centered at the stifle and measured instantaneous temperature at the given time. Orthogonal views  were obtained to determine mean temperature from a distance (between 50 and 150 cm) to include the entire hip and leg in the frame of the image with dogs in a standing position . ThermalAfter equilibration, ice compression was applied to the medial and lateral sides of the operated stifle for 20 min using clear plastic bags filled with crushed ice. Immediately following cryotherapy, thermographic images of the operated stifle were obtained in triplicate. Additional thermographic images were obtained every 15 min for the first hour after cryotherapy, followed by every 30 min until 2 h after cryotherapy. If mean temperatures of stifles remained below pre-cryotherapy levels at 2 h, data collection continued every 30 min until instantaneous temperature returned to within 1°C of pre-cryotherapy temperature for two consecutive time points .Analyses were conducted in SAS 9.4 TS1M7 . Differences in mean skin temperature between sides were analyzed using a repeated measures mixed effect model with group (side), time, and a side by time interaction. Due to statistical significance, the interaction term was kept in the model and individual differences between sides were subsequently evaluated using least squares means with Tukey's adjustment for multiple comparisons. Statistical significance was identified at the level of 0.05.n = 1) or inadvertent premature bandage removal (n = 1). Therefore, seventeen dogs underwent data analysis , Australian Shepherd (n = 2), Boxer (n = 3), Brittany Spaniel (n = 1), German Shepherd (n = 1), Jack Russell Terrier (n = 1), Labrador Retriever (n = 2), Shih Tzu (n = 1), and mixed breed (n = 5). There were 10 left and 7 right TPLOs.There were 7 spayed females, 8 castrated males, and 2 intact males. The mean age of dogs was 6.27 ± 2.75 years, with a range of 2.5 to 10.5 years of age. Mean body condition score was 6.47 out of 9 ± 1.28. The average weight was 27.53 ± 10.28 kg, ranging from 13.2 to 45 kg. Breeds represented included Akita (p < 0.001) between equilibrium to immediately after cryotherapy on all views. Minimum temperatures reached immediately after cryotherapy were 20.8, 19.8, and 21.0°C for medial, lateral, cranial and views, respectively. At 15 min, the mean temperature elevation of all views significantly increased (p < 0.001). There was no significant temperature difference (p = 1.00) between the lateral and the cranial views; however, the mean temperature in the medial view was higher compared to the lateral and cranial views, except at 0 min where the cranial was the highest (Mean skin temperature decreased by 7.4°C immediately after cryotherapy (0 min) and returned to baseline 45 mins after application on the medial aspect of the limb . For the highest . Mean sk highest . The meaThe results of this study revealed that dogs undergoing TPLO for cranial cruciate ligament injury have a similar cooling and rewarming period after cryotherapy compared to previous studies . Mean su2 (PGE2) and cellular energy consumption with increased temperature in knees  and the body size of dogs. The dogs in this study had varying BCS, from 5 out of 9 to 9 out of 9. There was no overall trend of temperature changes among dogs with different BCS. Due to higher adipose in dogs with higher BCS, there were concerns that dogs with higher BCS may have greater cooling effect due to low thermal conductivity and thermal diffusivity of adipose tissues . HoweverFuture work should be performed to evaluate the effect of cryotherapy on deeper tissue temperatures after TPLO. In addition, the method of cryotherapy following surgery should be further evaluated. Although cold compression is an easily accessible method of cryotherapy, it has also been shown to be among the more short-lived in duration. Full immersion and compressive cooling sleeves result in greater tissue temperature reduction and longer duration of action. However, if the inflammation and edema that result from surgical trauma are the cause of the more rapid rewarming, this may also occur with these other modalities. Further study of non-invasive thermal imaging is warranted to better understand the effect of cryotherapy in the clinical setting.We concluded that cryotherapy is a beneficial mode to reducing superficial tissue temperature in dogs undergoing TPLO but also acknowledge that these dogs may require more frequent cryotherapy post-operatively due to more rapid rewarming time compared to unoperated dogs. The results also support the belief that the medial aspect may be warmer due to inflammation from the incision and surgical site, and differences in the composition of tissues under the skin.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by University of Tennessee Institutional Animal Care and Use Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.MD and DM conceptualized the study design. JL, MD, and DM contributed to the acquisition of data. SW and DM analyzed and interpreted the data. All authors publicly accountable for relevant content, drafted and revised, and approved submitted manuscript.This study was funded by the University of Tennessee Small Animal Canine Arthritis Rehabilitation Exercise & Sports Medicine (CARES).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.


Three typical endpoints were used to assess the bioaccumulation potential: the bioconcentration factor (BCF), the bioaccumulation factor (BAF), and the biomagnification factor (BMF). Our results suggest that zooplankton has greater mean logarithmic BCF and BAF values than phytoplankton (3.31 vs. 1.42) and fish (2.04). The ENMs are biomagnified in zooplankton, with a mean BMF of 17.4, whereas trophic transfer from primary consumers (zooplankton) to secondary consumers (fish) was not observed (mean BMF of 0.13). No clear dependency was identified between the physicochemical characteristics of ENMs  and bioaccumulation, except for coated versus uncoated particles accumulated in phytoplankton. Carbonaceous ENMs were found to be more bioaccumulated than the other ENMs we considered, except for TiO2. A meta‐analysis of bioaccumulation data can (1) deepen the understanding of bioconcentration, bioaccumulation, and biomagnification of ENMs, (2) be used to support grouping strategies as a basis for a safer‐by‐design approach for ENMs, (3) be integrated into comprehensive hazard and risk assessments, (4) promote the standardization of testing guidelines, and (5) enhance future kinetic bioaccumulation modeling. Environ Toxicol Chem 2022;41:1202–1214. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.Understanding the bioaccumulation of engineered nanomaterials (ENMs) is essential for making regulatory decisions on potential environmental risks. Research in the field of ENM bioaccumulation has increased in recent years, but the compilation and statistical analysis of the available experimental data have not been updated. We therefore performed a meta‐analysis of the existing literature on the bioaccumulation of eight types of nondissolvable ENMs , the bioaccumulation factor (BAF) and the biomagnification factor (BMF). Engineered nanomaterials (ENMs) are manufactured for a variety of products and applications  nanoparticles in fish and fish organs. A centralized mesocosm database management system for ENMs  was developed, covering silver (Ag), CuO, cerium(IV) dioxide (CeO2), and TiO2 ENMs and including bioaccumulation data , primary consumers (zooplankton), and secondary consumers (fish) were considered. To allow a comparison of the results of different empirical studies, a database on ENM bioaccumulation was created by grouping experiments with different exposure and elimination conditions into different scenarios. Moreover, we converted the units of body burden to the same unit (mg/kg dry wt of organisms). Key factors affecting changes in bioaccumulation were analyzed, including exposure scenario, functional feeding group, exposure concentration, type of ENM, and physicochemical characteristics of the ENMs . Studies conducted under an environmentally relevant exposure concentration (less than 1 mg/L) were examined separately. In addition, challenges and knowledge gaps were identified. Finally, recommendations were made for experimental design and modeling.2, aluminum dioxide [Al2O3], Au, fullerene [C60], CNTs, iron oxide [FeOx], graphene, polystyrene) and three organism groups representing different trophic levels . These ENMs are usually assumed to undergo no dissolution and transformation under environmental conditions  was measured and documented, or if it could be calculated based on the given data. In addition, only adult animals were included and studies with embryos were excluded. In the last step of PRISMA, the database was complemented by identifying additional articles by analyzing the reference lists in various review articles. Finally, 98 publications were considered relevant studies, as listed in the Supporting Information, Table The database entries can be divided into six groups: (1) basic information on the publications; (2) material characterization including name, manufacturer, purity, crystal form, nominal diameter, length, coatings, shape, hydrodynamic diameter, and surface charge  in the aqueous test medium; (3) taxonomic group, full name and common name of species, and functional feeding group (only for zooplankton); (4) experimental conditions containing exposure routes, diet, exposure type, test location, illumination, and pH; (5) bioaccumulation results including nominal concentration, measured concentration, dose regime, acute or chronic studies, form measured, detection method, body burden in mg/kg organisms dry weight, endpoint , depuration duration, depuration with food or not, body burden after depuration, and remaining accumulated percentage after depuration; and (6) tissue distribution. In addition, each study was analyzed to find information concerning (1) trophic transfer among freshwater aquatic organisms, (2) the kinetic processes involved with bioaccumulation and depuration, and (3) particle size effects on bioaccumulation. When the data points were only presented in figures in the publications, they were extracted by using WebPlotDigitizer Ver 4.2 Rohatgi, . All theWe collected data points representing different exposure conditions, sizes, zeta potentials, or surface coatings. To minimize the over‐representation of kinetic studies reporting data under the same conditions at multiple time points, we only collected the data at the end of the uptake and elimination period. Thus, each row in the database represents an independent observation.Because studies varied in reported units of body burden, we converted them all to mg/kg dry weight of the organisms to enable the comparison of bioaccumulation data. The conversion factor of whole‐body weight from wet to dry weight is 10 for phytoplankton  and phytoplankton (n = 81). Thirty percent of the relevant studies investigated trophic transfer among freshwater aquatic organisms, 46% of the relevant studies presented the kinetics of bioaccumulation, and 25% evaluated the particle size effect on bioaccumulation. More details on parameter availability are plotted in the Supporting Information, Figure In total, 525 endpoints were collected from 98 laboratory‐based (or mesocosm‐based) studies with 38–60 entries for each endpoint, depending on how detailed the experimental conditions, the physicochemical parameters of the ENMs, and the number of tissues that were reported. Zooplankton was the most studied organism group, with 335 endpoints, followed by fish (Particle concentration section. Elimination regimes included no depuration (Sx.1), depuration in a clean water environment (Sx.2), and depuration in clean water with clean food (Sx.3). A distinction was also made between acute studies and chronic studies (Sx.4).We considered four types of uptake regimes and three types of elimination regimes, as presented in Figure Daphnia magna) relative to the mass of the whole organism. The gut is the main organ where zooplankton accumulate ENMs  allows us to gain more insight into the elimination process of ENMs based on the body burden before and after elimination in the same study  are organisms that feed by passing water through a specialized filtering feature that filters suspended matter as food. Gathering collectors catch food suspended in the water by using net‐like structures. Scrapers graze biofilm and algae that are attached to surface of rocks and plants. Shredders chew large pieces of material such as twigs and shrubs that fall into the water  on bioaccumulation and body burden (Corg) of zooplankton. Environmentally relevant exposure concentrations (less than 1 mg/L) are shown separately. When the measured Cw was low, the variation in Corg and bioaccumulation endpoints (BCF and BAF) was substantial. As depicted in Figure org ranged from 0.002 to 60,000 mg/L. Bioaccumulation endpoints calculated based on measured Cw ranged from 6 to 214,000 L/kg. When Cw increased above 15 mg/L, Corg, and bioaccumulation endpoints decreased significantly, below 300 mg/kg and 10 L/kg, respectively.Figure org, BCF or BAF first increased and then decreased. The turning point was at an approximate concentration of 0.15 mg/L, where organisms seem to reach the maximum amount of ENMs they can take up. This turning point could be attributed to the agglomeration, aggregation, and sedimentation of ENMs at high exposure concentrations  has a significantly lower BCF than Au and TiO2, this is only based on one single study. The BCFs of CNTs and C60, with log BCF of 1.8 and 3.1, are based on just one single data point, and thus their BCF values should not be overinterpreted.The bioaccumulation of different ENMs is shown in Figure 2, Au, C60, and CNTs, with more than 40 data points collected for each of them. The lowest bioaccumulation in zooplankton was found for Al2O3 and Fe2O3, with log BCF or log BAF in the range of −0.6 to 3. The carbonaceous ENMs (median in a range of 4–5) were more bioaccumulative than the other ENMs (median in a range of 0–3) except for TiO2. The carbonaceous ENMs compiled in our database include C60, CNT, and graphene. We investigated the effects of hydrodynamic diameter and zeta potential on bioaccumulation for these two groups of ENMs, carbonaceous and other (except for TiO2). As shown in the Supporting Information, Figure Compared with phytoplankton, the bioaccumulation level (log BCF or log BAF) of zooplankton plotted in Figure The influence of physicochemical characteristics on bio‐uptake is presented in Figure 2 nanoparticles with different nominal sizes  aggregated to similar hydrodynamic sizes of approximately 900 nm.As shown in the Supporting Information, Figure Figure 2) and aluminum hydroxide (Al(OH)3) layers enhance the dispersion of nano‐TiO2 in an aqueous medium  was low, with a median log BAF value of approximately 0. No study compiled in our database showed evidence of high ENM transport across the gut tract. Thus, nondissolvable ENMs seem to pose a negligible food safety threat to humans through the pathway of fish consumption.We explored the relationship between the bioaccumulation of ENMs and several extrinsic factors (exposure scenarios and concentrations) and intrinsic factors . Based on the current knowledge, the different uptake and elimination scenarios did not affect the bioaccumulation of ENMs in aquatic organisms. The results of the meta‐analysis showed no significant difference between the mean values of bioaccumulation endpoints from acute and chronic studies, with and without food. In addition, no clear dependency was found between some of the physicochemical characteristics  of ENMs and bioaccumulation.2. However, this may be an indirect effect of different particle sizes of the two groups because we found that the hydrodynamic diameter of the carbonaceous ENMs was in the range of 140–600 nm, where higher bioaccumulation level was observed, whereas the hydrodynamic diameter of other ENMs clustered outside this range, where the bioaccumulation was lower. We further observed that surface treatment plays a role in the bioaccumulation in phytoplankton because bare ENMs accumulated more in the organisms than coated ENMs. Moreover, our results imply that the use of nominal concentrations would underestimate the actual level of bioaccumulation, especially under environmentally relevant aqueous concentrations (less than 1 mg/L). This clearly suggests that future empirical research should report both nominal and measured concentrations of ENMs in the medium during exposure.The identity of ENMs was found to influence the bioaccumulation in zooplankton to some extent. Carbonaceous ENMs were found to be more bioaccumulated than the other ENMs we considered, except for TiOOur database revealed a few gaps in the current knowledge of ENM bioaccumulation, which were also reported in other reviews (Comandella et al., To better understand the actual bioaccumulation behavior of ENMs, more environmentally relevant exposure scenarios via the use of different exposure methods and systems are needed, especially employing mesocosm and field studies (Ayadi et al., https://doi.org/10.1002/etc.5312.The Supporting Information is available on the Wiley Online Library at Yuanfang Zheng: Conceptualization; Methodology; Software; Data curation; Visualization; Writing—original draft preparation. Bernd Nowack: Conceptualization; Supervision; Writing—review and editing; Project administration.This article includes online‐only Supporting Information.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Asthma control, patients’ awareness level and adherence to treatment in Kazakhstan have never been studied. The aim of this study was to verify the prevalence of controlled, poorly controlled and uncontrolled asthma in a large sample of three largest cities of Kazakhstan.We recruited 600 (median age 54 (interquartile range (IQR) 22) years, 64% females) patients with diagnosis confirmed earlier in the outpatient facilities in Almaty, Astana and Shymkent in 2020–2021. We offered a structured questionnaire on asthma control and risk factors as of GINA and performed spirometry. We report the prevalence of asthma control, knowledge and skills and pharmacological treatment with 95% confidence interval (CI) and the association of selected predictors with asthma control.With the median of 9 (IQR 13) years of diagnosis, 40% of patients had comorbid COPD and 42% had allergic rhinitis, whereas 32% lived with pets. Asthma was well-controlled in only 12.3% (95% CI 9.7–15.0), partly controlled in 29.8% (95% CI 26.2–33.5) and uncontrolled in 57.8% (95% CI 53.9–61.8) patients. ACQ-5 score  equaled 0.2 (IQR 0.85) in well-controlled asthma patients, 1.4 (IQR 1) in partly controlled and 2.8 (IQR 1.4) in uncontrolled asthma patients. Knowledge and skills levels were very low. Only 54% were on inhaled corticosteroids . 39% used steroids per os or parenterally within a period of 12 months (51% of patients with uncontrolled asthma).Asthma control, knowledge and skills levels of asthma patients in the largest cities of Kazakhstan remain unacceptably low, whereas pharmacological treatment is far from optimal. Urgent action should be taken to support doctors’ training, and we call to launch a national asthma program to coordinate asthma care in Kazakhstan. In Central Asia, including five countries of the former Soviet Union , the burden of chronic respiratory diseases (CRD) remains very high, whereas mortality from CRD is one of the highest in the World Health Organization European Region . Economi1)/forced vital capacity (FVC) below lower limit of normality (LLN), COPD prevalence was 8.7% in men and 3.4% in women in the age group 40 years and older [Chronic asthma is adults and chronic obstructive pulmonary disease (COPD) are the two leading causes of CRD in Kazakhstan, because the detection of other less prevalent diseases, including interstitial, is hampered by poor infrastructure and insufficient vigilance of primary healthcare doctors. Data on COPD prevalence in the epidemiological studies differ dramatically from the official data , 5. Thusnd older , which wAs with COPD, very little is known about the true prevalence of asthma in the country, and the only population-based study was completed in Almaty , leavingThe study was planned and implemented in only those patients, who had previous and current history of asthma combined with typical clinical presentation at present, received treatment and were listed as asthma patients in the medical facility registry. In three participating Kazakhstan cities, Almaty, Astana and Shymkent, we recruited patients from the lists of asthma patients, who attended local family medical centers (or polyclinics), adhered to a regular follow-up plan and came to obtain medications from the pharmacies in these family medical centers. In Astana and Shymkent, family doctors also communicated with pulmonologists to organize referral for this study. In addition, in Almaty patients were also referred from the Republican Allergy Center by allergy specialists. Two family medical centers in each city participated in the project. A family doctor, a pulmonologist or an allergy doctor in a polyclinic of Republican Allergy Center invited patients by phone and ask to attend examination in a specially designated room of the Faculty of Healthcare and Medicine of Al-Farabi Kazakh National University (for Almaty), Astana Medical University (for Astana) or the Central Provincial Hospital of Turkestan Province (for Shymkent). We enrolled patients with all types of asthma, including allergic and non-allergic asthma, in May 2020 through January 2021.Patients were instructed to take their regular medication in the morning, abstain from smoking 12 h prior to the examination, and attend the clinic in the morning to fill in the questionnaire and perform spirometry. We gave patients 30 min to rest after their arrival to allow for shortness of breath to lessen. A qualified internist assisted patients to fill in the questionnaire, which was offered either in Russian (about 90% cases) or Kazakh. Once the questionnaire was filled, we measured patients’ height and weight using electronic calibrated scales. Each patient signed a written informed consent to participate in the study, and the study protocol was approved by the Committee on Bioethics of Al-Farabi Kazakh National University.The questionnaire comprised 53 questions, was a combination or our original questions with standardized tools and included demographic and personal data, questions on medical history, We ascertained asthma control with Asthma Control Questionnaire of five questions (ACQ-5), validated in Russian , which aTreatment section started with a question whether a patient used any inhaled corticosteroid alone or in combination with a beta-agonist regularly for the last week, and a daily dose. A number of all available brand names on the market were offered in this question. We then asked if a patient used any corticosteroid in pills or injections during the last 12 months. The next question was on a preferred rescue medication and whether a patient has the medication with him/her at all times. we also recorded the number of rescue medication doses during the last 24 h and the last month. We than asked what medication a patient took on a day of examination. This section ended with a question on any other treatments practiced by a patient other than inhaled corticosteroids and beta-agonists.Risk factors for exacerbations verified in the questionnaire, as listed in GINA , include1) in liters and calculated FEV1/FVC from the best curve. All three included volumes were reported as actual in liters along with their percent to predicted values. Because predicted values for spirometry in Kazakh populations have never been published, we used Global Lung Function (GLI)-2012 reference equations for European population [Because the indication for spirometry in this study was to assess treatment effect and disease control, no bronchodilation was performed . Patientpulation . In Almap < 0.05 was a cut-off to consider non-random difference in comparisons.Sample size estimation was guided by the preceding similar studies, but we also considered even more patients with the aim to enroll the maximum number of patients referred from the participating facilities. We tested all continuous variables for normality using Shapiro–Wilk test. Most data were non-normally distributed; therefore, tests used in this study to compare groups were non-parametric, such as Mann–Whitney U test for two-groups comparisons and Kruskall–Wallis test for three or more groups. Groups of binary data were tested for differences using χ2 test from contingency tables, including for three or more groups, such as smoking status of highest attained education. The primary end-point in this analysis was asthma control, expressed as the prevalence of patients with well-controlled, partially controlled and uncontrolled asthma. Prevalence in most groups is expressed as mean with the corresponding 95% confidence interval (CI). Means of normally distributed data are stated as arithmetic means ± standard deviation; alternatively, we report medians with the corresponding interquartile range (IQR). We additionally tested whether such predictors as sex, age, pets at home, comorbid diagnoses, BMI, smoking, years of asthma, exposure to SHS and some other were associated with uncontrolled asthma in bivariate analyses. Because this study mainly planned as descriptive, we did not test predictive role of risk factors in regression models. All tests were completed in NCSS 2020 , and We enrolled 600 patients with bronchial asthma  with the median age 54 (IQR 22) years and with the median of 9 IQR 13) of diagnosis (Table 3 of diagp = 0.02) . In a bivariate analysis, uncontrolled asthma was associated with greater BMI (Mann–Whitney p = 0.03), pets at home  and comorbid COPD . The remaining predictors, such as age (Mann–Whitney p = 0.14), years of asthma (Mann–Whitney p = 0.05), daily smoking (χ2 p = 0.44), exposure to SHS (χ2 p = 0.96), comorbid allergic rhinitis (χ2 p = 0.92) were not associated with uncontrolled asthma.Based on four questions of GINA asthma control, asthma was well controlled in only 74 , partly controlled in 179  and uncontrolled in 347  patients. When comparing three cities using 2*3 χ2 test, we found significant difference between them ; in partly controlled 1.4 (IQR 1); in uncontrolled asthma patients the corresponding ACQ-5 score was 2.8 (IQR 1.4)  readings for their green, yellow and red zones; only 1% performed peakflometry at home daily; and only 4% had a diary of symptoms. 54% of patients possessed a personal nebulizer at home, but that did not affect asthma control .Only half of the group N = 324; 54%) were on inhaled corticosteroids alone or in combination with a long-active beta-agonist a week prior to examination. The fraction of inhaled steroids users significantly increased from 31% in a group of well-controlled asthma to 62% in uncontrolled asthma  with worse lung function. Their median FEV1% was 71% versus 82% predicted (Mann–Whitney p < 0.001), median FVC 80.5% versus 88% predicted (Mann–Whitney p < 0.01) and median FEV1/FVC 70% versus 77% (Mann–Whitney p < 0.001).Patients receiving inhaled steroids regularly had generally more severe disease. Thus, their ACQ-5 score was greater ; GERD was reported by 72 (12%) patients; confirmed food allergy was found in 100 (17%) and exposure to allergens in 214 (36%). Severe asthma exacerbation during the preceding 12 months was reported by 262 (44%) patients and was associated with greater ACQ-5 score , lower FEV1% predicted , FVC% predicted  and even FEV1/FVC . Twelve percent (N = 71) were ever-intubated or treated in an intensive care unit, and that predicted worse ACQ-5 score , lower FEV1% predicted , FVC% predicted  and even FEV1/FVC .Chronic rhinosinusitis was present in 137 (23%) patients  , up to 5Insufficient asthma control in Kazakhstan presented in this study is likely the outcome of poor training and unacceptable follow-up of patients. Asthma patients get admission to the outpatient facilities along with all other numerous reasons of admission for other patients, thus find themselves in a situation of very limited time and almost no opportunities to be trained how to use an inhaler, how to monitor asthma, what to do in case of symptom worsening, etc. Very poor awareness level on asthma symptoms management, use of inhalers and the value of peakflowmetry in our patients result from such improper time management of a general practitioner. However, very limited asthma knowledge was earlier reported elsewhere, such as in Turkey . With a One of surprising findings of this study was a frequent use of formoterol/ipratropium combination for asthma attack relief. The rationale for highly prevalent ipratropium use in asthma patients in Kazakhstan lies in the axis of medications procurement by the state medical facilities. Pharmacological treatment of both asthma and COPD patients listed as chronic patients is free of charge for patients and covered by the state programs. Therefore, buying one rescue medication instead of a range of other options for both patient groups saves time for personnel responsible for procurement. High prevalence of peroral corticosteroids and aminophylline is explained by inability to attain asthma control with inhaled steroids resulting from a combination of reasons  . PeroralPoor asthma control and very low skills and awareness levels of asthma patients necessitate further speculation . We beliWe consider inclusion of all three major cities with a population close to or more than one million people in Kazakhstan a strength of this presentation. The use of both GINA asthma control and risk factors questionnaire with ACQ-5 test, supplemented by spirometry is also an advantage of our study. Nevertheless, our study was limited to urban population only, whereas rural population may have even worse asthma control due to limited access to learning resources, direct access to medical facilities and medications . TherefoIn conclusion, this first report of asthma control in Central Asia demonstrated that yet very low fraction of adult asthma patients has their asthma under control, knowledge and skills of asthma patients are unacceptably poor, whereas pharmacological treatment is far from optimal and is not always consistent with GINA recommendations. Given that asthma pharmacological treatment in Kazakhstan is paid by the state, effort should be made to attain better asthma control with more resources allocated to doctors’ and patients’ training, improvement in spirometry quality and access to it, as well as sustainable research in asthma care, including the countryside. These findings might be more generalizable to the country’s urban areas and that more research is needed to understand the prevalence of asthma control in the rural regions of the country.


Multiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system. Changes in lipid metabolism have been suggested to play important roles in MS pathophysiology and progression. In this work we analyzed the lipid composition and sphingolipid-catabolizing enzymes in erythrocytes and plasma from MS patients and healthy controls. We observed reduction of sphingomyelin (SM) and elevation of its products—ceramide (CER) and shingosine (SPH). These changes were supported by the detected up-regulation of the activity of acid sphingomyelinase (ASM) in MS plasma and alkaline ceramidase (ALCER) in erythrocytes from MS patients. In addition, Western blot analysis showed elevated expression of ASM, but not of ALCER. We also compared the ratios between saturated (SAT), unsaturated (UNSAT) and polyunsaturated fatty acids and suggest, based on the significant differences observed for this ratio, that the UNSAT/SAT values could serve as a marker distinguishing erythrocytes and plasma of MS from controls. In conclusion, the application of lipid analysis in the medical practice would contribute to definition of more precise diagnosis, analysis of disease progression, and evaluation of therapeutic strategies. Based on the molecular changes of blood lipids in neurodegenerative pathologies, including MS, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine. Multiple sclerosis (MS) is an immune-mediated, neurodegenerative, demyelinating, chronic disease of unknown etiology with a possible genetic predisposition and effect of certain environmental factors ,2. This CERs with short carbon chains were found to stimulate oxygen species accumulation in hippocampal glial cells, which leads to oxidative stress and neuronal death ,10,11. TThe other member of the SL family, SPH, has received less research attention, compared to CER and S1P. However, the role of SPH in degeneration of oligodendrocytes and neurons in MS patients has been documented . There aOxidative stress is harmful in MS and results in formation of oxidized lipid products. Lipid peroxidation plays significant role in MS pathogenesis which is associated with stimulation of apoptotic events . PolyunsReports show augmentation of lipid peroxidation products in cerebrospinal fluid (CSF) and plasma of MS patients . AccumulErythrocytes usually have bigger diameters in MS, which is positively correlated with the progression of the disease. The erythrocytes lipid content, lipid metabolism and molecular profile are different in MS patients, which makes these cells an object of special interest. Red blood cells are assumed as a major store and source of S1P, which, as already mentioned, plays a specific pathogenic role in MS. Since S1P has been largely studied due to its ubiquitous functional activity, in this work we focused our attention on the mechanism of accumulation of two other important sphingolipid metabolites—CER and SPH, the latter being recognized as a precursor of S1P.Studies were carried out on the SM–S1P pathway and were focused particularly on CER and SPH and on the enzyme activities responsible for their accumulation—sphingomyelinase and ceramidase. As already mentioned, the SM signaling cascade is composed mainly of SM, CER, SPH and S1P, in which one component emerges from the other by sequential enzymatic degradation. The obtained results showed that erythrocytes and plasma of MS patients contain less SM and PUFA, as well as higher levels of CER and SPH, compared to controls. Also, blood plasma of MS patients was observed to contain upregulated sphingomyelinase (SMase) activity, which we presume is responsible for the reduction of SM, the latter acting as a natural membrane antioxidant .The data showing the clinical course of MS according to Kurtzke’s Expanded Disability Status Scale (EDSS) for the eighteen patients under investigation are shown in The characteristics of the patients and the controls are presented in In this work we studied the alterations in the phospholipid (PL) composition of erythrocyte membranes (ghosts) and plasma form patients with MS . We wereSince there is permanent lipid exchange between erythrocytes and plasma, we also analyzed the PL composition of plasma from MS and healthy individuals B. In MS It should be noted that in plasma and ghosts of both analyzed groups we tested the phospholipid, lysophosphatidylcholine (LPC) that is missing in most of the reports devoted to blood PL composition in MS patients. Since LPC, which was elevated in MS patients, is a hydrolytic product of PC, we were interested in analyzing of the phospholipase activity responsible for degradation of PC to LPC and arachidonic acid (AA) which is discussed below.The reduction of SM is an interesting finding, which deserves special attention. SM is the source of the functionally active CER and SPH, which have been related to demyelination and oligodendrocyte death in MS . We founp < 0.05). Since ceramide in erythrocyte membranes and plasma is a product of the secreted acid sphingomyelinase (ASM), we analyzed its activity in blood plasma . The resThus, it seems likely that both upregulation and higher expression of ASM underlie the elevation of CER, which through ceramidase, yields SPH, the letter being phosphorylated by sphingosine kinase to S1P. To analyze in detail whether the sphingolipid catabolic pathway is responsible for accumulation of sphingosine, we measured the activity of alkaline ceramidase (ALCER), which is located preferentially in erythrocytes . ALCER wThe relatively high content of LPC in blood plasma and erythrocytes ghosts of MS patients  focused Our studies confirmed the already known fact, that the level of saturated fatty acids (SAT) was higher in erythrocytes and plasma of MS patients . We comp2α. These are prostaglandin F2α-like compounds, which are produced by free radical–catalyzed peroxidation of arachidonic acid and are assumed as markers of in vivo oxidative stress. As evident from SM acts as an intrinsic antioxidant which prevents the PL fatty acids from oxidative destruction due to its tight association with the acyl chains. Thus, its reduction could render the polyunsaturated acyl chains more vulnerable to oxidative attack. As already mentioned, oxidative stress is considered as a pathogenic factor, participating in the onset and progression of MS . StudiesMultiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system, where the body’s immune system attacks the nerves and the myelin sheath. These processes result in a variety of symptoms involving disturbed movement, fatigue, pain, changes in vision, cognitive difficulties, etc. . The etiSphingolipids, together with glycerophospholipids, are involved in many cellular functions, including cell proliferation, signaling cascades, apoptosis, etc. Changes in lipid metabolism and level of separate lipid molecular species have been suggested to play important roles in multiple sclerosis pathophysiology and progression. Especially sphingolipids, which are largely expressed in the Central Nervous System (CNS), have been implicated in the pathogenesis of MS . The majIn this work we analyzed the lipid composition of erythrocyte membranes (ghosts) and plasma from MS patients and healthy controls A,B. AlthFurther research was focused on the accumulation of SPH, which is precursor of S1P, the latter being the most functionally active member of sphingolipid metabolites. A large number of studies has been concentrated on S1P and much less interest has been devoted to SPH as a member of the sphingolipid family. This is why we studied the MS-induced changes in the mechanism of SPH production, which occurs through degradation of CER by alkaline ceramidase (ALCER), the latter being reported in human erythrocytes . Our stuThe major membrane phospholipid, PC, which serves as PLA2 substrate, showed a trend for reduction in both erythrocyte membranes and plasma from MS patients, although the observed differences were not statistically significant A,B. In aAs mentioned above PUFA are products of PLA2, the latter circulating in human plasma. PLA2 plays critical roles in the pathogenesis of neurodegenerative diseases such as multiple sclerosis by enhancing oxidative stress and initiating inflammation. The levels of PLA2 activity in MS patients and the effect of inhibiting PLA2 on the severity in different experimental models of neurodegenerative pathologies have not been elucidated. Inhibiting sPLA2 leads to lower clinical severity or no signs of experimental autoimmune encephalomyelitis (EAE) in mice, and a lower incidence of EAE lesions compared to animals without PLA2 inhibition . The samChanges in plasma FA have been reported to correlate with the progression of MS. Most epidemiological studies state that diets rich in saturated FA correlate with MS progression, provoking the development of this pathology. On the other hand, diets rich in PUFA seem to decrease the risk of MS development , may eveReports show that not only unsaturated fatty acid products, but also CERs play role in development of oxidative stress. There is evidence that CERs can significantly increase reactive oxygen species liberation in glial cells . MS pathWe measured the plasma content of lipid-derived markers indicating oxidative destruction such as lipid peroxides and F2-isoprostanes . The conThere are certain limitations concerning the conclusions drawn, which are related mainly to the restricted number of the tested cohort. The reported results were observed in patients with relatively high disability and long disease duration. Further studies should be performed to confirm the validity of the present results for patients with lower disability and disease duration.In conclusion, multiple factors are reported to increase the risk of onset and progression of multiple sclerosis, but the etiology of this pathology remains largely unclear. As typical for other inflammatory neurodegenerative pathologies, oxidative stress and lipid peroxidation are closely related to MS development. Alterations in the lipid profile seem to be specific for this disease which is associated with dysregulation of the lipid homeostasis and lipid metabolism, this being valid especially for sphingolipids. Lipid analysis presented in this work demonstrates the changes of lipid molecules and their metabolism in erythrocytes and plasma of MS patients. Clinical lipidomics has the potential to be applied in MS diagnosis as well as in evaluation of therapeutic approaches by providing a detailed analysis of the lipidome profile of MS patients. Finally, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine.All patients and healthy individuals gave written informed consent before being included in this study. The designed investigations were approved by the local ethics commission. Eighteen healthy non-smoking individuals (nine men and nine women) in the range 35–57 years of age participated in the investigations. The 18 patients (nine men and nine women), aged 34–59 years were clinically diagnosed with relapsing-remitting multiple sclerosis (the most common form of MS), according to the McDonald revised diagnostic criteria for MS (2017) and by neuroimaging. The degree of disability was determined according to the scale of Kurtzke’s Extended Disability Status Scale (EDSS) . The MS g for 10 min at 4 °C. The supernatant from this spin was centrifuged at 10,000× g for 10 min at 4 °C to pellet any remaining cells or platelets. The supernatant thus obtained was used in the experiments listed below. Ghosts were prepared by freezing and thawing the red cells. Any intact cells which remained after three times freezing and thawing were pelleted by centrifugation. Erythrocyte membranes were kept frozen at −70 °C until used.Blood samples of 10 mL were collected by venipuncture of the peripheral forearm vein around 8 AM after overnight fasting. The obtained blood was anticoagulated with sodium citrate. The erythrocytes were pelleted by centrifugation at 2000× Lipids from erythrocyte ghosts and plasma were extracted with chloroform/methanol according to the procedure of Bligh and Dyer . The orgThe phospholipid extracts were saponified with 0.5 N methanolic KOH and methylated with boron trifluoride-methanol complex . The fatty acid methyl esters were separated by GC-MS on a column 60 m × 0.25 mm ID BPX70 × 0.25 µm.v/v) at flow rate of 2 mL/min. The HPLC was equipped with an automatic injector with an injection loop between 50 and 1000 μL. Under these conditions, the typical elution time for NBD-ceramide was about 10 min. The excitation wavelength was 455 nm and emission was detected at 533 nm [Separation of NBD-ceramide was performed on a disposable reverse phase column  using methanol: water: 85% phosphoric acid  according to the manufacturer’s instructions.Plasma sphingosine level was determined using sphingosine ELISA kit  according to the manufacturer’s instructions.3/CH3OH . The samples were vortexed for 10 s and centrifuged at 5000× g. An aliquot of the aqueous phase was measured for fluorescence. The hydrolysis of NBD-SM by ASM results in release of NBD-phosphocholine into the aqueous phase, whereas ceramide and the unreacted NBD-SM remain in the organic phase. Results were expressed as pmol hydrolyzed SM/min/µL plasma [ASM activity in plasma was measured using NBD-SM as substrate. Before use, the substrate was dried under nitrogen, resuspended in 200 mM sodium acetate (pH 5.0) and sonicated for 10 min to obtain micelles. Incubations were performed for 30 min at a final volume of 0.5 mL. The reaction was terminated by extraction in 0.5 mL CHClL plasma .2 and 5 mM MgCl2) at 37 °C for 30 min. The reaction was terminated by addition of 0.5 mL CHCl3/CH3OH . After the extraction, sphingosine was determined by sphingosine ELISA kit . Results were expressed as pmol hydrolyzed CER/min/µg ghost protein [Ceramidase activity was determined by the release of sphingosine from ceramide. Briefly, erythrocyte ghosts (50 µg protein) were incubated with ceramide substrate (100 mM) in buffer 25 mM Tris-HCl bufer, pH 9, containing 5 mM CaCl protein .2 and 0.1% fatty acid free bovine serum albumin. Incubation was carried out for 30 min at 37 °C with continuous shaking in a total volume of 0.5 mL. The reaction was stopped with 0.5 mL chloroform/methanol (2:1 v/v) and the liberated fatty acids were extracted. After methylation the fatty acid methyl esters were determined by GC-MS. PLA2 activity was calculated as pmol fatty acids/min/µL plasma and the alteration was expressed as % compared to control values.Phospholipase A2 activity was assayed by the method described below. Plasma samples were incubated with 100 nmol egg yolk phosphatidylcholine as substrate in 100 mM Tris-HCl pH 8.6 with 5mM CaClLipid peroxidation in plasma was measured by a fluorimetric method using thiobarbituric acid (TBA). The plasma lipids containing lipid peroxides were precipitated with phosphotungustic acid and then were incubated with TBA. The fluorescence of the reaction product was measured with excitation at 515 nm and emission at 553 nm. The concentration of lipid peroxides was expressed in terms of malondialdehyde (ng/mL plasma) using tetramethoxypropane as a standard.2α levels were determined using ELISA isoprostane kit ABCAM, (ab175819) according to the manufacturer’s directions.The 8-iso-PGF™  according to the manufacturer’s instruction. Erythrocyte ghosts and plasma samples  and heated for 4 min at 95°C. Proteins were resolved on SDS-PAGE, transferred to nitrocellulose membrane and blocked for 1 h in 5% non-fat dry milk in TBST . Membranes were than incubated with the appropriate primary and secondary antibodies, including polyclonal anti-alkaline ceramidase 2 , monoclonal anti-acid sphingomyelinase , monoclonal anti-vitronectin , monoclonal anti-GAPDH , anti-mouse IgG (Fab) HRP conjugate , anti-rabbit IgG (Fab) HRP conjugate . Immunoblots were visualized using the ECL system (Santa Cruz).Prior to electrophoresis, plasma samples were depleted from albumin and IgG by using Albumin IgG Depletion SpintrapThe content of protein was determined according to the method of Bradford and colleagues .Statistical processing of the data was made by one-way analysis of variance (ANOVA), using In Stat software, Graph Pad In Stat 3.1, developed by Graph Pad Software, San Diego, CA, USA.The first member of the sphingolipid cascade, sphingomyelin (SM), is reduced and ceramide (CER) is increased in erythrocyte membranes and plasma of MS patients.The expression analyzed by Western blot and the activity of acid sphingomyelinase (ASM) were elevated in plasma of MS patients compared to controls.The activity of alkaline ceramidase (ALCER) is upregulated but the expression is unchanged in erythrocyte membranes of MS patients compared to controls.We suggest that the unsaturated/saturated (UNSAT/SAT) fatty acid ratio could serve as a marker to differentiate between erythrocytes and plasma of MS patients and controls.Our results confirmed that the lipid-derived markers of oxidative stress, lipid peroxides and isoprostanes, are higher in the plasma of MS patients than in control individuals.