Acute lymphoblastic leukemia (ALL) is associated with higher levels of pro-inflammatory cytokines and oxidative stress. Recently, the levels of extracellular heat shock protein 72 (eHSP72) were found to be elevated in ALL, and its elevation associated with poor prognosis. Therefore, considering the possible role of eHSP72 as a modulator of the immunological system and metabolism, the aim of this study was to describe the response of eHSP72 to the induction phase of chemotherapy, along with metabolic, inflammatory and oxidative stress markers, in children and adolescents newly diagnosed with ALL.Nineteen patients were recruited and analysed before and after the induction phase of chemotherapy (with 28 days of duration). Blood samples were taken for the analysis of C-reactive protein (CRP), levels of lipoperoxidation, insulin (and HOMA-IR), cortisol, glucose, lipid profile and eHSP72.We found that induction phase of chemotherapy leads to a drop in glucose levels (from 101.79±19 to 75.8±9.7 mg/dL), improvements on inflammation  and oxidative stress , reduction on eHSP72 (p=0.03) and improved insulin sensitivity .Our results indicate that eHSP72 may have an immune and metabolic role and could be used as a marker of the treatment success and metabolic changes in children with ALL. Cancer development is often associated with chronic states of inflammation, hormonal unbalance, hyperinsulinemia and insulin resistance , 2. TherAcute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, but it can occur at any age. ALL is a disorder caused by an abnormal expression of genes, which are usually a result of chromosomal translocations . LeukemiIn addition to the well characterized pro-inflammatory cytokines involved on ALL, an emerging role for the heat shock proteins (HSPs) family has been suggested. HSPs are considered part of a family of proteins known as “stress proteins” since their expression is induced by a wide range of stressors, such as oxidative stress , thermalA vital aspect regarding HSP72 physiology is its versatility to induce antagonistic actions, depending on the location of the protein . For exaTherefore, considering the possible role of eHSP72 not only as a marker of ALL development, but also as a modulator of the immunological system and metabolism, the aim of this study was to describe the response of eHSP72 to the induction phase of chemotherapy in children and adolescents newly diagnosed with ALL. In addition, we attempt to understand the relation of eHSP72 with metabolic, inflammatory and oxidative stress parameters.Patients had age between one and fifteen years-old, most were under 10 years of age (children), and ten (52.6%) were male. Table The results of routine biochemical analysis in the first phase of the treatment are presented in Table Results from CRP (as a marker of inflammation) and TBARS (a marker for lipoperoxidation) indicates that induction phase (for 28 days) decrease the levels of inflammation and oxidative stress (CRP decreases from 7.3 (3.3 – 2.9) mg/dL to 2.5 (1.3 – 4.4) mg/dL, and TBARS decreases from 3.83 ± 0.80 nmol/mL to 2.34 ± 0.63 nmol/mL, p<0.05 for both). Regarding the levels of eHSP72, when analyzing the remaining patients  we found a similar pattern, where eHSP72 tends to decrease from 1.40 (1.00 – 3.39) ng/mL to 1.23 (0.69 – 1.97) ng/mL, however no statistical significance was reached. Saying that, we decided to analyze the individual responses to see the general behavior of eHSP72 to chemotherapy. Interestingly, 70% of the subjects decreased their plasma levels of eHSP72. For this reason, we decided to go on a new type of analysis (multiple imputation data - described on the methods) to see if the results would indicate a significant statistical difference, when considering the missing data. In fact, the results from this analysis have indicated that eHSP72 can significantly decrease after the induction phase of chemotherapy.Cortisol levels was the only biochemical marker of stress that did not suffer significant reduction with treatment , demonstrating the efficiency of induction phase of chemotherapy to reduce oxidative stress and inflammation in a relatively short period of time  spectroscopy [Metabolically, the different types of leukemia's can present completely distinct profiles. Recently, metabolic alterations associated with two subtypes of acute leukemia, ALL and acute myeloid leukemia (AML), were investigated in the serum of these patients and compared with a healthy control group, using troscopy . Remarkatroscopy .Following that, the question that arises is: if transient hyperglycemia may be induced by the use of corticosteroids and asparaginase during the course of chemotherapy, what may be the cause of hyperglycemia in ALL patients observed prior to any therapy? The answer may be connected with the increased sympathetic tone that occurs in this condition . Heart rMoreover, children with ALL often present fever. Curiously, the inflammatory response involved in fever occurs even in the absence of infection. The levels of TNF-α, IL-6, IL8 chemoattractant protein-1 (MCP-1) and IL-10 are higher in patients with ALL when compared to the control children. Moreover, the levels of the T helper 1 (interferon-γ and IL-12) cytokines are higher in patients with ALL while transforming growth factor β is lower in ALL. These results indicate that the circulating levels of cytokines are elevated in patients with newly diagnosed ALL without apparent infection, reflecting a strong and deregulated inflammatory state in this disease, with a Th1-polarization profile . Since tMany other factors are suggested to modulate the immune function, e.g., the release of HSP72 (by hepatocytes and immune cells) and also the activation of sympathetic corticotropin-releasing hormone (CRH)–histamine axis, both dependent on catecholamine action. The production of eHSP72, for example, is mediated by a noradrenergic stimulation via of α1 receptors in the liver, the main organ responsible for its release, but also by immune cells , 41. TheLow-grade inflammation is well known to induce insulin resistance. Therefore, increased cytokine levels, along with eHSP72 may induce to oxidative stress pathways activation in plasma and several tissues that, in the case of skeletal muscle, may blunt the insulin signaling. Recent data from our laboratory showed that the levels of intracellular HSP72 (iHSP72) is positively correlated with insulin signaling (sensitivity), while the eHSP72 may induce inflammation and insulin resistance , 41, 42.Summarizing our data, we found that induction phase of chemotherapy in lower age children leads to a drop in glucose levels, improvements on inflammation and oxidative stress markers, reduction on eHSP72 and improved insulin sensitivity  and activation of immune system, inducing pro-inflammatory pathways activation and eHSP72 release. eHSP72, along with other cytokines and their stimulation on the ROS/RNS production will induce skeletal (and other tissues) insulin resistance, that together may explain hyperglycemia prior to the treatment. Induction drugs used on chemotherapy will decrease inflammation  between March and December 2015. Exclusion criteria were metastasis, Down´s syndrome, hypothyroidism, presence of other chronic disease or having started chemotherapy. In total, nineteen consecutively patients were eligible for inclusion. Patients were evaluated in the beginning (before chemotherapy - “before induction” and at the end of the induction phase (28 days after - “after induction”). The goal of induction chemotherapy is to achieve a remission. Independent of risk, children received following chemotherapy drugs: L-asparaginase, methotrexate, prednisone, vincristine and daunorubicin. Ethical approval was obtained from Ethics Committee of the Federal University of Rio Grande do Norte (protocol 976.388).® weighting scale and recorded to the nearest 0.1 kg. Standing height was measured using a calibrated stadiometer for infants (Filizola®), and recorded to the nearest 0.1 cm. Body mass index-for-age were calculated according to World Health Organization [Body weight was measured using a Filizolanization .Venous blood samples were obtained during the morning hours after an overnight fast. Glycaemia, blood cell count, hematocrit, leukocytes, lymphocytes and platelet counts, plasma urea, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, sodium, potassium and calcium were determined by colorimetric enzymatic assay. All measurements were performed at the hospital as part of the routine in the clinical treatment.®, Assay Designs Cortisol ELISA® kit, Labtest). Insulin resistance was calculated by the homeostasis model assessment of insulin resistance (HOMA-IR) and β-cell function by using HOMA-β [C-reactive protein (CRP) was determined by nephelometry . Insulin (UI/mL) and cortisol (μg/dL) were analyzed by chemiluminescence  were calculated as appropriate, and compared with independent t-test between sexes. There were missing data for eHSP72. Therefore, methods of multiple imputation data were applied, which follow three main steps: a) imputation of missing data to obtain complete databases; b) estimation based on n complete databases, and c) a combination of methods. Data were assumed to be missing at random [Data were analyzed by SPSS, v.22 . t random . ResultsWe found that induction phase of chemotherapy, in lower age children, lead to a drop in glucose levels, improvements on inflammation and oxidative stress markers, reduction on eHSP72 and improved insulin sensitivity. Our results indicate a possible key involvement of eHSP72 as an immune and metabolic controller that may be used as a marker of the treatment success and metabolic changes. However, the pattern of change in our variables  needs to be measured, during and after the other stages of the disease and chemotherapy treatment, and with a larger patient number.


Head and neck Synovial sarcoma (SS) accounts for 3–10% of all total body SS. It is rare to find it in the oral cavity, especially on the floor of the mouth.We present a 44-year-old Chinese male, who had been misdiagnosed as fibroadenoma, with a swelling on the right submandibular region for more than 3 months. The radiology examinations and the pathology results indicate the diagnosis of SS of the floor of the mouth.The patient only had a surgical operation, without radiotherapy and chemotherapy. At the first follow-up, the patient exhibited no clinical or radiographic complications, and the patient was asymptomatic on subsequent visits.Misdiagnosis results the delay of diagnosis and treatment of SS. Immunohistological analysis might be the most important tool to confirm the diagnosis of SS. Synovial sarcoma (SS) is a rare malignant neoplasm of unknown histological origin, accounting for 5.6% ~ 10% of all soft tissue sarcomas .. Most sThe tumor usually occurs in close association with tendon sheaths, bursae, and joint capsules, primarily in the para-articular regions of the extremities. Head and neck SS accounts for 3–10% of all total body SS, with high incidences in the hypopharynx, the postpharyngeal region, and the parapharyngeal space , 6. It’sA 45-year-old male reported that he had a swelling approximately 5.2 × 2.8 × 5.9 cm on the right submandibular region for more than 2 months and was admitted to the local hospital. At the local hospital, the diagnosis of fibroadenoma on the floor of mouth was made. An incisional biopsy was performed under guidance of ultrasound and was submitted for histopathologic examination, the examination revealed that expansion of the lymphatic vessels could be seen in the right sublingual. After an intra-oral excision in the floor of mouth of the right submandibular region (Inferior to the mylohyoid), the immunohistochemistry showed Vimentin was positive, Ki-67 percentage was about 35%, CD34, S-100, CK, P63 and LCA were negative. After discharged from that hospital, he felt that the swelling had grown again in the same area, even rapidly in the near week.One month after the first operation at local hospital, the patient was referred to our hospital for a growth nodule on the floor of the mouth, associated with pain, numbness, and dyspnea. Intraoral examination revealed a proliferative and ulcerated mass measuring approximately 6.0 × 1.0 cm in the right sublingual involving the right floor of the mouth extending from the alveolus of the left mandibular cuspid to the right mandibular 2nd molar teeth and extending anteriorly crossing the midline of the tongue. The mass was firm, with an unclear border, and the surface covered with black pseudomembranous Fig. . The immThe surgical treatment included primary tumor resection, cervical lymph node dissection, and anterolateral musculocutaneous flap reconstruction Figs. , 5, 6, 7Within 1 year after surgery, the patient was seen in our out-patient clinic every month to check the recovery and whether there was recurrence or lymph node metastasis. Six months later, the first radiographic postoperative follow-up showed no recurrence. One year after surgery, PET-CT examination was performed, and the patient had no recurrence and distant metastasis. Then the patient was followed up at six monthly intervals and is still alive with no evidence of recurrence after 46 months since undergoing curative intent surgery.Synovial sarcoma (SS) was first documented by Simon in 1865  and was SS usually occurs in the lower limbs and trunk, especially in the periarticular soft tissues . Head anFor tumors that have recurred multiple times or diameter > 5 cm, the growth rate tends to accelerate and may cause an emergency. In this case, the patient’s tumor rapidly increased with surface bleeding after admitted to our hospital. Moreover, he even suffered from severe dyspnea due to the tumor and clot blocking the airway. According to the postoperative tumor anatomy, the central part of the tumor showed liquefaction necrosis with some dark brown liquid Fig. , which mIn our case, the patient was previously diagnosed as fibroadenoma because of the rarity of SS and lack of typical clinical and imaging manifestations. Although Pantomography, CT, MRI, and PET-CT can be used as diagnostic tools, smaller SSs often show similar imaging features to benign tumors on CT and MR imaging , 17. DesSynovial sarcoma can be classified into three major histopathological subtypes: 1) monophasic SS containing uniform spindle cells or epithelial cells, 2) biphasic SS composed of epithelial cells arranged into glandular structures with spindle cells arranged into fascicles, 3) poorly differentiated SS characterized by the presence of spindle and/or round blue cells . In histThe other cause of misdiagnosis might be the previous immunohistochemistry lacked some key immune marker detection such as EMA, CD99, TLE-1, and Bcl-2, etc. So far, there is no single immunological marker specific to synovial sarcoma has been found. SS shows positive of TLE-1, AE1 / AE3, EMA, CK7, CK19, Vimentin, Bcl-2, CD99, and S-100 while generally shows negative express of CD34, CD31, actin (HHF-35) or Myoglobin , 21. SomFor some cases with atypical histological morphology or confusing results of immunohistochemistry, detection of SYT-SSX fusion gene by molecular biology or cytogenetic technology can be used to help diagnose with synovial sarcoma . It was The optimal approach to the treatment of SS is still unclear and there is no standard treatment protocol . It is rIn our case, chemotherapy was not performed. The importance of chemotherapy has not been widely acknowledged due to the lack of evidence that chemotherapy was associated with improved overall survival. According to the Yang’s report , after oThe prognosis of synovial sarcoma is often associated with tumor location, size, patients’ age, surgical procedure, degree of differentiation . GeneralIn this case, as the rarity SS tumor, the patient was misdiagnosed at the local hospital. Immunohistological analysis might be the most important tool to confirm the diagnosis of SS. Surgical treatment focus on primary tumor resection without chemotherapy might be considered according to the patient’s situation.


The primary outcome was the body mass index (BMI). After treatment, metformin group had a mean decrease in BMI of 1.46 ± 0.14 (p < 0.001) and insulin resistance index (IRI) of 4.30 ± 2.02 (p < 0.001). The betahistine group had no significant alteration in BMI or IRI. However, placebo group had a mean increase in BMI of 1.27 ± 0.77 (p < 0.001) and IRI of 0.45 ± 0.86 (p < 0.001). Between the two treatment groups, metformin significantly decreased weight, BMI, fasting glucose, insulin level, and IRI but not waist circumference when compared with betahistine. Moreover, metformin significantly decreased weight, BMI, waist circumference, fasting glucose, insulin level, and IRI when compared with placebo, whereas betahistine significantly decreased body weight, waist circumference, BMI, insulin level, and IRI but not fasting glucose when compared with placebo. In this study, we found that both metformin treatment and betahistine treatment were efficacious in improving antipsychotic-induced weight gain and insulin resistance, and metformin was more efficacious in preventing and revising the weight gain induced by antipsychotics.Antipsychotic-induced weight gain is one of the most common adverse effects of antipsychotic treatment. However, there are no well-established interventions for the weight gain yet. In this study, we pooled the data from two clinical trials, which were originally examining the efficacy of betahistine and the efficacy of metformin in treating antipsychotic-induced weight gain and insulin resistance. A total of 67 people with schizophrenia or bipolar disorder treated with antipsychotics were assigned to 36 mg dayClinical Trial Registration:www.ClinicalTrials.gov, NCT00451399(Study 1), NCT00709202(Study 2) Antipsychotics, especially second-generation antipsychotics , could l1 and H3 receptors, has been widely used to treat vertigo symptoms in Meniere's disease. Interestingly, a previous study suggested that histaminergic H1 and H3 receptors are crucial to the potential mechanism of antipsychotic-induced weight gain  was to examine the efficacy of metformin in the treatment of antipsychotic-induced weight gain; the second trial (NCT00709202) originally was to examine the efficacy of betahistine in the treatment of antipsychotic-induced weight gain. The data from STUDY 2 have not been published elsewhere, though the primary outcomes of STUDY 1 have been published before  were eligible for our study , and theFor STUDY 1, the participants were administered with the drug in a double-blind placebo controlled fashion. For the first 4 days, the participants took 250 mg of metformin or placebo at their evening meal, after which a second or third dose was added before breakfast and lunch, respectively, for another 80 days. Each participant was given a record sheet to log the number of trial medications taken daily. Only trihexyphenidyl for extrapyramidal symptoms or lorazepam for insomnia or agitation were allowed as needed. For STUDY 2, the participants took 6 mg of betahistine at their evening meal for the first 2 days after enrollment, then 6 mg of betahistine at lunch and evening meal for the third day, after which 6 mg more of betahistine was added each day till the 11th day. The participants took 18 mg of betahistine at lunch and evening meal at 11th day and for another 73 days. Any antipsychotics taken by the participants before enrollment remained at the same dosage throughout the course of the study.The primary outcome was body mass index (BMI). The secondary outcome was body weight, waist circumference, fasting glucose, insulin level, and insulin resistance index (IRI). To calculate the BMI, weight in kilograms was divided by height in meters squared. To calculate the IRI, insulin level (mIU/L) × fasting glucose (mmol/L)/22.5 was determined, in accordance with homeostasis model assessment .Baseline data included related demographics, a comprehensive medical history, a physical examination with the measurement of weight and height, and a related laboratory test. Research nurses were blind to the type of treatment to perform all assessments. Related laboratory tests included fasting insulin and fasting glucose. Fasting blood samples were confirmed by the patients or their caregivers.Follow-up visits were made at week 12 after starting the treatment, and all baseline evaluations including physical examination, laboratory test, and weight and height measurements were repeated. Serum insulin level was measured with a solid phase radioimmunoassay. Weight and height measurements were taken after removing their shoes and upper garments and donning an examination gown.t-tests, chi-squared analyses, and one-way analysis of variance (ANOVA) as appropriate. Fisher's exact test was used when necessary. For the comparison of the three groups at baseline, ANOVA was used to compare continuous variables with homogeneity of variance, Kruskal-Wallis test was conducted for the variables without homogeneity of variance, and chi-squared analysis was used for categorical variables.All analyses were conducted by using the Statistical Package for Social Sciences, version 23 . Continuous variables were described as mean (SD) and confidence interval. Categorical variables were described by frequencies and percentages. We used post-hoc tests (least significant difference procedure) were conducted to compare between the two groups. For the variables without homogeneity of variance, the ANOVA was conducted after ranking. The difference was considered statistically significant if a two-tailed P-value was < 0.05. The difference between baseline data and 12 weeks data was analyzed by a paired t-test.For the follow-up data were all continuous variables, analysis of covariance (ANCOVA) was the main strategy used when we compared among the three groups, while the corresponding baseline values and those variables found to be significantly different at baseline were regarded as covariates. To compare the difference among the three groups, Demographic and baseline outcomes were compared among the three groups Table . The 25 After 12 weeks of treatment, statistical analysis showed that, in metformin group, the mean body weight and BMI value decreased significantly, while in placebo group, there's a significant increase of body weight and BMI; however, in betahistine group, no significant differences were found in the mean body weight or BMI value Table .At the end point, patients in metformin group had a significant decrease in body weight by 5.79% compared with baseline, while body weight in betahistine group decreased by 1.32% without significance; however, patients in placebo group significantly gained 5.51% of their body weight. Correspondingly, in metformin group, the mean BMI significantly decreased by 1.46, while the mean BMI decreased by 0.33 in betahistine group with no significance, and the mean BMI increased by 1.27 in placebo group significantly.p = 0.058).As shown in Table Over the 12-week period, there was a significant decrease in the values of mean fasting glucose level, insulin level, and IRI in metformin group; however, in betahistine group, no significant change in the value of fasting glucose level, insulin level, or IRI was found compared with baseline, while in placebo group, there was a significant increase in insulin level and IRI but not fasting glucose level Table .There were no significant differences in the frequency and types of adverse events reported among the three groups. There were six serious adverse events that affected more than 5% of the entire sample Table .This study was designed to test the comparative efficacy of metformin and betahistine on preventing further weight gain or causing weight decrease in people with schizophrenia or bipolar disorder who had gained more than 10% weight in the first 3 years of treatment with antipsychotics. To our knowledge, this is the first research to compare metformin and betahistine in treating antipsychotic-induced weight gain. After a 12-week trial, we found that betahistine treatment effectively controlled antipsychotic-induced weight gain, while metformin significantly relieved antipsychotic-induced weight gain. Both metformin and betahistine were found to have a significant advantage when compared with placebo.Current interventions to minimize antipsychotic-induced weight gain and metabolic syndrome include pharmacologic and non-pharmacologic ways . PharmacThe potential clinical effect of betahistine on reducing antipsychotic-induced weight gain and its mechanism has gained more attention in recent years. The histamine system has played a crucial role in the regulation of energy homeostasis , 30, 31.Metformin mainly increases the function of insulin in the liver and decreases the rate of hepatic glucose production . MetformIn addition, our study showed that metformin significantly decreased body weight, BMI, fasting glucose level, insulin level, and IRI but not waist circumference when compared with betahistine. These findings suggested that the treatment with metformin could be more efficacious than betahistine in preventing and reversing the weight gain induced by antipsychotic agents in people with schizophrenia or bipolar disorder, while both treatments were found to have a significant advantage over placebo. Few studies had compared the effect of betahistine and metformin before. According to our study, although betahistine group failed to decrease the body weight significantly, it prevented further weight gain with a decreasing tendency. Therefore, we suggest metformin as the first consideration for antipsychotic-induced weight gain while betahistine as an alternative if metformin was not tolerated or adhered.This study has some limitations. First, the data were collected from two independent studies, thus, the sample error was inevitable and STUDY 2 was not a randomized placebo controlled clinical trial. Second, this study was based on schizophrenia or bipolar disorder participants with four different antipsychotics: clozapine, olanzapine, risperidone, or quetiapine. Previous studies suggested that the type of antipsychotics affects the plasma adiponectin level and also affects body weight significantly , 53. HowIn conclusion, despite these limitations, this study has clearly shown that metformin could be more efficacious than betahistine in increasing insulin sensitivity and reversing the weight gain induced by antipsychotics with 12 weeks of treatment, while both could significantly improve the body weight and insulin sensitivity induced by antipsychotics. We suggest metformin as the first consideration for the treatment while betahistine as an alternative if not tolerated or adhered.RW and ZJ designed and conducted the research. DK analyzed and interpreted data and drafted the article. RW, ZJ, and RL provided critical revision of the article, while GH, TS, ZJ, RL, LL, MS, YY, YW, XW, and YL collected and analyzed the data. XH analyzed the data and drafted the data. RW are responsible for the final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this paper, for the first time, inexpensive waterglass solutions are exploited as a new, simple and ecofriendly chemical approach for promoting the formation of a silica-based coating on hemp fabrics, able to act as a thermal shield and to protect the latter from heat sources. Fourier Transform Infrared (FTIR) and solid-state Nuclear Magnetic Resonance (NMR) analysis confirm the formation of –C–O–Si– covalent bonds between the coating and the cellulosic substrate. The proposed waterglass treatment, which is resistant to washing, seems to be very effective for improving the fire behavior of hemp fabric/epoxy composites, also in combination with ammonium polyphosphate. In particular, the exploitation of hemp surface treatment and Ammonium Polyphosphate (APP) addition to epoxy favors a remarkable decrease of the Heat Release Rate (HRR), Total Heat Release (THR), Total Smoke Release (TSR) and Specific Extinction Area (SEA)  as compared to untreated hemp/epoxy composites, favoring the formation of a very stable char, as also assessed by Thermogravimetric Analysis (TGA). Because of the low interfacial adhesion between the fabrics and the epoxy matrix, the obtained composites show low strength and stiffness; however, the energy absorbed by the material is higher when using treated hemp. The presence of APP in the epoxy matrix does not affect the mechanical behavior of the composites. In the last ten years, the interest in natural fiber-reinforced polymer composites exhibited a significant growth as far as fundamental research and their industrial applications are considered: indeed, these materials are not expensive, fully or partially recyclable and also biodegradable ,2,3,4. AAs compared to synthetic fiber-reinforced composites, the natural fiber-containing counterparts are more environmentally friendly, hence deserving use in a wide range of applications, including transportation , military purposes, building and construction industries , packaging, consumer products, etc. Among the different natural fibers, despite its current limited availability, hemp is particularly raising interest for green composite manufacturing, as it is an inexpensive renewable resource and shows low density, high specific strength with respect to glass or aramid fibers and good biodegradability, as well ,9.Until the early 1900s, industrial hemp was a valuable crop used all over the world for its strong fibers and oil seeds. Indeed, the Yearbook of the United States Department of Agriculture documents well the significant spread of hemp cultivation all over the world in 1913 . By the One of the major drawbacks of the aforementioned composites, including those containing hemp, is their ease of flammability when exposed to a heat flux or a flame source : this beThe natural fiber-reinforced composites, when exposed to fire or any other high-intensity heat source, undergo thermal decomposition and combustion processes according to the adopted experimental conditions. In particular, the time to ignition of the composites and the intensity of combustion process are significantly affected by such parameters as heat and mass transfer from and to the composite material, oxygen concentration and the circulation of gas in the area where combustion takes place. Important issues comprise the rate of flame spread, Heat Release Rate (HRR), mass loss and carbonization rates. The ease of the combustibility of a composite material depends on a number of factors, including the nature of the composite and its components , its density, structure, thermal conductivity, humidity content, and so on.The fire retardancy of composite materials can be enhanced exploiting different strategies. First, it is possible to add different kinds of fire retardants in liquid or solid form during the manufacturing process: as a result, they are embedded within the composite structure. More specifically ,13,14:(1) the natural fibers can be impregnated with a solution of the flame retardant;(2) the flame retardant can be incorporated into the adhesive system ;(3) the fibers can be subjected to a surface treatment;(4) the fibers can be mixed with the flame retardant before the addition of an adhesive.In these conditions, the performed treatments should exhibit an acceptable thermal stability that prevents the activation of the flame retardant during the fabrication step of the composite material. Second, it is possible to apply fire retardant finishing treatments at the end of the fabrication process: this is very often performed when intumescent flame retardant coatings are exploited .The treatment with non-combustible components provides natural fibers or lignocellulosic particles with a fire retardant coating: in these conditions, fire retardants tend to migrate inside the flammable materials, thus resulting in the fire protection of the latter.The different flame retardant additives (5–10 wt % in relation to dry mass) used for lignocellulosic materials comprise ammonium salts of phosphoric acid , zinc chloride, boric acid, salts of sulfuric acid, zinc borate, vermiculite, aluminum hydroxide, magnesium hydroxide, expandable graphite and pentaerythritol (as the carbon source for intumescent systems) ,16,17,18Unlike thermoplastic matrix-natural fiber composites, for which the open scientific literature reports several nice examples ,20,21,22Manfredi et al.  investig2. A significant decrease of the peak of the heat release rate was found for the systems containing aluminum trihydrate: this finding demonstrated that the obtained composites can compete with current building materials in terms of fire behavior.Hapuarachchi and co-workers  assessedChai et al.  comparedVery recently, Szolnoki et al.  preparedIn this paper, we prepared hemp fabric/epoxy composites by using vacuum bag molding. This technique has minimal impact on tool cost and imposes no limits on the part size process for producing large-scale composites; thus, the reduction in laminate flaws to improve part quality can be obtained at a competitive cost.In particular, a new, inexpensive, simple and ecofriendly chemical strategy is exploited to coat hemp fabrics with a silica-based fire retardant layer, obtained by using a waterglass coating prepared in acidic conditions. In parallel, Ammonium Polyphosphate (APP), a very well-known flame retardant, is added to epoxy resin, aiming at assessing the possible joint effects of the flame retardant with the waterglass treatment. Very recently, this additive was used by our group  for confPursuing this research, in this work, we investigate the effect on the concurrent presence of surface-treated hemp fabrics and APP on the fire behavior of the obtained composites, assessed through cone calorimetry tests. The effect of the modification of hemp fabrics with the waterglass treatment is assessed through Scanning Electron Microscope (SEM), Fourier Transform Infrared (FTIR) spectroscopy and solid-state Nuclear Magnetic Resonance (NMR). Furthermore, Thermogravimetric Analysis (TGA) performed in nitrogen are exploited for assessing the thermal stability of both the waterglass-treated hemp fabrics and of their epoxy composites.2, supplied by MAEKO S.r.l., Milan, Italy) and a two-component epoxy resin system  were used for fabricating composite laminates. The two components are reported by the supplier to be modified bisphenol A resin and modified cycloaliphatic polyamines. The epoxy resin was added with APP particles (white free-flowing powder) supplied by Tecnosintesi S.p.A. .Sodium metasilicate and hydrochloric acid (37% ACS) reagents were purchased from Sigma Aldrich . Plain weave hemp fabrics  to the laminate volume. Four different composites were produced by infiltrating hemp fabric sheets or treated hemp fabric sheets with epoxy or APP-charged epoxy. The obtained samples are coded as reported in After impregnation, in order to increase the volumetric percent of reinforcement and to eliminate entrapped air, the laminates were enclosed in a vacuum bag, and polymerization was accomplished through a 48-h cure cycle at room temperature. The 3 mm-thick laminate contains 25 wt % of reinforcing agent; the 22 vol % was obtained as the ratio of the hemp fiber volume .FTIR transmittance spectra were recorded with a Nikolet 5700 FTIR spectrometer  using a single reflection Attenuated Total Reflectance (ATR) accessory with a resolution of 4 cmSEM images of samples were obtained on a Leica Stereoscan 440 Microscope (20 kV) , equipped with an energy Dispersive Analytical System (EDS) from Inca Energy 200, by using AZtecEnergy EDS Software .A digital Optical Microscope (OM), HIROX , was employed to observe the morphology of the fractured composites in bending tests (see below). The observations were carried out at room temperature and by using KH-8700 Software .TGA were performed using a Netzsch TG209  apparatus, from room temperature up to 800 °C, at a heating rate of 10 °C/min under a nitrogen atmosphere  and by using Proteus Software .29Si and 13C frequencies of 59.62 and 75.47 MHz, respectively. Samples of Hemp (H) and Treated Hemp (HT) (80 ± 1 mg) were loaded into 4-mm zirconia rotors, closed with Kel-F caps and spun at a rate of 10,000 ± 1 Hz. 13C NMR spectra were acquired by applying a cross polarization technique and consisted of 1814 time domain points, a spectral width of 300 ppm , a recycle delay of 2 s, 4000 scans and 1 ms of contact time. The 13C CPMAS pulse sequence was conducted by using a 1H Ramp pulse to account for the non-homogeneity of the Hartmann-Hahn condition. 29Si NMR spectra were acquired by using a direct polarization and consisted of 2048 time domain points, a spectral width of 500 ppm , 40 s of recycle delay and 2730 scans.NMR spectra were acquired with a 300-MHz (7.0 Tesla) Bruker Avance magnet , composed of a wide-bore system and equipped with a CPMAS (Cross-Polarization Magic-Angle-Spinning) probe, working at 29Si and 13C spectra were Fourier transformed by applying a two- and four-fold zero-filling and adopting an exponential filter function with a line broadening of 350 and 50 Hz, respectively.Free Induction Decays (FIDs) were processed by BrukerTopSpin  and MestreNOVA  Software. Prior to be phase and baseline correction, 3), with a heat flux of 35 kW/m2, in horizontal configuration. Time To Ignition , Total Heat Release , peak of the Heat Release Rate  were measured. Total Smoke Release , carbon monoxide yield , carbon dioxide yield  and Specific Extinction Area  were evaluated, as well. For each sample, the experiments were repeated at least three times in order to ensure reproducible and significant data.To investigate the combustion behavior of the aforementioned materials subjected to a constant heat flux, cone calorimeter tests  were performed following the ISO 5660 standard, by using squared samples  Alliance RT/50 universal testing machine in stroke control, setting the crosshead speed at 1 mm/min in accordance with the ASTM 790M standard and by using MTS TestWorks Software . The tests were performed on specimens of 60 × 25 × 3 mmThe washing fastness of the treated hemp fabrics was carried out with a solution containing 4 g/L commercial detergent; the liquor ratio was 50:1. Each wash was performed at 40 °C for 5 min. After each washing cycle, the fabric was removed, gently squeezed and rinsed with tap water. Then, repeated washing was carried out until a total of 250 min was reached.−1 may be assigned to the stretching of the –Si–O–cellulose and –Si–O–Si– bonds, respectively [ectively ,28.Therefore, a silica-based coating probably anchored to the hemp substrate through the formation of covalent bonds should have been formed. This finding may be ascribed to the low pH value (pH = 2.5) of the acidified waterglass solution: indeed, it is well known that when silicates are dissolved in acidic solutions, the formation of silicic acid would be expected, according to the following equation: However, as soon as the reaction proceeds, the molecular units become larger, slowly giving rise to thickening phenomena that finally lead to the formation of a gel ,30,31. T(1) formation of the particles from the precursors;(2) subsequent growth of the particles;(3) creation of links among the particles, which give rise to chains and networks extending throughout the liquid medium.Harris and coworkers  hypothesThe pH = 2.5 of the acidified waterglass solution, which is slightly higher than the silica isoelectric point (2.0–2.5), but lower than that reported for hemp (>3.0) ,34,35, iTherefore, the formation of a silica-based coating anchored to the hemp surface through covalent bonds is reasonably supported. A mass increase of 5 wt % was observed after the hemp fabric treatment described in the Materials and Methods section. In order to further confirm this hypothesis, washing fastness tests have been performed according to the procedure described elsewhere : the sil13C spectrum of the H sample  Stage I (between 300 and 400 °C): this involves two competing pathways that yield aliphatic char and volatiles.(2) Stage II (between 400 and 800 °C): some of the aliphatic char converts into an aromatic form.According to the reported scheme, the TG curves recorded in an inert atmosphere  show one5% and T10% values of It is worthy to note that the waterglass treatment, because of the acidic characteristics of the deposited coating, anticipates the thermal degradation of the cellulosic fibers ; pkHRR (kW/m2); THR (MJ/m2); TSR (m2/m2); mass residue at the end of the cone calorimeter test; SEA(m2/kg); carbon monoxide and dioxide yields (kg/kg).The first two lines refer to untreated (H) and waterglass-treated (HT) fabrics. It is worthy to note that the waterglass coating, despite an anticipation of the ignition of the fabric, is responsible for a slight decrease of HRR, pkHRR and THR and for a limited increase of the final residue, as well, hence further showing its protective effect exerted on the underlying fabric.2 . A similar trend is observed as far as pkHRR is considered: its decrease is as much as −14.9% . On the other hand, THR does not seem to be affected by the treatment of the fabrics with the waterglass coating, while the latter shows a detrimental effect on TTI, which is reduced from 55 down to 39 s.Conversely, the hemp fabric surface treatment seems to be very effective in improving the fire behavior of the prepared composites. Indeed, the presence of the waterglass coating significantly reduces the HRR by 35%, namely from 402 down to 260 kW/mA significant role is played by APP: indeed, this flame retardant additive, despite a reduction of TTI, favors a further remarkable decrease of HRR , pkHRR  and THR , with respect to the unfilled composite counterparts .In addition, the waterglass coating, also in combination with APP, seems to limit the smoke formation of the epoxy composites: in particular, APP turns out to significantly decrease both TSR and SEA parameters. It is noteworthy that the phosphorus additive seems to be more effective when added to the composites where hemp fabrics have not been subjected to the treatment with waterglass. As an example, TSR is decreased by 58.4% and SEA by 46.4%, as well, when 15% of APP is added to the composite (compare the H/E and H/E-15APP samples).The very high residues after cone calorimetry tests for H/E-15APP and HT/E-15APP  and in plane (100×) view, are shown in As shown in Furthermore, the presence of APP in the epoxy matrix does not seem to affect the overall mechanical behavior of the obtained composites.In this paper, hemp fabrics have been treated with a new, inexpensive, simple and environmentally-friendly silica-based coating, able to protect the fabrics from heat sources, hence improving their fire behavior when utilized as reinforcing agents in epoxy-based composites. To the best knowledge of the authors, this is the first time that inexpensive waterglass solutions were exploited for this purpose: indeed, the proposed approach can be considered as an application of sol-gel chemistry using a precursor that does not need hydrolysis and is performed in water solutions, avoiding the organic solvents usually required by the alkoxy precursors. FTIR and solid-state NMR spectroscopies have confirmed the formation of –C–O–Si– covalent bonds in between the waterglass silica coating and the underlying fabrics.The waterglass treatment, which is resistant to washing, seems to be very effective at improving the fire behavior of hemp fabric/epoxy composites, also in combination with ammonium polyphosphate. In particular, the concurrent presence of hemp surface treatment and APP significantly improves such cone parameters as HRR, THR, TSR and SEA, which turn out to decrease respectively by 83%, 35%, 45% and 44% as compared to untreated hemp/epoxy composites. At the same time, the formation of a very stable char is promoted, as also assessed by TG analysis performed in an inert atmosphere.Conversely, the low interfacial adhesion between the fibers and the epoxy matrix promotes a brittle behavior of the composites, which show a slightly lower stiffness as compared to the theoretical one. However, the fracture energy absorbed by the material reinforced by the waterglass-treated silica coating is higher. Finally, the presence of APP in the epoxy matrix does not seem to affect the mechanical behavior of the obtained composites.


Psoriasis is considered to result from the interaction of genetic factors and environmental exposure. The evidence for familial aggregation in psoriasis has been reported but population-based studies related to the magnitude of genetic contribution to psoriasis are rare. This study aimed to evaluate the relative risks of psoriasis in individuals with affected relatives and to calculate the proportion of genetic, shared, and non-shared environmental factors contributing to psoriasis. The study cohort included 69,828 patients diagnosed with psoriasis enrolled in National health Insurance in 2010. The adjusted relative risks (RR) for individuals with an affected first-degree relative and affected second-degree relative were 5.50 , 5.19–5.82) and 2.54  respectively. For those who have affected first-degree relatives, their RR was 1.45  for Sjogren’s syndrome and 1.94  for systemic sclerosis. This nationwide study ascertains that family history of psoriasis is a risk factor for psoriasis. Individuals with relatives affected by psoriasis have higher risks of developing some autoimmune diseases. Psoriasis is a chronic inflammatory cutaneous disorder that results from dysregulated immune reaction mediated by immune cells and keratinocytes ,2. The pStrong evidence has suggested familial clustering in psoriasis. Twin studies reported higher concordance rates for monozygotic twins of 0.20–0.73 compared with 0.09–0.2 for dizygotic twins, and heritability estimates ranging between 66 and 90% ,11,12. EThis population-based study used a nationwide genealogy reconstruction technique to identify family members of over 20 million people. The individual-level family relationships were linked to health information obtained from Taiwan’s National Health Insurance (NHI) Database, which contains phenotype data regarding health and diseases in essentially the whole population of Taiwan. The aims of this study were to estimate the relative risks (RRs) of psoriasis in individuals with affected relatives and to calculate the proportion of genetic, shared and non-shared environmental factors contributing to psoriasis. In addition, we also examine the RRs of other autoimmune diseases associated with a family history of psoriasis. This study was approved by the Institutional Review Board of the Chang Gung Memorial Hospital (104-9794B). We obtained the medical information of study subjects from the Taiwan NHI system, which was established in 1995 and provides health insurance to over 99% of population in Taiwan . The NHIThe methods of identifying relatives and genealogy reconstruction were described in our previous studies ,22,23. TThe identification of siblings was based on sharing one or more common parents. If a couple did not have offspring, they were not assigned to the same family because they were not related based on a common blood relative. Twins were full siblings who had the same birth date (±1 day) but their zygosity could not be identified from the database.The registry of beneficiaries was renewed biannually. All the changes including employment, residence, insurance status (insurant or dependent), and relationships between insurant and dependents could incur an individual record in the registry. We used this dynamic nature to maximize the identification of possible family link by incorporating the entire registry records from 1995 to 2010.We then used the previous links to build pedigrees. We defined a family as a cluster of individuals who were related to each other by blood or by at least one common blood relative. Those subjects without any identified parent were considered founders. We established a pedigree for each founder and then pedigrees were linked if they had common descendants. It should be understood that spouses without a child were not classified into one pedigree. We constructed 4,229,301 families encompassing 21,009,551 parent-child relationships; 17,168,340 full sibling pairs; and 342,066 twin pairs.The case definition of incident psoriasis was based on dermatologist or rheumatologist diagnosis  codes: 696.1). To ensure the validity, only patients with at least three records in outpatient or inpatient care in 2010 were included in this study. We further identified patients with autoimmune diseases by the specific codes from the NHI database including the following: rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis, dermatomyositis, polymyositis, Sjögren’s syndrome, multiple sclerosis, and inflammatory bowel diseases.We adjusted covariates that might confound the familial association including age, gender, socioeconomic status , occupation and family size. The income level was calculated approximately based on individual’s insurance premium categories and ranked into gender-specific income quartiles. Occupation is classified into four categories: (1) public servants, teachers, military staff, veterans and their family members; (2) non-manual workers and professionals; (3) manual workers; and (4) others. The residential location for each individual was categorized based on its level of urbanization . We defined an individual as a prevalence case when he/she registered in NHI in 2010 and met the case definition of psoriasis between 1 January 1996 and 31 December 2010. The prevalence of psoriasis was estimated for both the general population and individuals with a family history. The relative risks (RRs) of psoriasis calculated in the present study were relative recurrence risks, which were determined as the prevalence of psoriasis among individuals with a specific type of affected relative, divided by the prevalence of psoriasis in the general population ,26,27. IHeritability is the proportion of phenotypic variance contributed by genetic factors, while familial additionally considered shared environmental factors. Both measures were calculated using the polygenic liability model ,29,30,31p-values less than or equal to 0.05 were considered significant. The statistical analysis was performed by SAS v.9.3 .Furthermore, we calculated the degree of familial co-aggregation of other autoimmune diseases in families affected by psoriasis using a marginal Cox proportional hazards regression model with an equal follow-up period for all individuals. RRs and 95% CI for rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, dermatomyositis, polymyositis, Sjögren’s syndrome, Behcet’s disease, inflammatory bowel disease, and vasculitis were calculated as adjusted prevalence ratio of specific autoimmune diseases between subjects with a first-degree relative affected by psoriasis and general population. RRs were adjusted for age, gender, family size, and socioeconomic status. Two-tailed The study population included 22,974,772 individuals enrolled in National Health Insurance in Taiwan in 2010. There were 69,828 patients who had a diagnosis of psoriasis in the general population, giving a crude prevalence of 0.30% with a prevalence of 0.22% for women and 0.39% for men . In the Prevalence (recurrence risk) of psoriasis in individuals with affected first-degree and second-degree relatives categorized by specific types and gender are shown in In comparison with the general population, individuals with one type of affected first-degree relative had a RR of 5.19  and those with two or more had a RR of 27.42 (22.07–34.08) for psoriasis.The RRs of psoriasis were 63.19  in individuals with an affected co-twin, 7.85  with affected siblings, 5.16  with affected offspring, 5.05  with affected parents, 2.85 (2.17–3.75) with affected grandchildren, 2.63  with one affected niece or nephew, 2.43  with one affected grandparent, 2.19  with one aunt or uncle. Overall, the tetrachoric correlation coefficients for first-degrees relatives, full siblings and spouse were 0.24 , 0.31 , and 0.16  respectively . The accThe relative risks of autoimmune diseases other than psoriasis in individuals with first-degree or second-degree relatives affected with psoriasis compared to general population were shown in This is the first nationwide population-based study to investigate the risk of psoriasis and other autoimmune diseases in individuals with affected first-degree or second-degree relatives. We found a 5.5-fold and 2.5-fold increased risk of psoriasis in individuals who have affected first-degree and second-degree relatives than general population, suggesting the association between genetic distance and the risk of psoriasis. Even though familial factors contributed to half of phenotypic variance with one-fourth of total variance attributing to genetic factors, more than 80% of patients were sporadic cases. Moreover, individuals with a family history of psoriasis are at increased risk of having RA, Sjögren’s syndrome, SLE, and systemic sclerosis, suggesting common genetic roots shared between psoriasis and these autoimmune diseases. Psoriasis is a common, chronic, immune-mediated skin condition, which is currently thought to result from the interaction of genetic factors and environmental exposures . SubstanSo far, GWASs have identified 86 susceptibility loci that are associated with psoriasis in European-origin and Chinese populations . HoweverOur study noted that individuals with a family history of psoriasis had higher risk for psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, and systemic sclerosis and uveitis. Previous evidence for familial co-aggregation between psoriasis and other autoimmune disease is rare. Nevertheless, one nationwide cohort study revealed first-degree relatives of multiple sclerosis patients were at increased risk of psoriasis  and multThere are some limitations for this present study. First, the definition of cases based on the diagnosis recorded in the registry of NHI database, lacking clinical presentation and severity findings. However, we only included cases diagnosed by experts including dermatologists and rheumatologist; therefore the case definition was rigorous and unlikely to affect the results. Second, we used spouse as a control to estimate heritability, which we might have underestimated due to the effect of assortative mating. However, Heun et al. has found assortative mating can only be of minor relevance in family studies . In addiIn conclusion, this nationwide study ascertains that family history of psoriasis is a risk factor for psoriasis and the effect of environmental factors contributing to this disease was stronger than previously expected. Individuals with relatives affected by psoriasis have higher risk to develop some autoimmune diseases. These results are informative for patient education and counseling as well as future familial studies for psoriasis.


Bifidobacterium with adhesion property (CMB1), the other combined multiple strains of Bifidobacterium without adhesion property (CMB2). It was found that CMB1 can alleviate constipation more efficiently by improving the water, propionate and butyrate content in feces, and overall gastrointestinal transit time. Meanwhile, from the perspective of fecal microbiota, CMB1 alleviated constipation mainly by increasing the relative abundances of genera  associated with rapid bowel movement. From the perspective of cecal microbiota, CMB1 alleviated constipation mainly by increasing the relative abundances of genera Lactobacillus, Bacteroides, unclassified S24-7, Dorea, Ruminococcus, Coprococcus, and Rikenella, and decreasing the relative abundances of genera Oscillospira, Odoribacter and Unclassified F16, which are associated with methane production and colonic transit. Overall, changes of microbiota in caecum by CMB1 reflect the stage of constipation in mice more comprehensively than that in feces.Constipation, which seriously affects living quality of people, is a common gastrointestinal disease. The engagement of the intestinal flora in the development of symptoms of constipation has been frequently hypothesized. In this study, constipated mice induced by loperamide were used to investige the alleviation of constipation by Bifidobacteria. Bifidobacteria was sorted out according to their adhesive properties into two groups. One group combined multiple strains of  Bifidobacterium or Lactobacillus and an increase in pathogens  worldwide and complicated etiology . Its cliathogens . Supplemathogens  or lactoathogens , either athogens .Bifidobacterium might be related to the adhesion property of the strains  neurotransmitters, motilin (MTL), gastrin (Gas), substance P (SP), endothelin (ET-1), somatostatin (SS) and vasoactive intestinal peptide (VIP) were purchased from Wen LE Bioengineering Institute .Loperamide was dissolved in sterile water and its ultimate density was 1 mg/mL. For acticarbon solution, gum arabic 100 g and water 800 mL were boiled until the solution was transparent. Acticarbon 50 g was then added and boiled three times. After cooling, the solution was diluted with water to 1000 mL and stored at 4°C. Shake well before use.Bifidobacterium strains from five species obtained from American Type Culture Collection (ATCC) or China General Microbiological Culture Collection (CGMCC) were stored at the Culture Collection of Food Microorganisms in Jiangnan University . All the strains were cultured under anaerobic conditions for 24–48 h at 37°C in modified MRS (cMRS) broth supplemented with 0.05% w/v L-cysteine-HCl (Merck). To prepare active cultures for all experiments, all strains were consecutively reactivated in an anaerobic atmosphere at least three times using 3% (v/v) inoculum in cMRS broth at 37°C for 24–48 h before use. To use these strains in the animal experiments, the bacterial culture was centrifuged at 5000 × g for 10 min, washed twice with PBS, and centrifuged again to obtain the bacteria. The bacteria were divided into two groups (CMB1 and CMB2) of five based on their adhesion properties  and performed at the Experimental Animal Center of the Jiangnan University [License No. SYXK(SU)2016-0045].Eight-week-old male BALB/c mice were obtained from Shanghai Laboratory Animal Center . The mice were kept in polyvinyl chloride (PVC) cages under environmentally controlled conditions with a 12-h light-dark cycle and standard commercial mouse feed and water were provided n = 8): normal , control (constipation mice without treatment), CMB1 (constipation mice treated with CMB with adhesion properties) and CMB2 (constipation mice treated with CMB with no adhesion properties).To examine the preventive effects of CMB on constipation, 32 mice were used after a week-long adaptive period. The mice were randomly separated into four groups  intragastrically from day 15 to day 17 to induce constipation  using intragastric administration once a day for 17 days. The CMB1 and CMB2 groups were intragastrically administered 0.25 mL of normal saline solution containing 4 × 10tipation . ChangesThe relative indices of constipation , stool collection and SCFAs in feces were measured as previously described . Feces wThe GI neurotransmitters levels in the serum were determined by an enzyme-linked immunosorbent assay (ELISA) instrument according to the manufacturer’s instructions . The experiments mainly include the preparation of standard curve and the detection of sample absorption value (OD450) according to the standard curve in units of ng/L.The enteric microorganisms in the fecal and caecal samples were measured using a metagenomics method . Microbi1. The representative sequences for each OTU were aligned to identify the species using PyNAST in QIIME. Rarefaction curves for alpha diversity were generated to assess the efficiency of the sequencing depth for representing and comparing microbial communities. Species richness was estimated using Chao-1 . With the intake of loperamide, the fecal water content showed a downward trend in all groups, which indicated that loperamide induced constipation in mice.Before constipation was induced, the water content of the feces was higher in CMB1, and CMB2 groups than in the control and normal groups (p < 0.05). Meanwhile, there was no statistical difference between the CMB2 and control groups. These results demonstrate that CMB2 had no effect on constipation induced by loperamide. There was no significant difference in weight gain, feed and water consumption between the mice throughout the experiment. This indicated that constipation had no effect on the weight and appetite of mice.Compared with the constipation group, the symptoms of constipation  were significantly improved in CMB1 group (p < 0.05). In CMB1-treated group, the Bifidobacteria combination showed preferable effect in relieving constipation. These results indicated that the difference in relieving constipation between CMB1 and CMB2 may be related to the levels of Gas and SP in serum.Constipation-related neurotransmitters, such as MTL, Gas, SP, ET-1, SS and VIP, play an important role in regulating gastrointestinal motility. In addition, the secretion of GI neurotransmitters is different in disease and normal state. Therefore, we studied these GI neurotransmitters in serum of mice after treated with CMB. The results showed that CMB1 obviously increased the levels of MTL, Gas and SP and decreased the levels of SS, VIP and ET-1. The variation tendency of GI neurotransmitters in CMB2 group were the same as that in CMB1 group, except for Gas and SP  classifier. Changes in the taxa that were obviously different between treatments are shown in Bifidobacterium and Adlercreutzia) were included in this phylum. At the genus level, the level of Bifidobacterium in CMB groups were significantly higher than in constipation group. There was no significant difference among groups in the level of Adlercreutzia (Bifidobacterium).Two genera .Seven genera Mucispirillum. There was no significant difference among groups in the level of Mucispirillum.This phylum included a single genus, Staphylococcus, Lactobacillus, Lactococcus, Streptococcus, Clostridium, Eubacterium, Blautia, Dorea, Oscillibacter, Ruminococcus, Sporobacter, Coprobacillus, Turicibacter and so on) were included in this phylum. At the genus level, the relative abundance of unclassified Lachnospiraceae, Clostridium, Sporobacter, Coprobacillus, Turicibacter, Oscillibacter and Ruminococcus decreased significantly in CMB groups compared to control group. Meanwhile, the relative abundance of Clostridium and Sporobacter were higher in CMB2 treatment group than that in CMB1 group. In contrast, the relative abundance of Lactobacillus was the highest in CMB1 treatment group, followed by normal and CMB2 groups and then the constipation control group .Akkermansia was the only genus found in this phylum. Its level was only increased in CMB1 treatment group, whereas there was no significant difference among other groups.Bifidobacterium, Lactobacillus, Akkermansia, Prevotella and unclassified Helicobacteraceae significantly increased and the levels of Clostridium and Anaeroplasma in feces samples decreased compared to CMB2-treated group. Moreover, the levels of Bifidobacterium, Lactobacillus, Akkermansia and Prevotella were negatively correlated with constipation, while Clostridium and Anaeroplasma were positively correlated with constipation. Therefore, after CMB1 intervention, the gut microbiota in constipated mice tended to be beneficial to host health.In brief, in CMB1-treated group, the relative abundances of Bifidobacterium combinations showed a certain overlap in the caecal microbiota regulation.A dataset containing 373,216 high-quality sortable 16S rDNA gene orders was produced from 32 fecal specimens using MiSeq sequencing. The average sequence read was 11,663 per sample. Representative sequences of all of the sequences were clustered, and a 97% sequence similarity cut-off was used. The number of OTUs per sample ranged between 412 and 1330. We performed association tests based on α- and β- diversity measures. The normal and constipation control groups had obvious differences in α- diversity based on species richness, the observed species and diversity . After CA total of 29,193 sequences were assigned to 1,396 OTUs that were clustered into 54 genera and 9 phyla using the RDP classifier. Changes in taxa are shown in Figures 6Adlercreutzia only increased in control group, whereas there was no significant difference among other groups. The relative abundance of Bifidobacterium was the highest in CMB2 treatment group, followed by CMB1 treatment group and then normal and control groups (Bifidobacterium).Two genera were found in this phylum, Bacteroides was the highest in CMB1 treatment group, followed by CMB2 and then the normal and control groups. The levels of Parabacteroides only increased significantly in control group, whereas there was no significant difference among other groups. The level of Rikenella was the highest in CMB1 group and the lowest in CMB2 treatment group, whereas there was no significant difference between normal and control group. The relative abundance of unclassified S24-7 was the highest in CMB1 treatment group, followed by CMB2 and control groups and then the normal group. The relative abundance of Odoribacter recovered to normal level after treated with CMB2, whereas there was no significant difference between CMB1 and control group. There was no significant difference among groups in the level of other genera  were found in this phylum. The abundances of Dehalobacterium, Dorea, Coprococcus, and Ruminococcus were significantly higher in CMB1group compared to CMB2 group, whereas the abundance of Oscillospira was obviously lower in CMB1group compared to CMB2 group. The relative abundance of Lactobacillus, and Clostridium increased significantly in CMB groups compared to control group. The relative abundance of unclassified Lachnospiraceae decreased significantly in all treatment groups, except the normal group. And there was no significant difference among groups in the level of other genera , immune system, intestinal microbiota and their metabolites. Any disorder or dysfunction in any of these factors can lead to intestinal motility disorders (constipation or diarrhea). Therefore, in this study, GI neurotransmitters levels were measured to determine the effect of Bifidobacteria on constipation-related intestinal nervous activities. The results showed that the reduced GI neurotransmitters in serum of constipation mice tended to be up-regulated in CMB1 intervention group, while CMB2 intervention had no effect on recovery of neurotransmitters . Thus, CMB1 may alleviate constipation by up-regulating neurotransmitters . Considering the adhesion properties of Prevotella and Lactobacillus in feces samples treated with CMB1, and increase in unclassified S24-7, Rikenella, Bacteroides, Dorea, Lactobacillus, Dehalobacterium, Desulfovibrio, Ruminococcus and Coprococcus in caecal content samples treated with CMB1. It has been widely reported that either Prevotella or Bacteroides dominates the human gut and they were proposed to be antagonistic . It also can be seen from the results that the ratio of Firmicutes to Bacteroidetes increased significantly in feces samples of CMB1-treated group (from 0.89 in control group to 1.55 in CMB1group). A higher ratio of Firmicutes to Bacteroidetes is associated with faster transit in the large intestine (Bifidobacterium) in feces sample of CMB1-treated group was also correlated with faster colonic transit and in agreement with an earlier study , Lactobacillus (Firmicutes) and Akkermansia (Verrucomicrobia), which have been shown to improve the motility of the intestine by provoking the release of 5-HT or by promoting cholinergic pathways (Bifidobacterium and Lactobacillus) caused by constipation. Recent studies in rodents indicate that Akkermansia in the gut might reduce obesity, diabetes and inflammation  and unclassified Helicobacteraceae . Some species of Anaeroplasma, Parabacteroides and Clostridium are pathogenic to humans and are associated with colitis and gastroenteritis (Helicobacter (unclassified Helicobacteraceae) are associated with peptic ulcers, chronic gastritis, duodenitis and stomach cancer  can alleviate constipation more efficiently. CMB1 noticeably increased the water content of feces, small intestinal transit rates, the first black stool defecation time and the concentration of propionic and butyric acids. Moreover, constipated mice treated with CMB1 had a unique profile of caecal microbiota that reflect the relief of constipation more comprehensively and accurately, in comparison with fecal microbiota which was only associated with the colonic transit.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA531550).The datasets (gut metagenome Genome sequencing and assembly. SRA accession: PRJNA531550) for this study can be found in the [Sequence Read Archive (SRA)]  and performed at the Experimental Animal Center of the Jiangnan University [License No. SYXK(SU)2016-0045].LW and GW conceived and designed the experiments. LW, CC, and SC performed the experiments. LW, GW, Y-kL, and HZ analyzed the data. JZ, HZ, and WC contributed reagents, materials, and analysis tools. LW, GW, and Y-kL wrote the manuscript. All authors contributed to the manuscript revision, and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Similarly, using a cell culture preparation, we have found that high doses of ferric ammonium citrate (FAC), a factor known to enhance iron accumulation, increased MPP+-induced cell death in U251 and SH-SY5Y cells, and even in control cells. However, at low dose FAC restored or increased the viability of U251 and SH-SY5Y cells in the absence or presence of MPP+. These observations imply that high levels of iron likely contribute to or heighten MPP+ toxicity in the later stages of PD. While we report reduced iron levels in the earlier stages of MPTP induced PD, the significance of these changes remains to be determined.Despite much efforts in the last few decades, the mechanism of degeneration of dopamine (DA) neurons in the substantia nigra (SN) in Parkinson’s disease (PD) remains unclear. This represents a major knowledge gap in idiopathic and genetic forms of PD. Among various possible key factors postulated, iron metabolism has been widely suggested to be involved with fueling oxidative stress, a known factor in the pathogenesis of PD. However, the correlation between iron and DA neuron loss, specifically in the SN, has not been described in experimental animal models with great detail, with most studies utilizing rodents and, rarely, non-human primates. In the present study, aiming to gain further evidence of a pathological role of iron in PD, we have examined the correlation of iron with DA neuron loss in a non-human primate model of PD induced by MPTP. We report a significant iron accumulation accompanied by both DA degeneration in the SN and motor deficits in the monkey that displayed the most severe PD pathology and behavioral deficits. The other two monkeys subjected to MPTP displayed less severe PD pathologies and motor deficits, however, their SN iron levels were significantly lower than controls. These findings suggest that high iron may indicate and contribute to heightened MPP Parkinson’s disease (PD) is a well-known progressive neurodegenerative disorder that involves significant degeneration of dopamine (DA) neurons in the substantia nigra (SN) . Such ceIron, a redox-active transition metal, is a critical player in many physiological processes in the nervous system, such as myelin synthesis, DA metabolism, and neurotransmitter regulation . However+. Utilizing this combination strategy, we have demonstrated in a limited number of monkeys (n = 3) that the highest level of SN iron accumulation correlated with the following: highest loss of DA neurons, greatest oxidative stress in the SN, and the most severe PD-like motor deficits. Furthermore, the monkeys that exhibited less severe neuronal pathology and motor deficits had lower SN iron levels than control monkeys, further implying a correlation between iron and PD pathology. Parallel in vitro studies showed that high doses of ferric ammonium citrate (FAC), which is known to enhance iron accumulation, increased cell death in U251 and SH-SY5Y cells with or without MPP+, while low levels of FAC did not exacerbate cell death (under the same conditions).Non-human primate models of neurodegenerative diseases, particularly related to PD, are very effective methods of investigation due to their close relationship with humans. In addition, very few non-human primate studies have begun to investigate this phenomenon. Therefore, the primary goal of this study was to further document the correlation of iron with oxidative stress and DA cell death in a monkey model of MPTP-induced PD. A parallel study using cell culture aimed to investigate the direct causality of iron accumulation and the death of a human glial and neuronal cell line in the presence of MPPad libitum via an automatic bubbler. A standard diet  was fed twice daily. Meanwhile, vegetables and fruits with equal nutrients were provided to each animal every day. The healthcare and maintenance of non-human primates was performed under the supervision of specialty veterinarians. All biohazard waste was autoclaved before disposal.All the experimental protocols were reviewed and approved by the Animal Welfare and Use Committee, and all the experimental procedures were in conformity with the guidance of the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the United States. In total, six healthy female rhesus monkeys  were obtained from Sichuan Primed Biological Technology Co., Ltd  (Certificate No SCXK (Chuan): 2013-105). Two weeks prior to the experiment, the animals were transferred from their home room to stainless steel monkey cages, one animal per cage, in a feeding room with controlled conditions of temperature (19 to 26°C), humidity (40 to 70%) and light . Tap water was provided n = 3) and MPTP group (n = 3). The MPTP group and normal group were administered with a small dose (0.2 mg/kg) of either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine  or the same dose of saline, respectively, by intramuscular injection daily for 45 days (detailed protocol see st MPTP) and the second for 30 days (2nd MPTP). Both MPTP-injection periods were followed by an interval of no injection, 8 and 7 weeks, respectively.The monkeys were randomly divided into two groups: normal (control) group , and the brains were rapidly removed on ice, divided into coronal slabs of 4 mm thickness and then fixed in 4% paraformaldehyde phosphate buffer solution (pH 7.4) for further use. Brain slabs were then cryoprotected by soaking in 20 and 30% sucrose solution at 4°C and sliced into 30 μM coronal sections.The sections were floated in 0.1% chrome alum-gelatin solution and mounted in slides. After drying at room temperature, the sections were processed for hematoxylin and eosin (H&E) staining, Nissl staining , Prussia2O2):V(methanol) = 1: 50] to eliminate endogenous peroxidase activity. After washing three times in PBS, sections were incubated in 10% normal goat serum for 30 min at 37°C, and then sections were reacted with primary antibodies  for 24 h at 4°C. After several washing steps, sections were incubated with biotinylated secondary antibody followed with washing steps and incubation with streptavidin peroxidase for 1 h at room temperature. Finally, the sections were stained in the ammonium nickel sulfate-DAB for no more than 30 min. The staining solution was prepared as described previously (Sections for immunohistochemistry were rinsed thrice in 0.01 M PBS (pH = 7.4) and then placed in hydrogen peroxide  and Mk3  when compared with control monkeys  = 43.5, P < 0.0001]  = 11.14, P < 0.0001]  = 16.46, P < 0.0001] and A11.6   = 4.138, P = 0.0096] and Mk3  when compared to controls . Tissue analysis using Nissl staining showed that DA neurons in the SN of MPTP-injected monkeys were atrophied, especially in Mk3 . Quantit monkeys . Meanwhi monkeys . Specifi monkeys . Quantit 0.0001] . However 0.0001] . There w 0.0001] . Similar 0.0126] . While Tcontrols , we onlyIn the present study, accumulation of brown dots was noted in the SN of MPTP-lesioned monkeys . The insF = 18.81, P < 0.0001]  = 13.04, P < 0.0001] and Mk2  to a lesser degree, when compared to control . When analysis was conducted using the data from entire SN, it appears that iron labeling in Mk3 was significantly greater than that in control monkeys [ 0.0001] . Interes control . The sam control . Therefo control –6. This  control , 6.+-induced DA cell death  = 9.295, P < 0.0001]  = 60.52, P < 0.0001] (Existing studies have found that oxidative stress is likely involved in the MPPll death . In addill death . We ther 0.0001] . While s 0.0001] . No sign 0.0001] . Therefo+ exposed cell cultures (U251 and SH-SY5Y). Ferric ammonium citrate (FAC), which has been shown to enhance iron accumulation, was utilized at concentrations consistent with the current relative literature (+-induced cell death on U251 cells. In the low level of MPP+ (0.5 mM) for which no significant cell death occurred in U251 cells, pretreatment (2 h) with a low dose (10 μM) of FAC led to an increased cell viability in the presence of MPP+  for which cell death did occur, lower level of FAC  could reverse the cell death caused by MPP+  that was non-toxic, low level of FAC (200 and 500 μM) increased cell viability, while high level of FAC  lowered the cell viability when compared to control or MPP+ only (+ (5 mM), which caused significant levels of cell death in SH-SY5Y cells, additional higher levels of FAC  exacerbated the cell death caused by MPP+ alone  of different proteins , and then dopamine could be converted into a stable compound, clamping toxicities in the DA cells . The majproteins . Specifigy in PD . In factgy in PD , leadingll death . Additioll death . The reso-quinone, iron may stimulate α-synuclein aggregation through direct binding. For example, In addition to encouraging α-syn aggregation though the pathway of DA-Despite strong evidence of iron neurotoxicity in PD, the function of iron is still far from clear. For example, based on our monkeys and cell culture investigation in the current study, the effect of iron on cell viability related to PD pathology does not appear to be detrimental in all cases. For example, compared to Mk3, Mk1 and Mk2 displayed significantly less severe motor deficits and neuronal cellular damage associated with PD pathologies. Interestingly, the overall iron content in the SN region of both Mk1 and Mk2 was decreased compared to that of control monkeys. This seems to suggest that iron levels in the SN of monkeys with PD may consist of two phases: an early, less severe phase, where iron levels are depressed, and a later, more severe phase with elevated levels of iron. Although the pathological significance of elevated iron levels in relation to neurodegeneration is supported by most of the existing studies, the significance of early stage iron depression is not clear. Regardless, as the PD pathology advances, iron levels appear to eventually elevate, which is likely due to the overwhelming oxidative stress and degenerative process .It is well established that iron is an essential metal that is critical for cellular energy metabolism in physiological processes, such as glucose metabolism, the Krebs cycle, and oxidative phosphorylation . Therefoin vivo studies are in good agreement with our parallel in vitro investigation. In the cell culture study, we noted that mild and moderate levels of FAC, a compound that enhances iron accumulation, when applied either alone or in the presence of MPP+ (FAC applied before the exposure of MPP+), did not cause or exacerbate cell death instigated by MPP+. However, at high concentrations, FAC increased cell death with or without MPP+. It is also clear that at higher levels iron can be toxic, even in the absence of or at low levels of MPP+, which is likely due to, at least in part, the stimulation of oxidative stress (a well-known pathology).These Lipofuscin is a known end product of oxidative stress . It has Following the demonstrating of the well-established iron toxicity in PD, iron chelation therapy in PD has been examined as a novel treatment option, first in animal models and recently in human studies. For example, clinical studies have shown that deferiprone (DFO), an iron chelator, could reduce iron content in the dentate and caudate nucleus  and prov+ toxicity leading to DA neuron cell death. The exact mechanism by which iron together with oxidative stress mediate the MPTP/MPP+-induced degeneration that leads to the SN specific neuron loss is not clear. However, the current results indicate that the interplay of excessive iron with neuromelanin, DA, α-syn, and the process of oxidative may be a key factor in causing cell death of DA neurons in the SN. Therefore, it is likely that excessive iron may increase the risk of PD in humans, along with various other identified environmental risk factors. Meanwhile, our in vivo and in vitro data also indicate that iron is suppressed during the early stages of PD, however its significance remains unclear and will be the topic of a future study. As such, any possible treatment strategies for influencing iron levels in PD may need to take the timing of treatment into consideration, likely by reducing iron during the late stages of PD when iron is high in the SN (to suppress degeneration).In a monkey model of PD using MPTP, we have gathered findings that support the notion that excessive iron is likely a causal factor mediating and accelerating MPTP/MPPAll datasets generated for this study are included in the manuscript and/or the All the experimental protocols had been reviewed and approved by the Animal Welfare and Use Committee, and all the experimental procedures were in conformity with the guidance of “the National Institutes of Health Guide for the Care and Use of Laboratory Animal” of the United States.ZC and RS designed the overall study and contributed to the writing of the manuscript. LS performed all of the experiments related to clinical motor evaluations, immunohistochemical analysis, data analysis, and contributed to the writing of the manuscript. ER assisted with statistical analysis, data analysis, figure preparations, and contributed to the writing of the manuscript. WM performed the experiments related to animal feeding and management, tissue collection and histopathology. CH and QL contributed to planning some of the experiments and contributed to revising the manuscript. WZ, LG, and AC provided the funds and reagents. WL, YX, JF, and LT coordinated and supervised the project. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.