This study was conducted to investigate the effects of wilting and microbial inoculant treatment on the fermentation pattern and quality of Italian ryegrass silage.6 cfu/g of Lactobacillus plantarum. The silages samples were collected at 1, 2, 3, 5, 10, 20, 30, 45, and 60 days after ensiling and analyzed for the ensiling quality and characteristics of fermentation patterns.Italian ryegrass was harvested at heading stage and ensiled into vinyl bags (20 cm×30 cm) for 60d. Italian ryegrass was ensiled with 4 treatments  in 3 replications, wilting time was 5 hours and additives were treated with 10in vitro dry matter digestibility and there were no significant differences in acid detergent fiber (ADF), total digestible nutrient (TDN), water-soluble carbohydrate (WSC), ammonia content, and pH (p>0.05). However, wilting treatment resulted in higher ADF and neutral detergent fiber content of Italian ryegrass silage (p<0.05), and the WNA treatment showed the lowest TDN and in vitro dry matter digestibility. The pH of the silage was higher in the wilting group (WNA and WA) and lower in the additive treatment group. Meanwhile, the decrease in pH occurred sharply between the 3–5th day of storage. The ammonia nitrogen content was significantly lower in the additive treatment (p<0.05), and wilting had no effect. As fermentation progressed, the lactic and acetic acid contents were increased and showed the highest content at 30 days of storage.Wilting treatment resulted in lower crude protein and The wilting treatment did not significantly improve the silage fermentation, but the inoculant treatment improved the fermentation patterns and quality of the silage. So, inoculation before ensiling is recommended when preparing high quality of Italian ryegrass silage, and when wilting, it is recommended to combine inoculation for making high quality silage. Lolium multiflorum Lam.) is an herbaceous annual or biennial grass that is grown for silage, and as a cover crop. In the United States, Lolium multiflorum is sometimes used as a winter cover crop to prevent erosion, build soil structure, and suppress weeds. It grows on about 1 million ha in the humid, southern United States, being used primarily for winter pasture in clear seeding and in dormant bermudagrass sods [Two types of ryegrass, perennial and annual, are being cultivated in worldwide. Italian ryegrass , which restrict the storage methods. Most farmers stored IRG in the form of silage, and some farmers are trying to store as hay. Produced silage  is wrapped in plastic vinyl and distributed throughout the country . HoweverEnsiling forage crop is well known method of conservation for a shortage season. Lactic acid bacteria (LAB) convert water soluble carbohydrates (WSC) under anaerobic conditions into lactic acid. As a result of the pH decline, the silage is well preserved. Acidification of well fermented silage inhibits undesirable microorganisms. During the fermentation process, competition takes place between LAB and undesirable microorganism, and fermentation quality always depend on the result of the competition .clostridial fermentation reducing voluntary intake [Dry matter (DM) content of raw materials has a great influence on silage fermentation, which affects all fermentation characteristics, pH level and quality parameter of silage. Ensiling with low DM content around 25% could cause inferior fermentation and high pH level deducing serious DM loss, compared with higher DM content. At less than 300 g/DM kg may also generate an increase in seepage loss and expedite y intake . But wilThe application of silage additives is normally recommended to ensure and improve silage fermentation. At present, LAB inoculants are the main additives in many parts of the world . LAB addGenerally, wilting results in lower WSC content, extensive protein breakdown and sometime higher total volatile fatty acid (VFA) during ensiling. Wilting also affects the chemical composition, DM losses, silage fermentation and animal performance .This study evaluated the effect of wilting and inoculant on fermentation dynamics and qualities of IRG silage.The experiment was split-plot design with three replications. The main plot was four treatments  and sub-plot was silage opening dates .Italian ryegrass (IRG) was cultivated in an experimental field of Pyeongchang campus, Seoul National University . “Kogreen” variety, developed by the National Institute of Animal Science, was seeded on about 1 ha area on September 27, 2016 at a seeding rate of 40 kg/ha. At seeding date, 40 kg/ha of nitrogen, 150 kg/ha of phosphate, and 75 kg/ha of potassium were applied as fertilizer. An additional 100 kg/ha of nitrogen and 75 kg/ha of potassium fertilizer were applied in early March 2017.Lactobacillus plantarum). Recommended level (106 cfu/g fresh matter) of inoculant was dissolved in tap water (1 L per 1 g of inoculant) and sprayed  into mixed samples. Thereafter, approximately 600 g treated material was packed into vinyl bag , air was taken out, sealed  and stored at the ambient temperature (22°C to 28°C) in the shade.IRG was harvested at 16 May 2017 using mower conditioner  and chopped into about 2 to 3 cm pieces using a forage cutter . The ryegrass harvested for wilting treatment was dried in the field for 5 hours. After manual mixing, chopped IRG was treated with silage inoculant , and WSC. Another subsample (about 300 g) was stored at −80°C in a refrigerator and subsequently used for sequential determination of silage acidity (pH), organic acids, microorganisms, and ammonia nitrogen.Three vinyl bag silos per treatment were randomly opened on 1, 2, 3, 5, 10, 20, 30, 45, and 60 days after ensiling, respectively. Two subsamples per vinyl bag silo were retained for further analysis. One subsample (about 300 g) was dried at 65°C in a forced-air drying oven for 72 h and then used to determine DM content and other chemical compositions, including acid detergent fiber (ADF), neutral detergent fiber (NDF), crude protein (CP), Dried samples were ground to pass through a 1-mm screen and kept in double-plug type plastic bottles for analysis. The CP was determined using the Kjeldahl method ; ADF and3-N concentrations were analyzed by the method of Chaney and Marbach [A frozen 10 g sample of each silage (three replications per treatment) was macerated with 90 mL of distilled water for 30 min. in a shaker and filtered, and the filtrates were used to measure pH with a pH meter . 10 g sample of each silage was macerated with 90 mL of distilled water for 24 h and filtered through filter paper (#6). The filtrates were analyzed for VFA and lactic acid contents. The VFAs were analyzed by Kim et al  using ga Marbach  using a Data on fermentation dynamics and chemical composition were subjected to two-way analysis of variance with the fixed effects of treatments, ensiling days and interaction (treatment ×ensiling days) using the general linear model procedure of SAS ver 9.1  . Least sThe chemical composition, pH, WSC, and ammonia nitrogen contents of pre-ensiled IRG are given in As presented in Wilting has a big impact on the fermentation pattern of silage. In this experiment, wilting decreased the CP, NDF, and IVDMD content of raw materials. Kim et al  reportedOn the other hand, the WSC content of the material was not significantly different among treatments, and the average level was 153.4 g/kg. Parker  stated tAfter 60 days of fermentation, the CP content of IRG silage was low in the wilting group, and the inoculant treatment increased CP content. This was judged to be the result of lowering protein degradation by improving fermentation pattern. Kennedy  also repOn the other hand, ADF and NDF contents were higher and IVDMD contents were lower in WNA treatment silage. The wilting treatment increased the NDF content of the raw material, which is believed to have increased since the high level was maintained even after fermentation.Comparing the IVDMD before and after silage preparation, the IVDMD of silage was lowered (763.4 vs 735.0 g/kg). Kim et al  also shoAfter 60 days of storage, the DM content of IRG silage tended to increase in wilted silage but the addition of the inoculant had no significant effect on DM. Keady and Murphy  also repThe most important changes in silage quality was in pH. Wilting resulted in higher silage pH and inoculant was lower. Many studies have reported that LAB treatment lowers the final pH of silage. Wilting increased final pH of silage by increasing the DM content of the raw material. Wilting has negative effect on silage acidity due to increasing DM content and restricted fermentation .The inoculant treatment increased the lactic acid content of the silage, but the wilting treatment resulted in a decrease. This is the result of limited fermentation due to the decrease in moisture content. Kim et al  also fou3-N/total nitrogen (TN) ratio, which indicates the degree of proteolytic degradation, was reduced by LAB treatment, and according to Haigh [3-N content was less than 10% of the TN, it was classified as high quality silage. So, the silage of this experiment can be classified as good quality. Sharp et al [3-N content produced by proteolysis is reduced by the treatment of LAB additives. According to the report of Dawson et al [The NHto Haigh , when thrp et al  also fouon et al , wiltingon et al . The conThe contents of CP, IVDMD, ADF, and NDF in IRG silage showed a significant difference with wilting, LAB treatment and number of fermentation days. The LAB treatment did not show any difference in CP content during fermentation, but CP showed a tendency to increase slightly in control (NWNA). IVDMD of silage showed a tendency to decrease with fermentation, but there was no significant difference in NWNA treatments. The contents of ADF and NDF increased with the fermentation progress, but there was no significant difference in NWNA treatment. Keady and Murphy  reportedDuring silage fermentation, the pH changes gradually decreased over time and significantly decreased from 3rd day. However, in the WNA treatment, the pH decrease sharply occurred from slightly late 5th day. However, Zhao et al  reportedWilting increased ammonia nitrogen content. Derbyshire et al  and HaigWilting resulted in lower CP and IVDMD, but no significant differences in ADF, TDN, WSC, ammonia content and pH (p>0.05) of raw materials. However, wilting treatment resulted in higher ADF and NDF content of IRG silage (p<0.05). The pH of the silage was higher in the wilting group (WNA and WA) and lower in the additive treatment group. The decrement in pH occurred sharply on the 3th to 5th day of storage. The ammonia nitrogen content was lower in the additive treatment (p<0.05). As fermentation progressed, the lactic and acetic acid contents increased and showed their highest content at 30 days of storage. In conclusion, the wilting did not significantly improve the silage fermentation, but the inoculant treatment improves the quality of the silage. So, inoculation before ensiling is recommended when preparing high quality of IRG silage, and when wilting, it was recommended to combine inoculation to make high quality silage.


The Hippo pathway plays an important role in embryo development, and separation of trophectoderm(TE) and inner cell mass (ICM) cell lines. Therefore, this study investigated effect of maternal age on activity of Hippopathway in human embryos. In this experimental study, the developed up embryos to the blastocyst stage and theembryos whose growth stopped at the morula stage were collected from women aged 20-30 years old  and >37 years . Expression of OCT4, SOX2, CDX2, GATA3, YAP genes and therelevant proteins, in the both groups were evaluated using respectively quantitative reverse transcription-polymerasechain reaction (qRT-PCR) and immunofluorescence methods. There was no significant difference in the expression level of OCT4, SOX2, CDX2, GATA3 and YAP genes inblastocyst and morula stages, between the two groups. However, SOX2 and CDX2 gene expressions in morula stageembryos of the old group was statistically lower than that of the young group .Additionally, in the embryos collected from women with >37 years of age, at the blastocyst stage, phospho-YAP (p-YAP)protein was found to be accumulated in the TE, but it was almost disappeared from the ICM. Additionally, in the oldgroup, contrary to the expectation, YAP protein was expressed in the ICM, rather than TE.The results of this study showed that YAP and P-YAP among the Hippo signalling pathway may be alteredby increasing age. Pre-implantation development starts from fertilization andcontinues with repeated divisions. It leads to the formationof a complex structure called blastocyst , 2. The CDX2 and GATA3 gene expressions together with TEAD4,which in turn induces trophoblast formation and OCT4 downregulation procedure as well as the oocyte and embryo quality, ovarian reserve and number of treatmentcycles. Inclusion criteria for the study encompass the quality of embryo, women’s age andlack of genetic diseases.This experimental study was in accordance with the Declaration of Helsinki, following theapproval of Ethical Committee of Royan Institute . The patients were selected based on age, body mass index (BMI),cause of infertility, type of control ovarian stimulation (agonist and antagonist), Numberof 1V5TM medium  for three days, before transferring to theG2V5TM medium (Vitrolife). The embryos (either those stopped at the morula stage orthose reaching the blastocyst stage) were collected on day 5 and divided into two groups:the embryos collected from women aged 20-30 (young group) and the embryos belonging to women>37 years of age (old group).Exclusion criteria include embryos of the patients which were not developed to morula andblastocyst stages. Fresh and frozen embryos from consenting couples attending RoyanInstitute for infertility treatment (after signing written consent) were cultured inG-ΔΔCt formula. Gene expressionanalysis was performed for pluripotency markers  and Hipposignalling marker (YAP). GAPDH was considered ashousekeeping gene. The oligonucleotide primers were designed using NCBI site, Perl Primerand Gene Runner software. The utilized primers in the present work are listed in Table1.Total RNA from embryos that either reached to the blastocyst stage or stopped at themorula stage, were extracted using the RNeasy micro Kit , before,synthesizing complementary DNA (cDNA) by Fermentase kit (Germany) . PCR reaThe embryos were fixed in 4% paraformaldehyde solution  for 15 minutes atroom temperature and placed in 0.25% Triton X-100  soluble inphosphate-buffered saline  for 30 minutes at room temperature. Then, theembryos were incubated with 5% BSA in donkey serum for 60 minutes at room temperature toblock unspecific binding of the antibodies, before being incubated with the followingprimary antibodies: OCT4 , CDX2 , YAP  or P-YAP  overnight at4˚C, as previously described . Then, tAll experiments were performed using fourindependent biological replicates. Data were analysedusing t test , chi-square(for protein expression and demographic information),or Mann Whitney tests (for comparison of the cyclenumbers) using the SPSS statistical software . On the other hand, number of cycles inthe young group was significantly lower than that of the oldgroup (P<0.05). Additionally, mean age of the participants inthese groups was significantly different (P<0.05). Based onthe demographic data, it seems that ovarian resources beginto decline in women with >37 years of age.YAP) and pluripotency genes(OCT4 and GATA3) at the Morula stage. However,SOX2 and CDX2 genes at the Morula stage hadsignificantly higher expression levels in the young group, compared to the old group at the blastocyst stage at the blastocyst stage did not showsignificant variation, by comparing the young and oldgroups .In the old group, at the blastocyst stage, P-YAPprotein was found to be accumulated in the TE, but itwas almost disappeared from the ICM. Additionally,contrary to the expectations, in the old group, YAPprotein was expressed in the ICM rather than TE.SOX2 and OCT4 genes (ICM genes) were significantlyreduced. On this basis, it can be said that Hippo pathway plays an important role in theearly embryo differentiation by affecting these gene. Moreover, in they showed thatSOX2 and CDX2 genes play an important role indevelopment of embryos. CDX2 gene is necessary for gastrulation. Themutation in this gene disturbs WNT, FGF and Hippo signalling. SOX2 isinitially expressed in the most of cells during morula stage. But, in the blastocyst stage,it is only expressed in ICM. Various factors can affect the embryo development from a zygoteto a blastocyst, including quality of gametes, type of culture medium and oxygenconcentration, as well as women’s age.During differentiation of the blastomeres, cell polarity and position govern formation ofTE and ICM . This prMVH/OCT4 genes in the mouse ovarian cortex. The level LATS2, MST1, MVHand OCT4 were significantly decreased with increasing the age. YAP was alsoobserved in the ovarian cortex of two-months old, but not 20-months old mice. In addition,YAP phosphorylation was found in ovarian cortex of seven-days old compared to 20-months oldmice. Moreover, the amount of P-YAP/YAP was decreased, as 7-days old mice grew to 20 months.These results show that expressions of Hippo signaling pathway proteins are altered withincreasing the age of ovary. A study performed by Pelissier et al.  group, YAP protein expression was expressed in ICM rather than TE. These datamay indicate the inappropriate functionality of Hippo signaling pathway at the advancedages. More accurate results could be obtained, if immunestaining was performed for all fourproteins in the embryo.The result of this study showed that although the levels of


Poly(lactic acid) (PLA) is a relatively brittle polymer, and its low melt strength, ductility, and thermal stability limit its use in various industrial applications. This study aimed to investigate the effect of poly(methyl methacrylate) (PMMA) and PMMA/silica hybrid particles on the mechanical properties, interfacial adhesion, and crystallization behavior of PLA/block acrylic elastomer. PLA/block acrylic elastomer blends exhibit improved flexibility; however, phase separation occurs between PLA and block acrylic elastomer domains. Valid time-temperature superposition (TTS) measurements of viscoelastic behavior were obtained and exhibited interfacial adhesion with the addition of PMMA or PMMA/silica in PLA/block acrylic elastomer blends. In particular, the phase separation temperature was increased by the incorporation of PMMA/silica hybrid particles, which suggests a potential role for these particles in improving the phase stability. In addition, PMMA inhibits crystallization, while PMMA/silica acts as a nucleating agent, thus increasing the crystallization rate and crystallinity degree. Poly (lactic acid) (PLA) is a thermoplastic aliphatic polyester that has gained substantial attention due to its potential to substitute for traditional petroleum-based polymers ,2,3. HowSilica nanoparticles have been used to improve the mechanical properties, adhesion, durability, and wear resistance of polymers. It has a strong polarity due to the presence of the hydroxyl group on the surface, rendering it difficult to disperse as agglomerates in polymer composite preparation. Numerous studies have reported the dispersion of silica nanoparticles in composites via surface pretreatment of modifications ,17,18,19In this study, we prepared polyvinylpyrrolidone (PVP)-functionalized poly(methyl methacrylate) (PMMA) via emulsion polymerization and used it as a template for adsorption of silica nanoparticles onto the surface via a sol-gel method. As a result, it was possible to obtain raspberry-like PMMA/silica hybrid particles. PMMA/silica hybrid particles reduced the initial agglomeration among silica particles. In addition, these PMMA particles, which are compatible with PLA, were introduced to disperse silica nanoparticles as a carrier system. In addition, the flexibility of PLA was given by using a triblock acrylic elastomer composed of PMMA and poly(n-butyl acrylate) (PnBA). It exhibits high dispersion in the PLA matrix because the PMMA region is compatible with PLA ,36,37. T4OH, 28 wt.%) as a catalyst, potassium persulfate (KPS) as an initiator, polyvinylpyrrolidone  as a stabilizer, and tetraethylorthosilicate (TEOS) as a precursor were purchased from Sigma-Aldrich Corp. and were used as received. Deionized water was used in all polymerization and synthesis processes after being purified in an Aquapuri system PLA (PLA4032D) was obtained from Nature Works (USA). Block acrylic elastomer  was obtained from Kuraray , and primary and secondary antioxidants  were purchased from Songwon, Korea. Methyl methacrylate (MMA) and methyl acrylate (MA) as monomers, 1-dodecanethiol as a chain transfer agent (CTA), ammonium hydroxide (NH4OH were added to the reaction medium (methanol/water), dispersed under stirring and sonicating. TEOS was added at a rate of 2 mL/h and then synthesized at room temperature for 24 h. The obtained colloid dispersion solution was washed by several cycles of centrifugation and re-dispersion. Finally, PMMA/silica hybrid particles were obtained after vacuum freeze-drying. The compositions of the PMMA and PMMA/silica hybrid particles are shown in The mechanical properties of the PLA composites were analyzed by a universal testing machine . The measurements were performed for a “dog-bone” injection specimen at a cross speed of 10 mm/min and a load cell of 10 kN. The results represent the mean and standard deviation of the five measurements. Tg), melting temperature (Tm), crystallization temperature (Tc), and cold crystallinity (Xcc). The crystallinity of the PLA was calculated as the crystallization enthalpy that arises during the heating process because no crystallization enthalpy arises during the cooling process:HLAm is the melting enthalpy (J/g), cH is the crystallization enthalpy, and DSC  was applied to investigate the thermal properties of the PLA composites, including the glass transition temperature , and the silica contents were confirmed by the residual amount. Measurements were performed using a weight of 10 ± 0.5 mg to raise the temperature to 800 °C under nitrogen gas. The morphology of the PLA composites was confirmed by a field-emission scanning electron microscopy  under an acceleration voltage of 3 kV. SEM images were collected for the side of a fractured tension specimen, a cryo-fractured surface in liquid nitrogen, and a surface extracted in xylene for PLA composites. The surfaces were coated with platinum (Pt) by an ion sputter .*|), and tan An oscillation rheometer  was applied to investigate the rheological properties of the PLA composites, including the storage modulus (G′), loss modulus (G″), complex viscosity (|𝜂d > d’). Therefore, the interfacial adhesion between PLA and block acrylic elastomer was decreased, resulting in breakage of the matrix like fiber strands during deformation. 1H NMR) analysis as shown in To verify the miscible between the PLA and the block acrylic elastomer, PMMA, and PMMA/silica particles, SEM analysis was performed to investigate the dispersion and shape of the block acrylic elastomer phase in the PLA matrix. The morphologies of the stretched surface, cryo-fractured surface, and fractured surface extracted in xylene for PLM7 and PLMS7 are shown in *| for PLM7 and PLMS7 with a shift factor To confirm the validity of the time-temperature superposition (TTS), modified Cole–Cole plots and master curves of tan perature . Therefoy region . Figure paration ,52,53,54paration ,39 whichparation ,56,57,58paration ,20,35. Tparation  PLA/PMMA the TTS . HoweverR is the gas constant of 8.315 J/mol∙K, and T is the absolute temperature. aE represented the effect of the temperature on the behavior of blends. These results indicate that the melting behavior of PLM7 is more sensitive to temperature compared with PLMS7. For comparison, the Ea of neat PLA, PMMA, and PLA/PMMA 70/30 have been reported in the literature as approximately 80, 182, and 110 kJ/mol, respectively  as a function of logarithmic time (ln(t)). The crystallization parameters are summarized in t0.5 increases with increasing PMMA content. The Avrami exponent, n, increases from 2.64 to 3.03. The t0.5 of PLM shows a rapid increase from 12.54 to 23.78 min compared to PL7/3. It can be seen that the addition of PMMA decreases the crystallization rate. PLMS samples have an Avrami exponent n of approximately 2.98 to 3.25. It can be expected to have a complete crystalline form. In the PLMS samples, the PLMS1 has the lowest t0.5, but Δt0.5 increases with increasing PMMA/silica content. These results show that PMMA/silica has an effect of inhibiting crystallization due to the PMMA, but the effect of promoting crystallization of silica particles is more largely attributed to it. In n and k can be obtained from the slope and intercept in the plot of ln [−ln(1−θ(t))] as a function of ln(t), based on Equations (4) and (5). θ(t)) vs. crystallization time (t), and thermal properties for the PLA composites at heating rates of 5, 10, and 15 °C/min, respectively. ln [−ln(1−θ(t))] as a function of logarithmic time (ln(t)) for the PLA composites. According to Equation (5), n and k can be obtained from the slope and the intercept, and n is approximately 2.0, whereas PL7/3, PLM, and PLMS have values of approximately 1.0–1.3. These results indicate that the non-uniform crystal was formed by one-dimensional crystal growth due to the addition of the block acrylic elastomers and PMMA. The addition of PMMA tends to increase kc and decrease t0.5, while the crystallinity decreases, indicating that the crystallization rate tends to increase with decreasing crystallinity. Moreover, the interactions between the PMMA and PLA molecular chains can disturb the folding of the PLA molecular chains, thus reducing the crystallinity [kc values that the crystal growth does not have a relatively large difference than that of PLM. Therefore, the addition of PMMA/silica has a greater effect on increasing the number of initial crystal nuclei than increasing the crystal growth rate. The Avrami equation has been used to describe isothermal crystallization behavior. By assuming that the crystallization temperature is constant, the Avrami equation can also describe non-isothermal crystallization. In a temperature-dependent non-isothermal process, the crystallization rate ( follows :(6)lnkc=allinity ,75. The In this study, the mechanical properties, viscoelastic behavior, and crystallization of PMMA and PMMA/silica particles in PLA/block acrylic elastomers were investigated. The block acrylic elastomer exhibits a microstructure phase in the PLA matrix and is excellent for enhancing flexibility. Interestingly, the PLA/block acrylic elastomers were transparent blends. However, the interfacial adhesion with PLA was limited by the low composition of PMMA (approximately 28%) compared with PnBA (approximately 72%). Here, the interfacial adhesion was increased by PMMA and PMMA/silica. The results confirm the validity of the TTS. In addition, the PMMA/silica increased the phase separation temperature of the PLA/block acrylic elastomer blends. However, the PMMA/silica remained in the form of a shell and partially acted as an additive defect with regard to the mechanical properties. The block acrylic elastomer and PMMA decreased the crystallization rate and crystallinity of PLA. In particular, a small amount of PMMA significantly decreased the crystallization rate and crystallinity. However, the addition of PMMA/silica has a greater effect on increasing the number of initial crystal nuclei, thereby increasing the crystallization rate and crystallinity.


The aim of the present study was to assess the prognostic value of hematological parameters of childhood obesity, as potential predictors of cardiorespiratory fitness (VO2max), using a sample of children and adolescents with obesity and risk for diabetes. A total of 84 clinically healthy children and adolescents were recruited, of which 21 were considered lean, 22 overweight and 41 obese, with a mean age of 12.0 ± 1.9, 11.4 ± 2.0, and 11.2 ± 2.1 years old, in each weight status category, respectively. Age and sex did not differ between groups. Hematologic testing was performed after 12 h of fasting including glucose, serum lipids, insulin, hc-CRP, adiponectin, leptin and fibrinogen levels. Cardiorespiratory capacity for exercise was assessed to determine VO2max, using a cycle ergometer. The VO2max was negatively correlated with progressive strength to the BMIz , hs-CRP , glucose  and insulin levels , the homeostasis model assessment of insulin resistance (HOMA-IR) , as well as to the leptin  and fibrinogen concentrations . The multivariate analysis revealed that only leptin and fibrinogen concentrations could predict the VO2max adjusted for the BMIz of participants. The receiver operating characteristic (ROC) curve for the diagnostic accuracy of leptin, hs-CRP and fibrinogen concentrations for the prediction of VO2max revealed a good diagnostic ability for all parameters, with leptin being the most promising one (area under the curve (AUC): 99%). The results verify that in children with obesity, VO2max may be predicted from hematological parameters (leptin and fibrinogen), possibly bypassing more invasive methods.Obesity is defined as abnormal or excessive fat accumulation that presents a risk to health. The ability to exercise is affected by adiposity, and this mechanism involves low-grade chronic inflammation and homeostatic stress produced mainly in adipocytes, which can result in abnormal adipokine secretion. To date, the gold standard for cardiorespiratory fitness assessment is considered to be the maximum oxygen uptake (VO Obesity is the result of a positive energy balance and/or physical inactivity, demonstrated by excessive body weight deposition for one’s height. The pathophysiology of obesity is complex and multifactorial, with the initial trigger stemming from homeostatic stress produced mainly in adipocytes, resulting in adipokine secretion attempting to balance energy homeostasis ,2. As a Moreover, obesity is associated with a plethora of coagulation factors, all setting the basis for the establishment of the atherosclerotic process and cardiovascular disease (CVD) ,8,9. Of 2max) [2max is a difficult procedure for children, who often fail to achieve the reported standards [2max is influenced by both the genotype [One of the most important results of obesity is the reduced cardiorespiratory fitness (CRF) observed due to exercise intolerance ,15. This2max) ,16. Overtandards ,17,18, rgenotype  and the genotype . In paragenotype ,22.2max among children with obesity and risk for diabetes, it would be extremely useful if it could be predicted by more assessable indicators.Studies have revealed a decreased CRF with increasing leptin and fibrinogen concentrations among children and adolescents ,25,26,272max levels among children of different weight status tiers and evaluate possible correlations between VO2max, serum leptin, adiponectin and fibrinogen levels.The aim of the present case-control study was to determine VOn = 21) were recruited from local schools, via advertisements, forming the control group. Thus, the number of participants totaled 84 children and adolescents, who were divided into three groups, according to body weight status. Participant characteristics are presented in 2max tests; (7) use of any medication.A total of 63 children and adolescents, 22 overweight and 41 with obesity , aged 8–Parents/guardians of participants signed informed consent forms prior to participation, after having the aims and method of the study explained in full detail. The protocol was approved by the Bioethics Committee of the Medical School of Aristotle University of Thessaloniki (Ref No. 91/21-12-2009). All data were handled with emphasis on anonymity and data protection, according to the Declaration of Helsinki and its latter amendments. This is a secondary analysis of a previous reported sample , with thBody weight and height of participants were measured at approximately 9:00 a.m. by an experienced pediatrician (K.T.) and a dietitian (A.V.) using a Harpenden wall-mounted stadiometer  and a Seca 700 mechanical column scale .2). Weight status was defined according to the World Health Organization (WHO) international growth reference data for children and adolescents >5 years of age [Body mass index (BMI) was calculated as the ratio of body mass (kg), towards the height of each participant, squared , based on the Tanner stages criteria . AccordiCRF was assessed with a cardio-pulmonary exercise test (CPET), performed on a cycle ergometer  using a spirometer  . The tesThe exercise protocol began with a resting period of 2 min for baseline measurements, followed by a 3 min warm-up period of unloaded cycling power. Exercise challenge consisted of progressively increasing increments of 10 Watt/min, followed by a recovery stage lasting for 3 min. All children were encouraged to exercise until the onset of exhaustion, throughout the test.Fasting venous blood was obtained from all participants at 9:00 a.m. and either assessed, or immediately centrifuged with the serum being stored at –80 °C. Stored serum samples were used for the analyses of triglycerides (TG) and high-density lipoprotein cholesterol (HDLC) levels, using Vitro Chemistry DT60 . Fasting blood glucose (FBG) levels were assessed with the hexokinase method .Citrated plasma samples were used for the assay of fibrinogen levels, using a commercially available kit , based on the Geftkin et al.  method.Insulin levels were assessed using a Human Insulin ELISA kit (ALPCO Diagnostics) with both inter- and intra-assay precision being below 15%. The HOMA-IR (homeostasis model assessment of insulin resistance) was calculated by (fasting insulin (μU/mL) × fasting glucose (mM)/22.5) for insulin resistance .Serum leptin and adiponectin levels were assessed with Human Leptin ELISA kits . The intra-assay coefficient of variation (CV) for adiponectin was 2.97–3.84% and the reported inter-assay ranging between 2.84 and 5.5%. The respective intra- and inter-assay CVs for leptin were 5.9 and 5.6%, respectively. High-sensitivity C-reactive protein (hs-CRP) was determined by a two-site chemiluminescent enzyme assay.R language, and the SPSS statistical software v. 23.0 . The level of significance was set at 5%. Initially, the general characteristics of variables were examined and, thereafter, we assessed all prerequisites for performing statistical analyses, including normality in distribution, variations, etc. Due to the uneven number of participants in each group, the bootstrap technique was applied for 10,000 identical items.All analyses were carried out with the Jamovi v. 1.2 (the Jamovi project) using the Differences between groups were assessed with ANOVA, using Bonferroni correction for normally distributed continuous variables. Such variables are presented as mean ± standard deviations (SD). For categorical variables, differences between groups were assessed with the Chi-square test. Correlations between variables were evaluated with the Pearson’s test.2max was also adjusted to the BMIz of each participant and the calculated adjusted values were also used in the analyses.Apart from crude parameters, the VO2max, receiver operating characteristic (ROC) curves were also plotted [For the assessment of the diagnostic ability of leptin, hs-CRP and fibrinogen in predicting VO plotted .Univariate and multivariate regression models were also designed, using quantitative variables. The effect of the degree of obesity in CRF was assessed with general linear models. Degree of obesity was converted to a dummy variable for use in multiple regression models, in order to better define the best model explaining the results, using stepwise and enter methods. Residual distribution was assessed in all cases, using the Kolmogorov–Smirnov test. Models were also assessed for tolerance and collinearity.2max was significantly higher. Overall, VO2max was significantly reduced at each increasing obesity tier. In parallel, leptin and fibrinogen levels were significantly increased at each higher adiposity level. The results remained similar for all of the aforementioned parameters, even after dividing the sample according to sex, with the exception of fibrinogen levels. 2max was decreased in each ascending adiposity level, whereas circulating leptin and fibrinogen levels were increased, in parallel to the weight status.In 2max (expressed as mL/Kg/min) demonstrated negative correlations of progressive strength to the BMIz , hs-CRP , glucose  and insulin levels , HOMA-IR , leptin  and fibrinogen levels  of participating children. On the other hand, a weak, positive correlation was observed with adiponectin .In the total sample, the crude VO2max was expressed as a percent (%), all aforementioned correlations either remained similar, or were strengthened, becoming more significant, with the exception of adiponectin . In more detail, the VO2max (%) was negatively associated with fibrinogen levels , leptin , glucose  and insulin levels , HOMA-IR , hs-CRP , and BMIz .When the VO2max adjusted for BMIz and the multivariate regression models predicting VO2max (adjusted) by biochemical parameters, are presented in 2max.Linear correlations with VO2max. The adjusted R2 of the multivariate regression model was equal to 0.527 and although the correlation between leptin and fibrinogen as independent variables was rather strong , no collinearity issues existed, as the portion of variance that was not explained by the other variable (tolerance) was greater than 0.73. In addition, the remaining multicollinearity indicators, including the variance inflation factor (VIF), Eigen values, the condition index and variance proportions, were within the accepted thresholds.According to the multivariate analysis, only leptin and fibrinogen levels could predict the VO2max is presented in The ROC curve for the diagnostic accuracy of leptin, hs-CRP and fibrinogen concentrations for the prediction of VO2max and may predict the latter.Our study revealed that in children with overweight and obesity, leptin and fibrinogen concentrations are strongly correlated to the VO2max decreased among overweight children, an increment in leptin levels and a parallel decrease in circulating adiponectin concentrations were observed, triggering the production of more energy.Adipokines and insulin have been shown to interplay with CRF . AccordiIn accordance with the literature, the present, secondary analysis of previous data verified the existence of associations concerning leptin, adiponectin and hs-CRP with CRF, irrespective of the weight status categorization applied. Studies to date have shown that reduced CRF is associated with increased leptin ,24,25 an2max. Lewitt and Baker [With regard to the fibrinogen concentrations, in the present analysis it was shown that they have a good diagnostic ability for the prediction of VOnd Baker  also revnd Baker . In parand Baker . In a fond Baker , increasnd Baker , observand Baker , lower fnd Baker  and consnd Baker . In parand Baker . On the nd Baker .Taken et al.  showed tNevertheless, the CRF assessment entails several restrictions in daily clinical practice. Personnel with expertise is required, as well as suitable equipment. As a result, few pediatric clinics have units equipped for assessing the CRF. Children and adolescents with obesity often find it difficult to respond to the CPET as a result of emotional, psychological and behavioral disorders associated with obesity , complim2max.A strong point in the present study was the favorable response of participants to the CPET, using the cycle ergometer. This could be due to the study design  but also to the method selection for the CRF assessment. In children, CPET using the cycle ergometer seems to outweigh that performed on the treadmill. Furthermore, an additional strong point in our study was the application of the CPET for assessment of the exercise ability and not only for defining the VO2max in a pediatric population, and its prediction by easy-to-perform assays.Limitations of the present study include the relatively small sample used and the cross-sectional design and indirect definition of the body fat. BMI was selected as the most widely accepted method for indirect body fat assessment in children and adolescents. However, important aspects of the study herein include the assessment of VOIntervention studies have showed that diet and exercise consist of cost-effective strategies in improving metabolic profile among children with obesity, as well as among those with concomitant respiratory issues, and, as such, they could be employed to tamper the problem and mitigate the prevalence of type 2 diabetes mellitus in the young ,81,82,832max can be predicted by hematological parameters (leptin and fibrinogen), bypassing more invasive methods. Early metabolic disorders due to childhood and adolescent obesity seem to affect cardiovascular fitness for exercise and their timely assessment is mandatory in order to protect children and adolescents with obesity from metabolic and cardiovascular complications, and reducing the risk for developing diabetes.Childhood obesity is accompanied by too many comorbidities ,85,86. T


Intra-group mean differences of BMI were not statistically different in both groups after 16 weeks of date consumption. Even HbA1c did not change, both within and between groups after date consumption . Between groups, mean difference changes (intervention minus control) showed a statistically significant improvement of quality of life index of ∆ = ± 30.66 points  due to the consequent improvement in mental health. Although the definitive effect of dose/intake response of date consumption on Hb1Ac, lipid profile, and BMI in T2DM subjects is still to be established, the study suggests that dates could potentially have a beneficial effect on lipid profile, especially in reducing total cholesterol and elevating HDL, because of its high polyphenolic content. In addition, a low–moderate consumption of dates did not impact glucose levels because of dates’ low glycemic index.Dates have a low glycemic index and are a source of antioxidants but, nevertheless, contain more than 70% sugar. This study aims to assess the effects of date consumption (three dates daily) on glycemic profile (HbA1c), body mass index (BMI), quality of life, and lipid profile, including total cholesterol, triglyceride, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in terms of safety for type 2 diabetic mellitus (T2DM) subjects. A randomized controlled trial was conducted with a sample of 100 T2DM subjects  randomly assigned in two groups. The first group received three dates daily for 16 weeks, and the control group avoided date consumption. After a 16-week follow-up period, the study results showed an improvement of lipid profile with a statistically significant decrease in total cholesterol of ∆ = −0.209 mmol/L  95% −0.358, −0.059;  Phoenix dactylifera L.) is one of the most important fruit crops in the Middle East and North Africa that produce edible and delicious dates. Date palms are spread across Iraq, Iran, Saudi Arabia, Egypt, Tunisia, Algeria, Libya, United Arab Emirates (UAE), Bahrain, and Oman. All date varieties have four distinct stages of maturity, namely, kimri (immature and inedible), khalal , rutab (soft and ripe), and tamar (ripe and dried) [Date palm (d dried) . The datd dried) .p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, and o-coumaric acid) were identified in Omani date varieties [Nutritional analysis showed that dates are high in carbohydrates and sugar. Dates consist of more than 70% sugar, consisting mainly of glucose, fructose, and a small amount of sucrose . Dates aarieties .Recent studies have shown that these constituents of dates act as potent antioxidants, antiglycemics, and anti-inflammatories, providing suitable nutritional therapy for different diseases such as cancer, diabetes, and dyslipidemia ,6,7,8. WDates, because of their high fiber and phenolic contents, could play a potent role in the prevention of chronic diseases such as cardiovascular disease (CVD), atherosclerosis, hypertension and T2DM. Prevention of CVD may be due to the inhibition of platelet aggregation as well as the oxidation of low-density lipoprotein (LDL). Phenolics, because of their anti-inflammatory and antithrombotic effects, may be able to reduce blood pressure ,10. SuchPreliminary data in the literature suggest that date fruit supplementation may exert maximum serum cholesterol, triglyceride, and LDL reduction potential through several mechanisms that modulate cholesterol absorption and metabolism ,15. In aPhytochemicals present in dates could also be used as antiobesetic agents, as demonstrated by the inhibitory effect of pits from various date palm varieties against lipase activity . The effThe aim of this research is to assess the effect of date consumption on glycemic profile (HbA1c), body mass index (BMI), quality of life, and lipid profile in Bahraini adult T2DM patients.This study was a randomized controlled trial. The protocol of the present study was approved by the Ministry of Health in Bahrain and the Ethics Committee of the Department of Biology at the University of Bahrain. The study was conducted in full compliance with the Declaration of Helsinki and Ethical Guidelines. The University Trial Registration Number is AURS/496/2018.Participants were enrolled from January 2018 to September 2018, and the end of the trial was in January 2019. Written informed consent was obtained from all subjects participating in the study.2, and an estimated glomerular filtration rate >50.0 mL/min/1.73 m2.Subjects with the following criteria were eligible: diagnosed with T2DM according to the diabetes diagnostic criteria (current HbA1c >6.0% and <10.0%), aged between 20 and 65 years, BMI >22.0 kg/mSubjects with the following criteria were excluded: type 1 diabetes, history of diabetic ketoacidosis, diabetic coma or precoma within 6 months prior to the date of consent, serious infections, surgery, serious trauma requiring insulin therapy, moderate or higher renal dysfunction  level ≥1.3 mg/dL and female Cre ≥1.2 mg/dL), hemodialysis treatment , severe liver injury, and history of serious vascular complications  requiring hospital admission.This study was a randomized, monocenter (University of Bahrain), not blinded, and controlled clinical trial. Assignment to the 3 dates daily group or the control group (no intake of dates) was according to a computer-generated randomization scheme (1:1) done by the nutritionist and statistician.T2DM subjects in the intervention group received 3 dates daily for 16 weeks, while those in the control group were initially advised to avoid date consumption.The variety of dates used in the study was Khudary cultivar dates, which is widely consumed among Bahrainis. The same batches of packaged dates (tamar stage) purchased from the local market, imported from Al-Qassim, Saudi Arabia, were used for this investigation.During the study period, diet and exercise therapy remained the same as before the study initiation.The primary outcome was to assess the change in HbA1c levels in the intervention group that received 3 dates daily for 16 weeks. Secondary outcomes included changes in BMI values, body composition, total cholesterol, LDL cholesterol, fasting, triglycerides, HDL cholesterol, and quality of life Short Form 36 (SF-36) index. We assessed how safe our study was by recording all adverse events seen during the study.A two-arm, randomized control trial was performed for a period of 16 weeks. Three dates daily were provided as part of a breakfast meal. The subjects were instructed to maintain their regular dietary habits, except for breakfast, and to record and take pictures of the contents of daily meals, snacks, and beverages during the intervention period.Subjects attended two visits to the laboratory, spaced 16 weeks apart. Participants were not permitted to eat or drink (except for water) and to smoke 12 h prior to the blood sample being taken between 7:00 and 8:30 a.m. Height and body weight were measured in order to calculate the BMI.Biochemical analyses on total cholesterol, HDL, and triglycerides were measured by commercially available enzymatic assays using an autoanalyzer. LDL was calculated using the formula of Friedewald . HbA1c aThe participants were tested with the SF-36 health survey to assess their quality of life . This quBasal dietary analysis was conducted a week before the test period. The diet diary was collected every weekday, and the daily intake of each component was calculated by an experienced dietitian on the basis of the food composition tables for the Bahrain . The levVolunteers were advised to continue current levels of physical activity during the clinical trial. Furthermore, physical activity was monitored on a weekly basis with self-assessment done by the International Physical Activity Questionnaire (IPAQ) .t-test at 0.05 level of significance, taking into consideration a dropout rate of 10%, and 90% power).The sample size was determined based on the previous study by Verma et al. . Based ot-tests for continuous variables, as appropriate. In the primary analysis, the baseline-adjusted means and 95% confidence interval (CI) estimated by analysis of covariance (ANCOVA) with the change in the primary outcome at 16 weeks were compared between the 3 dates daily group and control group. The comparisons were adjusted for age and gender, ANCOVA was also used for the secondary outcomes at each time point (16 weeks). The adverse events analysis and safety analysis were performed by Fisher’s exact test. All p values were two-sided. A p value <0.05 was considered statistically significant. All analyses were performed by SPSS 21 software .The statistical analysis and reporting of this study were conducted in accordance with the consolidated standards of reporting trials (CONSORT) guidelines , with thn = 50) and the control group (n = 50). Only 46 subjects in the intervention group were analyzed, as four patients were lost in the follow-up, while all subjects in the control group completed the follow-up.As shown in 2.The baseline parameters of the study are displaced in p = 0.311, while with the control group, ∆ = −0.044%  and p = 0.554. Between-group analysis showed a slight reduction  of ∆ = –0.087%  between the groups.As shown in p < 0.05; versus nondates consumers that found ∆ = 0.024 mmol/L , p = 0.115) and a suggestive, but not statistically significant decrease in LDL in the intervention group of ∆ = −0.171 mmol/L . No effects on other biomarkers tested, such as on glycemia, were detected either within groups or between groups.After a follow-up period of 16 weeks, the findings showed an improvement of lipid profile with a statistically significant decrease in total cholesterol in the group receiving 3 dates daily , No relevant side effects were observed in the intervention group.p = 0.405; control: p = 0.302).IPAQ score of the overall groups showed a non-statistically significant difference in physical activity for each weekly follow-up. The mean level score of physical activity intragroup (time × interventions) did not statistically differ significantly  is shown in 2) for the intervention group before and after the intervention. R2 is computed as the partial correlation, adjusted for gender. No statically significant associations were detected according to the level of R2.The present study shows for the first time in literature that a daily low intake of dates in T2DM subjects did not affect an increase in HbAlc levels, and there was no evidence to discourage individuals with diabetes from avoiding dates in moderation. Furthermore, the daily low intake of dates did not affect the BMI, and it seems promising in the enhancement of the lipid profile.The study findings revealed that date consumption in T2DM subjects was associated with a statistically significant decrease in total cholesterol and a suggestive (but not significant) decrease in LDL after the consumption of three dates per day for 16 weeks in the intervention group. This was probably because dates are a good source of dietary fiber, which has been shown to be hypocholesterolemic in animals . A similFurther, we found that date consumption for 16 weeks had no effect on body mass index in both groups either before or after date consumption. This could be attributed to the high polyphenol content present in dates. Similarly, Rock et al.  showed tMoreover, the BMI of healthy subjects consuming approximately 70 g dates daily was not accompanied by any weight change at the end of the three-week study period . InteresIn addition, this study showed that the consumption of dates boosted various measures of function and quality of life, particularly mental health as assessed by SF-36. Indeed, our study confirmed that the overall quality of life of subjects supplemented with three dates daily improved significantly. At the same time, the lack of date consumption in the control group affected the quality of life index of T2DM subjects. The probable explanation of this finding is that utilization of dates has cultural and religious functions among Muslims. The fruit is mentioned more than 20 times in the Quran, and it is usual to break the daily fast with dates during Ramadan. Moreover, it has a special social status among Bahrainis and Arabs in general, as dates and date-based foods are served during every auspicious occasion and event, such as weddings, births, family gatherings, and religious holidays .In the present study, the consumption of dates showed no statistically significant increase or decrease of levels of HbA1c, LDL, and triglycerides between date consumers and nondate consumers, either before or after the intervention. This goes in line with the fact that fructose is less rapidly absorbed than glucose, which results in a relatively low glycemic index (GI) . In addiLimitations of the study were that the amount of dates consumed were limited to only three dates, which is on the low side, considering that, traditionally, large amounts of dates are consumed because of their religious and nutritional significance. In addition, we used only one variety of dates at the tamer stage. Last but not least, the overall macronutrient distribution and quantity of each group’s diet was not evaluated specifically for quality of fats, carbohydrates, and proteins (and relative foods). Consequently, conclusions from this study may not be applicable to all types (fresh and sun-dried) and varieties of dates.The daily low intake of dates in T2DM subjects did not increase HbAlc levels, and there is no evidence to support discouraging people with diabetes from consuming dates in moderation. Our results showed clear benefits of date consumption on the lipid profile of diabetic individuals, particularly a significant reduction in total cholesterol and an increase in HDL cholesterol and quality of life. Furthermore, we have shown that date consumption does not result in increased triglyceride and LDL levels, or BMI. However, more long-term randomized clinically controlled intervention trials are needed to evaluate the effects of date consumption on serum glucose and insulin levels in T2DM individuals.


This work studied the impact of three types of styrene-butadiene (SB and SBS) block copolymers on the morphology, electrical, and rheological properties of immiscible blends of polypropylene:polystyrene (PP:PS)/multi-walled carbon nanotubes (MWCNT) with a fixed blend ratio of 70:30 vol.%. The addition of block copolymers to PP:PS/MWCNT blend nanocomposites produced a decrease in the droplet size. MWCNTs, known to induce co-continuity in PP:PS blends, did not interfere with the copolymer migration to the interface and, thus, there was morphology refinement upon addition of the copolymers. Interestingly, the addition of the block copolymers decreased the electrical resistivity of the PP:PS/1.0 vol.% MWCNT system by 5 orders of magnitude . This improvement was attributed to PS Droplets-PP-Copolymer-Micelle assemblies, which accumulated MWCNTs, and formed an integrated network for electrical conduction. Molecular simulation and solubility parameters were used to predict the MWCNT localization in the immiscible blend. The simulation results showed that diblock copolymers favorably interact with the nanotubes in comparison to the triblock copolymer, PP, and PS. However, the interaction between the copolymers and PP or PS is stronger than the interaction of the copolymers and MWCNTs. Hence, the addition of copolymer also changed the localization of MWCNT from PS to PS–PP–Micelles–Interface, as observed by TEM images. In addition, in the last step of this work, we investigated the effect of the addition of copolymers on inter- and intra-cycle viscoelastic behavior of the MWCNT incorporated polymer blends. It was found that addition of the copolymers not only affects the linear viscoelasticity  but also dramatically impacts the nonlinear viscoelastic behavior under large deformations .] Filled polymer blends are among the most attractive options to develop new materials for electrical conduction. The mixture of two or more polymers can be miscible or immiscible. Miscible polymer blends are not common, and most of the polymer blends are immiscible. For immiscible blends, depending on the number of species incorporated, two or more phases form and different morphological structures are generated ,2,3,4,5.When a conductive nanofiller (CN) is mixed with an immiscible biphasic polymer blend, the nanofiller can be localized in one of the phases, in both phases, and/or at the interphase. This localization is driven by thermodynamic ,9,16,17 The addition of CNs into multiphase polymer blends produces morphological changes, such as a decrease or increase in domain size ,26,27,28Compatibilizers or copolymers are often added to the immiscible polymer blends to stabilize the blend morphology ,35,36,37The results presented in this manuscript illustrate how the addition of styrene-butadiene (SB) block copolymer can alter the localization of multi-walled carbon nanotubes (MWCNT) in polypropylene:polystyrene (PP:PS) immiscible blends. This modification with block copolymers concurrently enhances the electrical and rheological properties and decreases the domain size of the polymer blend nanocomposite. It was found that the addition of block copolymers decreased the electrical resistivity and increased the storage modulus by 5 and 1 orders of magnitude, respectively, for PP:PS/70:30/MWCNT 1.0 vol.%. The results were explained using MWCNT selective localization in the blend system and morphological changes (decrease in domain size) upon the addition of block copolymers. To justify the results, molecular simulation and solubility parameters were employed to quantify the interactions between the polymers, copolymer, and MWCNT. Additionally, as the materials experience large and rapid deformation in real processing conditions in industry, we studied the effects of copolymer addition and morphology on inter- and intra-cycle rheological response of the MWCNT incorporated blends under large amplitude oscillatory shear (LAOS) flow. Stress decomposition method was utilized to explain the intra-cycle viscoelastic response of the blends in nonlinear region. It is shown that the stress waveform considerably depends on the type of the copolymer, signifying that Lissajous–Bowditch plots can be used as a strong tool to distinguish the polymer blends.The molecular simulation was performed using the GAUSSIAN 16 software   to underThe simulation process consisted of the geometrical optimization of the CNTs, polymer and copolymer segments, and polymer/CNT and copolymer/CNT assemblies. This optimization was based on the energy minimization, which consisted of changing the spatial arrangement of the atoms of the molecules until the inter-atomic forces at each atom are close to zero. To confirm that the obtained structures corresponded to the global minimum in energy, a frequency calculation  was performed. All the calculations were performed employing the semi-empirical parameterized model 6 (PM6) by ignoring diatomic differential overlap (NDDO) .To study the interactions between the polymer and copolymer segments and the CNTs, binding energy, and molecular electrostatic potentials were evaluated. The binding energy parameter is defined in Equation (1).w = 209,300 g/mol, MFI = 5 g/10 min and viscosity at 40 s−1−1) was provided by Flint Hills Resources®. Polystyrene (PS) Styron® 615APR  was obtained from Americas Styrenics LLC. Styrene-butadiene (SB) diblock and styrene-butadiene-styrene (SBS) triblock copolymers were kindly donated by KratonTM Corporation, and their specifications are provided in Polypropylene (PP) H0500HN (MTM NC7000) were purchased from Nanocyl S.A. . According to the manufacturer, the MWCNTs were produced with the catalytic carbon vapor deposition process, and have an average diameter of 9.5 nm, a length of 1.5 µm, and a surface area of 250–300 m2/g.Multi-walled carbon nanotubes (MWCNTs)  provided better mechanical properties, while PS is known to generate MWCNT nanocomposites with low percolation threshold and high electrical conductivity ,46,47. Tylinders . The volTransmission electron microscopy (TEM) was performed to determine the selective localization of MWCNT in the blend and blend/copolymer systems. 100 nm sections were cut using a Leica EM UC6 ultramicrotome  equipped with a diamond knife. The ultramicrotomy was performed at −90 °C in a nitrogen atmosphere. A Tecnai TF20 G2 FEG-TEM  electron microscope equipped with a Gatan UltraScan 4000 CCD camera  at 2048 × 2048 pixels was used to image the sections.Scanning electron microscopy (SEM) was used to study the morphology of the neat PP:PS/70:30, PP:PS/70:30/MWCNT and PP:PS/70:30/MWCNT/copolymer nanocomposites. The samples were etched using tetrahydrofuran (THF) to remove the PS phase. This process was performed for 2 days at ambient temperature. Then, the samples were cryo-fractured in liquid nitrogen and dried in a vacuum oven at 60 °C for 1 day. Before SEM visualization, all samples were coated with platinum in an argon atmosphere to avoid scanning problems, i.e., electrostatic charge accumulation on the surface. The SEM microscope FEI XL30  scan was carried on at an acceleration voltage of 20 kV.−1. Measurements were taken by a load cell, and the samples were extruded through a 0.508 mm (L/D = 20) die (entrance angle 120°) at 200 °C. The results were analyzed by KARS software, and the Weissenberg–Rabinowitsch correction [The shear viscosity for the neat type of PP, PS, SB-D1431P, SBS-D1102K, and SB-D0243K systems was measured using a Kayeness (Dynisco) capillary rheometer , Model LCR600 D8052M-115 2046 WVS, in the range of 0–1000 srrection  was perf6 Ω∙cm, a Keithley 6517 A electrometer connected to a Keithley 8009 test fixture  was used to test the samples. On the other hand, for resistivities lower than 106 Ω∙cm, according to ASTM 257-75 standards, a Loresta GP resistivity meter  connected to an ESP four-pin probe  was used. The four-pin probe, which has an inter-pin distance of 5 mm and a pin diameter of 2 mm, was used to minimize the effect of the contact resistance. The applied voltage for all the electrical resistivity measurements was 90 V. Three samples per each composite assay were measured, and the results were averaged.Electrical resistivity measurements were conducted using two setups according to the resistivity range. For electrical resistivities greater than 10The rheological behavior was evaluated using a rheometer . The set-up was equipped with a 25 mm parallel-plate geometry at a gap-size of 0.3 mm. All the oscillatory tests were performed at 200 °C.A summary of the system energies and orbitals is found in It was noticed that when the PS segment was connected to the butadiene part of the copolymer, the PS molecules did not take the stiff linear configuration; rather, they took a coiled configuration . Thus, wThe localization of the MWCNTs in the immiscible blend systems was directly determined using TEM. The TEM image of the blend without copolymer in −1). The low viscosity of the PS might have facilitated the wetting of MWCNTs, thus decreasing the probability of interaction between MWCNTs and the copolymers, as well as with PP. In addition, it is important to consider that the formation of micelles or the possible assembly of copolymers at the interface might have altered the localization of MWCNTs in the blend. Thus, the mutual interactions between the copolymers, PP, PS, and CNT should also be considered. We used the Hildebrand solubility parameters   for PP, 1 cm−3). Hence, it can be inferred that during mixing, the copolymer chains preferred to interact with PP or PS than remaining with the CNT. Hence, according to the simulation results, CNTs not covered by copolymer chains can migrate to PS phase, which is energetically more favorable than PP. In fact, according to the solubility parameter data, CNTs showed a higher affinity to the styrene block and PS phase; thus, they can preferentially localize in PS and the styrene block of the copolymer and form micelles. TEM results  projections on the elastic  and shear thickening ratio (T) indices for samples, which is defined as ectively . GL′ . In the nonlinear regime, a positive S index and negative T index revealed intra-cycle strain-stiffening and intra-cycle shear-thinning behavior, respectively. The deviation of these indices from zero at higher strain amplitudes showed the extent of the non-linearity. Filipe et al. [T and S indices in the deep nonlinear region for blends containing copolymers compared to polymer blends without copolymer verified their findings  on the morphological, electrical, and rheological properties of PP:PS/70:30/MWCNT blend nanocomposites. The copolymers effectively decreased the PS domain size in the blend nanocomposites. The copolymers at the interphase provided steric hindrance against the coalescence of PS domains. The increase of MWCNT content induced co-continuity in PP:PS blends; however, this did not affect the migration of the copolymer to the interphase. Molecular simulation and solubility parameter analyses were employed to understand and predict the MWCNT localization in the PP:PS/70:30/copolymer system. The simulation predicted that diblock copolymers had higher binding energy towards CNT surface, in comparison to PP and PS, and indicated that the triblock copolymer/CNT interaction was thermodynamically unfavored. Hildebrand solubility parameters showed that the block copolymer segments preferred to interact with PP or PS rather than with MWCNT. This implies that when copolymers migrate to the interphase, they would expel MWCNTs, allowing them to migrate to the PS phase. However, some MWCNTs were observed in the micelles, the interface, and PP phase. This localization might be attributed to the migration of MWCNTs between the phases due to copolymer assembly in micelles and at the interface.The rearrangement of MWCNTs in the blend upon addition of the copolymers enhanced the electrical conductivity  of the PP:PS/70:30/copolymer/MWCNT nanocomposite. The integrated network formed by the copolymer, PS droplets, PP, and micelles containing MWCNTs led to a significant decrease of 5 orders of magnitude in electrical resistivity  for the MWCNT concentration of 1.0 vol.%. The significant improvement in electrical conductivity made these nanocomposites promising candidates for applications such as electromagnetic interference shielding and electrically conductive films. Moreover, the rheological results of this work verified that the stress waveform considerably depends on the type of the copolymer, signifying that Lissajous–Bowditch plots can be used as a strong tool to distinguish polymer blends with different morphologies.


Kineococcus species. Moreover, 5 out of the 13 genera represented by one isolate were also possible new species. Specifically, the sequences of 80 isolates held a percentage of identity below the 98.7% threshold considered for potentially new species. These strains belonged to 20 genera. Our results reveal a clear link between medium dilution and isolation of new species, highlight the unexploited bacterial biodiversity of the Tabernas Desert, and evidence the potential of simple strategies to yield surprisingly large numbers of diverse, previously unreported, bacterial strains and species.One of the most diverse ecological niches for microbial bioprospecting is soil, including that of drylands. Drylands are one of the most abundant biomes on Earth, but extreme cases, such as deserts, are considered very rare in Europe. The so-called Tabernas Desert is one of the few examples of a desert area in continental Europe, and although some microbial studies have been performed on this region, a comprehensive strategy to maximize the isolation of environmental bacteria has not been conducted to date. We report here a culturomics approach to study the bacterial diversity of this dryland by using a simple strategy consisting of combining different media, using serial dilutions of the nutrients, and using extended incubation times. With this strategy, we were able to set a large (254 strains) collection of bacteria, the majority of which (93%) were identified through 16S ribosomal RNA (rRNA) gene amplification and sequencing. A significant fraction of the collection consisted of Actinobacteria and Proteobacteria, as well as Firmicutes strains. Among the 254 isolates, 37 different genera were represented, and a high number of possible new taxa were identified (31%), of which, three new  Only a small fraction of the microbial diversity of our planet can be cultured under traditional microbiological tools, media, and strategies . The useThe generally accepted “1% culturability paradigm,” which refers to the fact that around 99% of environmental microorganisms are in fact unculturable, was recently revised by One of the reasons behind the obstacles in culturing environmental bacteria is the difficulty in mimicking the particular environmental conditions that certain microbes require for growth, as standard laboratory conditions may differ from the natural habitats . Small vMulti-omic data have broadened our knowledge on previously unknown microbial communities and taxa . SpecifiMetagenomic data have been used in order to design tailor-made culture media for specific samples , but thein situ, overcoming the problem of mimicking the environmental conditions , is a particular, not very much studied region to date in terms of bacterial culturability. It is formed by marls, sandstone, and scarce vegetation. The annual rainfall is below 250 mm, its climate is considered hot semiarid to desert climate, depending on the altitude, and it has interesting, well-developed biocrusts.Biological soil crusts are multi-organism consortia worldwide—spread in dryland landscapes, being estimated that around 12% of the total terrestrial surface is covered by biocrust . SpecifiIn the present work, we have characterized the microbial communities of the Tabernas Desert by using improved culturomics techniques that have allowed the identification of an unprecedented rich diversity of bacterial strains from this unique European dryland. We hypothesized that by combining culture laboratory techniques and increasing incubation times, a higher diversity of bacterial taxa would be isolated. The results obtained confirm that simple strategies in culturomics can lead to a better understanding of the culturable fraction of microbial communities, including the isolation of new and potentially new microbial taxa.Biocrust samples were obtained in September 2018 at the Tabernas Desert, in the vicinity of the Natural Park. Sampling was carried out by taking in Falcon tubes the upper 1–2 cm of biocrust-covered surfaces in three different locations , and a total of eight samples were collected of different biocrusts in terms of color and appearance , which w–3–10–5 samples was spread on Petri dishes containing either Tryptic Soy Agar (TSA) medium  or Reasoner’s 2A (R2A) medium  at concentrations 1 × (standard concentration), 0.1×, and 0.01 × . TSA is a rich media used for general purposes, whereas R2A is a nutrient-poor media that favors the isolation of slow-growing and oligotrophic bacteria. Media were sterilized by autoclaving at 121°C for 20 min. Agar was autoclaved separately to the nutrient solution of each medium and added just before pouring the media into the plates. After 2 weeks of incubation at room temperature (23°C), individual colonies were selected based on their color and morphology and isolated by independent re-streaking on fresh media in order to obtain them in pure culture. New colonies were also selected a month of incubation, in order to identify, small, slow-growing microorganisms. A total of 254 isolates were cryo-preserved as a glycerol stock  at −80°C until required.The samples were homogenized by mixing 1 g of biocrust with 1 ml of sterile phosphate buffer saline (PBS) 1×. Then, serial dilutions of the suspensions were prepared, and 50 μl of the 10A loopful of 3 μl of microbial biomass from grown plates was suspended in 100 μl of Milli-Q sterile water and then boiled for 10 min before the PCR in order to ensure cellular lysis. Colony PCR was used for taxonomic identification through 16S rRNA gene sequencing by using the universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′)  and 1492® Terminator v3.1 Cycle Sequencing Kit  was used to tag amplicons, which were sequenced with the Sanger method by the Sequencing Service  of the University of Valencia (Spain). All sequences were manually edited with Trev  (100 V for 30 min). Amplicons were precipitated overnight in isopropanol 1:1 (vol:vol) and potassium acetate 1:10 (vol:vol)  at −20°C. DNA was pelleted by centrifugation at 12,000 rpm for 10 min, then washed with ethanol 70%, and resuspended in the required 15 μl of Milli-Q water. BigDyeith Trev  to elimiith Trev  and 1055ith Trev  were useIn order to analyze the closest environmental clone or isolates of our strains, an extensive blast was carried out against the NCBI Nucleotide collection (nr/nt) databases optimized for highly similar sequences.Culturing different biocrust samples from the Tabernas Desert yielded a large number of different colonies in terms of color, shape, and morphology . A totalArthrobacter spp., which was the most abundant genus representing 24.8% of the collection. The second most represented genus was Mycolicibacterium, holding a 7.09% of abundance, similar to the abundances of Microbacterium and Roseomonas. In contrast, the less abundant genera were Agrococcus, Amycolatopsis, Azospirillum, Bacillus, Caulobacter, Enterovirga, Flaviflagellibacter, Herbaspirillum, Micrococcus, Noviherbaspirillum, Paenarthrobacter, Staphylococcus, and Variovorax, being represented only by one isolate each  were identified, belonging to 37 different genera, and only 18 isolates remained not identified due to either a lack of amplification in PCR or failure in sequencing. A total of 62 isolates were identified as ate each .Arthrobacter spp., whereas the rest were distributed among half of the genera detected. Interestingly, between the genera that had at least one possible new species, five of them were represented by just one isolate   . For exasp. nov. . Moreovecription . This pomezawaea .Arthrobacter and Microbacterium, isolates were identified in all six media but, most frequently, in the most concentrated ones. Others, such as Actinoplanes, were isolated from the most diluted concentration on TSA and R2A, and the possible new genus was isolated from TSA 0.01×. Non-identified strains were also isolated from all six media, although the higher abundance was detected in the less concentrated ones, especially on R2A 0.01×. Interestingly, the five Blastococcus isolates grew on R2A 1× , whereas 15 genera were isolated just from one of the tested conditions . The quan R2A 1× .The co-isolation of genera in the different nutrient conditions was compared through Venn diagram representations, which are shown in Planctomonas, which was represented by six (five from TSA 1× and one from TSA 0.01×). In the case of the unique R2A taxa, apart from the five Blastococcus and three Massilia strains, the rest were also represented by one isolate.The comparison, regardless of the dilution factor, of TSA and R2A media in terms of genera diversity revealed that one fourth of the groups (as non-identified isolates and the possible new genus were included) had been exclusively isolated in TSA, whereas around 20% were unique taxa for R2A . This reMoreover, the distribution of the possible new species and the non-identified isolates was different among the media dilution used . The oneCaulobacter, Geodermatophilus, Azospirillum, and Blastococcus  and the Badain Jaran Desert (China), among others. Others (8.66%) were closely linked to clones or isolates inhabiting extreme environments, such as oil or heavy metal-contaminated soils, or cold environments, such as glaciers and permafrost samples. Furthermore, 8.3% of our strains were related to environmental clones or isolates found in aquatic habitats, mainly rainwater but also lakes, fish ponds, and freshwater sediments. Interestingly, 8.7% of the closest relatives corresponded to plant-associated clones, either associated with the rhizosphere, phyllosphere, or endophytic bacteria, and a small fraction representing 4.7% of the collection was similar to animal-associated bacteria, such as the gut of insects or corals.Although drylands and cold and hot deserts are a source of biotechnologically-relevant bacterial strains , the uniThe distribution of phyla in the collection resembles the microbial communities studied in other desert environments, such as the Atacama, the Gibson, or the Sahara Desert, with Actinobacteria as the most abundant phylum, followed by Proteobacteria and, to less extent, Firmicutes, among the culturable fraction . These tMicrobacterium and Lentzea isolates, as well as other represented genus to lesser extent, such as Streptomyces, Amycolatopsis, and Actinoplanes, may be playing a role in the modulation of communities through competitors’ growth inhibition   . Moreoveed soils , and Artompounds , which iompounds .Arthrobacter is the most abundant taxa in the collection established from the Tabernas Desert, with as many as 62 strains assigned, 26 of which being possible new species. Arthrobacter spp. do not only play a major role in the maintenance of natural processes in soils, such as the degradation of organic matter  generated by UVA and UVB stresses  studies and the–up to date–still very low fraction of culturable microorganisms. This has, well beyond microbial ecology, important biotechnological implications.https://www.ncbi.nlm.nih.gov/genbank/, MT749781–MT750013 and MN069867–MN069869.The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: MP conceived the work. EM-M, HG-V, and MP carried out the sampling. EM-M and HG-V performed all the experiments. EM-M, HG-V, JuP, JaP, and MP analyzed the results, wrote, and approved the manuscript. All authors contributed to the article and approved the submitted version.MP and JuP are founders of Darwin Bioprospecting Excellence S.L. HG-V is an employee of Darwin Bioprospecting Excellence S.L. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Perimenopause is a period that every woman must go through, most people are more or less affected by perimenopausal symptoms, it to affect women's health, work, life, and economy. As acupuncture treatment is more and more increasing in perimenopausal symptoms, there have also been many clinical trials about it. But the results of the trials are inconsistent. Therefore, we will conduct a systematic review and meta-analysis of the safety and efficacy of perimenopausal symptoms treated with acupuncture.The protocol followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols. RCT study on different acupuncture interventions for perimenopausal symptoms will be searched in 8 databases . Besides, the search will also be performed on the clinical trial research platform if necessary. The primary outcome that will be extracted: the Flushes per 24 hours, the Frequency of hot flashes, the severity of hot flashes, the menopause-related symptom score, the treatment efficacy, the adverse event. Endnote software X8 will be used for study selection, STATA 13.0 and Review Manager software 5.3 will be used for analysis and synthesis. These studies selection, data extraction, and risk of bias assessment will be conducted by 2 independent reviewers.This study will provide the results: 1. the primary and secondary outcome indicators of different acupuncture intervention measures  for perimenopausal symptoms. 2. The effects of different control groups  on the analysis results will be reported, especially the effects of different sham acupuncture control (invasive/noninvasive) on the analysis results.This systematic review and meta-analysis study hopes to provide useful evidence for better use of different types of acupuncture in treat perimenopausal symptoms and better design of control groups in related clinical trials. In addition, the research conclusion will be published in peer journals.OSF REGISTRATION NUMBER DOI 10.17605/OSF.IO/VZCKU Ethics and dissemination This conclusion of the study will be published in peer journals. The ethical approval is not required because there is no direct involvement of human. These chain reactions may cause or promote the occurrence of diseases such as high blood pressure, diabetes, obesity, cardiovascular diseases, psychological diseases and so on while accelerating aging. Hot flashes can affect women's work and quality of life, Of course, sleep will also be affected.–5,7,8,21 In addition, hard to falling sleep has been shown to associate strongly with anxiety,,23 Insomnia and anxiety of menopausal women have a chain effect and cause depression. So it can be seen that the symptoms of perimenopause are not single, there may be multiple symptoms. It may even promote or cause other diseases.–27Perimenopause syndrome, referred also as a climacteric syndrome. The perimenopausal symptoms include premenopause, menopause and postmenopause. Perimenopause is a normal and important aging phenomenon that occurs in women's life. Although some women have no obvious symptoms during this period, Most women still suffer from perimenopausal symptoms that affect health, work, life, and economic status.,17,28,29 Therefore, hormone replacement therapy has become the most effective treatment for the perimenopausal syndrome.,17,30–32 But the Women's Health Initiative (WHI) reported an increase in cases of Cardiovascular disease after hormone therapy.–35 Considering the duration of perimenopausal symptoms, hormones are not suitable for long-term use.–39 Therefore, more people choose to try non-hormonal replacement therapy,–44 acupuncture is one of them.–47 Acupuncture treatments include traditional hand acupuncture, electroacupuncture, ear acupuncture, laser acupuncture, acupressure, moxibustion, etc. Research reports that acupuncture can relieve perimenopausal symptoms and improve quality of life.,46,54 Although according to research reports that the efficacy of acupuncture has obvious advantages compared with the waiting group or the usual care controls, it also has certain effects compared with western medicine control, it compared with fake acupuncture is highly controversial.,57,58 The related meta-analysis studies also showed it.,60 A meta-analysis study by Li et al Showed that The number of RCTs that compared the acupuncture control to a sham group is too small, and some studies had a flawed methodology, making it difficult for them to generate reliable conclusions about the efficacy of acupuncture. Therefore, the systematic review and meta-analysis hope to further analyze the efficacy and safety of acupuncture in the treatment of perimenopausal symptoms, focusing on the analysis of different acupuncture interventions and different controls, especially sham acupuncture controls.What is the cause of such a wide-ranging, long-lasting disease? The etiology of perimenopausal syndrome, which is now known to be due to the decline or disappearance of ovarian function, hormone levels fluctuations, and instability.22.1,63 The ethical approval is not required because there is no direct involvement of human.This meta-analysis and systematic review will be based on the guidance of the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols (PRISMA-P) for systematic reviews of interventions.2.22.2.1 These women are 40 to 55 years and have perimenopausal symptoms .The patients in this study will include the woman who transitional period before and after menopause.2.2.2The acupuncture treatment methods of the intervention group included traditional hand acupuncture, electroacupuncture, auricular acupuncture, moxibustion, laser acupuncture, acupressure, etc. There are no acupuncture points, frequencies, and needle retention times.2.2.3,58 and invasive control (the needle penetrates the skin but does not work. It does not require the feeling of “Deqi” Or acupuncture in places without acupoints).,65The control group included waiting, routine care, medicine control, Sham acupuncture (invasive/noninvasive), etc. The waiting control and routine care control will not be treated with any acupuncture; the medicine control includes Chinese medicine control and western medicine control; the sham acupuncture control includes non-invasive control 2.2.42.2.4.1(1)The flushes per 24 hours.(2)The frequency of hot flashes.(3)The severity of hot flashes.(4)The menopause-related symptom score .(5)The treatment efficacy.(6)The adverse event.2.2.4.2(1)Hormone level .(2)Menopausal quality of life score (WHOQOL).(3)Depression scale assessment.(4)Sleep quality assessment.(5)The expected value of acupuncture.(6)Intervention acupuncture experience.2.2.5The design method is the literature of randomized controlled trials (RCTs), no date or language limits for publication were set.2.3The following conditions will be excluded:(1)(a) Received any other alternative therapies 4 weeks before enrollment; (b) ovarian cyst, tumor, oophorectomy, or hysterectomy; (c) severe metabolic, thrombo-embolic or endocrine disease, uncontrolled hypertension or use of sedatives, anxiolytic or antidepressant medication, or use of narcotics.(2)While this study will exclude any literature regarding acupuncture combined with drugs as the intervention group, as well as the literature that other drugs can be used at the same time during acupuncture treatment.(3)Non-randomized controlled trials will be excluded.(4)We will exclude the study that data cannot be extracted, duplicate data, and cannot be provided in full text. However, for studies with insufficient data, we will try to contact the authors of these studies to provide more complete data.2.4We will perform a systematic clinical trial research search in databases of PubMed, Embase, Cochrane, Web of Science, CBM, CNKI, WAN FANG, and VIP. If necessary, we will also Search some trial research registration platforms. No date or language limits for publication were set. Search terms for this study include perimenopause, hot flashes, acupuncture, acupressure, electroacupuncture, ear acupuncture, RCTs, etc. The search strategy is designed by the combination of MeSH words, free words and truncation search. The specific search strategies are listed in Table 2.5All results retrieved from the database will be imported into the EndNote software (X8), and an automatic search for duplicate studies will be set up. After excluding duplicate studies, the 2 authors will independently screen the remaining literature by title and abstract. If the title and abstract include exclusion criteria or do not include inclusion criteria, create a “Title Abstract Exclusion ” in endnote and move the clinical trial study into it. The remaining clinical trials will be searched for full text. Two authors will assess eligibility for the full text of these studies, and create “Publication type” “Study aim/design” “Intervention” “secondary analysis” in endnote according to the reasons for exclusion. The excluded clinical trial studies were moved into the corresponding group according to the reasons for exclusion. Two authors should contact the author of the study if the clinical trial studies are classified as an “insufficient” category due to unclear information or missing data. In addition, all processes will be performed independently by the 2 authors, then discuss the results together. the third review author will identify the study When the opinions of the 2 authors diverged. The select study flowchart Figure 2.6Data will be extracted independently by reviewers using a standardized form. Extracted information included:(1)Publication features ;(2)Characteristics of research objects ;(3)Measures of intervention/control group ;(4)Outcome indicators ;(5)The key information of biased risk assessment;(6)Outcome data will be divided into the pre-treatment data table, the in-treatment data table, and the follow-up data table according to the clinical study time so that the change value can be calculated later. .2.7In this study, 2 researchers will conduct risk assessments independently. After the assessment, the results of the cross-check can be discussed and resolved by other researchers and 2 researchers. The final decision will be made by the third author When the 2 authors have a controversy with the assessment. The bias risk assessment included in the study will conduct using the bias risk assessment tool recommended by Cochrane manual version 5.1.0 for RCT. the valuation content includes random method design; assignment concealment; whether to use blind method for subjects and researchers; whether the result data is complete, whether there is a selective report of research results, etc. The rating is divided into high risk, low risk, and unclear. The assessment will be performed using Review Manager software 5.3 software and a risk assessment form will be drawn.2.8We will contact the first corresponding authors of the included studies to get missing or insufficient trial data by email. If the data cannot be obtained. We will analyze whether the missing data has an impact on the meta-analysis, and if the impact is large, this clinical trial study will be excluded.2.9In this study, Revman 5.3 and STATA 13.0 statistical software will use for analysis. If the includes outcome indicator include 10 or more articles, we will use a funnel chart to test the risk of publication bias.2.9.1The confidence intervals (CIs) will be set to 95% for both continuous outcomes and dichotomous outcomes. The dichotomous outcomes (the effective rate and adverse events) will analyze the rate ratio. For continuous variables, the mean and SD of the change value were will calculate according to the baseline value and endpoint value of the extracted data. If the included data is not mean and SD, calculate and analyze according to the formula.2.9.2X2 test (the test level was α = 0.1), and evaluate with I2 statistics. The fixed effects model will be used for the analysis of this outcome indicator, when Its heterogeneity test result is I2 < 50% (No heterogeneity). if the heterogeneity test result is I2 ≥ 50% (have heterogeneity), after excluding obvious clinical and methodological heterogeneity, the random-effects model will be used for the analysis of this outcome indicator.The heterogeneity in the study will be analyzed by the 2.9.3The subgroup analysis is key in this study design.(1)We will perform subgroup analysis on outcome indicators with different controls. The subgroup analysis of the control group includes waiting, routine care, medicine control, Sham acupuncture (invasive/noninvasive), etc.(2)We will also perform a subgroup analysis of outcome indicators with different interventions. The subgroup analysis of the intervention group includes ear acupuncture, electroacupuncture, moxibustion, acupressure, traditional hand acupuncture, etc.2.9.4When there is an insufficient sample size, missing data, quality of analysis and research, methodological elements, etc, we will perform sensitivity analysis. The specific implementation is to eliminate the single study, then analyze again, and evaluate the difference between the eliminated results and the original combined results3,17,28,29 but some studies have shown that hormones are not suitable for long-term use.–39 Therefore, more people choose to try non-hormonal replacement therapy,–44 acupuncture is one of them.–47 There are more and more clinical trials of acupuncture treatment for perimenopause, and some meta-analysis researches related to it have also appeared.,59–61 These studies demonstrate that acupuncture has a certain effect on one or more of the outcome indicators of perimenopause symptoms compared with the waiting group or the routine care group. But the comparison with sham acupuncture is still controversial.,58 At the same time, these studies have analyzed the effects of acupuncture on perimenopausal symptoms, but there is no comprehensive analysis comparing the effects of different acupuncture intervention methods on the outcome indicators of perimenopausal symptoms, which may be the reason for the insufficient sample size at that time.Hormone replacement therapy has become the most effective treatment for perimenopausal syndrome,,58 and invasive control (the needle penetrates the skin but does not work. It does not require the feeling of “Deqi” Or acupuncture in places without acupoints).,65 On the other hand, intervention subgroup analysis includes ear acupuncture, electroacupuncture, moxibustion, acupressure, traditional hand acupuncture, etc. The ultimate objective of this study is to hope that the conclusions drawn from the analysis can provide more useful evidence for the clinical operation and clinical research of acupuncture treatment of perimenopausal symptoms.Therefore, it is necessary to further analyze the safety and efficacy of acupuncture in the treatment of perimenopausal symptoms. Except for routine efficacy and safety analysis, this study focused on subgroup analysis. On the one hand, the subgroup analysis of the control group includes waiting, routine care, medicine control, Sham acupuncture, etc. The sham acupuncture control includes non-invasive control Conceptualization: Qiujun He.Data curation: Qiujun He, Yajing Ren, Yanqiu Wang.Formal analysis: Qiujun He, Yanqiu Wang, Feng Zhang.Methodology: Qiujun He, Yajing Ren.Project administration: Qiujun He.Supervision: Sanyin Zhang, Peng Yang.Validation: Qiujun He, Yanqiu Wang, Feng Zhang.Writing – original draft: Qiujun He.Writing – review & editing: Qiujun He, Yanqiu Wang.Qiujun He orcid: 0000-0002-1679-9625.