Hypoxic-Ischemic Encephalopathy (HIE) is one of the most relevant contributors to neurological disability in term infants. We hypothesized that clinical outcomes of newborns with (HIE) can be associated with changes at plasma metabolic level enabling the detection of brain injury. Plasma samples of a cohort of 55 asphyxiated infants who evolved to moderate/severe HIE were collected between birth and completion of therapeutic hypothermia (TH). Samples were analyzed employing a quantitative gas chromatography–mass spectrometry method for the determination of lactate and pyruvate and an untargeted liquid chromatography–time-of-flight mass spectrometry method for metabolic fingerprinting. Brain injury was assessed employing magnetic resonance imaging (MRI). A critical assessment of the usefulness of lactate, pyruvate, and pyruvate/lactate for outcome prediction was carried out. Besides, metabolic fingerprinting identified a dynamic perturbation of eleven metabolic pathways, including amino acid and purine metabolism, and the steroid hormone biosynthesis, in newborns with pathologic MRI outcomes. Although data suggest the usefulness of lactate and pyruvate monitoring during 72 h for discerning outcomes, only the steroid hormone biosynthesis pathway was significantly altered in early plasma samples . This study highlights pathways that might potentially be targeted for biomarker discovery or adjuvant therapies to be combined with TH. Hypoxic-Ischemic Encephalopathy (HIE) is the neurological consequence of impaired blood flow and/or gas exchange during birth . DecreasHIE is one of the most relevant contributors to neurological disability in the pediatric age. In high-income countries, the incidence of HIE is around 1.6 per 1000 infants, while the risk is around 10-fold higher in low-income countries . TherapeAt present, no bedside test is available for an accurate and early diagnosis of HIE. In the delivery room, physicians rely on prenatal clinical information and serial Apgar scores with special emphasis on neurological assessment. In addition, cord blood gases, metabolic acidosis, and increased lactacidemia further reflect the severity of anaerobic metabolism . The modHIE has been associated with damage to the integrity of the blood–brain barrier and, hence, brain injury could be reflected in peripheral blood samples . In the Blood lactate, a hallmark of anaerobic metabolism, has been traditionally employed as surrogate marker for tissue hypoxia and/or ischemia. Pyruvate dehydrogenase controls the metabolic flux between the tricarboxylic acid cycle and glycolysis. Under hypoxic conditions, glucose is metabolized into lactate and exported to the extracellular compartment . In infa1H-nuclear magnetic resonance was used to characterize umbilical cord blood samples from infants with perinatal asphyxia or HIE compared to matched healthy controls, revealing significant changes in 18 metabolites [Comprehensive metabolic phenotyping has been increasingly employed for identifying possible therapeutic targets, and the discovery of biomarkers to support treatment selection, patient stratification, and enhanced diagnosis . Howeverabolites . A panelabolites . Umbilicabolites . Eight pThe aim of this study was to elucidate dynamic metabolic changes in a cohort of newborn infants evolving to moderate/severe HIE undergoing TH in which hypoxia–ischemia-induced brain injury was assessed employing MRI. This study describes for the first time the evolution of the neonatal plasma metabolome of HIE covering the time period between birth and completion of TH treatment. The objectives of the present study were focused on preselecting disease-specific pathways at the phenotypic level that might be suitable for enabling an identification of those infants at risk of developing brain injury within the infants that qualify for hypothermia treatment as well as for monitoring the individual response to treatment and elucidating new potential therapeutic targets.p-value = 0.05). Although the analysis of the effect of topiramate vs. placebo was out of the scope of this study, the inclusion of a similar proportion of individuals from both treatment groups  allowed compensating for the impact (confounder) of potential effects of pharmacological treatment on the metabolome.Population characteristics are shown in The evolution of lactate and pyruvate plasma concentrations as well as lactate/pyruvate with time are shown in t-test, α = 0.05) comparing normal vs. pathologic outcomes as determined by MRI. It can be observed that the number of altered features increases with time. Whereas 208 (2.6%) of the total of 8122 detected features showed different mean concentrations in blood before the initiation of TH, 24 h after the initiation of TH, already 491 (6.0%) were altered; 48 h and 72 h after the initiation of TH, this value further increased to 1228 (15.1%) and 1330 (16.4%), respectively. Furthermore, the pattern of affected metabolites shifted over time with only 65 of the altered features shared between blood before the initiation of TH and 24 h samples, 279 between 24 and 48 h samples, and 776 between 48 and 72 h samples. This indicates that the alteration of the metabolome in newborns with pathologic MRI outcomes rapidly changes at early time points and slowly stabilizes thereafter. In The HYPOTOP trial was designed to minimize shortcomings of single center trials, thus improving the generalizability of the observations. This is underpinned by the homogeneity of the clinical and demographic parameters between both study groups , as shown in A complementary analytical approach aimed at exploiting the potentials of untargeted metabolomic fingerprinting for enhancing the current understanding of the complex perturbations of hypoxia–ischemia on the individual, phenotypic level and its impact on the pathophysiology of HIE. The importance of repeated phenotypic monitoring should be highlighted as it is well known that HIE passes through different phases and TH induces a reduction in cerebral and whole-body metabolism . FurtherWe demonstrated that the early evolving blood metabolome provides useful information for the discrimination of newborns who will show pathologic MRI patterns after TH. In the first blood sample analyzed in this study, collected before initiation of TH, levels of two metabolites of the purine metabolism, i.e., xanthine and its derived nucleoside xanthosine, were found to be altered. Purine metabolism has been described repeatedly to be affected in animal experiments on hypoxia/reoxygenation  as well At later time points in samples collected during TH, a total of eight pathways involving amino acid metabolism, synthesis, and degradation have been observed to be perturbed when comparing between normal vs. pathologic groups. In the literature, several reports on the perturbation of amino acids in hypoxia and hypoxia–ischemia in experimental models and neonates can be found  and the This study has some limitations. Although TH is a routine clinical practice, patients need to be transferred to level III NICUs to receive this treatment. Consequently, umbilical cord blood could not be collected from these patients. This is an intrinsic problem of studies on infants with HIE and the exclusion of outborn infants would dramatically increase the time needed for the recruitment of a representative number of subjects. In addition, this study does not include a healthy control group or newborns with asphyxia or classified as mild HIE (Sarnat 1), as only newborns subjected to hypothermia treatment were included. There is risk of adverse neurodevelopmental outcome of mild HIE newborns, but, up to now, there is no evidence to support cooling in these babies, and therefore we consider that the study of the metabolic fingerprint of those infants in correlation with the clinical outcomes would be of interest . FurtherWe report the first metabolomic study involving human subjects and serial sample collections in HIE for modeling brain injury as confirmed with MRI. Herein, the time-dependent evolution of lactate and pyruvate levels in newborns with HIE undergoing TH is shown. Our data underpin the usefulness of blood lactate with a cut-off value of 1.1 mM between 24 and 48 h and pyruvate with a cut-off value of 51.4 μM at 72 h for anticipating favorable/unfavorable MRI outcomes. Although these findings need to be validated in independent cohorts, these biomarkers could be easily implemented within clinical routine care, as portable devices for point-of-care testing are commercially available. However, at very early sampling time points (before initiation of TH), the measurement of those parameters does not support outcome prediction. Metabolic pathway analysis revealed the time-dependent perturbation of several pathways. Purines  were found to be altered in blood samples from newborns with pathologic MRI outcomes drawn before the initiation of TH and the steroid hormone biosynthesis pathway has been significantly altered during the whole study period comprising early samples collected shortly after birth and until completion of TH after 72 h. Hence, this study provides evidence of the usefulness of metabolites from both pathways as candidate biomarkers to be evaluated in future clinical validation studies as they might allow an early prediction of central nervous system impairment reflected in an anatomic alteration detectable by MRI. In depth knowledge of metabolic alterations might furthermore support studies targeting the development of adjuvant therapies to be combined with TH, e.g., the administration of allopurinol and steroid hormones.The study was approved by the Ethics Committee for Biomedical Research of the Health Research Institute La Fe  and registered as EudraCT 2011-005696-17 under the abbreviation HYPOTOP. Informed consent was obtained from parents of all participants and all methods were performed in accordance with relevant guidelines and regulations.A cohort of 62 newborns enrolled in the HYPOTOP trial was included in this study according to pre-established inclusion and exclusion criteria. The HYPOTOP trial is a randomized, controlled, multicenter, double-blinded clinical trial aiming to assess the efficacy of topiramate vs. placebo in patients with HIE undergoing TH. A detailed description of the HYPOTOP trial including the study design and sample-size estimation can be found elsewhere .®, General Electric Healthcare, IL, USA) and always included 3D Gradient Echo T1 weighted MR images, coronal and axial Fast Spin Echo T2 weighted MR images, diffusion weighted images , and susceptibility weighted imaging. In most cases, single voxel MR spectroscopy in thalamus, white, and grey matter was acquired using PROBE sequence, as well as diffusion tensor imaging (32 directions). The score used to differentiate mild from moderate and severe HIE was based on the MR score described by Barkovich [N = 22) or “pathologic” (N = 33) neuronal outcomes following the criteria explained elsewhere [MRI was performed between Days 4 and 8 after birth. It was carried out using a 3T magnet system  and at 24 (N = 50), 48 (N = 50), and 72 h (N = 52) after the administration of the first dose of medication or placebo from a peripheral vein using heparinized syringes (1% sodium heparin). Blood samples were centrifuged immediately at 1800× g and 20 °C during 10 min. Plasma was collected and stored at −80 °C until analysis.A 0.5 mL sample of whole blood was extracted at birth  95:5 containing phenylalanine-D5, caffeine-D9, leucine enkephalin, and reserpine, each at a concentration of 2 µM. Blank extracts were prepared by replacing whole blood with 500 µL of H2O. A quality control (QC) sample was prepared by mixing 5 μL of each plasma sample and a total of 10 QC sample aliquots were processed as described for plasma samples.Plasma samples were thawed on ice and homogenized on a Vortex S0200 mixer  during 10 s at maximum speed. Seventy-five microliters of cold (4 °C) CH−1. Autosampler and column temperatures were set to 4 and 40 °C, respectively, and an injection volume of 4 μL was used. Initial conditions of 98% of mobile phase A (H2O (0.1% v/v HCOOH)) were kept for 0.5 min, followed by a linear gradient from 2% to 20% of mobile phase B (CH3CN (0.1% v/v HCOOH)) in 3.5 min and from 20% to 95% B in 4 min. Conditions of 95% B were held for 1 min and a 0.25 min gradient was used to return to the initial conditions, which were held for 2.75 min.Chromatographic separations were performed on an Agilent 1290 Infinity UHPLC chromatograph using a UPLC ACQUITY BEH C18 column  and a flow rate of 400 μL min+ and TOF MS mode. The electrospray ionization parameters were set as follows: gas T, 200 °C; drying gas, 14 L min−1; nebulizer, 37 psig; sheath gas T, 350 °C; sheath gas flow, 11 L min−1. For automatic MS spectra re-calibration during analysis, a mass reference standard was introduced into the source via a reference sprayer valve using 149.02332 (background contaminant), 121.050873 (purine), and 922.009798 (HP-0921) m/z as references.Full-scan MS data between 100 and 1700 m/z with a scan frequency of 6 Hz (1274 transients/spectrum) were collected on an iFunnel quadrupole time-of-flight Agilent 6550 spectrometer operating in ESISystem suitability was checked employing a standard mixture containing internal standards. The analytical system was conditioned by injecting the QC extract 9 times at the beginning of the batch. Data acquired during system conditioning were discarded from data analysis. In total, 188 plasma sample extracts were analyzed in random order in a single analytical batch. QC samples were analyzed every 5th sample and twice at the beginning and end of the batch for assessment and correction of instrumental performance . FurtherRaw UHPLC-TOFMS data were converted into centroid mzXML format using ProteoWizard . Metabol5. C was defined for each feature as the median value in QCs. Then, features detected in blanks  and those with a RSD% in QC samples ≥20% were excluded. The final peak table contained 8122 features. Systematic identification of detected features was carried out by matching m/z values against the Human Metabolome Database (version 4.0) [The obtained peak table was imported into MATLAB R2017b  for further data processing and analysis. To reduce intra-batch variation, the Quality Control-Support Vector Regression algorithm  and the ion 4.0)  with 5 pion 4.0) ,39.t-test . MetaboAnalyst (version 4.0) [t-test p-values  and fold changes were computed for each metabolic feature comparing normal and pathologic outcomes for each sampling time point separately. Pathway analysis was carried out using MetaboAnalyst with the “MS peaks to pathways” tool  and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway library (Homo sapiens). Pathways with p-values from Fisher’s exact t-test < 0.05 were considered as altered. Putative identifications based on m/z from pathway analysis were corroborated by manual inspection of raw data and MS/MS data when available (“level 2” identification as defined by The Metabolomics Standards Initiative [MATLAB 2017b inbuilt functions as well as in-house written scripts (available from the authors upon reasonable request) and the PLS Toolbox 8.0 from Eigenvector Research Inc.  were used for PCA, non-parametric Wilcoxon rank sum test, and ion 4.0)  was useditiative ).


Many studies have shown that mesenchymal stem cells have the ability to restore function in models of premature ovarian insufficiency disease, but few studies have used stem cells in the treatment of ovarian physiologic aging (OPA). This experimental study was designed to determine whether human amniotic fluid mesenchymal stem cells (hAFMSCs) have the ability to recover ovarian vitality and to determine how they function in this process. Mice (12–14 months old) were used in this study, and young fertile female mice (3–5 months old) were the control group. Ovarian markers for four stages of folliculogenesis and DNA damage genes were tested by qPCR and western blot. hAFMSCs were used to treat an OPA mouse model, and the animals treated with hAFMSCs displayed better therapeutic activity in terms of the function of the mouse ovary, increasing follicle numbers and improving hormone levels. In addition, our results demonstrated that the marker expression level in ovarian granular cells from patients with OPA was elevated significantly after hAFMSC treatment. In addition, the proliferation activity was improved, and apoptosis was dramatically inhibited after hAFMSCs were cocultured with hGCs from OPA patients. Finally, in this study, hAFMSCs were shown to increase the mRNA and protein expression levels of ovarian markers at four stages of folliculogenesis and to inhibit the expression of DNA damage genes. These works have provided insight into the view that hAFMSCs play an integral role in resisting OPA. Moreover, our present study demonstrates that hAMSCs recover ovarian function in OPA by restoring the expression of DNA damage genes. Ovarian physiologic aging (OPA) is a worldwide reproductive health problem . A graduvia gap junctions (Human granulosa cells (hGCs) are a significant class of ovarian cells. They are situated outside of the zona pellucida in oocytes and are connected to oocytes unctions . They plunctions . Moreoveunctions . It has unctions . The desunctions .Persistent senescent cells are generated by different situations related to human health throughout organismal life: age-related disease, natural aging, and therapeutic intervention-induced aging . DNA damThe therapeutic activity of human amniotic fluid mesenchymal stem cells (hAFMSCs) has been demonstrated in preclinical investigations. Previous researchers have used many types of MSCs to repair damaged ovaries in rats or act as therapeutic agents to treat ovarian insufficiency disease, such as human amniotic MSCs, human adipose MSCs, and human umbilical cord MSCs . HoweverNevertheless, there is little known regarding whether hAFMSCs contain the potential to oppose OPA and how they might perform this function. Therefore, the aim of the current study was to determine whether hAFMSCs have the power to restore normal ovarian function in OPA disease.via amniocentesis that were performed under sterile conditions, and the residual fluid from prenatal diagnoses was used for research. Amniotic fluid samples with abnormal karyotypes were excluded. The range of maternal age was between 30–35 years old, and the range of gestational age was between 15–22 weeks . Follicular fluid samples (2–5 ml from every sample) were collected separately. This study was executed with the approval of the Ethics Committee of Suzhou Hospital, and informed consent was acquired from each participant. To obtain hAFMSCs, we centrifuged amniotic fluid samples at 3000 × g for 5 min and discarded the supernatant. Then, hAFMSCs were seeded and cultured in DMEM/F12 medium  supplemented with 1% glutamine, 1% penicillin/streptomycin , 10% FBS , and bFGF  at 37°C in a 5% CO2 atmosphere. After reaching 70% to 80% confluence, cells were passaged following incubation in 0.25% trypsin  at 37°C for 5 min. Approximately 3×107 cells were obtained, and the cells were plated on a 10 cm cell culture dish. hAFMSCs at the fourth–eighth passage were tested.All samples of amniotic fluid cells were collected The expression of CD34, CD105, CD73, CD90, and CD29 was tested to characterize the phenotype of hAFMSCs by means of FACS. The exact method used was the same as the FACS analysis described elsewhere in the methods section. Differentiation kits were used to test the ability of hAFMSCs to differentiate .As described in a previous study of ours , young pOvarian physiologic aging female C57BL/6J mice  and young, fertile female C57BL/6J mice  were purchased from Nanjing Medical University. All experiments were approved by the Institutional Animal Care and Use Committee and were conducted following institutional guidelines. The mice were bred in conditions with a 12-hour light/dark cycle at a temperature of 28 ± 2°C. To determine the estrous cycle, mouse vaginal smears were checked at 9:00 am every day. Only young mice with normal estrous cycles and OPA mice with disorganized estrous cycles were used in this study. Aging mice were randomly distributed into the control group (young mice) and treatment group (n = 15 per group).hGCs were divided into three groups, namely, the OPA hGCs group, the hAFMSCs cocultured with OPA hGCs group, and the young hGCs group. Tests were performed 7 days after culturing the hGCs with hAFMSCs. There were three mouse groups: the hAFMSC-injected OPA mouse model group, the OPA mouse model group, and the young mouse group. A total of 4 weeks after the hAFMSCs were injected into the ovary with OPA, hematoxylin and eosin (HE) experiments were conducted to calculate the follicle number.p < 0.05 was considered statistically significant.The mice (with or without transplanted cells) were euthanized between 0 and 4 weeks after hAFMSC treatment. Ovaries from both sides of each mouse were extracted and fixed in 10% formalin. Then, ovaries were paraffin embedded, serially sectioned to produce 5 μm thick sections, and stained with HE. Primary, primordial, secondary, and antral follicles were classified, and their numbers were calculated by choosing three typical sections from each ovary. Only follicles containing an oocyte were counted to avoid calculating a follicle twice. All experiments were conducted three times. The results are presented as the mean ± standard deviation, and p < 0.05 was considered statistically significant.hAFMSCs or hGCs were digested separately using 0.25% trypsin-EDTA for 5 min before being mixed well to produce a suspension of single cells. A Cytofix/Cytoperm Fixation/Permeabilization Solution kit  was used to permeabilize and fix the samples according to the manufacturer's instructions. PE- or FITC-conjugated antibodies or their relevant isotype controls were used to characterize hAFMSCs and hGCs. Finally, cells were analyzed using a fluorescence-activated cell sorter . Information concerning the primary antibodies used is provided in The levels of FSH, E2, or AMH in the plasma of the OPA mouse model were evaluated with an ELISA kit  following the manufacturer's instructions. In brief, 50 μl of serum sample was added to each well of the test plate before being sealed with a membrane and incubated at 37°C for 30 min. Then, the wells of the plate were dried and washed with wash buffer five times. Subsequently, 50 μl of an HRP-conjugated reagent was added to each well of the plate, and then the plate was incubated at 37°C for 60 min. The plate was washed again with wash buffer five times. Afterwards, each well received 50 μl of substrate A solution and 50 μl of substrate B solution before being incubated at 37°C for 15 min. Finally, 50 μl of stop solution was added to stop the reaction, and the plate was scanned with a spectrophotometer  to measure and record the value of light absorbance.p < 0.05 was considered statistically significant. Information regarding the primary antibodies used for western blot analyses is listed in hGCs and ovaries were gathered and dissolved in lysis buffer as previously discussed. Total protein was extracted and loaded on 10% gels. Then, the protein was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and the proteins were then electrotransferred to polyvinylidene difluoride membranes . The membranes were incubated overnight at 4°C with primary antibodies. Then, appropriate secondary antibodies  were incubated separately with the membranes. After washing three times with Tris-buffered saline, a chemiluminescence kit was used to enhance specific signals . Finally, the membrane was examined with an imaging detection system , and ImageJ software  was used to analyze immunoreactive bands. All experiments were repeated at least three times. The results are presented as the mean ± standard deviation, and p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, and ***p < 0.001. Primer Premier 5.0 was used to design the primers for qPCR. The primer sequences are listed in All results are shown as the means ± standard deviation. SPSS 17.0 and GraphPad Prism 8 software were used in this study, and First, to evaluate the therapeutic outcomes of hAFMSCs in OPA disease, FACS was used to characterize the hAFMSCs. The results revealed that CD29, CD90, and CD73 were highly expressed, while the levels of other markers, CD105 and CD34, were reduced to a minimum in hAFMSCs , as shown in +CYP19A1+ cells than what was observed in the OPA group (20%). Western blot experiments indicated that hAFMSCs produced a more dramatic increase in the expression of FSHR (102%), AMH (96%), FOXL2 (107%), and CYP19A1 (105%) than the OPA group did , as shown in To examine the therapeutic outcome of hAFMSCs on patients with OPA in the clinic, hGCs were collected from the young and OPA groups to analyze the effects of marker expression after coculture with hAFMSCs; these experiments were performed in our reproductive center as previously reported . FACS anIn summary, powerful recovery effects were exhibited by hAFMSCs for hGCs with OPA.To examine the effects of hAFMSCs on apoptosis and cell proliferation, hGCs and hAFMSCs were cultured together for 7 days. A FACS assay was applied to evaluate cell activity. As shown in In conclusion, hAFMSCs could enhance proliferation and prevent apoptosis of hGCs with OPA.To determine how the ovarian function of the OPA mouse model was enhanced by therapy with hAFMSCs, hAFMSCs were injected into the mouse OPA model ovary for 1 month. The follicular regulation of gene expression levels at different stages was tested by qPCR and western blot methods, and it included the assessment of the following genes and stages: PGC oogonia stage (FOXL2 and CYP19A1), follicle formation stage (MSH4 and STAG3), follicular growth stage (GDF9 and AMH), and follicular maturation stage (FSHR and BMP15). The mRNA assay results demonstrated that hAFMSCs decreased the expression of the PGC  and oogonia markers (FOXL2 and CYP19A1) to 90% and 96%, respectively, which was a more dramatic effect than that observed in the OPA group  when compared to the levels in the young group , BRCA1 (70%), PARP1 (90%), and XRCC6 (105%) to levels that were higher than those in the OPA group , relative to those of the young group . Our findings indicated that hAFMSCs augmented the level of proliferation and repressed apoptosis in OPA hGCs to normal levels . All the patients provided written informed consent prior to participation in this study. Our investigation using experimental animals was conducted on the basis of the Nanjing Medical University Animal Center's specific guidelines and standards (20170330).in vivo and in vitro. QZ participated in the statistical analysis and revised the manuscript. BH contributed to hGC collection and purification. QZ contributed to hGC purification culture. CD carried out the partial immunoassays. WW carried out the partial HE assays. BH and JL participated in the mice feeding. BH planned the experiments and drafted part of the manuscript. HL planned the experiments and wrote the manuscript. All the authors read and approved the final manuscript.BH and CD performed the cellular and molecular assays This work was supported by grants from National Natural Science Foundation of China (81801515), Suzhou science and technology for people's livelihood (SYS2018081), the Suzhou expert team of clinical medicine (SZYJTD201708), and the Suzhou talent training program (GSWS2019005).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.


Women experience drastic hormonal changes during midlife due to the menopausal transition. Menopausal hormonal changes are known to lead to bone loss and potentially also to loss of lean mass. The loss of muscle and bone tissue coincide due to the functional relationship and interaction between these tissues. If and how physical activity counteracts deterioration in muscle and bone during the menopausal transition remains partly unresolved. This study investigated differences between premenopausal, early perimenopausal, late perimenopausal, and postmenopausal women in appendicular lean mass (ALM), appendicular lean mass index (ALMI), femoral neck bone mineral density (BMD) and T score. Furthermore, we investigated the simultaneous associations of ALM and BMD with physical activity in the above‐mentioned menopausal groups.2) and neck T scores calculated. Current level of leisure‐time physical activity was estimated by a validated self‐report questionnaire and categorized as sedentary, low, medium, and high.Data from the Estrogen Regulation of Muscle Apoptosis study were utilized. In total, 1393 women aged 47–55 years were assigned to premenopausal, early perimenopausal, late perimenopausal, and postmenopausal groups based on follicle‐stimulating hormone concentration and bleeding diaries. Of them, 897 were scanned for ALM and femoral neck BMD by dual‐energy X‐ray absorptiometry and ALMI (ALM/heightP < 0.001), ALMI , neck BMD , and T score . After adjusting for potential confounding pathways, a higher level of physical activity was associated with greater ALM among the premenopausal , late perimenopausal , and postmenopausal  women. The positive association between femoral neck BMD and level of physical activity was significant only among the late perimenopausal women .Appendicular lean mass, appendicular lean mass index, femoral neck bone mineral density, and and T score showed a significant linear declining trend across all four menopausal groups. Compared with the postmenopausal women, the premenopausal women showed greater ALM (18.2, SD 2.2 vs. 17.8, SD 2.1, Skeletal muscle and bone losses were associated with the menopausal transition. A higher level of physical activity during the different menopausal phases was beneficial, especially for skeletal muscle. Menopause‐related hormonal changes predispose women to sarcopenia and osteoporosis and further to mobility disability and fall‐related fractures in later life. New strategies are needed to promote physical activity among middle‐aged women. Longitudinal studies are needed to confirm these results. Female sex hormones have wide‐ranging effects on women's health and well‐being over the lifespan. During midlife, women experience drastic hormonal changes due to ovarian aging and the consequent menopausal transition. The menopausal transition phase includes elevation of the serum follicle‐stimulating hormone (FSH) concentration and a decline in the estradiol concentration, both of which exhibit wide interindividual variation.Muscle and bone mass decline with aging, increasing the risk for sarcopenia and osteoporosis in later life. The contribution of menopause‐related hormonal changes, especially in oestrogens, is known to lead to bone loss through increased bone turnover with a net deficit in bone formation relative to bone resorption.Muscle and bone tissues have a close developmental and functional relationship.This study investigated differences between 47 to 55‐year‐old premenopausal, early perimenopausal, late perimenopausal, and postmenopausal women in appendicular LM (ALM), appendicular LM index (ALMI), femoral neck BMD, and T‐score. We also investigated simultaneous associations of ALM and femoral neck BMD with current physical activity in the four menopausal groups.2, being currently pregnant or lactating, conditions affecting ovarian function, oral or transdermal oestrogen‐containing hormonal preparations or other medications affecting ovarian function, and chronic diseases or medications seriously affecting muscle function. A written invitation to take part in the study was sent to 6878 potential participants. The response rate was 47%. Eligible participants (n = 1627) were invited and 1393 came to the laboratory for a health interview and to give a fasting blood sample. Participants who had reported serious or unclear health conditions were examined by a physician to ensure safe participation in the measurements. All study participants provided a written informed consent. The study was approved by the ethics committee of the Central Finland Health Care District (K‐SSHP Dnro 8U/2014).Data from the Estrogenic Regulation of Muscle Apoptosis (ERMA) study were utilized. A detailed description of participant recruitment and categorization into menopausal groups has been reported earlier.g. FSH and 17β‐estradiol (E2) levels were immunoassayed using IMMULITE® 2000 XPi . A participant was categorized as premenopausal if she had FSH < 9.5 IU/L or a regular menstrual cycle and FSH < 17 IU/L; early perimenopausal if she had FSH 17–25 IU/L or an irregular menstrual cycle and FSH 9.5–30 IU/L; late perimenopausal if she had FSH 25–30 IU/L or had experienced occasional menstrual bleeding during the past 3 months and FSH > 30 IU/L; and postmenopausal if she had experienced no menstrual bleeding during the past 6 months and FSH > 30 IU/L or no menstrual bleeding during the past 3 months and FSH > 39 IU/L or very high FSH (>130 IU/L) with or without occasional menstrual bleeding. For women with incomplete menstrual cycle information, the categorization was based solely on FSH level and stricter cutoff values were applied . The numbers of participants in each category were premenopausal 235, early perimenopausal 180, late perimenopausal 193, and postmenopausal 289.Participants' menopausal status was determined based on serum FSH concentration and menstrual cycle reported by monthly diaries and following the Stages of Reproductive Aging Workshop criteria.2) and ALM as a surrogate for muscle mass by dual‐energy X‐ray absorptiometry  after overnight fasting. Participants were scanned in a supine position in the centre of the table using the default scanning mode for total body and proximal femoral region automatically selected by prodigy software (Lunar Prodigy Advance Encore v. 14.10.022). To calculate ALMI, ALM was divided by height squared (kg/m2).2  was assessed by a questionnaire with a seven‐point scale ranging from household chores to competitive sports.Level of education was assessed by a single question and categorized as primary, secondary, bachelor's, master's, or doctoral level. Because of the small number of participants in the primary and doctoral levels, the primary and secondary levels were combined and the bachelor's, master's, and doctoral levels were also combined. Body mass was measured with a digital scale and height with a stadiometer. Body mass index was calculated as body mass divided by height squared (kg/m2). Total body fat mass (kg) was measured by DXA.Participants self‐reported their musculoskeletal diseases or conditions, previous bone fractures, gynecologic history, and use of medications. Data related to the use of hormonal contraception and other gynecological hormonal treatments  were collected with standardized questions. Current users and those who had used oestrogen‐containing medication  during the last 3 months were wholly excluded from the ERMA study. Smoking status was self‐reported with standardized questions and categorized as current, former, and never.Hand grip force was measured with an adjustable dynamometer chair  from the dominant hand in a sitting position with elbow flexed at 90°. The participant was instructed to squeeze the handle as forcefully as possible for 2 to 3 s, and the peak value out of three to five maximal trials was taken as the result.2 test. To analyse linear trends in the continuous background variables and in muscle and bone mass over the menopausal groups, the Welch test [a modification of one‐way analysis of variance (ANOVA) accommodating unequal group variances] was used. Adjustment for age was performed using univariate analysis of covariance. Tamhane's post hoc test was used to localize differences in the background variables between the menopausal groups. To explore indications for the timing of bone and muscle loss during the menopausal transition, the ALM, ALMI, BMD, and neck T score of the premenopausal women (reference) were compared with the corresponding values for the other three study groups using Dunnett's post hoc testing procedure.Means and standard deviations for the continuous variables and frequency distributions with percentages for the categorical variables were calculated. Differences between the groups in the categorical variables were assessed by cross tabulation and χFigure2, P = 0.002); fat mass differed significantly between the physical activity  and education  groups; fat mass was associated with ALM  and BMD ; body height was associated with ALM  and BMD ; and a significant increasing linear trend was observed in the ALM and the BMD from the non‐users of the contraceptives to the former users and to the current users .To investigate the simultaneous association of ALM and BMD with physical activity, a four‐group multivariate linear model was constructed. In addition, a prespecified contrast was used to explore the differences between the premenopausal and early perimenopausal women combined and the late perimenopausal and postmenopausal women combined. Theoretically, meaningful and available confounders, which were significantly associated with the predictor or the outcome, were included in the models shown in Although smoking status showed no statistically significantly association with either ALM or BMD, we decided to retain it in the model. Smoking is a known risk factor for osteoporosis, and it is suggested that smoking is inversely associated with peak torque and fat infiltration into muscle tissue among postmenopausal women.TableP < 0.001). Thus, the serum E2 concentration was the highest and FSH concentration lowest among the premenopausal women followed, in descending order, by the early perimenopausal, late perimenopausal, and postmenopausal women . No significant between‐group differences were observed in anthropometrics, total body fat mass, smoking status, musculoskeletal problems, previous bone fracture, or level of physical activity. As expected, the distribution of the use of hormonal contraception differed significantly between the groups; nearly half of the premenopausal women were current users, whereas among the perimenopausal and postmenopausal groups, the corresponding percentage varied from 26% to 32%. Because use of oestrogen containing medication was the exclusion criteria of the current study, the current users of hormonal contraception include only users of intrauterine device (95%) or progestin‐only pills (5%). Grip force was significantly lower in the postmenopausal than premenopausal and early perimenopausal women (P < 0.001 for both comparisons).As expected, the premenopausal women were the youngest and the postmenopausal women the oldest Table. No signTableP < 0.001 for both comparisons). The difference between premenopausal and early perimenopausal women in ALM (P = 0.27) and ALMI (P = 0.66) and between premenopausal and late perimenopausal women in ALMI (P = 0.09) were not statistically significant. The ALM was significantly higher among premenopausal compared with late perimenopausal women (P = 0.03). The relative proportion of participants with ALM below the sarcopenia cutoff value of 15.02 was 4–5% in the premenopausal and early perimenopausal groups and 7–8% in the late perimenopausal and postmenopausal groups. Accordingly, 2–3% of premenopausal and early perimenopausal women had ALMI below the sarcopenia cutoff value of 5.45 while the corresponding percentages in the late perimenopausal and postmenopausal women were 3–4%.Appendicular LM and ALMI showed a significant linear trend across the four menopausal groups Table. ALM andTableP < 0.001 for both comparisons). The differences between premenopausal and early menopausal women in BMD (P = 0.42) and in T score (P = 0.28) were not statistically significant. Similarly, the difference between premenopausal and late perimenopausal women in BMD and in T score (P = 1.00 for both comparisons) were not statistically significant. The relative proportion of participants with a T score below −1 (cutoff for osteopenia) was 17% in the premenopausal and 13% in the early perimenopausal women and 24% in the late perimenopausal and 27% in the postmenopausal women.BMD and T score also showed a significant trend over the four menopausal groups Table. Neck BMFigureThe differences in ALM, ALMI, and neck BMD between the physical activity groups are shown in FigureTableFigureP = 0.02).P = 0.30). All the path coefficients of the adjusted models are shown in the Supporting Information, Table S1.In the adjusted models that included ALM, BMD, and the above‐mentioned covariates, a higher level of physical activity was significantly associated with greater femoral neck BMD only in the late perimenopausal women; in the postmenopausal women, the association was borderline significant. No significant differences were observed in the path coefficients between the premenopausal and early menopausal groups and the late perimenopausal and postmenopausal groups , early perimenopausal (ePERI), late perimenopausal (lPERI) and postmenopausal (POST) groups.Click here for additional data file.


Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes, which together accounted for > 78% of the all phyla in vegetation restoration. The soil microbial diversity was higher in RG than in NG. Two types of vegetation restoration had significantly different characteristics of soil microbial community (P < 0.001). Based on a molecular ecological network analysis, we found that the network in RG had a longer average path distance and modularity than in NG network, making it more resilient to environment changes. Meanwhile, the results of the canonical correspondence analysis and molecular ecological network analysis showed that soil pH (6.34 ± 0.35 in RG and 7.26 ± 0.28 in NG) was the main factor affecting soil microbial community structure, followed by soil moisture (SM) in the Songnen meadow, China. Besides, soil microbial community characteristics can vary significantly in different vegetation restoration. Thus, we suggested that it was necessary and reasonable for this area to popularize reseeding grassland in the future.Microorganisms have important ecological functions in ecosystems. Reseeding is considered as one of the main strategies for preventing grassland degradation in China. However, the response of soil microbial community and diversity to reseeding grassland (RG) and natural grassland (NG) remains unclear, especially in the Songnen Meadow. In this study, the soil microbial community compositions of two vegetation restoration types (RG vs NG) were analyzed using a high-throughput sequencing technique. A total of 23,142 microbial OTUs were detected, phylogenetically derived from 11 known bacterial phyla. Soil advantage categories included  However, ecosystem services and biodiversity have decreased by the human activities around the world6. Among the serious issues, the loss of soil microbial diversity is considered as the main threat to the balance of ecosystems. Moreover, the composition of soil microbial communities (SMC) varies widely across different biomes have been reported7. For instance, Zhang et al.8 found that the distribution of SMC is dependent on latitude gradient, while other studies revealed that it is also dependent on altitude gradient10, soil pH11, nutrients12, and temperature13. However, other factors like climate change, which currently disrupts the structures and functions of global ecosystems14, could also modulate SMC composition.Soil microorganisms, involved in various important ecological functions, are an important index for evaluating the response of various belowground biogeochemical processes of ecosystems to environmental changes15. As climate becomes warmer and drier in Songnen Meadow area, together with increasing degeneration of permafrost, there is continuous decrease in soil surface water content, change in plant community, and/or significant succession, to the extent that the natural grassland has successively transformed into degraded grassland16. Consequently, Chinese Government launched the Grain-for-Grassland policy (or vegetation restoration project) largely contributed to maintaining soil and water conservation, which widely improved carbon sequestration and reduced floods17. For example, Yang et al.18 found soil carbon storage was closely related to soil bacterial diversity after vegetation restoration in Loess Plateau, and then Yang et al.19 confirmed that vegetation restoration greatly improved the physiological activity of soil microorganisms. Hence, according to the actual situation of the Songnen Plain, a lot of degraded grassland or abandoned farmland has been converted to reseeding grassland via natural succession. In fact, the vegetation restoration changes environment condition, by directly modifying root systems, and the exudates as well indirectly affecting soil quality and even soil microbes21. Concurrently, some studies have suggested that plant succession could significantly alter the structure of SMC and have a large-scale effect on soil carbon and nitrogen content23. Reseeding grassland is also considered as an effective measure for ecological restoration in this region24, which could restore ecosystem functioning and soil quality, but also influence soil microbe dynamics26. Therefore, a better understanding of the unique microbial geographical flora in this region will contribute to predictions of the impacts of vegetation restoration on ecosystems.The Songnen Meadow of China is an ecologically fragile ecosystem vulnerable to various environmental disturbances due to its high altitude and extreme climatic conditions27, plant community structure28, plant primary productivity30, and soil carbon cycle or nitrogen cycle32. As a consequence, our knowledge regarding the structure of SMC in the ecosystem, particularly in the Songnen Meadow, and how the linkage between environment and microbial ecology may respond to the changing vegetation, is limited. With a growing body of biological evaluations at molecular levels, high-throughput sequencing technology has been widely and effectively applied in the research field of microbiology33. It can also be rapidly and effectively used to study the composition of microbial community and identify soil microorganisms via comparative analysis of total DNA. However, due to the present limitations in research techniques and analytical methods, few microbial diversity studies have thus far investigated the interactions between microbial species and population in the Songnen Meadow. Molecular ecological networks can provide important information about the complex ecological interactions between different species and reveal changes in the topological structure of microbial networks38. Statistical analyses showed that the approach was reliable, sensitive and robust in withstanding noise inherent in high-throughput data sets40. Hence, the molecular ecological network analysis method can be instrumental in predicting the microbial community composition41 and unraveling the microbial diversity response to different vegetation restoration.Previous research on vegetation restoration has mainly focused on vegetation coverageHere, we investigated soil microbial community and diversity about two types of vegetation restoration  on the Songnen Plain. We hypothesized that soil microbial community characteristics have significant differences in different types of vegetation restoration and vegetation restoration strongly affects soil microbial community and diversity in this unique region.24.The experiment was carried out at the Frigid Forage Research Station located at Heilongjiang, China Fig. , runned −2 including both types of degraded and undegraded meadow. For this reason, both types of degraded and undegraded meadow has been used to evaluate different methods for vegetation restoration  in the effects of vegetation restoration on soil microbial community and diversity. The paired meadows, namely Dongsanbaishang (DSBS) and Xisanbaishang (XSBS), are now covered by different vegetation, respectively, but have similar soil, topography, and climate conditions , reseeding grassland (RG) was adopted by reseeding forage crops  in 2008–2018, followed by prohibition of any cultivation and fencing until the present. There was no significant logging, grazing, or human activities. In XSBS meadows , natural grassland (NG) was adopted by natural grass restoration without human disturbance since 2008. Now the dominant plant species belong to Leymus chinensis Tzvel, Carex duriuscula, Radix sanguisorbae, and Potentilla anserina L. After 10 years of vegetation restoration and construction, these paired meadows have formed completely different vegetation landscapes, providing an ideal platform to examine the effects of different types of vegetation restoration on soil microbial community and diversity on Songnen Meadow. More information on soil type and doIn DSBS meadows  in DSBS (reseeding forage vegetation) and NG  in XSBS , with nine sample sites of each vegetation type. In each sample sites, we established three subplots located at least 100 m apart from each other. These subplots were a permanent quadrat (1 × 1 m) by a modified Whittaker plot. We gathered the surface soil samples (0–30 cm) in each subplot. Four replicates were obtained using a soil corer (6 cm in diameter) along an S-shaped curve, and then mixed to one sample. Additionally, we divided soil samples into two parts: one part was to analyze soil properties; for the other part was stored at − 80 °C immediately using liquid nitrogen for later DNA analysis.We selected RG  before oven-drying at 65 °C for 72 h. Species diversity of the above-ground communities were estimated via Shannon–Wiener index (H) as described by Wang et al.43:i is species, and Pi is the relative coverage of species i.All samples were collected at the peak of biomass growing season (mid-August) in 2017. We estimated species-level cover, community-level cover, community-level above (ANPP) and below-ground biomass (BNPP) in one 1 m44  were examined following the procedures described in Bao et al.44 . The oth18. As described by Yang et al.18, soil available P (Olsen P) was obtained by shaking 1.5 g of dried soil for 30 min at 20 °C in 100 mL of a 42% NaHCO3 solution (pH 8.5). Finally, we determined the available N concentrations (AN) and NO3-N (ANN) in each plot after initial extraction of 10 g soil subsample with 50 mL of 2 mol/L KCl using a Flow-Solution analyzer .First, we determined soil organic carbon (SOC) and total N in the soil in each plot with the aid of FOSS Kjeltec 2300 Analyzer Unit . For total P, we followed the sodium hydroxide melting molybdenum antimony colorimetric method45. According to the overlap relation between paired end (PE) reads, the number of sample sequences in each stage was processed by statistical data, and the data quality was evaluated. Table High-throughput soil sequencing was performed using a method described in Zhou et al.41, Illumina HiSeq was used. The website of Institute of Environmental Genomics, University of Oklahoma (https://ieg2.ou.edu/MENA) was used to construct the molecular ecological network of the soil microorganisms with and without nitrogen application and to calculate its characteristic parameters. The construction process of the molecular ecological network is as follows: the operational taxonomic unit (OTU) data of soil microbial species were first converted into lg matrix, and then Pearson correlation moment was constructed and further converted into similarity matrix. According to the random matrix theory40, the adjacency matrix was derived from the similarity matrix by applying the appropriate threshold value, and the adjacency matrix was used to encode the connection strength between each pair of nodes. The characteristic parameters used for the description of the network information include: (1) fluidity, which is one of the most commonly used network indicators and in our study was used to calculate the total number of connections between each gene (node) and other connected genes, and to express the connectivity strength between a gene and other genes; (2) path length, that is, the shortest distance between two genes; (3) the clustering coefficient, which represents the degree of connectivity between one node and other nodes; and (4) the modularity of each network and description of the modularity of molecular ecological network. The ecological network was divided into several modules, and each module was a functional unit of the biological system. The function of node was represented by the connectivity within the module (Zi) and the connectivity between the modules (Pi). Zi measures the role of a point in all modules, and the higher the value, the greater the role in the module. Pi measures the degree to which a node participates in other modules. A higher value indicates a closer relationship with other modules. Cytoscape software was used to draw the network diagram and compare the structure of soil microbial network in the two alpine meadow types.Based on the results of high-throughput sequencing of soil bacteria41.Linkages between network topology and environmental variables were also examined. Gene significance (GS) was defined as the square of Pearson correlation coefficient between relative abundance of genes in networks and environmental variables. Then, Mantel tests were used to measure the correlation between GS and connectivityhttps://ieg2.ou.edu/MENA); SigmaPlot 12.0 was used for mapping and the molecular ecological network was visualized using the software of Cytoscape 3.0.All preliminary statistics and the different test data analyses were done with the SPSS 19.0 software. Microbial diversity was characterized by Shannon-Wienner and Simpson diversity indices, and microbial species richness was calculated. The characteristics of soil microbial community were sequenced by the detrended correspondence analysis (DCA). The relationship between community distribution pattern and environmental factors was estimated by the canonical correspondence analysis (CCA). Further statistical analysis was carried out using Vegan packages in R software; non-similarity test was analyzed using online websites  with these parameters being higher in RG. The species richness of plant in NG was significantly higher than in RG (P = 0.001), but their Shannon–Wiener of plant diversity were similar (P > 0.05) (Table The results showed that the dominant species in the RG were 5) Table .Table 3SP < 0.05), while the proportion of soil sand was similar (P > 0.05). Soil pH was significantly higher in NG than in RG (P < 0.05), and unlike soil moisture (SM). Soil total and available N concentrations, ammonium nitrogen, and soil total P concentrations were similar (P > 0.05) in the two types of vegetation restoration. Soil organic carbon and nitrate nitrogen were significantly higher in RG, while soil available P concentrations was higher in NG (P < 0.05) (Table The proportions of soil clay and silt were significantly different in the two types of vegetation restoration (5) Table .Table 4SProteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were more abundant than others. The relative abundance of Proteobacteria, Acidobacteria and Bacteroidetes were significantly higher in RG than in NG, but Actinobacteria was lower in RG (P < 0.05). The relative abundance of soil microorgan in RG and NG at the level of bacterial phyla was shown in Fig. Detected OTUs can be divided into 32 kinds of bacteria and 2 categories of archaea, among which the P < 0.05), Shannon–Wiener showed that there was no significant difference between NG and RG . RG network has high connectivity, positive link percentage and average aggregation coefficient, while NG network has longer average distance (P < 0.05). As shown in Fig. Proteobacteria and Chloroflexi, respectively, while NG contains 3 module hubs of Actinobacteria. RG contains 5 connectors, but NG has none.Based on the RMT algorithm, the network topological properties are shown in Table 3–N) and the composition of soil microbial communities was performed simultaneously as presented in Fig. Canonical Correspondence Analysis (CCA) between environmental factors  was defined as the square of Pearson correlation coefficients between relative abundance of genes in a network and environmental variables. Then, Mantel tests were performed to examine the linkages between gene connectivity and GS, which showed that environmental variables were significantly correlated with network connectivity. In the network from RG presented in Table P = 0.005), soil moisture , soil available N , soil total organic carbon , and aboveground plant richness (P = 0.050). In the network from NG, node connectivity was significantly correlated with GS of pH (P = 0.000), soil moisture , soil available N , soil available P , soil total organic carbon , and aboveground plant richness (P = 0.020).The importance of environmental variables to network topology was examined as described previouslyProteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were more abundant than others  on the Songnen Meadow. We found these four groups of microbial as the dominant flora in the two experimental meadow types, accounting for > 78% of all the phyla. ers Fig. .These re49. In our study, RG needs replanting plants and soil disturbance, which makes it easier to reduce soil bacteria and microbial diversity. On the contrary, there was no disturbance in NG, soil bacterial community rapidly increased in nondisturbed conditions, resulting in the higher soil bacterial diversity50.There was a higher Simpson indices and Shannon–wiener index in RG Fig. , support32. In this case, RG network has high connectivity, positive link percentage and average aggregation coefficient, while NG network has longer average distance (p < 0.05)  were automatically chosen to avoid ambiguity in the network reconstruction56, so it renders networks more robust to noise40. From our network analyses, the shorter average path distance and the higher connectivity, which showed that the interaction between soil microbial is more complex in RG network than in NG network. Meanwhile, the shorter average path distance communicated to the entire network may be more sensitive to interference, so it means more stable in RG than in NG. In fact, NG network hub of module type is relatively singular, with most OTUs belonging to Proteobacteria, while the NG network is less resistant to the outside world because of the simplifying community composition57. Our results also were confirmed by this report that network modularity can be used as a resistance indicator of network system against external changes58.Molecular ecological network analysis play an increasingly important role with the high-throughput sequencing technology providing a powerful information of microbial community compositione p < 0.0 59. Our results indicated that pH was the main factor determining soil microbial community features, followed by soil moisture (SM) in the Songnen meadow of Northeast, China. These results are partly consistent with some studies. For instance, Shen et al.11 found that soil pH controls soil microbial diversity and community composition and concluded that soil pH can affect the distribution of microbial widespread influence. Zhang et al.1 also found pH to be the main factor affecting SMC structure and the magnitude of microbial distribution patterns. Based on the method of constructing network analysis, soil pH value had the highest connection in the network interactions between environmental factors and microbial taxonomic community OTUs. Thus, it was not difficult to understand that Soil pH and soil moisture may have indirect influence on other soil physical and chemical properties60, understanding its potential relationships may be an important question for future research to improve our predictability of the vegetation restoration effects on terrestrial ecosystems.Soil pH is usually one of the important factors that affect microbial diversity, and it has a close relationship with soil microbial community structure and compositionvs. NG) on the Songnen Meadow. There were different molecular ecological networks of vegetation restoration, which were characterized as scale-free, small-world, modular and hierarchical. The RG network has a longer average path distance and higher modularity, which make it more resistant to external environmental changes, including climate variability, than the NG network. Consistently, soil microbial community characteristics are significantly different across vegetation restoration. The composition of soil microbial community change is important for evaluating the responses of ecosystems to vegetation restoration. This will be special use for anywhere to improve vegetation restoration in further.Overall, our finding underscores the importance of soil microbial community structure and diversity in different vegetation restoration (RG Supplementary Information.


Spices are the esoteric food adjuncts that are used for enhancing the sensory quality of the food in Punjabi diets and add many health benefits. Estimating the intake of spices at the individual level is a challenging task as they are consumed in very small quantities as compared to other foods. The present study aimed to assess the intake and spices consumption level of spices among urban and rural households.A study was carried out among 100 households each from urban and rural areas from Ludhiana district of Punjab, India to collect the information regarding frequency of spice usage and portion sizes using a questionnaire. The information pertaining to sociodemographic characteristics of female respondents from urban and rural households were also collected. The commonly used 25 spices in Punjabi diets were selected to assess their dietary intake at the individual level among households.>dhal > curry preparations among urban and dhal > vegetable>curry preparation among rural households. Average amount of spices consumed by urban adult women was 10.04 g per day which was higher as compared to spices consumed by rural adult women per day (7.68 g).Spice consumption frequency was more in urban households in comparison to rural households. The maximum mean consumption frequency score among urban and rural households was observed for red chilli powder (5.00) and turmeric powder (5.00). Maximum percentage (76 and 72%) of urban and rural households preferred to use the unroasted form of spices, respectively. The highest mean intake and range was observed for red chilli powder (3.19 g with range 0.35–5.23 g) among urban women and (2.41 g with range 0.25–3.75 g) for rural women. Spice intake from individual dishes showed the maximum number of portion sizes for red chilli powder that were from vegetableThe study concluded that the urban households showed higher consumption of spices as compared to rural households thus assessing the quantifying intake of spices. Urban adult women consumed more spices per day as compared to rural women. Therefore, more encouragement for increased use of spices is required to reap various health benefits of spices in combating metabolic disorders. In the culinary arts, spices are the esoteric food adjuncts that are used for enhancing the sensory quality of the food. The leaf or the herbaceous part of a fresh plant used for flavoring the food preparation is often referred to as a culinary herb where as, any dried part of the plant is known as a spice . Thus, sIn the present study, an attempt was made to assess the intake and consumption level of spices among adult women of urban and rural households based on the frequency of spice usage and portion size of spice consumed.The study population consisted of selected 100 urban and 100 rural households in different areas of Ludhiana district of Punjab, India. The households belonged to different socioeconomic groups and to different religions. One day intake was noted from adult women from urban and rural households. The commonly used 25 spices in Punjabi diets, were selected to assess their dietary intake among households. Standard household measuring spoons that measure 1 teaspoon, 1/2 teaspoon and 1/4th teaspoon were used to standardize the weight of each selected whole or powdered form of spices as most of them are used in small quantities. Further, some of the spices used in a minute quantity were verified with an electronic balance.An interview schedule cum questionnaire was developed to elicit the information pertaining to the sociodemographic characteristics, the pattern of spice use and intake, frequency of usage, the quantity of spices in routine dishes in each household. The questionnaire was pretested in ten households and was found satisfactory for collecting the required information. The final questionnaire was prepared by undertaking a pilot study involving suitable urban and rural female respondents to ensure the validity of the questionnaire which was excluded from the final sample. The required data were collected using a pre-structured non-disguised questionnaire cum interview schedule from female respondents in the households, who were involved in a cooking activity to elicit information regarding the spice consumption as described in the questionnaire. An individual if does not cook most of their own food, they may not know all the food items contained in mixed dishes, therefore the food preparer was selected to provide information on items that added during cooking.dhal, vegetable and curry were also noted separately from adult women of urban and rural households.Spice intake pattern in urban and rural households was assessed based on the spice usage in the households which include the information regarding the type of spice used, intake and frequency of usage with options of ‘daily’, ‘twice’ or ‘thrice’ a week, weekly and occasionally options as described in the questionnaire. For the easy identification of various spices, common names were also mentioned in the questionnaire. Mean consumption frequency scores were also calculated by giving scores: daily (5), thrice a week (4), twice a week (3), weekly (2) and occasionally (1). For the spice intake, the detailed recipe was noted regarding the type and quantity of different spices added to routine dishes. The most frequently used spices in these dishes were identified by the number of households using them. For each meal consumed by responding women herself, name of the dishes and portion size of spices in individual dishes were elicited using a 24-h recall method. To calculate the portion size of the added spice consumed, the quantity of prepared dish consumed by an individual and number of portions obtained from that quantity was noted. One day 24-h recall method determined the quantity of spice intake by an individual based on the spice intake per portion size consumed from all dishes in 1 day. Spice intake per portion size of each individual dish consumed like p < 0.01, p < 0.05. For the analysis of data SPSS  and SAS  were used.The intake of individual spices was presented as frequency intake. The quantity of spices intake was expressed as mean ± standard deviation (S.D), median, 90th percentile and ranges. Statistically, results were analyzed using Chi-square test of independence of attributes. Wilcoxon-Mann-Whitney test was used to compare the mean consumption frequency of spices among the adult women of urban and rural households. Results were considered statistically significant at In recent times, consumption frequency and measurement of dietary intake of spices is gaining much significance due to various phytochemicals and presence of antioxidant potential in spices that have been recognized to have health-promoting benefits and protective role against chronic diseases.The sociodemographic characteristics of female respondents from urban and rural households is given in Table The consumption frequency of spices among urban and rural households is shown in Table p < 0.01) higher among urban households for cumin seeds, asafoetida, black pepper, gooseberry powder, mustard seeds, pomegranate seeds, dill, nigella, cinnamon and saffron when compared to rural households. The mean frequency score of bay leaf, black salt, black cardamom, carom seeds, green cardamom, fennel seeds, coriander seeds, dry mango powder and clove showed non-significant difference among the adult women of urban and rural households.The mean consumption frequency score of major spices has been depicted in Table p < 0.05) was found in using a different form of spices namely green cardamom, black pepper, asafoetida, cinnamon, fenugreek seeds, coriander seeds, bay leaf, clove, carom seeds and black cardamom among urban and rural households. The overall result, it showed that a maximum of 76% of urban and 72% of rural households preferred to use the unroasted form of spices followed by 19% of urban and 21% of rural households using roasted form of spices and remaining using both roasted and unroasted form of spices.The form of spices used among households is presented in the Table p < 0.01 level of significance. The percent of spice median intakes of spices representing below 1 g were 72 and 89% of urban and rural median intakes, respectively. Out of 18, 10 and 7 spices among urban and rural were having 90th percentile above 1 g, respectively. The highest 90th percentile value was observed for red chilli powder (4.41 g) among urban and turmeric powder (3.23 g) among rural. Overall, an average adult woman consumed 10.04 g of spices per day as compared to 7.68 g per day for rural women indicating higher consumption of spices among urban households.Spice intake among adult women based on the portion size and quantity of individual spice consumed from all dishes and foods expressed as mean, median, 90th percentile levels and ranges are presented in Table The distribution of the level of spice intake based on portion sizes is depicted in the Table dhal, vegetable and curry were evaluated given in the Tables Dhal (legume preparation), vegetable (dry vegetables) and curry . Maximum number of portion sizes consumed were highest from dhal followed by vegetable and curry among rural households.The spice intake from individual dishes like dhal among urban households and turmeric powder (1.04) from dhal among rural households elicited in Table dhal were red chilli powder, turmeric powder, mustard seeds, carom seeds, cumin seeds, garam masala, black pepper and asafoetida among urban and rural households. Median intakes above 0.5 g were observed for red chilli powder and turmeric powder among urban households while red chilli powder, turmeric powder and mustard seeds among rural households. The significant difference was obtained for turmeric powder, mustard seeds, carom seeds, black pepper and garam masala intake among urban and rural households (p < 0.05). While, red chilli powder, asafoetida, cumin seeds and dry mango powder showed a non-significant difference between both households. The highest 90th percentile intake level was observed for turmeric powder 1.66 and 2.71 from dhal among urban and rural households, respectively.Mean intakes were highest for red chilli powder (1.88 g) from Data on the intake of spices from vegetables was depicted in Table rajmah, black channa, curry, etc., their spice intake has been presented in Table curry preparation was highest among urban (1.08 g) and rural (1.12 g) households. The non-significant difference was seen among all spices mentioned with exception of red chilli powder and garam masala, which showed a significant difference (p < 0.05) among urban and rural households. Median values obtained above 0.60 g were observed by red chilli powder, turmeric powder, carom seeds, asafoetida, cumin seeds, coriander seeds, black pepper and garam masala among urban households while red chilli powder, turmeric powder, carom seeds, asafoetida and garam masala among rural households. The highest 90th percentile of 1.91 and 1.89 g for turmeric powder among urban and rural households, respectively.Another routinely prepared dishes like  are considered as the main contributor of spices apart from other dishes in 1 day intake. A maximum number of portion sizes for red chilli powder and turmeric were from vegetables>dhal > curry preparation among urban households. While among rural households the maximum number of portion sizes were from dhal > vegetables>curry preparation for red chilli powder and turmeric powder.Legumes and vegetables In Indian cuisine, the usage and consumption of spices are mainly due to their extrinsic flavour. Various methods such as food frequency questionnaires supplemented with weighed records, dietary recall method, and portion size estimation were used in previous studies , 8. The lassi and curds during summers and sprinkled on salads. Carom and cumin seeds are frequently used in vegetable tadkas and paranthas among both households. In Norway, it was observed that out of 27 different herbs and spices investigated only eight were consumed by one-third of the population which indicated the lower consumption of spices [The study showed that out of 25 spices studied, most frequently consumed by more than 80 households were 12 and 9 spices among urban and rural households, respectively. Earlier studies in India also showed that consumption frequency of turmeric powder and red chilli powder was higher than most of the other spices which are consistent with the observations in the present study . Black sf spices . Ferrucif spices .The usage form of spices in the households also varied indicating that the unroasted form of spices consumed by the majority of urban and rural households. Generally, spices preferred for roasting are black pepper, asafoetida, carom seeds, cumin seeds, fenugreek seeds, mustard seeds, cinnamon, clove and black cardamom during food preparations. Susheela stated that some spices are processed for their microbial stability and removal of extraneous matter and roasting is one of the cooking processes to release characteristic flavour volatiles and undesirable particles . Moreovegaram masala (33.3%), coriander (33.3%) and turmeric powder (28.6%) in New Delhi. While in Mumbai, chilli powder (58.3%) had the highest median per capita consumption (g/month/person), followed by turmeric powder (21.7%) and cumin seeds (20.0%). Trivandrum was characterized by high consumption (g/month/person) of chilli powder (166.7%) coriander (102.0%) and ginger (373%). Further study revealed that the per capita monthly median intake level of 0.17–4.6 and 3.1 g for cinnamon and cloves, respectively in different regions of India which were found to be higher than stated in the present study [Spice intake determination is being acknowledged as they possess bioactive compounds and antioxidant properties. Frequency data of intake together with portion size estimations provided a good quantitative estimation of spice intake at individual level rather than opting for the frequency of spice intake alone. The spice intake varies substantially between different countries, states, geographical regions within the same country possessing different dietary habits and patterns and cuisines within the same region. At an individual level, spice intake measurement is a tough and challenging task than estimating the intake of staple foods like cereals and other foods that are consumed in large quantities daily. Sherman and Hash reviewed through traditional cookbooks which revealed the mean number of spices used in 36 countries were 6.5 and 9.3 spices through vegetable and meat-based recipes in India while in European countries mean number of spices ranged from 1.6 to 4.5 and 0.6 to 4.2 spices through meat and vegetable based recipes, respectively . The meant study . This maoriander 2.0% and owder 21.% and cum powder 2.6% in Nent study . In mostnt study . Severaloriander 2.0% and nt study . Similarin seeds .0%. Trivnt study  while innt study .curry which is prepared daily among all households in Southern India. Further, the highest 90th and 97th percentile values for chillies  were observed from chutney and dhal [These observations assume relevance for quantifying spice intake since all spices are not consumed on daily basis and intake of individual spices varied with frequency of consumption of dishes. Dishes are considered as main contributor of spices on daily basis. In the present study, intake portion size of spices was estimated not herbs and it depends upon the number of spices used and consumed on a daily basis as monthly intake could not provide relevant results. An average portion size intake of total spices and herbs from habitual dishes which ranged from 4.9 g to 26.1 g in Thailand . Turmeriand dhal . These oThe intake levels of spices were relatively much lower than the other foods. It does not necessarily mean that spices are of little value. It is pertinent to mention that spices do not only add flavor and taste but also have high polyphenolic content and antioxidant potential of vital importance. The frequency of spice intake and portion size at the individual level of adult women in urban and rural households will provide a quantitative estimate of spice intake. The present study conducted in Ludhiana district of Punjab (India) showed the most frequently consumed spices among rural and urban households in Punjabi diets were red chilli powder, turmeric powder, cumin seeds, carom seeds, black pepper, asafoetida, green cardamom, black salt, fennel seeds and coriander seeds. The spice consumption was higher in urban adult women as compared to rural adult women. Further studies are required to explore the importance and intake of spices.