Exoskeleton-assisted walking (EAW) is expected to improve the gait of spinal cord injury (SCI) individuals. However, few studies reported the changes of pulmonary function (PF) parameters after EAW trainings. Hence, we aimed to explore the effect of EAW on PF parameters, 6-min walk test (6MWT) and lower extremity motor score (LEMS) in individuals with SCI and to compare those with conventional trainings.1), forced expiratory flow (FEF), peak expiratory flow, and maximal voluntary ventilation, 6MWT with assisted devices and LEMS were reported pre- and post-training.In this prospective, single-center, single-blinded randomized controlled pilot study, 18 SCI participants were randomized into the EAW group (n = 9) and conventional group (n = 9) and received 16 sessions of 50–60 min training . Pulmonary function parameters consisting of the forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1 (p = 0.013) were significantly greater in EAW group  compared with conventional group  after training. Participants in EAW group completed 6MWT with median 17.3 m while wearing the exoskeleton. There was no difference in LEMS and no adverse event.Values of FVC (p = 0.041), predicted FVC% (p = 0.012) and FEVTrial registration: Registered on 22 May 2020 at Chinese Clinical Trial Registry (ChiCTR2000033166). http://www.chictr.org.cn/edit.aspx?pid=53920&htm=4.The current results suggest that EAW has potential benefits to facilitate PF parameters among individuals with lower thoracic neurological level of SCI compared with conventional trainings. Additionally, robotic exoskeleton helped walking. The online version contains supplementary material available at 10.1186/s12984-021-00880-w. Spinal cord injury (SCI) is a worldwide life-disrupting pathological condition with estimated 17,810 injuries occurred in the United States in 2017 . RespiraPulmonary function parameters are affected by the paralyzed degree  of the respiratory muscles  which co2 max [1), forced expiratory flow (FEF25/50/75), peak expiratory flow (PEF), and maximal voluntary ventilation (MVV). Besides this, previous trials were non-randomized controlled trials. Therefore, this randomized controlled study primarily aimed at finding out whether the EAW trainings are different from conventional rehabilitation trainings in improving PF parameters among SCI individuals. Our hypotheses were that EAW training can improve the PF parameters and maintain the lower extremity motor score (LEMS) which does not make a difference relative to that made by conventional training. Second, individuals could complete walking while wearing the exoskeleton. To our knowledge, this is the first clinical trial to concern about the PF parameters by EAW training.Recently, exoskeleton-assisted walking (EAW) has been confirmed to help individuals with thoracic and lumbar SCI to walk safely –22. Desp2 max –25 durinThis was a single-blinded, randomized controlled efficacy trial with 2 parallel groups and intention-to-treat analysis. The study protocol has been registered at Chinese Clinical Trial Registry (ChiCTR2000033166) and approved by the medical ethics committee of West China Hospital of Sichuan University (#19-667). All patients were informed of the procedure, the use of their data and images for research. They understood the purposes and provided written informed consent according to the 1964 Declaration of Helsinki prior to their participation.From May 2020 to August 2020, we prospectively enrolled all adult individuals with a diagnosed SCI below T3 and above L2 at least 1 month. Participants were recruited from inpatients in 3 units of Rehabilitation center, West China Hospital, Sichuan University. None of them had EAW training experience before. In addition, the eligible individuals met the following inclusion criteria: (1) American spinal injuries association impairment scale  (ASIA) cThe individuals were randomly divided into the EAW group or conventional group in a 1:1 ratio by simple randomization method, using computer-generated simple random tables. The sequences were preserved using closed envelop method by one researcher who did not participant in the trainings and assessments (H.C.H). PFT and LEMS assessment of individuals pre- and post-training were performed by the same two clinical researchers (X.Y. and Y.O) who were blinded and did not know whether the individual was in EAW or conventional group. 6MWT was performed by two researchers who participated in EAW training (H.C and C.P.D) and conventional training (H.Y.Z). Clinical data was recorded after averaging.max = 220—age)[® China).The AIDER  powered robotic exoskeleton  was used for the EAW training. All subjects were individually fitted to the robotic exoskeleton according to pelvic width, thigh length, and shank length. Exoskeleton-assisted walking training program was conducted consecutive 4 days a week, 16 training sessions in total. Every training session lasted 50–60 min containing interval rest as needed by standing, leaning on the wall, or sitting while wearing the device. Training session included sitting, standing, walking, climbing stairs and slope with maximal assistance-walking mode  that is max, 50–60 min/session, 4 days/week, 4 weeks). Medications and rehabilitation nursing were ordered based on the medical condition.For the conventional group, the rehabilitation program included strength training using dumbbell between 5 and 20 kg, aerobic exercise, such as walking training with brace as well as static and dynamic balance training in sitting or standing position. The conventional rehabilitation training had the same intensity, duration, and frequency as EAW training  based onthe standardized procedures as the American Thoracic Society  describe25/50/75 means forced expiratory flow at 25, 50 and 75% of the FVC. Peak expiratory flow reflects the intensity of respiratory muscles. Maximal voluntary ventilation refers to the maximum volume of air, a subject can breathe over a specified period [Forced vital capacity refers to the total capacity of air that can be blown out by maxima forced expiration following maximal inspiration. Forced expiratory volume in 1 s refers to the capacity of air that is blown out for a single second. Force expiratory flowd period .2). In additional, the level of effort at the end of the test was reported by the rate of perceived exertion (RPE) based on the Borg scale [The 6MWT  was performed in door that is aimed to determine walking ability. Additionally, it can manifest cardiorespiratory endurance through evaluating the walking distance in accordance with the guidelines of the American Thoracic Society . At the rg scale . Particirg scale  and ASIAThe statisticians (X.N.X and J.D) were blinded to the program and completed analyses utilized SPSS version 25 . The Shapiro–Wilk test was used to determine if data were normally distributed. These were recorded as means ± standard deviations (SDs), others were described as median and inter quartile range (IQR) where necessary. The mean values (Δ) of post-intervention minus pre-intervention were recorded. Independent Student t-test was used to compare continuous data related to clinical features between two groups. Paired Test Student t-test was used to compare varies between pre- and post-intervention. Furthermore, the Fisher’s exact and Pearson’s Chi-square tests were used if the data were categorical variables. Wilcoxon rank-sum test and Mann–Whitney U test were used if the data was not normally distributed. Pearson correlation test was performed to discuss the relation between the distance of 6MWT and changes of PF parameters. In all statistical tests, p < 0.05 was defined as significant.A total of 87 individuals with SCI were screened enrollment, of which 69 were excluded as per exclusion criteria n = 61) or declining to commit the full participation (n = 8). Eighteen eligible individuals were randomized to either EAW group (n = 9) or conventional group (n = 9). The Consolidated Standards of Reporting Trials (CONSORT) diagram is showed in Fig.  or decliThere were no adverse events related to treatment in either group.1  between groups. Nevertheless, there were no statistically significant differences in FEF75 , FEF50 , FEF25 , PEF , and MVV  , FEF75 , PEF , and MVV . Howbeit, there was no statistically significant differences in FVC , FEF50 , and FEF25 . Nonetheless, there was no evidence of statistical improvements from pre- to post-intervention for individuals who received conventional training in FVC , predicted FVC% , FEV1 , FEF75 , FEF50 , FEF25 , PEF , and MVV .Participants who treated with EAW training had statistical improvements from pre- to post-intervention in mean change in predicted FVC% , FEV2  than conventional group. There were differences in HR , and RPE  between groups. Moreover, participants who treated with EAW training had differences from pre- to post- intervention in HR , and PRE . Howbeit, distance  and SpO2  showed no differences. For conventional group, no statistical differences were reported for distance , HR (Z = 0.351 = 0.725), SpO2  and RPE . The outcomes of correlation between the distance of 6MWT and evert item of PFT are shown in Additional file 1  and FEF75 .Of the 10 participants who completed the final 6MWT, 2 were in the conventional group. All participants in EAW group completed it while wearing the exoskeleton in door. A summary of secondary outcomes is provided in Table For LEMS, there was no statistical difference between two groups . Additionally, neither groups showed improvement in LEMS . More detailed data are provided in Additional file 1 compared with conventional trainings. Nonetheless, there were no differences in FEF, PEF, and MVV between two groups. Moreover, EAW trainings offered statistically different results of PFT than the beginning, except FVC, FEF50 and FEF25.This study focused on the changes of PFT and 6MWT using EAW and conventional trainings in individuals with T4 to L1 SCI. Robotic exoskeleton has been explored and applied in rehabilitation treatment after SCI. At present, the robotic exoskeleton has demonstrated some advantages in assisting walking and improving physical functions , 35. We 2 peak among the incomplete SCI individuals during gait training. We reported changes in PF parameters among complete and incomplete SCI individuals. In the present study, it was reported better results in FVC, predicted FVC% and FEV1 compared with the conventional trainings that were widely used in SCI rehabilitation program. There were statistical differences in post-predicted FVC%, FEV1, FEF75, PEF and MVV after EAW compared with pre-outcomes. The improvements in PF parameters might reflect the increasing of respiratory muscle strength and pulmonary ventilation. Alamro et al. [Some previous studies –25, 35 ho et al.  has demoo et al.  also repo et al. , 39.1 and FVC associate with higher inflammatory factors that reflect systemic inflammation. Hence, better results of FEV1 and FVC are beneficial to reduce systemic inflammation and manifested better functions of principal bronchus. Although there was no difference in pre- and post- FVC in 2 groups, we reported a higher mean value after EAW training. At least, both trainings helped to maintain the FVC, and EAW might improve the FVC. Moreover, the minimal clinically important difference for predicted FVC% is 2–6% by distribution-based method [1 is 0.1 L according to previous study by the anchor-based method [The increasing or maintenance of PF parameters has crucial meanings for many patients with SCI. Hart et al.  has repod method . The mead method , which i2 max improves exercise capacity among individuals with C7 to L5 SCI. In this study, the HR values during the trainings reached a moderate-intensity level based on the ACSM guidelines for exercise testing and prescription [As for the conventional trainings which are recommended and wildly used for many years –45, thiscription . The incRobotic exoskeleton was achievable to enhance the walking capacity. Furthermore, no improvement in LEMS also manifested that EAW effect on walking capacity, not no motor function or lower limb muscle performance. Our result of 6MWT was similar to McIntosh et al.  and SaleFor individuals with SCI, the results of 6MWT might not report the cardiopulmonary endurance completely. In this study, the distance was partially related to PFT. There was no statistically significant collection between the distance and most item of PFT. This might be influenced by the primary disease, different assisted devices and the using duration. Longer using duration may result in longer distance. Benson et al.  found thThis study was limited to the small sample size and number of training session, although our feasibility study has proved this training period realized the application of EAW and was most achievable in inpatient rehabilitation in the health care system. The results in this study may not be generalized to larger population. It is necessary to explore the sample size and future investigation on the effectiveness of EAW. Although we tried to avoid the detection bias, it was inevitable during the 6MWT. A majority of subjects from the EAW group was AIS A level that may influence the results of 6MWT, although there was no difference of LEMS at the baseline. Moreover, further study needs to have control groups composed of healthy participants with EAW training or conventional training, and compare with individuals with SCI.max by the formula: HRmax = 220—age might be inaccurate. HRmax is individual and related to sex and physical activity status [max in older adults [Furthermore, the intensity calculated by the HR was rough and imprecise in some degree. The predict HRy status . In thisr adults . MoreoveThe study successfully manifested that EAW training has the potential to improve performance in PF parameters among individuals with SCI, especially in ventilation. Additionally, robotic exoskeleton is a device that can assist individuals with SCI to stand up and walk. The results suggested that the effectiveness of EAW might be similar, or better than conventional training. Further research is essential to confirm these results in a larger sample size and better study protocols. The data in this study will be helpful for further efforts towards improving the design and clinical application of EAW.Additional file 1. Outcomes of correlation between the distance of 6-minute walk test (6MWT) and every item of pulmonary function test (PFT).Additional file 2. Individual-participant data.


The FTIR analysis of PPE-CSNPs demonstrated no spectral changes indicating no possible chemical interaction between the PPE and CSNPs, which confirms that the PPE was physically entrapped within NPs. Moreover, FTIR spectra of pure PPE showed specific absorption bands (at 3293–3450 cm−1) attributed to the incidence of phenolic compounds, such as tannic acid, ellagic acid and gallic acid. Total phenolic content (TPC) and antioxidant analysis of selected CSNPs revealed that the encapsulated NPs had significantly lower TPC and antioxidant activity than those of pure PPE, indicating that CSNPs successfully preserved PPE from rapid release during the measurements. Antibacterial tests indicated that pure PPE and PPE-loaded CSNPs effectively retarded the growth of Gram-positive S. aureus with a minimum inhibitory concentration (MIC) of 0.27 and 1.1 mg/mL, respectively. Whereas Gram-negative E. coli, due to its protective cell membrane, was not retarded by pure PPE and PPE-CSNPs at the MIC values tested in this study. Gas chromatography-mass spectroscopy analysis confirmed the incidence of various phytochemicals, including phenolic compounds, fatty acids, and furfurals, with possible antioxidant or antimicrobial properties. Overall, CSNPs can be regarded as suitable nanomaterials for the protection and controlled delivery of natural antioxidants/antimicrobials, such as PPE in food packaging applications.The encapsulation of pomegranate peel extract (PPE) in chitosan nanoparticles (CSNPs) is an advantageous strategy to protect sensitive constituents of the extract. This study was aimed to develop PPE-loaded CSNPs and characterize their physical, structural morphology, antioxidant and antimicrobial properties. Spherical NPs were successfully synthesized with a mean diameter of 174–898 nm, a zeta potential (ZP) of +3 – +36 mV, an encapsulation efficiency (EE) of 26–70%, and a loading capacity (LC) of 14–21% depending on their loaded extract concentrations. Based on these results, CSNPs with chitosan:PPE ratio of 1:0.50 ( Punica granatum L.) comprise roughly half of the fruit’s weight and are not directly consumed and discarded as waste. The peels are rich in bioactive compounds, such as polyphenols, flavonoids, proanthocyanidins, and hydrolyzable tannins compared to pomegranate juice and seeds [The food industry produces copious amounts of agricultural waste with potential application as the substrate for the recovery of added-value compounds ,2. In thnd seeds ,8,9,10. nd seeds , antimicnd seeds ,12, antind seeds , anti-innd seeds , antimutnd seeds , and antnd seeds  and coulMost natural bioactive compounds and bio-preservatives are sensitive to oxidative reactions and, therefore, can be easily decayed by extreme environmental or processing conditions  ,18. ThisChitosan is a linear polysaccharide with cationic nature and high potential to encapsulate natural ingredients. This would result in developing different forms of chitosan matrix . These systems can be used for the encapsulation of medicinal herbal extracts and essential oils for potential applications in the food industry, pharmaceutical and cosmetics . ChitosaArrabidaea chica extract [Tridax procumbens leaf extract [Many reports describe the encapsulation of various herbal extracts in CSNPs and their potential applications . The enc extract , flavono extract , phenoli extract , and Tri extract  in CSNPsEscherichia coli (PTCC 1163) and Staphylococcus aureus  as stock cultures were obtained from Persian Type Culture Collection .Low molecular weight (LMW) chitosan (Mw 55–180 kDa) with deacetylation degree of 74–84%, acetic acid, sodium tripolyphosphate (STPP), Folin–Ciocâlteu phenol reagent, DPPH , methanol, ethanol, sodium carbonate, and Mueller-Hinton (MH) broth and MH agar media were prepared from Merck . Punica granatum L.) of Rabab-e-Neiriz cultivar , characterized by thick peels [Fully ripened pomegranates  to achieve a 0.5% w/v concentration, and it was then kept overnight under magnetic stirring at ambient temperature to get a clear solution, and its pH was adjusted to 4.6 using a 1 M NaOH. Chitosan solution was then filtered through a 0.45 µm syringe filter. PPE at different loading contents was added to the CS solution according to formulations shown in CSNPs were prepared by ionic gelation method using STPP as a crosslinking agent. LMW chitosan (0.2 g) was added to 40 mL acetic acid (1% w/v); it was then filtered through a 0.22 µm syringe filter, its pH was adjusted to 5.6 by 1 M HCL, and it was stored at 4 °C before use. Chitosan nanoparticles were spontaneously produced by gradually adding 10 mL of cold STPP solution using a syringe pump (with a flow rate of 0.25 mL/min) into 10 mL of chitosan solution containing PPE under vigorous stirring (1400 rpm) for 50 min while holding in an ice-bath. The obtained suspension was then centrifugated at 9000 rpm for 30 min [Chitosan solution containing PPE was magnetically stirred (at 500 rpm) for 60 min at ambient temperature (23 °C). A stock solution of STPP was prepared in double-distilled water to achieve a concentration of 0.2% (r 30 min . The supw/v) was added and shaken for another 5 min followed by standing for 30 min in a water bath at 40 °C. The absorbance of the samples was determined with a UV-vis spectrophotometer  at 765 nm. Standard curves were prepared using GA (0–250 μg/mL), and the results were stated as mg GA equivalence (GAE)/g dry matter [TPC was determined using the Folin–Ciocâlteu (Fol–Ci) method  with they matter . The amoThe mean diameter (MD) of nanoparticles, zeta potential (ZP), and polydispersity index (PDI) of freshly prepared CSNPs with and without PPE loading were studied using dynamic light scattering (DLS)  ,49. The To observe the surface morphology of CSNPs, the samples were dried on glass slides and mounted on aluminum stubs, followed by gold thin layer coating using vacuum ion sputtering device . Then, nanoparticles were observed using scanning electron microscopy (SEM)  .The encapsulation efficiency (EE) is defined as the real content of material loaded into the NPs. A predetermined amount of freshly prepared PPE-loaded CSNPs was centrifuged at 9000 rpm for 35 min at 25 °C. The supernatant was analyzed for PPE using UV-vis spectrophotometry, as mentioned in −1  according to the method described previously [To study the potential interactions between chemical structural elements of the components used in the preparation of loaded CSNPs, Fourier-transform infrared (FTIR) spectra were obtained from 4000–400 cmeviously .ABlank and ASample are the absorbances of the control and the sample.DPPH  free radical scavenging activity (RSA) of PPE and PPE-loaded CSNPs was determined according to the method reported by Shetta et al.  with somEscherichia coli and Gram-positive Staphylococcus aureus using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests [8 CFU/mL of McFarland turbidity standard at 0.5. The standardized suspensions were then further diluted by 1:100 in MH broth and kept incubated at 37 °C for 24 h. Microtiter plates were prepared according to stages 1–4, as shown in Antibacterial activity of PPE before and after loading in CSNPs was evaluated against Gram-negative C) tests ,55. The C) tests  with somThe reduction of blue resazurin to pink resorufin indicates the viability of microbial cells because of their reductase enzyme action. By lasting the incubation time (4 h), the column with blue/purple color (no color change) indicated no microbial growth and thereby scored as MIC value. The MBC is considered as the minimum concentration of the material, which kills 99.9% of bacterial inoculum  by assayMeasurement of PPE components was done using a GC  connected to a mass spectrometer (Model QP-5050A) equipped with a DB-5 column   and a flame-ionized detector, which was operated in the EI (electron ionization) mode at 70 eV. Helium as the carrier gas was used, and the flow rate was adjusted to 1.2 mL/min. The oven temperature was increased from 50 to 240 °C by a 3 °C/min rate. Injector and detector temperatures were set to 230 and 270 °C, respectively. The split ratio was adjusted to 1:10, and the injected volume was 1 μL of 10% PPE in n-hexane. The components were identified by comparing their mass spectra with reference chemicals available in the literature using computer matching with NIST107 and WILEY229 libraries .p < 0.05) difference between the means.Statistical Analysis System (SAS) software  was used to establish Pearson’s correlations between the independent variable  and dependent variables . For dependent variables, such as SEM particles’ MD, TPC, DPPH, and MBC values, a one-way analysis of variance (ANOVA) was performed. Tukey’s multiple range test was used to determine the significant  also showed unimodal size distribution. However, higher PPE loading concentrations shifted the size distribution curves to larger particle size domains showing a bimodal distribution with higher polydispersity. For example, PPE-loaded CSNPs with chitosan:PPE ratios of 1:0.75 and 1:1.00 exhibited large numbers of aggregated particles. This may be attributed to a possible leakage of PPE to the surface of these NPs, which could cause sticking and aggregation of NPs. p < 0.05) increased from 198 nm in chitosan:PPE ratio of 1:0.25 to 898 nm in chitosan:PPE ratio of 1:1.00. The zeta potential (ZP) of the nanoparticles indicates their surface charge and, thereby, their stability. As can be seen in This figure shows the intensity-weighted particle size distribution, which can be sensitive to small numbers of aggregated particles since the intensity is related to 10p < 0.05) decreased ZP values from 26.5 mV in chitosan:PPE ratio of 1:0.50 to 2.95 mV in chitosan:PPE ratio of 1:1.00. There was no significant difference in ZP values of CSNPs with loading ratios of 1:0.25 and 1:0.50. A similar range of zeta potential was found in other studies, where CSNPs were developed and loaded with other herbal extracts [p < 0.05) increased from 0.260 in chitosan:PPE ratio of 1:0.25 to 0.682 in chitosan:PPE ratio of 1:1.00.Incorporating PPE into these nanoparticles decreased their ZP values, indicating the weakening of their physical stability. Increasing PPE concentration significantly  correlated with the independent variable (CL) with R2 values of 0.95, 0.85, and 0.76, respectively. LC and MD variables with R2 values of 0.57 and 0.56, respectively showed less significant correlations at p = 0.013 and p = 0.0044, respectively compared to those of aforementioned variables. These results indicate that encapsulation efficiency and zeta potential are highly and negatively dependent on PPE concentration in chitosan nanoparticles. The polydispersity index and the particles’ mean diameter positively depended on the concentration level and showed increased values with increasing concentration of loaded PPE in chitosan nanoparticles. Scatter plot matrix data of all independent variables showed a similar significant (p < 0.001) correlation with concentration levels (data are not shown).Effect of PPE concentration at five levels  on variations of NPs mean diameter (MD), zeta potential (ZP), poly-dispersity index (PDI), encapsulation efficiency (EE), and loading capacity (LC) was evaluated by constructing a Pearson’s correlation. The results are shown in w/w). The results are shown in 1,b1. SEM micrographs showed spherical morphology and homogenous size distribution in the case of both CSNPs. ImageJ  was used to quantify particle size diameter and distribution [2,b2 [SEM was used to analyze the surface morphology and size distribution of CSNPs and PPE-loaded CSNPs  provides the highest ZP value indicating good physical stability with a high loading capacity and the second-best encapsulation efficiency, and thereby it was selected for the rest of the analysis. Further increase of PPE in CSNPs decreased EE values. The decrease of EE for the sample prepared using higher concentrations of PPE may be explained by the encapsulation limitation in these NPs. In addition, the LC of PPE-loaded CSNPs was in the range of 13.8–20.7% , 2930 , 1655 (C=O bond vibrations in the amide I molecules), 1373 , 1157 and 1080 (correspond to the stretching vibrations of C–O–C bonds), and 607 cm−1 (correspond to the vibration of pyranoside rings), which are also confirmed by Shetta et al. [−1 (–NH2 groups stretching vibration of in CS) was shifted to 3410 cm−1 in CSNPs attributing to the occurrence of STPP molecules [−1 in CS (relating to C=O stretching of the amide I) were shifted to 1646 and 1550 cm−1 in CSNPs, indicating that the amine groups of CS and polyanionic phosphate groups of STPP may take part in the reaction [−1 related to –COOH groups of TTP was not seen in the FTIR spectrum of CSNPs, indicating that chitosan was completely crosslinked with STPP [Fourier-transform infrared (FTIR) technique was used to characterize the chemical structure of components involved in pomegranate extract loaded CSNPs. FTIR spectra of CS, STPP, PPE, CSNPs and PPE-loaded CSNPs are shown in a et al. . It is e−1 is ascribed to stretching vibration of N–H and O–H bands, which can be possibly found in tannic acid, ellagic acid and gallic acid, which has been confirmed in other studies [−1 were Attributed to C–H, C=O, and carbonyl groups. The relatively small bands that appeared at 1437, 1350 and 1230 cm−1 are assigned to vibrations of the aromatic ring in PPE. The strong band at 1030 cm−1 ascribed to C–O stretching of carboxylic acid. FTIR analysis was used to evaluate whether the encapsulation nature of PPE within CSNPs is chemical or physical entrapment. If no or minimal changes of FTIR spectrum were observed than the parental compounds, one could expect a physical entrapment, whereas spectral shift would be attributed to possible chemical interaction between PPE and CSNPs. Comparing FTIR spectra of CSNPs  lower than that of pure PPE. The amount of pure PPE used in TPC analysis was similar to its concentration used to prepare PPE-loaded CSNPs (at chitosan:PPE ratio of 1:0.50 w/w). Thus, the lower TPC value of CSNPs compared to pure PPE shows a good encapsulation efficiency of these NPs that preserve PPE from leaching out during the measurement. However, based on previous studies [Carum copticum essential oil preserve encapsulated bioactive material during TPC assay.The results of TPC determination for pure PPE, CSNPs and PPE-loaded CSNPs (at chitosan:PPE ratio of 1:0.50) are presented in  studies ,52, it c studies  also indw/w). Thus, lower DPPH value of CSNPs than pure PPE might be due to the protective effect of CSNPs in restricting the rapid leakage of PPE from chitosan nano-capsules during the DPPH assay, as also was seen for TPC data  showed no color change representing the sterility of the MH broth medium. As can also be seen in E. coli, while MIC and MBC values of 0.27 and 0.55 mg·mL−1 were obtained for S. aureus, respectively , making them susceptible to cell lysis.It has to be mentioned that pure PPE was used at a concentration equal to that of loaded in CSNPs at a CS:PPE ratio of 1:0.50. No antibacterial effect of PPE against crobials . Such meAccording to S. aureus . The resulting NPs exhibited spherical morphology, unimodal and narrow size distribution with an average particle diameter of 208 nm, and possessed desirable encapsulation efficiency (EE) and positive surface charge , indicating their physical stability. However, increasing the loading concentration of PPE increased the average particle size, polydispersity and exhibited large particle aggregates. The loading capacity (LC) and EE were increased and decreased, respectively, as a function of PPE loading concentration. FTIR results indicated no spectral change between the components of PPE-CSNPs, confirming physical entrapment or encapsulation of PPE within CSNPs. PPE-loaded CSNPs showed significantly lower TPC and DPPH RSA values compared to those of pure PPE, confirming a successful encapsulation of PPE within CSNPs. MIC and MBC antibacterial test revealed the antibacterial activity of PPE against S. aureus. Overall, CSNPs provide an effective nanomaterial to encapsulate and protect the sensitive extract of pomegranate peels.Pomegranate peel extract (PPE) was successfully encapsulated within chitosan nanoparticles (CSNPs) using the ionic gelation technique. It was shown that the loading concentration PPE affects the physical characteristics of the obtained NPs. The optimal concentration of PPE was obtained at a chitosan:PPE ratio of 1:0.50 (


Barley contains abundant soluble beta-glucan fibers, which have established health benefits. In addition, the health benefits conferred by the gut bacteria have attracted considerable interest. However, few studies have focused on the barley consumption and gut bacteria of the Japanese population. In this study, we aimed to identify the relationship between the barley consumption and gut bacteria composition of the Japanese population.In total, 236 participants were recruited in Japan, and 94 participants with no complications of diabetes, hypertension, or dyslipidemia were selected for the study. We analyzed fecal samples from the participants, their medical check-up results, and responses to questionnaires about dietary habits. The participants were grouped according to their median barley consumption. Then, we assessed the relative abundance of 50 genera. Characteristic bacteria were evaluated for their relationship with barley consumption by multiple regression analysis, adjusted for disease and dietary habits, in all participants. We also analyzed the networks and clustering of the 20 selected genera.Bifidobacterium, Butyricicoccus, Collinsella, Ruminococcus 2, and Dialister were characteristic candidate bacterias of the group that consumed large amounts of barley (P < 0.05). The relationship between barley consumption and Bifidobacterium remained after adjusting for disease and dietary habits, and that of Butyricicoccus remained after adjusting for disease. Furthermore, network and cluster analyses revealed that barley consumption was directly correlated with Bifidobacterium and Butyricicoccus.According to the comparison between barley groups, Bifidobacterium and Butyricicoccus abundance was positively associated with barley consumption.Barley consumption generates changes in the intestinal bacteria of the Japanese population. We found that The online version contains supplementary material available at 10.1186/s40795-022-00500-3. The gut microbiome is important for health, and thus, several investigations have focused on the human gut microbiome. It has been reported that diet can alter the gut microbiome . MicrobiBlautia [Barley is an important cereal that contains the soluble fiber beta-glucan . Barley Blautia . AdditioBlautia  and diabBlautia  and loweBlautia . TherefoBlautia abundance and improves host cholesterol levels [Prevotella/Bacteroides and improves host blood glucose metabolism [Barley consumption affects the gut bacteria and host health , 7, 8. Fl levels . AnotherBlautia and Bifidobacterium, which utilize the dietary fiber from seaweed, a common item in the Japanese diet [Indeed, the effects of barley on the gut bacteria have been established in several countries , 7, 8. Fese diet . Barley ese diet , 11; howThe study was approved by the Yamanashi University Ethics Committee , the National Institutes of Biomedical Innovation, Health, and Nutrition Ethics Committee , and the Chiyoda Paramedical Care Clinic Ethics Committee . This study was conducted in accordance with the Declaration of Helsinki (2013) and was based on a registered study (UMIN000033479). Cross-sectional data were evaluated from the first year of the study to obtain an exploratory overview of the gut microbiome of a population that consumes barley. Sampling was conducted from August 2018 to March 2019.We enrolled 272 participants, which were employees of the barley processing company Hakubaku Co., Ltd. Our target sample included at least 100 participants. We excluded those with disorders (Risk 2) and pre-disorders (Risk 1) of diabetes, hypertension, and dyslipidemia from the main analysis. Although gastrointestinal disorders were not included in the exclusion criteria, none of the participants had a history of ulcerative colitis or Crohn’s disease. There were also four participants with a history of irritable bowel syndrome, but none were undergoing treatment. Details of the exclusion criteria for disorders are shown in Table S1  was calculated by dividing the weight by the square of the height. Dietary habits other than barley consumption were assessed using a brief self-administered diet history questionnaire . Barley consumption  was calculated using a questionnaire and the daily energy value from the BDHQ. Rice bowl size , proportion of barley mixed with white rice , barley-mixed rice consumed per month , and barley consumption rate (g/d) were determined. Medical history, including medication , and consumption of fermented foods and supplements were determined using questionnaires.g for 5 min at 25 °C, and the DNA was extracted from the supernatant using a Gene Prep Star PI-80X automated DNA isolation system (Kurabo Industries Ltd). DNA concentration was determined with the ND-1000 NanoDrop Spectrophotometer . The samples were stored at − 30 °C. The 16S rRNA gene was amplified from fecal DNA and sequenced [Fecal samples were collected at home with guanidine thiocyanate (GuSCN) solution, and DNA was extracted and stored at 10–30 °C for up to 30 d . Brieflyequenced . The V3–The sequence reads from Illumina MiSeq were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) software package (version 1.9.1) . We usedData were exported as BIOM files and imported into R (version 3.6.0). Diversity was analyzed using the phyloseq R-package. Alpha-diversity indices of observed OTUs, Chao1, Shannon, and Simpson indices were calculated using the estimate_richness function.t-test. The alpha-diversity and relative abundance of each genus were analyzed using Mann-Whitney U-test. P values were adjusted using false discovery rate (FDR) methods. To confirm the reliability of the analyses, we explored the relationship between barley consumption groups (0 = low and 1 = high) and each bacteria using multiple regression analyses with all participants. We expressly set the amounts of Bifidobacterium, Butyricicoccus, Collinsella, Ruminococcus 2, and Dialister as outcomes. We adjusted the model for age, sex, risk of diabetes, dyslipidemia, and hypertension for model 1. In addition to model 1, we adjusted the model for consumption rate  of cereals, sugar and sweetener, legumes, and beverages for model 2 and for cereals, sugar and sweetener, legumes, beverages, green vegetables, other vegetables, fish, and confectionery for model 3. We used the vif function of the car R-package to evaluate variance inflation factors (VIFs). All VIFs were < 5 and considered acceptable for these analyses [To compare the results of the medical check-ups and dietary habits between the high and low barley groups, we used Student’s analyses .We classified the participants into enterotypes A, B, and C using the pam function of the cluster R-package. We then summarized the composition of the intestinal bacteria by principal coordinate analysis (PCoA) using the vegdist function of the vegan R-package and the quasieuclid and dudi.pco functions of the ade4 R-package. Data were calculated using the Bray–Curtis method. Subsequently, the environmental factor arrows were fit to the PCoA figure using the envfit function of the vegan R-package. Significant genera were assessed using permutations of environmental variables.P < 0.1, we implemented a network analysis. The network is shown with lines of correlation with |r| > 0.15 . Different colors on the plots indicate different community groups. We fit the correlation data frame to the cc.df function of the igraph R-package using the reshape2 R-package and calculated bacteria community groups using the leading.eigenvector.community function. All analyses were carried out in R (version 3.6.0), and tests were two-sided; P < 0.05 was considered significant. All graphs except for those from the network analysis were created with the ggplot2 R-package [To visualize the associations between barley and 20 genera selected based on a -package .We obtained informed consent from 272 individuals, of which 30 participants resigned and six were excluded because of non-compliance and data loss. Ninety-four participants had no disorders and were included in the main analyses, and 236 were included in the multiple regression analyses Fig. . AdditioP = 0.054). Disorder risk did not significantly differ between the groups , and therefore, a male had a higher risk of disease and a higher percentage was excluded. Table P < 0.001), cereals (P = 0.048), sugar and sweetener (P = 0.03), and beverages (P = 0.047) were significantly different between the barley groups, and those of legumes (P = 0.056) tended to be higher in the high barley group. Therefore, we selected these four dietary categories for adjustment in the multiple regression analyses (model 2). Additionally, green leafy vegetables, carrot and pumpkin in the green vegetable category, boiled fish in the fish category, and Japanese confectionery and ice cream in the confectionery category had a significant or slight difference between the barley groups. Moreover, seaweed and mushroom, which are known to contain soluble fiber, were in the “other vegetable” category. Therefore, we added these four variables to model 2 and conducted multiple regression analyses (model 3).The characteristics of the barley groups did not significantly differ Table . HoweverP = 0.20). Figure Bacteroides (A), Blautia (B), and Prevotella 9 (C) . The most common enterotypes of the 94 participants were in the following order: B (60%), A (28%), and C (13%). The distribution of enterotypes was similar between the barley groups (B > A > C) and tended to be in the positive direction of PCoA2 in the high barley group, but the values were not significant , Collinsella (P = 0.03), Butyricicoccus (P = 0.002), Dialister (P = 0.04), and Ruminococcus 2 (P = 0.04) without FDR adjustment (Table Subdoligranulum (P = 0.08), Anaerostipes (P = 0.0502), Acidaminococcus (P = 0.07), and Ruminococcaceae UCG-013 (P = 0.06) tended to be high in the high barley group. Additionally, considering reproducibility in other analyses , B (driven by Blautia), and C (driven by Prevotella 9). However, the influential genus of enterotype B, Blautia, was different [Bacteroides) and C (Prevotella 9) were robust between cohorts, but for B it depended on the cohort. Furthermore, Blautia was reported as one of the major driving genera [The consumption of barley tended to be positively related to PCoA2, but this result was not significant . However, the average consumption of this cohort was as high as 4.6 g, and the range was 0–28 g. Therefore, this is a reasonable population to use for the evaluation of the effects of barley consumption. Second, a detailed barley questionnaire might influence the participants’ dietary and lifestyle habits. Although the participants were instructed not to change their dietary and lifestyle habits before and after the medical check-up, the possibility of bias remaining cannot be denied. Finally, we did not measure SCFAs or microbiome functions, which restricts the usefulness of our results. A follow-up intervention study with another cohort is warranted to overcome these limitations. In conclusion, barley might change the intestinal bacteria of the Japanese population. We selected Additional file 1: Table S1. Criterion of risk group of diabetes, hypertension, and dislipidemia. Table S2. Comparison of diet between low barley group and high barley group on cross-sectional study (n 94). Table S3. Alpha-diversity, compared low barley with high barley group on cross-sectional study . Table S4. Relative abundance(%) of the frequent 50 genera, compared low barley with high barley group on cross-sectional study . Table S5. Results of Multiple regression analysis (dichotomy and continuous) and Kendall rank correlation between barley consumption  and intestinal bacterias on cross-sectional study . Table S6. Association between the relative abundance of intestinal bacteria and barley intake group  by multivariate linear regression analyses (N = 236). Fig. S1. PCoA result. Color means high or low consumption of barley (P = 0.08). The arrow means high barley group . Fig. S2. The result of network analysis. Line means correlation |r| > 0.15 .


Adenomyosis (AM) and endometriosis (EM) often coexist. Laparoscopic surgery is one of the main methods for diagnosing and treating these conditions. This study aims to investigate the pregnancy outcomes of women with infertility with both AM and EM after laparoscopic surgery and to identify the relevant associated factors.This is a retrospective study involving women with infertility diagnosed with EM and AM. All patients had undergone laparoscopic surgery and were divided into two groups according to their pregnancy outcomes. Demographic data, operation records, and pregnancy outcomes were collected.p = 0.044). The group that did not proceed to have a live birth had a higher percentage of ovarian and peritoneal endometriosis (p < 0.05), while the distribution of deep infiltrating endometriosis and adenomyosis types were similar. Mean uterus diameter  and endometriosis fertility index (EFI)  were significantly correlated with live birth in the multivariable analysis.A total of 226 female patients with infertility diagnosed with both AM and EM underwent laparoscopic surgery. Of these, a total of 176 patients completed follow-up. Ninety-seven patients had live births, including 81 full-term and 16 preterm deliveries. The clinical pregnancy and live birth rates were 67.4 and 55.11%, respectively. One hundred thirty-five patients received in vitro fertilization (IVF), with 70 (51.85%) of these patients having live births. Age, endometrioma size, and uterus size were significantly lower in those who had a successful delivery. There was no statistically significant difference in symptoms, except that those who achieved live birth had a lower rate of anaemia (13.40% vs. 25.32%, Endometriosis and adenomyosis appear to have an adverse effect on pregnancy outcome. These might be related to the size of the uterus and EFI. Obstetricians and gynaecologists should be alert to this potential adverse effect and manage these patients accordingly. Adenomyosis (AM) and endometriosis (EM)  are beniAM and EM both have adverse effects on fertility, complicating treatment , 9. In aLaparoscopic surgery is one of the primary ways  to diagnThis study was approved by the Ethics Committee , and all procedures involving human participants followed the ethical standards of the institutional review board (IRB) from the study centre. Informed verbal consent was obtained from all patients at their follow-up interviews.We identified 257 patients who underwent surgery for the first time in our hospital with a diagnosis of both EM and AM between January 2013 and December 2017. Among them, 226 (87.94%) patients with infertility underwent laparoscopy and who wished to conceive after surgery were followed-up for a minimum of 2 years after the operation. The exclusion criteria were as follows: (1) age < 20 or > 40 years; (2) bilateral oophorectomy or hysterectomy; (3) intraoperative conversion to laparotomy; and (4) concomitant diseases that clearly affect fertility, such as submucosal fibroids, premature ovarian failure, systemic lupus erythematosus, malignant tumours, polycystic ovary syndrome, male factor and reproductive malformation.Infertility  was defiUltrasonographic exams were performed after the menstrual cycle immediately prior to surgery. Features on ultrasound that suggest AM were as follows: asymmetrical myometrial thickening, myometrial cysts, linear striations, hyperechoic islands, or irregular and thickened endometrial–myometrial junctional zones . If moreDuring the surgery, endometriomas were removed, and peritoneal endometrial tissue was treated with bipolar electrocoagulation. Deep infiltrating endometriotic nodules and adenomyoma lesions were resected after consultation between doctors and the patient, especially for those who had severe symptoms or repeated in vitro fertilization and embryo transfer (IVF-ET) failure. Pre-surgical symptoms and surgical data, including postoperative complications, were retrieved from patient admission and operative databases. The revised American Fertility Society (rAFS) score, staging from the revised American Society for Reproductive Medicine (rASRM), and EFI  were colIn this study, independent variables were classified into three groups: demographic factors, including age, body mass index (BMI), history of abortion and assisted reproductive therapies (ART), menstrual cycle length, dysmenorrhea, anaemia, CA125 and follicle stimulating hormone (FSH). Surgical factors included mean uterus diameter, mean cyst diameter, type of EM and AM, blood loss, surgical time, resection of AM and deep infiltrating endometriotic (DIE) nodules, rAFS score and EFI. Medical treatment and IVF after surgery were also examined as potential variables related to pregnancy outcome. The dependent variable for all these was live birth.P value less than 0.2 were subjected to multiple logistic regression (stepwise). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated as measures of the impact of the variables on live birth. All analyses used a two-tailed α of 0.05 and were performed using SPSS software , and p < 0.05 was considered significant.Continuous data are presented as the median (interquartile range) or mean ± SD and were compared using the t-test or ANOVA. Categorical data are described by the number of patients (including percentages) and were compared with Fisher’s exact test or the chi-square test. Variables identified with univariable analysis with a n = 32) or insufficient follow-up data (n = 11). Seven patients who became pregnant did not deliver and were thus also excluded. To report the factors associated with successful live births, we divided the 176 remaining women into two groups: Group A (n = 97) contained 81 women who give birth to full-term infants and 16 who birthed preterm infants, and Group B (n = 79) consisted of those patients who did not achieve a successful delivery. The average BMI (kg/m2) was 21.17 ± 3.86. A flowchart describing the group selection process for the patients is provided in Fig. During the study period, 226 patients with infertility with both AM and EM were identified and underwent laparoscopic surgery. Forty-three patients were excluded due to either age criteria (data n = 1. Seven p = 0.011) and higher EFIs . Blood loss during laparoscopy in group A was also less than that in group B , as was surgical time . Seventy-six patients underwent adenomyosis/adenomyoma resection. We also found that the non-live birth group had a significantly higher proportion of ovarian endometriosis (OEM) and peritoneal endometriosis (PEM) than the live birth group. However, the differences in DIE, AM types, leiomyoma, endometrial polyps, or obstruction of the oviduct between the two groups were not significant , 35 (19.13%) patients had failed to conceive, and 13 (7.10%) patients stopped trying to conceive: four due to disease relapses, two due to depression, four due to premature ovarian failures (POFs), one due to endometrial intraepithelial neoplasia (EIN), and two due to repeated IVF failures. One hundred thirty-five (73.77%) patients underwent IVF, and 70 patients had live births (51.85%). The mean time from surgery to pregnancy was 12.89 ± 8.66 months, and the mean time from the first ovulation after surgery to pregnancy was 8.56 ± 8.48 months. The clinical pregnancy rate, or the presence of a foetal heartbeat at 12 weeks of gestation, was 67.4, and 55.11% ultimately achieved a successful delivery. One hundred forty-eight (80.87%) patients had a positive pregnancy test. Forty (27.03%) patients experienced spontaneous pregnancy losses, two (1.35%) had ectopic pregnancies, and five (3.38%) experienced intrauterine deaths . Among the women who gave live birth, 16 (16.50%) had preterm labour, and 12 (12.37%) had abnormal placental conditions . Other detailed information regarding natural/IVF pregnancy outcomes is provided in Fig. p values < 0.20) in the univariable analysis were included in the multivariable model. When two variables were highly correlated, we introduced only one of them into the model and suppressed the other; for example, when examining age and FSH level, the latter was suppressed. Surgical time (p = 0.016) and blood loss (p = 0.011) showed a tendency of association, but they were excluded because they were highly correlated with the AFS score and type of AM. EFI includes age, infertility type, years of infertility, function of the fallopian tube/fimbria/ovaries, and AFS; therefore, they were not included as candidate factors. EFI (p < .001), anaemia (defined as haemoglobin < 110 g/L) (p = 0.047), VAS (p = 0.112), CA125 (p = 0.093), mean uterine diameter (p = 0.002), ART (p = 0.122), and medical history (p = 0.017) were included as covariates in the final model. AM type (p = 0.159) and AM surgery (p = 0.110) were also included, as they were reported to be related to the pregnancy rate [To determine the factors associated with a successful delivery, univariable analysis and multivariable logistic regression analysis were performed. Variables showing a tendency of association with live birth rate , respectively.We observed that EM phenotypes might directly impact pregnancy outcomes, as there were significant differences between the live birth group and the non-live birth group. Our previous results demonstrated the presence of follicles in the cyst walls of endometriomas on histopathological examinations , and thep = 0.89), while placenta previa was found to be more common in women with EM than in the control group . In our study, the preterm birth rate was 16.50%, and the incidence of abnormal placenta  was 12.37%. A possible explanation for this might be that our patients had both EM and AM. In a multi-centre retrospective questionnaire survey [In addition to the reduced oocyte yield, reduced fertilization rate (FR), and increased spontaneous pegnancy loss rate , 32, AM e survey  reportede survey  reportedThe aetiology of infertility is thought to include disordered inflammatory factors such as irregular prostaglandin  and cyclooxygenase 2 (COX-2) production , abnormaThe major strength of this study is the diagnostic approach used for AM and EM. The gold standard for AM diagnosis is based on histopathological analysis, which is an invasive technique with a potential risk of uterine rupture during pregnancy and delivery . TherefoOverall, our study suggests that the coexistence of EM and AM has adverse effects on both reproductive performance and the outcome of pregnancy. Age, BMI, and the sizes of endometrioma and the uterus were related to prognosis. IVF is an important technique for improving the pregnancy rate following surgery. These findings may prove useful in developing treatment plans for infertile patients with AM and EM. Further prospective studies with larger sample sizes are required to draw firmer conclusions.


Intake of snack foods has been previously associated with an elevated risk of chronic disease; however, studies on snack foods and metabolic syndrome (MetS) while considering the modifying effect of socioeconomic status (SES) and lifestyle factors on this association are lacking. We aimed to investigate the association between snack foods intake and the MetS risk, and the mediatory effects of SES and lifestyle factors on the forenamed association among adults who participated in the Tehran Lipid and Glucose Study (2006–2018).This is a prospective study of 1915 participants , aged 19–74 year who were free of MetS at baseline. Dietary intakes were gathered using a validated, semi-quantitative food frequency questionnaire at baseline (2006–2008), and with 3-year intervals afterwards. Alternative approach was used for snack foods from all available questionnaires during follow-up. Snack foods were divided into 4 categories, including total snacks, biscuits and cakes, candies and chocolate, and salty snacks. Total snack foods intake and its subgroup (serving/week) were modeled as tertiles. MetS was diagnosed according to the Joint Interim Statement criteria. Physical activity level (PAL) categorized as low/medium and high levels. Information regarding smoking (Smoker/Non-smoker), education (higher/lower education), and occupation (employed/non-employed) was gathered using questionnaire. The Cox regression was used, regarding interaction between snack foods, SES, and PAL on the MetS risk.A total of 591 incident MetS cases were diagnosed during 8.9 years of follow-up. The median total snack foods intake was 5.2 serving/week (IQR: 3.0–9.1). Total snack foods intake was positively associated with the MetS risk after adjusting for potential confounders . After adjustment for confounders, among snacks’ subgroups, candies and chocolate intake was positively associated with MetS risk. Moreover, among lower-educated and non-employed participants, candies and chocolate intake was positively associated with the MetS risk, by 38 and 43% respectively. Stratification based on PAL resulted a positive association between intake of total snack foods and candies and chocolates and risk of MetS among participants with low PAL.Snack foods, especially candies and chocolate, increased the risk of MetS, among individuals with a low PAL.The online version contains supplementary material available at 10.1186/s12937-021-00728-y. It is well established that metabolic syndrome (MetS), a cluster of interrelated metabolic abnormalities, can occur from early childhood to late adulthood . The NatSnack foods intake has recently been emphasized as a potentially unhealthy eating habit . Intake It is also well known that lifestyle factors alongside the diet may play an important role in the incidence of chronic diseases . SocioecTo our knowledge, no prospective studies have considered SES and lifestyle factors  as mediators of the relation between snack foods intake and MetS risk. Because of that, the current study aimed to, firstly, investigate the association between snack foods intake and risk of MetS, and secondly, investigate the mediatory effect of SES and lifestyle factors on the association of snack foods and MetS, among adult population during 8.9 years of follow-up.This study was conducted within the framework of the Tehran Lipid and Glucose Study (TLGS), a long-term prospective population-based study initiated in 1998 to determine the prevalence of non-communicable disease risk factors and its outcomes among the urban Tehranian population. The design and other details of TLGS were described elsewhere . Briefly2, compared with 35.4 years and 26.1 kg/m2 in participants who did not have nutritional data, in the phase III of the study. Among non-included participants, 32.9% had academic education and 21.5% were smokers, compared with 29.5 and 21.7% subjects who completed the FFQ.The current study used baseline examination data from phase III of the TLGS (2006–2008) and followed participants up to phase VI of TLGS (2016–2018), during an 8.91 (Interquartile Range: (IQR): 7.98–9.69) year follow-up. During the phase III of the TLGS, a representative sample of 4920 participants was randomly selected, for gathering their dietary information. From the 4920 selected participants in the present study, 3462 number of individuals, agreed to complete the FFQ. For the current study, 3265 adults aged 19–74 years with complete demographic, anthropometric, biochemical, and dietary data were selected. The characteristics of participants who completed the dietary assessment were similar to those of the total population in the phase III of TLGS . The mean = 872), those who were pregnant or lactating at baseline and during follow-up (n = 28), those with under-or over-reporting of energy intakes  (n = 115), those were on any specific diets as result of diabetes, hypertension or hyperlipidemia (n = 26), and participants with missing biochemical and anthropometric measures related to diagnosis of MetS during follow-up (n = 309). Final analysis was conducted on 1915 participants until 2018 with a response rate of 66%, over the 8.9 (IQR: 7.98–9.69) year follow-up  n = 15, thoseDuring face-to-face interviews, the regular dietary intakes of participants over the previous year were collected using a validated and reliable food frequency questionnaire (FFQ) –20. FreqSnack foods intake was collected using the FFQ reported a valid estimate against multiple dietary records. A good correlation coefficient was observed between FFQ and multiple 24 recalls  and between two FFQs  biscuits and cakes, 2) candies and chocolate, 3) salty snacks. Biscuits and cakes included biscuits, cakes, cookies, crackers, confectionery, and traditional Iranian confectionaries including Gaz and Sohan. Candies and chocolates included chocolates, candies, cubed sugar, Noghl, and Nabat. Salty snacks consist of potato chips and puff (a corn snack or crisp coated with a mixture of cheese or cheese flavored). Total snack foods was calculated by the sum of them.For assessing the biochemical measurements, after 12–14 h of overnight fasting, venous blood samples were obtained from all study participants placed into vacutainer tubes and centrifuged within 30–45 min of collection; fasting plasma glucose (FPG), high density lipoprotein cholesterol (HDL-C) and triglyceride (TG) analysis was done at the TLGS research laboratory on the day of sample collection, using a selector 2 auto-analyzer  and commercial kits . FPG was analyzed using an enzymatic colorimetric method with the glucose oxidase technique; inter- and intra-assay coefficients of variation (CV) at baseline and follow-up phases were both less than < 2.3%. HDL-C was analyzed after precipitation of apo-lipoprotein B-containing lipoproteins with phosphotungstic acid. TG was assayed using an enzymatic colorimetric method with glycerol phosphate oxidase. Both intra- and inter-assay CVs were below 3.0 and 2.1% for HDL-C and TG, respectively, in all baseline and follow-up assays.In terms of socioeconomic information, participants were divided into two groups according to their education levels. Subjects with a university degree were included in the higher-educated group and subjects with a degree less than a diploma were included in the lower-educated group. Moreover, based on economic status, participants were divided into two groups: 1) employed with income and 2) non-employed without income. The latest information on SES was used to classify individuals.Participants were divided into groups of smokers or non-smokers. The smoker group included ex-smokers and current smoker’s participants and other participants were in the non-smoker group. PAL was assessed using the modifiable activity questionnaire (MAQ) that included a list of activities including leisure time, job, and household activities; the frequency and amount of time spent per week engaged in physical activity over the last year were recorded . The valThroughout face-to-face interviews by trained interviewers, age, sex, and medical history data were gathered using a demographic questionnaire. Weight was recorded in minimally clothing to the nearest 0.1 kg on an SECA digital weighing scale , and height was measured in a standing position, without shoes to the nearest 0.1 cm. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m2). Waist circumference (WC) was measured at the umbilical level using an un-stretched tape measure, and was recorded to the accuracy of 0.5 cm. After a 15 min resting, arterial blood pressure was measured manually, using a mercury sphygmomanometer with a suitable cuff size for each participant. Systolic blood pressure (SBP) was determined by the onset of the tapping Korotkoff sound, while diastolic blood pressure (DBP) was determined as the disappearance of the Korotkoff sound. Blood pressure was measured twice on the right arm twice at least 30 s apart; and the average was considered as the participant’s blood pressure.According to the Joint Interim Statement, a MetS diagnosis requires the presence of three or more criteria  includinAll analyses were performed using SPSS software version 18  and Stata software version 14.0 . Normality of variables was tested by the histogram and Kolmogorov–Smirnov test. Quantitative variables were described as mean ± standard deviation (SD) or median (25th percentilee75th percentile), and qualitative variables were reported as percentage. In order to adjust energy, we used the residual method for all dietary factors . The eveTotal snack foods intakes and its subgroup (serving/week) were modeled as tertiles. Baseline characteristics and dietary factors were stated across tertiles of total snack foods by general linear models and chi-square for continuous and categorical variables, respectively. Cox proportional hazards regression analyses were used to estimate the hazard ratios (HRs) and 95% confidence interval (CI) for incidence of MetS across tertiles of biscuits and cakes, candies and chocolates, salty snacks, and total snack foods. In the model 1, we used a univariate analysis. Model 2 was adjusted for age at baseline and gender. Model 3 was additionally adjusted for energy intake, total fiber intake, smoking status, PAL, education levels, family history of diabetes, and family history of CVD events. Finally, Model 4 was further adjusted for BMI at baseline. The proportional hazard assumption of the multivariable Cox model was assessed using Schoenfeld’s test of residuals , the participants were categorized into low PAL (< 3 MET hour-week) or medium/high (≥ 3 MET hour-week) PAL . ParticiP < 0.20 [P-values < 0.05.Stratified analysis based on lifestyle and SES factors were done when P < 0.20 . InteracWe documented 591 new cases of MetS during the median follow-up of 8.9 years. The baseline mean (SD) age and BMI of the participants  was 36.5 (13.3) years and 25.6 (4.5), respectively. The median total snack foods intake was 5.2 serving/week (IQR: 3.0–9.1). From the total snack foods intake mentioned above, 2.2 (IQR: 1.2–3.8) was in form of cakes and biscuits, 1.6 serving (IQR: 0.95–2.4) in form of candies and chocolate and 1.7 serving/day (IQR: 0.6–3.8) in form of salty snacks.Baseline characteristics of study participants are shown in Table Table The association between total snack foods intake and its subgroups and risk of MetS are shown in Table In order to determine the modification effect of SES, lifestyle factors, and age groups on MetS risk across tertiles of different snack foods intake, stratification analysis was used. As illustrated in Table In addition, based on stratification by age, second and third tertiles of biscuit and cakes, candies and chocolate and total snack foods positively associated with risk of MetS among participants with aged ≥ 41 year old and low PAL  for metabolic syndrome across tertiles of snack foods intake among participants with aged < 40 year old. Supplementary Table 4. Multivariable adjusted hazard ratio  for metabolic syndrome across tertiles of snack foods intake among participants with aged ≥ 40 year old.


Globalisation continuously produces novel economic relationships mediated by flows of goods, services, capital, and information between countries. The activity of multinational corporations (MNCs) has become a primary driver of globalisation, shaping these relationships through vast networks of firms and their subsidiaries. Extensive empirical research has suggested that globalisation is not a singular process, and that variation in the intensity of international economic interactions can be captured by ‘multiple globalisations’, however how this differs across industry sectors has remained unclear. This paper analyses how sectoral variation in the ‘structural architecture’ of international economic relations can be understood using a combination of social network analysis (SNA) measures based on firm-subsidiary ownership linkages. Applying an approach that combines network-level measures  in ways yet to be explored in the spatial networks literature, a typology of four idealised international network structures is presented to allow for comparison between sectors. All sectoral networks were found to be disassortative, indicating that international networks based on intraorganisational ties are characterised by a core-periphery structure, with professional services sectors such as Banks and Insurance being the most hierarchically differentiated. Retail sector networks, including Food & Staples Retailing, are the least clustered while the two most clustered networks—Materials and Capital Goods—have also the highest average degree, evidence of their extensive globalisations. Our findings suggest that the multiple globalisations characterising international economic interactions can be better understood through the ‘structural architecture’ of sectoral variation, which result from the advantages conferred by cross-border activity within each. Network approaches to understanding global connectivity have emerged as a significant theme in political economy , and relIn this paper, we analyse sectoral variation in the ‘structural architecture’ of international economic networks. Our aim is to elaborate a comparative typology of international economic network structures for analysing the ‘multiple globalisations’  shaped bThis study sits among a growing number of papers using detailed datasets to study MNC networks –19 and iStructural transformations in the global economy since approximately the 1970s have produced novel geographical relationships guided primarily by the activities of large MNCs . The majCross-cutting the evolving meaning and substance of MNC headquarters, subsidiaries, and headquarter-subsidiary relations, scholars have emphasised the importance of thinking about how places  are structurally embedded in the global economy, particularly in relation to the increasing salience of MNCs in shaping geographical and economic relations , 36. Firad hoc conceptualisations of the global core prevailed, Johnston [ proportional to 1/Eα][ (disassortativity) of the industry networks highlight the tendency for an sector to have highly connected countries linked to poorly connected countries and vice versa. Contrarily, positive values are found when countries are mostly connected to countries of the same degree class. Larger values of negative r are interpreted as industries that exhibit strong international hierarchical differentiation [Finally, the of nodes , gives antiation , 82 and α.After the computation of each, these measures were used to compare the differentiated network structures against idealised network typologies. The combination of the four network metrics indicates how well-connected each sector graph is, and accordingly the level of globalisation of the entire network as well as the individual countries within it. In this analysis, the multiplex nature of our data set is not considered. Therefore, each metric is independently calculated for each layer Different classes of definitions have been developed to characterise the network structures in various fields such as complex networks, sociology, biology and economics –85. The Star-like network type , indicating a tendency to the idealised clique-like type of network structure. The sectors in this group are well connected across the world , including Technology Hardware & Equipment; Food, Beverage & Tobacco; Diversified Financials. Higher clustering and relatively higher assortativity in these networks are linked to less hierarchically differentiated organisation and denser global connectivity.Specifically, the top-right quadrant of the plot in C⟩ higher and r lower than the medians, indicating a polycephalous type. The sectors in this group are characterised as high-technology sectors , but very diverse, with network density D ranging from 0.04  to 0.11 (Semiconductors & Semiconductor Equipment), and average degree ⟨k⟩ ranging from 6.65  to above 11 . Sectors in this group are generally clustered but more highly disassortative, indicating a tendency for a small number of countries to emerge as highly but more selectively connected hub nodes.The top-left quadrant in C⟩ and r both below the medians and represents the idealised star-like type. This group includes relatively few sectors, characterised by strong hierarchical differentiation that is centralised around one key node or a small, centralised clique. This includes Automobiles & Components, Retailing, Food & Staples Retailing, which also exhibit very low network density (D less than 0.41) and average degree (⟨k⟩ less than 5.20) indicative of less extensively connected global networks.The bottom-left quadrant in r higher than the median but ⟨C⟩ lower than the median. These networks are segmented-decentralised and include sectors with no clear core and with less extensive global connections. Network density D and average degree ⟨k⟩ are rather low and many of the sectors are state-related  or highly localised  sectors.Finally, the bottom-right quadrant in Comparing the network properties, a striking pattern is that all sectors display a negative assortativity coefficient, indicating that nodes with a higher degree tend to be most connected to nodes with a lower degree, and vice versa. One explanation of the negative assortativity is that lower-degree countries such as Costa Rica and Cameroon are connected mostly to higher-degree countries such as the United States and France rather than one another, indicating a core-periphery structure of corporate connections. Negative assortativity has also been found in the global input-output network  as high-The resulting sector network graphs are shown in C⟩ = 0.70, r = -0.58, D = 0.07)  . Like in⟩ = 9.61  suggesti⟩ = 9.61  specialiC⟩ = 0.75, r = -0.67, ⟨k⟩ = 6.59, D = 0.04)  . It is c = 0.04)  providinC⟩ = 0.20, r = -0.73, D = 0.04)  . The netountries , specialC⟩ = 0.6447, r = -0.571, D = 0.06) .Real Estate exemplifies the segmented-decentralised network type (⟨ = 0.06) . The ind = 0.06)  operatinAs the global economy becomes more integrated through multinational firm activity, analysis of sectoral variation in global networks reveals the ‘structural architecture’ that underlies international economic structures. The characterisation of the different sectors, and the position of national economies, within global networks can be better achieved through a refined understanding of the processes and practices that scaffold the global economy , 52, 56.This paper has developed a novel method for comparing international economic networks by sector based on the ownership linkages of MNCs. How MNCs have connected across space has been long associated in studies of globalisation and internationalisation with differences in how specific sectors operate  or how iBased on the analysis, we add to the literature on variation by industry sector in shaping multiple globalisations , 16 in cC⟩ often form dense, interconnected networks with regionalised components, which we argue can be classified into either polycephalous or clique-like types. The international networks of Capital Goods, Commercial & Professional Services, Materials, Pharmaceuticals, Biotechnology & Life Sciences are all polycephalous, while other highly clustered international networks are Food, Beverage & Tobacco and Technology Hardware & Equipment, whose networks are clique-like. Both polycephalous and clique-like types reflect tightly bound global networks, with the difference being that the former are more hierarchically differentiated in nature, featuring greater disassortativity within international firm networks. This aligns with the findings in the ‘white goods’ sector [Sectors with a relatively high average clustering coefficient ⟨’ sector , with a ’ sector , 101 is ’ sector —a highlyC⟩ often operate within more regional, or local, subnetworks. Segmented-decentralised international industry structures such as Real Estate, Energy, and Telecommunication Services—although global in scope—have fundamentally regionalised components, for example, based on the relationships between suppliers, manufacturers, and distributors. The role of large state-owned, and state-related, enterprises in certain energy [Sectors with a relatively low average clustering coefficient ⟨n energy  and telen energy  sub-sectn energy , the staThe results suggest that both firm- and country-level dynamics explain the degree to which a sector is more or less extensively globally connected. A multilayer network perspective shows that countries may be specialised in some sectors but not in others, while a small number of countries comprise the core of most networks. Such findings provide additional insights into observations that economic specialisation is responsible in part for globalisation at the national scale, as are economies of scale that produce externalities tied to the size of a national economy. Thus, larger economies such as the United States, Germany, Japan, and China are often highly central to networks while smaller economies are often less central and less extensively connected across the world. Exogenous orientation explains much of the positionality within the international network, particularly in countries whose position is core to some sectors but not others. As Breul , MartinuUltimately, the typology of four idealised networks provides a framework for understanding variation in the structural architecture of international networks by sector. Though alternative network-level measures may be substituted, we find that the combination of clustering and assortativity helps explain core-periphery structures by sector, particularly insofar as they can be global in multiple ways. Some sectors feature relatively well-defined core that may be rather more  or less  globally connected. We find that advanced service sectors  exhibit strong hierarchical differentiation through extensive ‘global’ networks, while others are less clustered and therefore are characterised as ‘localised’ and ‘regionalised’ sectors. When combined, the typological framework helps identify sectoral variation in the structural architecture of international economic networks, which serves as a critical dimension to understanding and conceptualising how multiple globalisations are shaped by the large number of complex intraorganisational relationships.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S17 Fig(TIF)Click here for additional data file.S18 Fig(TIF)Click here for additional data file.S19 Fig(TIF)Click here for additional data file.S20 Fig(TIF)Click here for additional data file.S21 Fig(TIF)Click here for additional data file.S22 Fig(TIF)Click here for additional data file.S23 Fig(TIF)Click here for additional data file.S24 Fig(TIF)Click here for additional data file.


In the current study, we investigated the morphological differences, proliferation capacity, population doubling time (PDT), surface marker profiling, trilineage differentiation potential, and immunosuppressive ability of BM Mesenchymal Stem Cells (BM-MSCs) from untreated aAA patients and in the same number of age- and gender-matched controls.  We observed similar morphology, proliferation capacity, phenotype, trilineage differentiation potential, and immunomodulatory properties of BM-MSCs in aAA patients and control subjects.  Our results confirm that the basic and immunosuppressive properties of BM-MSCs from aAA patients do not differ from normal BM-MSCs. Our data suggest that BM-MSCs from aAA patients might not be involved in disease pathogenesis. However, owing to a smaller number of samples, it is not conclusive, and future studies with more exhaustive investigation at transcriptome level are warranted. The characteristic features of acquired aplastic anemia (aAA) are peripheral blood (PB) pancytopenia and bone marrow (BM) hypocellularity. It is believed that autoreactive T cells destroy hematopoiesis in aAA . Apart fThe underlying pathogenetic mechanism of acquired aplastic anemia involves inefficient hematopoiesis and abnormal immune responses. Hence, alterations in bone-marrow-derived mesenchymal stem cells could primarily or secondarily lead to acquired aplastic anemia. Features of altered BM microenvironment have been described in acquired aplastic anemia , 9. PrevTo understand the difference between the characteristics of acquired aplastic anemia BM-MSCs and normal healthy BM-MSCs, the following study was designed. In this study, we have evaluated the morphology, proliferation capacity, population doubling time, surface marker profiling, and differentiation potential of BM-MSCs from aAA patients compared to normal BM-MSCs. We have also evaluated the immunomodulatory potential of these MSCs as it is one of the important mechanisms by which MSCs show their repairable and regenerative potential.Five untreated aAA patients diagnosed as per the standard international criteria   were enrμg/ml) . The cells were incubated in a humidified atmosphere at 37°C with 5% CO2. Medium was changed every 3 days until the cell confluency reached 80%. Adherent cells were then passaged with 0.25% trypsin-EDTA  and reseeded at 1 × 104 cell/cm2.MSCs were isolated and cultured as described previously , 23. UnmMesenchymal Stem Cells are multipotent cells which can be differentiated into osteocytes, adipocytes, and chondrocytes. Therefore, the healthy and aAA MSCs were characterized for their trilineage differentiation potential as per the previous established protocols of our laboratory .N = 3) were seeded at a density of 50 × 103 cells per 35 mm petri dish . After 72 hrs, MSCs were enumerated and assessed for viability using Trypan Blue dye exclusion  assay. The PDT was obtained by the following formula [T is the time of harvesting, To is the time of seeding, N is the number of cells harvested, and No is the number of cells seeded.MSCs for each sample ( formula :(1)PDT=TN = 3) was performed at days 1, 3, 5, 7, and 14 and measured by 3--2,5-diphenyltetrazolium bromide (MTT)  assay. The technique was performed as per the previous established protocol [Proliferation rate of hMSCs . Acquisition and data analysis were performed using flow cytometry BD-LSR-II  and FACS Diva Software Version 6.2.2. Once confluency reached within 40–50%, the existing media from the dish were removed and cells were washed with PBS. Cells were fixed with 1 ml of 4% paraformaldehyde for 20 mins at 4°C. Permeabilization and blocking was done with 0.05% Tween-20 followed by 2% BSA in PBS for 30 mins at room temperature. MSCs were incubated with the Mouse monoclonal vimentin  primary antibody (1/100) for overnight at 4°C. Next day, after washing with PBS, cells were incubated with the secondary fluorescent antibody (1/500) for 40 mins at room temperature (RT). To visualise nuclei, slides were stained with diluted 1/4000 DAPI  for 3 mins at RT followed by thorough washing of the cells with PBS. The acquisition and imaging of the cells were performed using Floid Microscopy (Life Technologies).Immunofluorescence studies were performed for the detection of intracellular Vimentin protein in the MSC samples. Trypsinised cells were seeded on coverslips kept in a 35 mm dish , and MSC expansion media were added and incubated at 37°C, 5% COμg/mL) was used to stimulate the activation of human peripheral blood mononuclear cells (PBMCs) before coculture [μg/ml and cocultured (1 × 104 cells/well) with PHA activated hPBMCs (5 × 104 cells/well) in 1 : 5 ratio in RPMI-1640 medium  containing 10% FBS for 3 days in 96-well plates . Proliferation of hPBMCs was assessed by MTS assay. The 200 μl cell culture supernatant containing hPBMCs was collected in prelabelled 0.6 ml Eppendorf tube, and 20 μl of MTS reagent  was added in each tube, followed by incubation for 3 h at 37°C and 5% CO2. Afterwards, the tubes were centrifuged at 300g for 5 mins. 200 μl of supernatant was collected from each tube and transferred into the fresh 96-well plate. The absorbance was taken at 490 nm using ELISA reader . Lastly, % decrease was calculated by calculating the difference between positive control (activated PBMCs) and test group (MLR). Then, we divided the decrease by the positive control and multiplied the answer by 100.The study was approved by the Institutional Ethics Committee (IEC) (Ref no. IECPG-345/07.09.2017 (RT-6/29.11.2017)). All the samples were obtained after taking the donor's informed consent. Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll-Paque  density gradient centrifugation from blood donated by healthy volunteers. Phytohemagglutinin A ; 35 oculture . For coct-test or one-way analysis of variance (ANOVA). The cutoff value of significance  was 0.05. Results were expressed as mean ± SD.Data analysis was performed using Prism 5 (GraphPad) and Excel (Microsoft). A statistically significant difference among groups was determined by A total of 10 subjects, five aAA donors and five controls, were enrolled in the study. Clinical data of these donors are outlined in in vitro  are multipotent cells that may be isolated from the bone marrow (BM), adipose tissue, dental pulp, umbilical cord blood, or umbilical cord and they are known to contribute to the organization and functioning of the hematopoietic niche. Endothelial cells are known source of MSCs in BM niche and regulate hematopoietic stem cell (HSCs) proliferation and differentiation. However, according to the International Society for cellular therapy, the cells should be able to adhere to plastic surface, express surface markers  . MSCs arin vivo defect or different in vitro conditions.We found spindle-shaped typical morphology of MSCs in aAA and healthy donors. Additionally, our aAA and healthy BM-MSCs expressed vimentin protein. Vimentin protein plays a vital role in cellular stability and is generally expressed in normal mesenchymal cells . SimilaraAA and healthy control MSCs were then characterized using cell surface marker profiling. MSCs were identified based on the minimal criteria of the International Society of Cellular Therapy (ISCT) for human MSCs . The MSCMichelozzi and colleagues reported no difference in population doubling time of aAA-BM and normal-BM , whereasTrilineage differentiation is a hallmark characteristic for identifying healthy and functionally active MSCs. Trilineage differentiation is comprised of osteocytes, adipocytes, and chondrocytes differentiation. Li et al. have reported easier adipogenic differentiation, while it was difficult to induce osteogenic differentiation in aAA-MSCs. Likewise, superior adipocyte differentiation was detected in aAA-MSCs, but osteocyte differentiation was same as controls or was repressed , 30. OthThe disparities in various studies can be attributed to the variations in patient population and the heterogeneity of acquired aplastic anemia. Overall, a plethora of studies have reported contradicting observations, where aAA-MSCs have been shown to behave like MSCs from healthy donors while others have shown functionally impaired MSCs , 27–31.In the present study, at basic parameters, these MSCs show similar trend in terms of cell morphology, proliferation, population doubling time, and trilineage differentiation potential. Apart from these, MSCs are unique in terms of their immune response during inflammation via immunomodulatory factors and release of growth factors, chemokine and anti-inflammatory cytokines .It is already established that MSCs help in tissue repair and prevention of graft-versus-host disease . AltogetIn the present study, the immunosuppressive potential of both the aAA and normal MSCs was maintained. Although minute differences were observed, the immunosuppressive ability of MSCs from aAA patients was not equivalent to those obtained from healthy donors.BM-MSCs have been used in multiple studies and are also being evaluated at clinical level. However, obtaining bone marrow samples from healthy donor is not an easy task. Bone marrow aspiration is a painful process. There are a lot of ethical concerns with respect to bone marrow aspiration from healthy donors for MSCs isolation and expansion. In a routine scenario, bone marrow is obtained from patients undergoing aspiration as a part of routine treatment. During this aspiration, a small amount of bone marrow is required for MSCs isolation. However, there are certain queries related to the MSCs isolated from these patients.In aAA condition, their will be a defective hematopoiesis which eventually leads to empty bone marrow . The proIn conclusion, we have shown that BM-MSCs obtained from aAA patients and controls share almost the same morphological, phenotypical, growth, and differential properties; However, they differ in their immunosuppressive properties. Our results suggest that mesenchymal stem cells might not be involved in the pathogenesis of acquired aplastic anemia. Despite being not conclusive owing to the small number of samples, this study can be used as a base for further investigations. With future in-depth analysis and preclinical studies, much more therapeutic potential of MSCs can be explored. The multifaceted pathophysiology of acquired aplastic anemia is responsible for differences in results across various studies. The crosstalk between various components of bone marrow and disease pathogenesis is a fascinating area of research. Therefore, further investigations on stromal components in bone marrow of acquired aplastic anemia patients would be necessary for better understanding of the mechanism of this disease.