Clozapine (CZP) is an atypical antipsychotic agent commonly used in the treatment of schizophrenia. It is metabolized primarily by CYP1A2 enzyme, yielding a pharmacologically active metabolite, norclozapine (NCZP). Significant intra- and interindividual pharmacokinetic (PK) variability for CZP and NCZP has been observed in routine therapeutic drug monitoring. So the goal of this study was to evaluate the magnitude and variability of concentration exposure to CZP and its active metabolite NCZP on pharmacokinetic parameters in Uruguayan patients with schizophrenia with a focus on covariates such as cigarette smoking, age, sex, caffeine consumption, brands available of CZP, and comedication using population PK (PPK) modeling methodologies. Patients with a diagnosis of schizophrenia treated with brand-name CZP (Leponex®) for more than a year were included in the study. Then these patients were switched to the similar brand of CZP (Luverina®). Morning predose blood samples for determination of CZP and NCZP using a HPLC system equipped with a UV detector were withdrawn on both occasions at steady state and under the same comedication. Ninety-eight patients, 22 women and 76 men, took part in the study. Mean ± standard deviation for CZP and NCZP concentration was 421 ± 262 ng/mL and 275 ± 180 ng/mL, respectively. After covariate evaluation, only smoking status remained significant in CZP apparent clearance, inducing a mean increment of 32% but with no clinical impact. The results obtained with the two brands of CZP should ensure comparable efficacy and tolerability with the clinical use of either product. Smoking was significantly associated with a lower exposure to CZP due to higher clearance. The results obtained with the two brands commercialized in our country hint a bioequivalence scenario in the clinical setting. Schizophrenia is a severe neuropsychiatric disorder characterized by a high degree of morbidity and mortality with a prevalence rate from 0.4 to 1.4 % of the population –4. CZP iFrom the pharmacokinetic (PK) point of view, the absorption of CZP is almost complete, with an oral bioavailability between 60 and 70% due to first pass metabolism. Food does not seem to affect the amount of drug absorbed. The time to peak concentration after oral dosing is about 2.5 hours. The elimination half-life is about 14 hours at steady-state conditions, plasma clearance between 8.7 and 53.3 L/h, and distribution volume between 1.6 and 7.3 L/kg. CZP is 95 % bound to plasma proteins, primarily alpha-1-glycoprotein [CZP undergoes extensive metabolism principally to the stable metabolites, desmethylclozapine (active metabolite) and clozapine N-oxide. CYP1A2 and to a lesser extent CYP3A4 catalyze the demethylation of CZP while N-oxide formation is catalyzed by CYP3A4. The isoenzymes CYP2D6, 2C9, and 2C19 appear to play minor roles as well –10. In mBoth CZP and norclozapine  predict the clinical outcome . Thus, rThere is evidence of P-glycoprotein involvement in CZP absorption across the intestine and the blood brain barrier (BBB), but according to some authors olanzapine and risperidone are the only antipsychotic agents that may inhibit P-gp activity in the BBB , 20.Despite the effectiveness of CZP as a standard drug for schizophrenia treatment, significant intra- and interindividual pharmacokinetic variability for CZP and even for NCZP has been observed in routine TDM , 22.The influence of sex, smoking, and other factors on CZP and/or NCZP plasma concentrations has been previously reported, though little information is available about the influence of these factors on the Hispanic population –26.In Uruguay, two brands of CZP were registered by the Health Ministry and are now available for use in our population: the brand-name drug  and the similar formulation , both of them containing 100 mg of CZP. Bioequivalence studies are required for drugs having a narrow therapeutic index; however, only a few studies have been carried out since the Uruguayan regulation was approved in 2007. CZP is among these drugs and its formulations have not been studied in vivo yet to demonstrate bioequivalence. For this reason, concern has arisen among psychiatrists when switching from one brand to the other, as this could have significant clinical implications. Initial in vitro evaluation of similar and brand-name drugs in USP-2 Apparatus (paddles) at WHO's biorelevant  media  carried out by the attending physicians of the psychiatric hospital in Uruguay . All patients were treated with the same brand of CZP (Leponex®) for more than a year. Then these patients were switched to Luverina® as the hospital purchase changed CZP brand. Patients were for two months on Luverina® before blood sampling. So morning predose blood samples for determination of CZP and NCZP were withdrawn on both occasions at steady state and under the same comedication. Oral CZP was administered twice a day with each brand. The study conformed to standards indicated by the Declaration of Helsinki and its later amendments, approval was provided by the Ethics Committee of Hospital Vilardebó, and all patients in the study gave written informed consent prior to participation.Demographic data including sex, age, bodyweight, medication history, dosage regimen, time of last dose, sampling time, concentrations of CZP and NCZP for both brands, smoking habit, information on concomitant medications, caffeine consumption as well as biochemical and hematological test results, and other relevant data were collected using a data collection form.μg/mL in methanol) were added to 1.0 mL of plasma. Then 500 μL of sodium carbonate 1 M and some drops of t-butanol were added and the extraction was performed with 6 mL of cyclohexane and vortex shaken for 1 minute. After centrifugation the supernatant was separated and dried under nitrogen stream at 37-40°C. Dry residue was dissolved with 50 μL of mobile phase and 20 μL injected into the equipment. The separation of the compounds was performed on a Phenomenex Luna C18 column . The mobile phase consisted of methanol:acetonitrile: buffer phosphate 50 mM pH 6.5 (20:20:60) and was pumped at a flow-rate of 1.6 mL/min. The column compartment was kept at 36°C and detection was performed at the wavelength of 230 nm. Under these conditions the retention times of analytes were 6.6, 8.2, and 9.5 min for NCZP, CZP, and medazepam, respectively. The height ratios of the compounds' peaks to the medazepam peak  were employed for all calculations. The HPLC method was linear between 54.8 (lower limit of quantification: LLOQ) and 1086 ng/mL for CZP and 72.3 (LLOQ) and 1085 ng/mL for NCZP. Inter and intraday precision and accuracy were below 15% for both compounds.Predose morning blood samples were withdrawn and placed in heparinized tubes. Plasma was separated by centrifugation and stored at -25°C until analysis. Drug quantification in plasma was performed using a validated method with minor modifications . A ShimaThe PPK analysis was performed using NONMEM® 7.4 (ICON plc.) together with the modeling and simulation workbench Pirana-PsN-Xpose . Given t subject .−1 as estimated by Jerling et al. [CZP = CLCZP/F) and norclozapine (CLapNCZP = CLNCZP/F/f), where F is the oral bioavailability of CZP and f the fraction of NCZP formed after CZP biotransformation (fixed to 1). F was fixed to 1 for Leponex® and evaluated for Luverina® as an estimate of the relative CZP bioavailability between both drug products. To account for the effect of body weight on clearance, a power model was included evaluating different coefficients and finally fixing this value to the allometric standard of 0.75; therefore,θpop stands for the mean population estimate, θi the estimate for the i-th subject, and ηi the difference between θi and θpop in the log-scale. Log-normal distribution is given by assuming η ~ N, where ω2 is the between-subject variability.CZP first-order constant rate for absorption (ka) was fixed to a value of 1.24 hg et al. . Both CZCik is the predicted trough concentration for the i-th subject under treatment k, Cpred is the population prediction, and εik is the proportional residual variability assumed to be normally distributed around zero with variance σ2.Interoccasion variability was not identifiable since only one observation was available per subject at each period (under treatment with Leponex® or Luverina®) and thus was not included. Residual unexplained variability was modeled with a proportional error: CZP, CLapNCZP, and F, evaluating the effect of sex, smoking status, CZP formulation, beginning of treatment, caffeine consumption, and concomitant treatments: valproic acid, benzodiazepines, antidepressants, antipsychotics, antidiabetics, and oral hypoglycemic drugs. The effect of daily CZP dose on both apparent clearances was also assessed. Covariate effect was evaluated separately for each possible source of variability, including as a second step in a full-model those factors which significantly reduced the objective function (OFV) considering a chi-square distribution for the OFV difference  and finally performing a backward elimination with a stricter p value of 0.001 (ΔOFV = 10.83 for 1 df), conserving only those factors with significant contribution to the overall variability. Graphic diagnostics were also obtained for the assessment of covariate inclusion and correlation between covariates. Numerical predictive check (NPC) and Numerical Predictive Distribution Errors (NPDE) versus predictions were used for assessing the final model. The NPC evaluates model misspecification at several percentiles of data distribution by contrasting the observed data with nonparametric confidence intervals obtained with data simulated by the model. The percentages of observed data above the upper and below the lower limit of a prediction interval built from these simulations are divided by the corresponding expected percentage . The difference between these ratios and the unity  is statistically evaluated throughout a coverage plot, allowing the assessment of the model predictions. NPDE is also a simulation-based diagnostic which estimates model prediction discrepancies with the observed data. A model describes the data well when the prediction discrepancies are evenly distributed [Covariate search was performed for CLaptributed . For estThe in vitro dissolution was performed at the following 3 media: (1) HCl/KCl pH 1.2 solution, (2) acetate buffer pH 4.5, and (3) phosphate buffer pH 6.8. Six units of each product were tested in Distek® dissolution system 2100C equipment. The conditions were USP-2 Apparatus according to WHO guidelines for biowaivers, 75 rpm stirring speed; 900mL of medium per vessel maintained at 37 ± 0.5°C. Samples were automatically withdrawn by the use of an Agilent 89092EO pump at: 5, 10, 15, 30, 45, 60 minutes. The drug release at different time intervals was measured by UV-visible spectrophotometer at 230 nm (Agilent 8453 and ChemStation® software). Cumulative percentages of dose dissolved were calculated.The final dataset included 98 Caucasian patients, 22 women and 76 men. A total of 171 trough observations were recorded for both CZP and NCZP, of which 146 corresponded to 73 patients who completed both periods receiving Luverina® and Leponex®. Sixty-eight (93%) of these patients conserved the dosage regime after the change of brand. For 25 patients, only one period was available: 17 under treatment with Luverina® and 8 under treatment with Leponex®. Demographic data and other characteristics of the patients for both brands as well as CZP and NCZP concentrations are summarized in The final model estimates are shown in After covariate evaluation, only smoking status remained significant in CZP clearance (p<0.05), inducing a mean increment of 32%. Sex did not produce a significant impact in these data: an increase in the AIC was observed after including it as a covariate on CZP and NCZP apparent clearance, and no significant differences between male and female estimates were obtained. This covariate was reevaluated after smoking factor was included to discard a masking effect, obtaining similar results. Smoking subjects represented 45% within each sex. The inclusion of caffeine intake as a covariate on compound elimination was also discarded, after estimating a very small (less than 1% increase) impact on CZP and NCZP CLap. Regarding drug-drug interactions, the most influential coadministered drug was valproic acid (VPA), increasing NCZP CLap by 10%, a relationship that was not retained for the final model because it was not found to be statistically significant. Among all subjects in the study, 28 also received antidepressants, sertraline, or escitalopram. However, no impact on CZP and/or NCZP CLap was observed with this medication. Inclusion of between-subject variability for the bioavailability factor significantly improved the fit. The relative bioavailability of Luverina® versus Leponex® was estimated as 0.892. The bootstrapped 95% confidence interval for the relative bioavailability, an estimate of bioequivalence ratio of means, was 0.769-1.02. The same adverse events  were reported for both brands of CZP.Mean dissolution profiles of both formulations at the three different media  are shown in Mean plasma concentrations of CZP and NCZP were within the therapeutic concentrations reported in the literature and mentioned in Introduction section.Smoking incidence on CZP clearance has been reported before with a similar magnitude of effect for different populations of patients , 23, 33.CZP and CLapNCZP are likely influenced by smoking status [CZP seemed to be affected. Interpreting our data correctly if CLapNCZP remained unchanged, an increase in both NCZP bioavailability and clearance would be the reason for this observation. Surprisingly, smokers were not exposed to larger dosages due to the higher apparent clearance of CZP. It is likely that the induction found in this work did not have a clinical impact. One limitation of the study is that smoking status was assessed using patient self-reporting. We dichotomized patients into smokers and nonsmokers but did not assess the magnitude of smoking. Some authors found that a daily consumption of 7-12 cigarettes is probably sufficient for maximum induction of CZP metabolism in 50 % [CZP observed in this work for smokers was 32 %. This increase could be because of an increase in systemic elimination or a decrease in bioavailability secondary to an induction in presystemic metabolism. As the isoenzyme CYP1A2 is practically not detected in the intestine, only an increase in systemic elimination could explain our results [Analysis of the plasma CZP: NCZP ratio can give us information about the metabolic status of CZP and patient's adherence . In our g status . Neverth in 50 % . The inc results .CZP between male and female [Although sex was also reported to be a significant covariate affecting the apparent clearance of CZP and NCZP explained by a different CYP1A2 activity between men and women, our study found no differences in the CLapd female –25. Accod female . MoreoveAntidepressants are frequently used in the treatment of depressive symptoms associated with schizophrenia. In patients taking CZP, choice of antidepressant is complicated by additive pharmacodynamics effects and by pharmacokinetic interactions. The various antidepressants differ in their potency to inhibit CYP enzymes , 40. PreCoadministration of VPA is very common in patients under CZP treatment as CZP can trigger convulsive seizures. 40 % of the patients in this study were treated with VPA at low doses (approximately 400 mg/day). No influence of VPA on the metabolism of CZP was found in this study. Literature had provided contradictory results concerning the effects of VPA on CZP metabolism. Studies with different designs indicated no effects, mild inhibition, or mild induction –46. One CZP or CLapNCZP with nonconsumers in the covariate analysis. Caffeine consumers were similarly distributed among smoking and nonsmoking groups. Clearance estimates for the nonsmoking group  were similar to the previously reported, including a controlled multiple-dose bioequivalence study, indicating that the presence of an inhibiting effect of caffeine over CYP1A2 is unlikely [It is important to know whether drinking coffee, in the amounts consumed by patients, has a clinically significant effect on steady-state serum CZP concentrations, especially because schizophrenia is linked to high caffeine intake. Some authors indicate that caffeine reduces CZP clearance most likely by inhibiting CYP1A2 , 50. Chaunlikely .As stated in Introduction section, bioequivalence requirements were not fulfilled for CZP in Uruguay. As a result, the two formulations available for the treatment of schizophrenia and bipolar disorder have never been studied in vivo and psychiatrists are forced to establish drug treatments without knowing drug product interchangeability. For this reason, switching from brand-name (Leponex®) to similar formulation (Luverina®) has raised concerns among physicians and patients as the evidence related specifically to the safety of switching from brand-name to similar CZP is scarce. Only three studies in the literature –53 have Our in vitro dissolution study is in accordance with our in vivo findings. CZP is a weak basic compound with a pKa of 4.5 and it can be classified as a class II drug according to the Biopharmaceutics Classification System, owing to its good permeation properties through biological membranes and low aqueous solubility . Its aquCovariate evaluation showed that only smoking status remained significant in CZP apparent clearance, inducing a mean increment of 32%. By means of population pharmacokinetics modeling, a comparative study of the bioavailability between these two products in the clinical setting with very sparse data (1 observation per subject per formulation) could be carried out. Given that bioequivalence regulation has confronted several obstacles in our country, this approach could be applied to assess multisource products being currently used, because their unknown characteristics could entail increased therapeutic failures (inefficacy/toxicity). The results obtained with the two brands commercialized in our country hint a bioequivalence scenario in the clinical setting as the same efficacy and the same adverse effects were reported by the clinicians.


Both the parasites and FCs detected in the effluents of assessed WSPs were of higher concentrations than World Health Organization and Tanzania Bureau of Standards limits, except for helminths in the Morogoro WSP and FCs in the Mwanza WSP. FCs were significantly correlated with protozoa (p < 0.01) and predicted protozoa occurrence well (p=0.011). There were correlations between physicochemical parameters, parasites, and FC bacteria in the WSP systems. Inadequate performance of these systems may be due to lack of regular maintenance and/or systems operating beyond their capacity. FC indicators were observed to be a good alternative for protozoa monitoring, but not for helminths. Therefore, during wastewater quality monitoring, helminths should be surveyed independently.In Tanzania, waste stabilization ponds (WSPs) are employed to treat wastewater, and effluents are used for urban agricultural activities. The use of untreated or partially treated wastewater poses risks of disease transmission, including parasitic and bacterial infections, to exposed communities. Little is known about the occurrence, concentration, and removal of parasites and fecal coliform (FC) bacteria in WSPs in Tanzania. This study evaluates the occurrence and concentration of parasites and FCs in wastewater, the efficiency of WSPs in removing parasites and FCs, and the validity of using FCs as an indicator of parasites. This was a cross-sectional study conducted between February and August 2018. Wastewater samples were collected from three WSPs located in the Morogoro, Mwanza, and Iringa regions. APHA methods were used to test physicochemical parameters. The modified Bailenger method and Ziehl–Neelsen stain were used to analyse parasites. Membrane filtration method was used to analyse FCs. Data were analysed using IBM SPSS version 20. Helminth egg removal ranged from 80.8% to 100%. Protozoan (oo)cyst removal ranged from 98.8% to 99.9%. The Mwanza WSP showed the highest FC reduction (3.8 log units (100 mL) In Tanzania, waste stabilization ponds (WSPs) have been used for municipal wastewater treatment for several decades . EffluenDespite the abovementioned advantages, the use of untreated or partially treated wastewater poses a risk of disease transmission to farmers and the community . Apart fIt has been hypothesized that lack of operation and maintenance of wastewater treatment systems, such as WSPs, decreases the removal of pathogens (parasites) and fecal indicators, thus creating uncertainty about the risks associated with wastewater use . In TanzThe aims of this study were to (1) determine the occurrence and concentrations of wastewater-related pathogenic parasites and fecal coliform (FC) indicator bacteria in three selected Tanzanian WSPs and (2) evaluate their performance in removing pathogenic parasites and FC indicator bacteria. The correlation between physicochemical parameters, FC indicator bacteria, and pathogenic parasites (helminths and protozoa) was also examined.This was a cross-sectional study conducted between February and August 2018. Three WSP treatment systems located in the Morogoro, Mwanza, and Iringa regions in Tanzania were evaluated.3·d−1 of wastewater. Effluent from this system is discharged into Lake Victoria.The site in Mwanza is located in Butuja sub-ward, at 2°28′09.37″S 32°54′35.49″E. The system is owned by the Mwanza Urban Water Supply and Sanitation Authority (MWAUWASA) and serves about 3,500 connected households. The design of this system consists of 2 septage lagoons receiving fecal sludge delivered by vacuum trucks, 3 anaerobic ponds connected in parallel, 4 facultative ponds in parallel, and 6 maturation ponds in a series . This trWSPs in Morogoro are owned and operated by the Morogoro Urban Water Supply and Sanitation Authority (MORUWASA) and are located in Mafisa, Morogoro (at 6°47′07.28″S 37°40′29.34″E). The treatment system consists of 2 septage lagoons, 1 anaerobic pond, 1 facultative pond, and 4 maturation ponds connected in a series . The effThe Iringa wastewater treatment plant is located at 7°45′31.47″S 35°40′12.34″E in Don Bosco. The treatment system is owned by Iringa Urban Water Supply and Sanitation Authority (IRUWASA). The system has 2 septage lagoons receiving fecal sludge delivered by vacuum trucks, 2 anaerobic ponds operating in parallel, 1 facultative pond, and 2 maturation ponds connected in a series . The effWastewater samples were collected for analysis of physicochemical 1 L), parasitological , and bacteriological (250 ml) parameters at intervals of one month for 4 or 5 visits. During each visit to Morogoro, Mwanza, and Iringa WSPs, a total of 4 samples were collected, one from each sampling point, as indicated in Figures  L, parasWastewater samples for chemical oxygen demand (COD) and total nitrogen (TN) tests were preserved by using sulphuric acid at pH of 2; those for parasitological analysis were preserved by using 10% formaldehyde. All samples were transported to the respective laboratory in a cool ice box (at a temperature of 4°C) within 6–36 h after collection. The samples for physicochemical and bacterial tests were transported to the University of Dar es Salaam water resources laboratory, and those for parasitological tests were transported to the Muhimbili University of Health and Allied Sciences parasitology laboratory. All samples were analysed immediately upon arrival at the laboratory.On-site measurement of wastewater flow rates, temperature, and pH was conducted during sample collection. Wastewater flow rates were measured using a stopwatch and tracer method to detect the velocity of wastewater within a channel. The velocities of wastewater were multiplied to the channel cross-sectional area. The procedure was repeated thrice and the average was computed. Hydraulic retention time (HRT) of each system during the study period was estimated based on the calculated flow rate and designed volume of the system. Wastewater temperature and pH were determined by using a pH meter  by directly inserting the probe into the wastewater.In order to determine the performance of WSP operations, physicochemical parameters were measured following “Standard methods for the examination of water and wastewater” . These pCryptosporidium oocysts are very small, they can be difficult to detect in the MBM, so the smears were stained to increase visualization, in accordance with the modified Ziehl–Neelsen (ZN) procedure . The predictor (FC) explains 15.5% (Nagelkerke R2) of the variability of protozoa occurrence, which is significant at the 5% level . The odds ratio is 1.733 (95% CI 1.104–2.720). The model correctly predicted 29.4% of cases where there was an absence of protozoa, and 92.3% of cases where there was the presence of protozoa, giving an overall percentage correct prediction rate of 73.2%. These results are in accordance with results obtained by Payment and Locas . A simple binary logistic regression test was conducted to assess the ability to use FC indicator bacteria to predict the presence of helminth eggs and protozoan (oo)cysts in WSP wastewater. Results indicate that FCs are a good predictor of protozoa occurrence in WSP wastewater (presence/absence) [Chi-square = 6.516, dnd Locas  and HarwThe studied WSP reduced pathogenic parasites and FC concentrations from wastewater. However, they did not meet either one or both parasitological and microbiological standards as stated in the WHO guideline and TBS regulations. The Mwanza WSP system achieved the WHO standard for FC bacteria effluent concentration for safe reuse of wastewater in both restricted and unrestricted irrigation. The effluent FC concentration was also in accordance with the TBS standard limit for wastewater discharge into the environment. But the system did not meet the helminth concentration of ≤1 egg/L, as described in the WHO guideline. The Morogoro WSP system meets the helminth standard set by the WHO, but did not meet the WHO and TBS effluent FC concentration standards. The Iringa Municipal WSP system did not achieve either parasitological or microbiological quality as described by both the WHO guideline and TBS regulations. In order to reduce the risk of parasitological and bacterial disease transmission to the exposed community, measures such as proper operation and maintenance of WSPs must be taken. These measures will enhance WSP efficiency and ensure that the parasitological and microbial quality of effluents is achieved and sustained. Some efforts were observed in the Iringa Municipal WSP, whereby two new maturation ponds were under construction during the study period. Perhaps the addition of these two ponds will improve the parasitological and microbiological quality of the effluent of this system to a level that adheres to the WHO and TBS requirements.FCs correlated with protozoan (oo)cysts, but not with helminth eggs. Binary logistic regression results indicated that FCs are a good predictor of protozoa occurrence in wastewater but not of helminths. Thus, FCs are not a reliable indicator of helminths in wastewater. Therefore, during wastewater monitoring for reuse or discharge into sensitive receiving water bodies, such as lakes, rivers, and streams, helminth quality must be surveyed independently.


Amana edulis from water and ethanol system had the best efficacy. At the concentration of 0.1 mg/mL, this component has the highest inhibition rate up to 70%. To investigate the underlying molecular reasons, we observed that the component can significantly induce the liver cancer cells apoptosis and retard the cell reproduction at G2/M stage. Verification experiments showed that this component also has apparent inhibitive effects on other liver cancer cells, such as HepG2 and Huh7. On the other hand, it has less effectiveness on another cell line HepaRG, which retains many characteristics of primary human hepatocytes. The results suggested that there might be highly efficient antihepatoma ingredient in the water and ethanol extraction of Amana edulis. The pure substances remain to be isolated and further research on their targets is required.HCC is one of the fastest-rising causes of cancer-related death. Novel therapeutic approaches for treatment are warranted. The goal of this study is to find effective components from Chinese herbal medicines, which is an important alternative source of anticancer medicine. To this end, six different herbs were selected from various traditional literatures. Soxhlet extractor was used to distill the strong polar and weak polar components of each herb. The inhibitive effect of each component was determined using liver cancer cells BEL7404. From total of 12 extractions, it was found that the combined crude lysate of Hepatocellular carcinoma (HCC) has become one of the leading causes of death and an important public health problem in China because of the increasingly higher morbidity and mortality rates. It was reported that the death number from HCC ranks second in China, while the current treatment and drug R&D remain backward . Many HC Shennong's Materia Medica (~220 CE). Herbal medicine is an important treasure of Chinese nation, which has been used for thousands of years and gained remarkable effects on the treatment of various diseases including tumor, due to accumulating abundant theoretical and practical knowledge through thousands of years [Traditional Chinese medicine (TCM) is a medical science studying disease prevention, diagnosis and treatment, rehabilitation, and health preservation. It has been used in China for thousands of years –6. It haof years , and theof years –11. The of years . HCC is  Amana edulis, Prunella vulgaris L., Vaccaria segetalis (Neck.) Garcke, Gnaphalium affine D. Don, Paeonia lactiflora Pall., and Bupleurum chinense DC by taking the words of “Jiju, Jiejie, and Zhengjia” as the basis and under the principle of “no repetitive, effectiveness, hypotoxicity, and nonscarcity”.Although there are few records of such disease names of “tumor” or “cancer” in traditional medical works, the description of occurrence, development, diagnosis, and treatment to many diseases, such as “Yingliu” (goiter), “Luoli” (scrofula), “Zhengjiajiju”, and “Yeshi” , is of high similarity to “tumor” and “cancer” in modern medicine. Hereby, in the present study, by consulting a massive of ancient literatures, including “Treatise on Febrile Diseases (Shanghan Lun)”, “Huangdi's Canon of Medicine”, and “Compendium of Materia Medica”, we selected these medicinal herbs of Amana edulis from water and ethanol system had the highest inhibition efficacy. Our results further showed that crude lysate of Amana edulis could significantly induce the liver cancer cells into early and late stage apoptosis and retard the cell reproduction at G2/M stage. These data suggested that there may be highly efficient antihepatoma component in Amana edulis.We identified that the combined crude lysate ofAmana edulis, Prunella vulgaris L., Vaccaria segetalis (Neck.) Garcke, Gnaphalium affine D. Don, Paeonia lactiflora Pall., and Bupleurum chinense DC were all purchased from Tongrentang Pharmacy and kept in an airtight device for a period of time before processing.The human hepatoma cell lines of BEL7404 were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum (FBS) , 14 and 100g of Chinese herbal medicines was extracted using ethanol-water solvent mixed system  and petroleum ether-ethyl acetate mixed system (100mL of petroleum ether and 100mL of ethyl acetate) for 120min by heat reflux extraction, respectively. Then solvents were removed with vacuum rotary evaporation. The extractions were kept at −20°C until used. Amana edulis, as described in a previous paper [ Amana edulis and incubated for 24h. CCK-8 solution was added to each well and the plate is placed in the incubator for 4h after which the absorbance was read at 450nm using VERSAmax tunable microplate reader .Cell Counting Kit-8 was used to evaluate the cytotoxicity ofus paper . Cells w Amana edulis, 3--2,3-diphenyl tetrazolium bromide (MTT) assay was conducted in accordance with a previously described method [4 cells in 100 μL medium were plated into each well of 96-well plates (five wells per group). 24h after plating, 10μL 0.5mg/mL MTT solution was added to each culture well, and then the cells were incubated at 37°C for 4h. Then, the MTT solution in each culture well was removed. Finally, 150μL of DMSO was added to each culture well, and the mixtures were shaken. The absorbance was measured on a microplate reader  at 540nm.In order to further verify the cell proliferation of crude lysate ofd method  in the f5 cells/mL. After 24-hour incubation, cells were treated with crude lysate of Amana edulis and then incubated at 37°C for 24h. Cells were harvested by trypsinization and centrifugation, and cell supernatant was abandoned. The cells were washed three times with the precold PBS buffer, and the cells density was adjusted to 1×106 cell/mL. Then Annexin V and PI solution were added to the cells at room temperature without light exposure for 15min. Cell apoptosis was determined using flow cytometer .Cell apoptosis was analyzed using the Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's protocol  with slight modifications. Briefly, BEL7404 cells were plated in 96-well plates at a density of 4×105 cells/mL and incubated at 37°C to allow cells to grow to 60%–80% confluence. Cells were treated with crude lysate of Amana edulis for 24h. Cells were collected by digestion and fixed with 70% ethanol at 4°C overnight. Then the cells were resuspended in phosphate-buffered saline (PBS) and incubated at 37°C for 1h. Finally, the cells were stained with propidium iodide (PI) for 30min without light exposure. Stained cells were analyzed by flow cytometer . ModFit Cell Cycle Analysis software was used to analyze the percentage of cells in G1, G2, and S phases based on DNA content.Cells were seeded in 96-well plates at a density of 4×10The experiments were repeated three times, and the data represent the means. The results were analyzed using one-way ANOVA followed by Student's t-test , 20. An We selected six kinds of Chinese medicine herbals, which were highly correlated with liver cancer, and with no repetitive, effectiveness, hypotoxicity, and nonscarcity . Prunella vulgaris L., Paeonia lactiflora Pall., and Amana edulis in water-ethanol solvent and Prunella vulgaris L. and Vaccaria segetalis (Neck.) Garcke in Petroleum Ether-ethyl acetate retained the ability at the concentration of 0.1mg/mL. We evaluated the effect of Amana edulis on the growth of the BEL7404 cells, and the data showed that IC50 of Amana edulis after being treated for 24h in BEL7404 cells was 0.052mg/mL  mixed system extract can significantly induce early and late-age apoptosis of BEL7404 cells.The flow cytometry data showed that early apoptosis values are 3.49% to 24.70% with beginning and ending difference of 7.08 times, and late apoptosis values are 2.41% to 18.70% with beginning to ending difference of 7.76 times . These r Amana Edulis mediated cell cycle arrest at the G2/M phase, cell cycle assay was carried out. Amana edulis (water-ethanol) extract significantly arrested BEL7404 cells at G2/M phase after 24 hours' action, which may inhibit related protein activity during the division cycle (To investigate the inhibition mechanism of the crude lysate ofon cycle . Amana edulis (water-ethanol) mixed system extract on liver cancer cells have extremely significant difference compared with normal cells.To investigate whether this ingredient would have effect on other cell lines, human liver cancer cells BEL7404, HepG2, and Huh7 and normal liver cell HepaRG were used to conduct the verification experiment through MTT assay and t-test. The survival rate of HepaRG cell was the highest, followed by HepG2 cell, and the survival rate of Huh7 cell was the lowest . The resHepatocellular carcinoma (HCC) is a common malignant disease and is famous for its high incidence and mortality rates . Moreove Paeonia lactiflora Pall. root had been proved to be a component of effective prescriptions for treatment of liver disease [ Radix Astragali, significantly induced apoptosis and inhibited invasion of tumor cells [Chinese herbal medicine has been actively researched through various approaches. It has been increasingly accepted that Chinese herbal medicine plays an effective function in fighting against various diseases with low side effects, including HCC. In addition, it had been widely reported that some Chinese herbal medicines had an ability to inhibit the HCC at preclinical and clinical levels , 26. Pae disease . Astragaor cells , 28. Prunella vulgaris L. have multitarget and multipathway effects on antilung adenocarcinoma due to the regulation of steady state of calcium ion, cell cycle, and its steady state and the inhibition of tumor cell proliferation and metastasis [ Gnaphalium affine D. Don is a folk medicine and used to treat antiinflammatory, and expectorant activities, and the extract of Gnaphalium affine D. Don showed significant antiinflammatory activity and reduced the paw swelling on MSU crystal-induced paw edema model [ Bupleurum chinense DC present anticomplementary activity in vitro [ Vaccaria segetalis (Neck.) Garcke [ Paeonia lactiflora Pall. [Some Chinese herbal medicines in this study have been widely studied and even have been used in clinics. The extracts oftastasis . The Gnama model . Polysacin vitro . Vaccari) Garcke  and Paeora Pall.  were alsAmana edulis has the effects of clearing heat, dissipating phlegm, and resolving masses with meridian distribution of liver and spleen, which is one of the anticancer Chinese medicinal herbs. In this study, our data indicated that six kinds of Chinese herbal medicines were certain antihepatoma effects, finding Amana edulis (water-ethanol) mixed system extract has the highest efficiency in the inhibition of the proliferation of liver cancer at the concentration of 0.1mg/mL. However, the effect of Amana edulis (water- ethanol) mixed system extract should be further investigated. Amana edulis (water-ethanol) mixed system extract. These extracts can also increase early and late-age apoptosis rates and induce cell cycle arrest at the G2/M phase, indicating Amana edulis (water-ethanol) mixed system extract that may induce apoptosis. As is known to all, the proliferation and apoptosis of tumor cells are closely related to the regulation of cell cycle. For cancer cells, the blocking effect is an important index to evaluate the effect of antitumor therapy [ Amana edulis (water-ethanol) mixed system extract may be involved in the inhibition of the antitumor effect.In this study, it was also observed that DNA showed a long tail in therapy . Cell cy therapy . Ophiopo therapy . In ophi therapy . In addi Amana edulis (water-ethanol) mixed system extract contained potential high-activity anticancer effects on liver cancer cells in vitro. However, the effects of Amana edulis in vivo need to be further studied and further separation of single active component is expected to research its effect target. Moreover, more attention should be paid to consideration to develop the new drugs derived from Amana edulis for the treatment of liver cancer. In the struggle against HCC, we need not only the wisdom but the knowledge to solve the fatal problems that have plagued the whole world.In conclusion, our findings indicated that


Japonica and indica are two important subspecies in cultivated Asian rice. Irradiation is a classical approach to induce mutations and create novel germplasm. However, little is known about the differential response between japonica and indica rice after γ radiation. Here, we utilized the RNA sequencing and Weighted Gene Co-expression Network Analysis (WGCNA) to compare the transcriptome differences between japonica Nipponbare (NPB) and indica Yangdao6 (YD6) in response to irradiation. Japonica subspecies are more sensitive to irradiation than the indica subspecies. Indica showed a higher seedling survival rate than japonica. Irradiation caused more extensive DNA damage in shoots than in roots, and the severity was higher in NPB than in YD6. GO and KEGG pathway analyses indicate that the core genes related to DNA repair and replication and cell proliferation are similarly regulated between the varieties, however the universal stress responsive genes show contrasting differential response patterns in japonica and indica. WGCNA identifies 37 co-expressing gene modules and ten candidate hub genes for each module. This provides novel evidence indicating that certain peripheral pathways may dominate the molecular networks in irradiation survival and suggests more potential target genes in breeding for universal stress tolerance in rice. Oryza sativa L.) has various nutrients with high digestibility and absorption rate, providing staple food to more than half of the world’s population, especially in Asia. Cultivated Asian rice consists of two major subspecies, japonica and indica, both are widely used in genetics, breeding, and production. The two subspecies have many different features including plant architecture, leaf color, tiller number, grain shape, nutrition quality, temperature adaptation, and cultivation region. Though many reports are focusing on their morphological, physiological, biochemical characteristics and molecular diversity [Rice  to compare transcriptomic differences between the rice varieties Nipponbare  and Yangdao6  in response to irradiation stress at the early germination stage. Our results suggest that the mechanism in the direct DNA repair related pathway responds similarly between the subspecies, but their universal stress responses are contrastingly different. Knowledge about this difference will help us to understand the general differences of japonica and indica subspecies in withstanding radiation adversity and possibly other stressors. Furthermore, this work may offer a starting point for the elucidation of molecular networks underlying irradiation stress response in rice.However, there is no report about the differences between japonica varieties generally show a faster reduction in the survival rate than that of indica varieties at a lower to moderate dose of irradiation, with a 50% lethal dose (LD50) ranging from 112.0 to 349.8 Gy in japonica (average 259.7 Gy), and 271.3 to 390.0 Gy in indica (average 336.8 Gy), respectively. Among them, the japonica rice cultivar Nipponbare (NPB) was the most vulnerable one to irradiation in the japonica sub-group, whereas Yangdao6 (YD6) and Huazhan were relatively the more sensitive varieties in the indica sub-group.Seedling survival rate directly reflects the severity of damage triggered by irradiation of seeds. The seedling survival rates of the twelve varieties at various irradiation doses are displayed in To elaborate their different germination responses to irradiation, we compared the germination potential, seed germination rate, seedling emergence rate and seedling survival rate in NPB and YD6 at three irradiation doses. All four indices were reduced significantly after irradiation C–F, withAfter subjected dry seeds to irradiation, the length of both shoot and root in germinating seedling was significantly reduced in all varieties . Shoot lR2 value) of these features were all significant (p≤ 0.01), with root length, root surface area and root volume showing more inhibited in NPB than in YD6  and 50 Gy treatments of NPB and YD6. An average 47.4 million clean reads (range 38.7–55.0) were generated in each sample, corresponding to a total of 7.1G bp clean bases (range 5.8–8.2) indicating evenly distributed sequencing depth across treatments . The proThe high-quality clean reads were assembled by the Cufflinks algorithm . The reaR2 value) of the qRT-PCR validation data against the FPKM data was positively significant at p ≤ 0.05 in 49 of the 50 genes . The consistency between the qRT-PCR validation and the RNA sequencing results confirms the reliability of the RNA-Seq analysis.To validate the RNA sequencing data, we selected 50 genes (41 DEGs and 9 hubgenes) , which rThe numbers of differentially expressed genes (DEG) are shown in The varietal DEGs directly reflect the genetic differences between the two varieties. Regardless of tissues and treatment groups, there were 2691 common DEGs in varietal comparison groups , which wTo further inquire the functions and pathways of irradiation responsive common 561 DEGs involved in, we calculated their transcription level changes and classified them into three categories: induced in root and shoot of both varieties  at 0.94 (p = 6 × 10−12) and 0.71 (p = 1 × 10−4), respectively; but these traits were negatively correlated with the royal blue module. The scale of average diameter showed a positive correlation with the turquoise module , but negatively correlated with the value of the royal blue module . Finally, the size of this volume was in high positively correlation with the pink module  and negatively correlated with the purple module . GO enrichment analysis for these selected modules are shown in To identify candidate pivotal genes associated with seedling growth traits, WGCNA  was perfTo construct the gene networks, the highest significant correlated modules (one each from positively and negatively correlated modules) were selected. Since the scale of length and diameter share the same selected modules, totally six most relevant modules associated with the four phenotypic traits were chosen for the analysis. The co-expression network diagrams were generated basing on the degree of association of the genes within the module . The canTo screen for candidate hub genes, the top ten connected genes with their KME value in the specific modules are shown in The scale of average diameter is related positively to the turquoise module and negatively to the black module, respectively . In the The scale of volume is related positively to the pink module and negatively to purple module . In the Interestingly, we find 21 hubgenes are DEGs in this study . The hubThe heatmap of the candidate hub genes from different modules shows that the response of genes subject to the variety and tissue A. In theJaponica and indica rice are two important subspecies in the cultivated Asian rice group for rice production, especially in Asian regions. Recent reports have renewed our understanding of their divergence, indicating that they are domesticated independently, with limited intergroup introgression [indica showed a higher seedling survival rate than japonica. The molecular mechanism underlying the differential responses is lacking.gression ,22. Thisjaponica rice was found to be more sensitive to radiation in terms of seed germination potential, seed germination rate, seedling emergence rate and seedling survival rate. Indica rice shows better tolerance and therefore has a higher seedling survival rate than japonica rice after irradiation. The effects of irradiation on the morphology of roots and shoots are different, and the root elongation is more affected. These indicates that the effect of irradiation on japonica from phenotype to DNA level is more serious than that of indica. Even at low dose range, we found irradiation caused more extensive DNA damage in japonica rice than to indica rice. Studies in onion have shown that main contribution to the overall frequency of chromosomal aberrations is made by the bridge-type aberrations, fragments, and lagging chromosomes after irradiation, in addition to a variety of aberrations [japonica and indica subspecies is still waiting for clarification?It has been demonstrated that irradiation has different comprehensive effects on the physical and chemical properties, besides structural properties of starch, the amylose content, carboxyl content and acidity increase after irradiation . After irrations . What tyjaponica and indicia rice subspecies. Tissue effect DEG analysis also corroborate the above view that oxidation reduction as well as metabolic differences may define a differential irradiation response, as we have seen in the contrasting root and shoot vulnerability to radiation.Gamma radiation induces a wide range of damage and the irradiated cells must counteract these damages by activating complex reaction pathways . Leaf grSimilarly, we found groups of genes that displayed opposite regulation in the two varieties, either in irradiation response  or betweSHORT INTERNODES  were all up-regulated in all combinations. The OsSIN gene encodes a small protein located in the nucleus [OsSIN is more than 2-fold in irradiation effect  proteins, DNA-binding proteins from plants and hBRG1 protein, a central ATPase of the humans witching/sucrose non-fermenting (SWI/SNF) remodeling complex . In thisadiation . All thePst [Transcription factors are thought to play a key role in the activation and fine-tuning of various stress defense responses in plants . WRKY gePst . In thisjaponica.The major transcriptional reprogramming associated with the plant defense response requires not only different transcription factors but also the action of plant hormones ,46. As aindica and japonica.These differences are the results of joint regulation of many genes. We found the hub genes in so many different genes through the co-expression network, and found that these hub genes have a high degree of connectivity with other key genes. Changes in the level of core gene expression mean that many other genes also change. We also found that the expression patterns of these core genes were different before and after irradiation among cultivars. The difference in these expression patterns led to the difference in the response of rice to irradiation, and the difference in germination potential, germination rate, emergence rate, and seedling rate between Yet, transcriptomic dissection like this work shows can only reveal an integrative coordinated general differential response to irradiation by many genes. Further confirmation of these universal stress responsive genes to irradiation effects may reveal us more detailed mechanisms and potential applications.indica and japonica rice varieties , Huaidao5, Yanjing11, Wuyunjing24, Xudao3, Yangjing4038, Yangjing4227 and five indica: Yangdao6 (YD6), R1310, R527, R838, Huazhan. NPB and YD6 were selected for RNA sequencing analysis.Totally 12 rice varieties were used in this study, including seven p ≤ 0.05) or ** (p ≤ 0.01).Dry seeds were exposed to gamma radiation at these dosages: 0, 50, 100, 150, 250, 300, 400, and 500 Gy. After irradiation these seeds were soaked in tap water for four days and germinated at 32 °C for 8 hrs and then at room temperature (25 °C) on the 5th day. The seed germination potential is enumerated on the 2nd day post-germination and the seed germination rate on the 5th day. Morphological investigation features of shoot and root was on the 10th day. About 3000 rice seeds from each treatment were sowed in nursery field for seedling survival test. The seedling germination rate in field was counted on the 7th day and the survival rate on the 30th day. Calculation equations are as follows: seed germination potential =  × 100%, seed germination rate =  × 100%, seedling emergence rate = (number of emerged seeds/number of sown seeds) × 100%, seedling survival rate = (number of seedlings survived on the 30th day/number of sown seeds) × 100%. Three biological replicates were investigated and each biological replicate contained one hundred seeds, statistically significant are labeled as *  was used to generate image before subject to analyze by software WinRhizo Pro 2004a . Morphological features include length, surface area, average diameter and volume. Treatment has at least three biological replicates and each biological replicate contains fifty seedlings.Nuclei were prepared from shoot and root separately. Approximately 0.1 g of shoot and root tissues was chopped quickly in 200 ul of pre-chilled 1× PBS. The extraction mixture was filtered via 200 μm nylon mesh after incubation on ice 8 min. Approximately 30 μL of resulting suspension of nuclei was mixed with 70 μL of 1% low-melting-point agarose and spread on the microscope slide, which was pre-coated with 1% normal-melting-point agarose. After solidification for 20 min at room temperature, the nuclei were subjected to lysis by incubation for 80 min at 4 °C in a high salt solution  and followed by neutralization at 4 °C using 1xTBE solution three times for 5 min each. The neutralized nuclei were electrophoresed in a pre-chilled 1xTBE solution for 30 min at room temperature. After electrophoresis, the slides were kept to clear the starch grains from the gels in 1% of Triton X-100 for 10 min and dehydrate in 75% and 90% ethanol for 5 min respectively and air drying. The nuclei were stained with propidium iodide , and images captured by using a fluorescence microscope  equipped with a CCD camera. DNA damage signals were quantified using the comet assay software project .For RNA sequencing sample preparation, uniformly germinated seeds of NPB and YD6 were prepared before the irradiation. On the 3rd day after germination, the plants were exposed to γ-irradiation of 50 Gy (treatment), and 0 Gy (control). A total of twenty-four fresh samples containing two cultivars (YD6 and NPB), two tissues (shoot and root), two treatments (0 Gy and 50 Gy) and three biological replicates were prepared. All samples were collected immediately after the irradiation.Total RNA was extracted using the mirVana miRNA Isolation Kit  following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer . The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit  according to the manufacturer’s instructions. Then these libraries were sequenced on the Illumina sequencing platform  and 150 bp paired-end reads were generated. Total RNA was isolated from 8 samples with three biological replicates, including root and shoot. These three biological replicates from the NPB and YD6, respectively. NG0R, NG0S indicate the control (0 Gy) of root and shoot in NPB, respectively. NG5R, NG5S indicate the treatment (50 Gy) of root and shoot in NPB, respectively. YG0R, YG0S indicate the control of root and shoot in YD6, respectively. YG5R, YG5S indicate the treatment of root and shoot in YD6, respectively.p-value ≤ 0.05 and fold change ≥ 2 or fold change ≤ 0.5 was set as the threshold for significantly differential expression. The genes with FPKM values that fit the above criteria were considered for further analysis. Hierarchical cluster analysis of DEGs was performed to explore transcripts expression pattern. AgriGO was used to perform GO enrichment analysis . GO enrichment and KEGG pathway analysis were respectively performed using R based on the hypergeometric distribution.FPKM value of each gene was calculated using Cufflinks algorithm , and thehttp://mapman.gabip.org, last accessed on: 31 August 2019). For classifying the DEGs , the RGAP gene model of 552 genes in radiation response and 2350 genes in varietal effect were uploaded to the MapMan tool. The remaining genes were not assigned to MapMan terms due to unknown functions.Mapman software was used to analyze DEGs through the metabolism, large enzymes families, cellular response and transcription overview  and chromosomal DNA contamination was removed using DNAase following the manufacturer’s instructions. DEGs were selected based on their possible functions and expression patterns in RNA-Seq. Primer sequences  were dest method . All PCRWGCNA (v1.29) package in R was used to construct the co-expression networks . From thjaponica and indica rice subspecies. The development of shoots and roots is co-regulating the final rice seedling growth after irradiation, and the stability of agronomic traits in shoot part is more important for survival than that in root part. The core genes related to DNA repair and replication and cell proliferation are similarly regulated by irradiation, however the universal stress responsive genes are in contrasting differential response pattern between the japonica and indica rice subspecies. We identified specific modules that are associated with the phenotypic traits and candidate hub genes for each module. In conclusion, these results shed light on the regulatory mechanism and the difference between japonica and indica in response to irradiation stress in rice.This is a pioneer study to reveal the transcriptomic changes in response to irradiation between


Firmicutes, Proteobacteria and Bacteroidetes regardless of gender and different ages. Higher proportions of Proteobacteria were found in adult male and juvenile female individuals. The composition of Bacteroidetes was stable with the gender and age of FMD. Interestingly, the relative abundance of genera Clostridiales and Bacteroidales were higher in the juvenile FMD. Conversely, proportions of Pseudomonas and Lachnospiraceae were abundant in the adult FMD. Higher proportions of Ruminococcaceae, Dore, and 5-7N15 were found in the juvenile male groups. They may reflect the different immune resistance of male and female individuals at different stages of development. This study explored the fecal microbiota composition of forest musk deer in relation to gender and age, which may provide an effective strategy for developing intestinal microecological preparations and potential musk deer breeding.Animal gut microbiota begins to colonize after birth and is functionally indispensable for maintaining the health of the host. It has been reported that gender and age influence the composition of the intestinal microbiome. However, the effects of gender and age on the intestinal microorganism of forest musk deer (FMD) remain unclear. The aim of this study was to establish the relationship between the structure and composition of fecal microbiota of male and female forest musk deer with age. Here, Illumina Miseq 300PE sequencing platform targeting 16S rRNA V3–V4 hypervariable region applied to define the fecal microbiota of male and female FMD with two age groups, juvenile (age 1–2 years) and adult (age 4–10 years). Alpha diversity index did not show significant difference in bacterial diversity between the males and females or among age groups. The intestinal microbiota of FMD was dominated by three phyla, the  Moschus berezovskii) is one of the six species belonging to Moschidae family which is widely distributed in Asia, except one of the earliest musk deer that have existed from Oligocene deposits in Europe. In China, most forest musk deer is found in Sichuan province [The forest musk deer , five adult  male forest musk deer (MF); and five juvenile (JFF1–JFF5), and five adult (AFF1–AFF5) female forest musk deer (FF) were reared at Chongqing institute of drug cultivation . The forest musk deer were fed with constant and balanced diet. Their daily diet consisted of green and fine feed and varied in proportion according to gender and age. The animals included in this study were not administered any antibiotics or other veterinary drugs in the past two months, and each musk deer was kept in its own enclosure. A total of 20 fresh feces sample from FMD were collected during February 2018. To avoid contamination the central portion of feces was placed in sterile polyethylene ziploc bag, wrapped in foil paper, and then quickly dropped into the liquid nitrogen container, finally transferred to −80°C refrigerator, until DNA extraction.μl reactions with 5 μl of 5X reaction buffer, 5 μl of 5X GC buffer, 2 μl dNTP (2.5 mM), 1 μl each forward and reverse primer (10 mM), 2 μl gDNA template, ddH2O 8.75 μl, and 0.25 μl Q5 DNA Polymerase. PCR amplification consisted of initial denaturation at 98°C for 2 min, followed by 20–30 cycle of 98°C for 15 s, 55°C for 30 s and 72°C for 30 s, and a final extension of 72°C for 5 min. PCR amplified products were detected by 2% agarose gel electrophoresis and the target fragments were digested and recovered by using AxyPrepDNA gel recovery kit (Axygen). Ultimately, the sequencing Library was prepared by TruSeq Nano DNA library preparation kit from Illumina. The samples were sequenced on Illumina Miseq 300PE sequencing platform at Novogene Bioinformatics Institute.Total genomic DNA was extracted from fecal samples using the TIANamp Stool DNA kit  according to the manufacturer's instruction. The DNA quality was accessed by 1% agarose gel electrophoresis, and DNA concentration was measured by Nano Drop 3300 (Thermo Scientific). The highly variable regions (V3 and V4) of the 16S rRNA gene were amplified using the 338F/806R bar-coded fusion primer set: . All PCR was carried out in 25 http://ccb.jhu.edu/software/FLASH/) and QIIME , respectively to acquire high-quality data. The potential chimeric sequences were removed using USEARCH algorithm . Then sequences were assigned to operational taxonomic units (OTUs) with a 97% threshold of pairwise identity, and species annotated based on Ribosomal Database Project (RDP)-classifier using the Greengenes reference Database .The raw data of sequencing was stored in FASTQ format. Sequence assembly and quality filtering were performed using FLASH (v1.2.7 : http://jvenn.toulouse.inra.fr/app/index.html). The bar iagram of alpha diversity indices (Chao1 and Shannon) and relative abundance (Phylum and genus) were drawn using GraphPad Prism7. In addition, we used the independent sample t-test to analyze the significant difference in diversity index and relative abundance using IBM SPSS Statistics version 19. The Heatmap was generated through R package. The non-metric multi-dimensional scaling (NMDS) was plotted based on the Unweighted Unifrac distance. ANOSIM analysis was conducted for the Unweighted Unifrac distance subject to gender and age. Linear discriminant analysis coupled with effect size (LEfse) was generated to identify the microbial communities differentially represented between the groups at genus level using LEfse software (LDA >2).The rarefaction curves and rank abundance curves were displayed with R software, Venn diagrams were created using online weblink (The Illumina MiSeq 16 s rRNA sequencing data of 20 samples (five from each FMD group of this study) were analyzed for gut microbiota. After performing a series of quality filter steps, a total of 1,076,941 (average length of 422 ± 40 bp) chimera-free high quality sequences were recovered, with an average of 54,392 ± 8145 sequences per sample, ranging from 37,057 to 71,233. These sequences were assigned to a total of 8310 OTUs based on 97% similarity sorted from 20 fecal samples. Each sample has 1906 ± 315 OTUs on average, ranged from 1231–2292 . The seqWe compared alpha diversity metrics across four sampling groups of musk deer  to explore the changes in intestinal microbiota. Each musk deer group was divided according to gender and age. The Chao1 and Shannon index were not significant between males, females, or among age groups. However, the patterns of proportional differences for Chao1 diversity index among the groups were recorded as, JMF < AMF, JFF < AFF, JMF > JFF, and AMF > AFF, likewise for Shannon index as, JMF > AMF, JFF < AFF, JMF > JFF, and AMF < AFF . Furtherp = 0.04). The number of OTUs shared by gender group JMF-AMF and JFF-AFF were 5890, 5319, respectively. Likewise for age group JMF-JFF and AMF-AFF shared OTUs were 5489, 5578, respectively , Proteobacteria (Enterobacteriaceae) and Bacteroidetes , as shown in Supplementary The Simpson index of JFF was found significantly lower than AFF group  followed by AFF (58.66%), JFF (55.90%), and AMF (55.66%). On the contrary, the Proteobacteria was abundant in AMF (28.67%) followed by JFF (19.43%), AFF (17.45), and JMF (10.35%). The enrichment of Bacteroidetes was observed in AFF (20.65%), JFF (19.17%), JMF (17.02%), and AMF (12.59%), as shown in  and AFF (12.31%) in average. The others such as Lachnospiraceae, Dorea, Bacteroidales, Enterobacteriaceae and 5-7N15 were classified with relatively low abundance. Furthermore, Enterobacteriaceae had the lowest abundance in AFF accounting for only 0.11% in average  and five genera were significantly abundant in AFF . Meanwhile, only Barnesiellaceae genus was significantly associated to the JMF group. However, we did not find any significantly abundant bacterial taxa in the JFF group. Heatmap results based on Unweighted Unifrac distances for bacterial abundance at genus level showed that each sample varied with the same bacterial genera, but no strong clustering was observed in the samples grouped by gender and age , which were supported by the NMDS ranking.A LEfSe analysis was performed with the pooled data and observed abundant microbiota composition across 4 musk deer groups with gender and age which might be used as biomarker. Cladogram results showed that a total of 13 genera were differentially represented between JMF, AMF and AFF with LDA scores >2 . Among t and age . NMDS di and age . FurtherAkkermansia and Ruminococcaceae in the large intestine and fecal samples, while Clostridiales and Bacteroidales were relatively abundant in the forestomach and small intestine [Bacteroidetes, Ruminococcus, Lactobacillus, Flavonifractor and Clostridium were dominant genera in the cecum and rectum, while Lactobacillus show a decreasing decreasing trend from jejunum to the cecum [Firmicutes, Bacteroidetes and Proteobacteria predominate in dairy cattle reveal significant spatial heterogeneity in composition, diversity and species abundance distributions of intestinal microbiota [This is the first comparative study to characterize the composition and structure of fecal microbiota of forest musk deer with gender and age. Differences in complex fecal microbial communities in male and female is assumed to play important role in intestinal disease development of the forest musk deer. Previously many reports have shown the changes in gut microbiota composition of ruminants, such as Bactrian camel , sheep , dairy cntestine . An invehe cecum . Firmicucrobiota . Althougcrobiota . In addicrobiota . It is mIn the four sampling groups of musk deer, we did not find a significant difference in bacterial diversity. However, the abundance and diversity of intestinal microbial community in AFF was higher than JFF, while the diversity in the JMF was higher than AMF in age groups. In gender groups, the abundance and diversity of intestinal microbes in JMF was higher than JFF, while the diversity in AFF was higher than AMF . PreviouFirmicutes, Proteobacteria and Bacteroidetes, which is consistent with previous observations in FMD and other ruminants. The only difference is that the proportion of Proteobacteria in this study is higher, but similar to Rex rabbits. In the entire gastrointestinal tract of Rex rabbits, the most dominant phylum was Firmicutes followed by the Proteobacteria in the foregut [Firmicutes accounted for more than 50% in all four sampling groups and up to 71.15% in the JMF group (Supplementary Tables S4 and S5). The ruminants' microflora such as lineages Ruminococcaceae, Clostridiales, Lachnospiraceae and Dorea (Supplementary Tables S6 and S7) were mainly existed in the intestinal bacteria, the Firmicutes of forest musk deer. Firmicutes were typically the most abundant bacterial phylum in the gastrointestinal tract of vertebrate, particularly in herbivore, which play an important role in the breakdown of the fiber cellulose and nutrient absorption. Most symbiotic bacteria have been reported for the maintenance of intestinal homeostasis and immunity [Firmicutes abundance in the intestinal tract of musk deer with diarrhea would likely accompanied by reducing the digestive physiological functions [Proteobacteria was another dominant phylum, but the colonization of this phylum may be variable in different individuals of four sampling groups. The Proteobacteria was identified as dominant species in giant pandas and red pandas, which played a key role in digestion of the main food sources of lignin. Proteobacteria are often found in the natural environment and individual wild animals may do accumulate more [Proteobacteria is a marker for an unstable microbial community (dysbiosis) and a potential diagnostic criterion for disease. Acute or chronic inflammation and Low fiber diets can lead to enrichment of Proteobacteria in the GI tract [Proteobacteria and individual differences. Therefore, we speculated that these two groups may have chronic enteritis, while AFF contains a relatively low level of Enterobacteriaceae, and the group was in a relatively healthy state.Bacteroidetes are strictly anaerobic and have the ability to degrade complex molecules  in the intestine, which can promote the development of gastrointestinal immune system to improve the nutritional utilization of the host. Therefore, a high-starch diet is beneficial to the enrichment of more bacteroides, making them important for both herbivorous and carnivorous diets [Bacteroidetes is steady in the individuals of FMD and plays an important role in intestinal digestion.At the phylum level, the core bacterial phyla belong to  foregut . Firmicuimmunity . Meanwhiunctions . Interesate more , 34, 35.GI tract . In addiGI tract . The AMFus diets , 38. FurFirmicutes and Proteobacteria. This relationship was reflected in AMF, which contains more Proteobacteria than JMF; JFF contains relatively higher proportion of Proteobacteria than JMF, and the AMF contains more Proteobacteria than AFF. For the initial three days, the Proteobacteria was dominated later on Firmicutes begin to rise into the main class in Peking ducks [Firmicutes to Bacteroides in feces of older pigs  were extremely higher compared to those from piglets at one month of age [Proteobacteria in healthy captive male FMD was 5.2% ± 5.6% and significantly differed among other individuals [Proteobacteria, reflecting similar differences in competitive relationship. Verrucomicrobia mainly had a higher proportion in JFF and lower in other groups and Tenericutes represented mainly in adults. An interesting finding of our study is that the Clostridiales and Bacteroidales were higher in juvenile than adult FMD. Conversely, Pseudomonas and Lachnospiraceae were higher in adult. Ruminococcaceae, Dore and 5-7N15 were higher in the JMF group than other groups. Members of Ruminococcus have been identified as important members of the gut communities in female subjects [We also found a competitive relationship between ng ducks . A priorh of age . In addiividuals . Adult msubjects  and thissubjects . A LEfSeFirmicutes, Proteobacteria and Bacteroidetes were predominant phyla in the microbial community. There was a competitive relationship between Firmicutes and Proteobacteria in four sampling groups. The two groups of AMF, and JFF, may be in a sub-healthy state in terms of the diversity and composition of intestinal bacteria with lower diversity and more Proteobacteria. This reflects the variable immune resistance of males and females at different growth stages. However, the proportion of Proteobacteria in our study was 2 to 4 times higher than previous studies. The present study may provide meaningful biological insights into the age and gender based alterations in fecal microbiota of forest musk deer, and will be useful in developing intestinal microecological preparations and potential musk deer breeding.Overall, this is the first study to demonstrate the impact of gender and age on the gut microbiota of forest musk deer to some extent. This work shows that


In the short life of broiler chickens, their intestinal microbiota undergoes many changes. To study underlying biological mechanisms and factors that influence the intestinal microbiota development, longitudinal data from flocks and individual birds is needed. However, post-mortem collection of samples hampers longitudinal data collection. In this study, invasively collected cecal and ileal content, cloacal swabs collected from the same bird, and boot sock samples and cecal droppings from the litter of the broilers’ poultry house, were collected on days 0, 2, 7, 14 and 35 post-hatch. The different sample types were evaluated on their applicability and reliability to characterize the broiler intestinal microbiota. The microbiota of 247 samples was assessed by 16S ribosomal RNA gene amplicon sequencing. Analyses of α and β measures showed a similar development of microbiota composition of cecal droppings compared to cecal content. Furthermore, the composition of cecal content samples was comparable to that of the boot socks until day 14 post-hatch. This study shows that the value of non-invasive sample types varies at different ages and depends on the goal of the microbiota characterization. Specifically, cecal droppings and boot socks may be useful alternatives for cecal samples to determine intestinal microbiota composition longitudinally. In the six- to eight-week life span of commercial broilers the intestinal microbiota composition changes rapidly ,2. SeverThe sample type has a large impact on the observed intestinal microbiota, because the diversity and functionality of the intestinal microbiota substantially differs per intestinal region in mammals and poultry ,14,15. ISalmonella [A non-invasive sample type used in several studies is the cloacal swab ,37,38. Tlmonella ,41. TheoTherefore, the aim of this study is to evaluate invasively collected samples and non-invasively collected samples with regard to characterization of the intestinal microbiota composition. On two different broiler farms cloacal swabs were collected intravitally and from the same birds cecal and ileal content was collected at post-mortem at 0, 2, 7, 14 and 35 days of age. At the same time points, cecal droppings and boot socks samples were obtained from the broiler house. We compared the microbiota composition of cecal and ileal content to cloacal swabs, and compared cecal content to cecal droppings, and cecal content with boot socks samples. In addition to the microbiota composition, also the discriminative properties of the sample types were assessed, by evaluating its ability to distinguish farms or the age of the broilers. The microbiota composition of 247 samples was assessed by 16S ribosomal RNA (rRNA) gene amplicon sequencing, to determine intestinal microbiota composition longitudinally in broiler flocks and in an experimental setting. Results from this study show that cecal droppings represented the cecal microbiota of the five individual birds across different time points quite well, even though we collected those samples in pools from the litter in the poultry house. Early in the production cycle, the composition measured with the boot socks was close to that of cecal content samples. Cecal and ileal content samples are, however, distinct from cloacal swabs. Therefore, cecal droppings and boot socks may be useful alternatives for cecal samples to determine intestinal microbiota composition longitudinally. This study contributes to expanding the toolbox for the collection of valuable data under field and experimental circumstances. With these tools, novel strategies can be developed to steer towards more resilient gut microbiota.The Dutch Central Authority approved the animal experiment for Scientific Procedures on Animals and the Animal Experiments Committee  of Utrecht University , and all procedures were done in full compliance with all relevant legislation.® G160, Elanco, Houten, The Netherlands) from day 0 onwards until day 28 followed by narasin  from day 28 to day of slaughter age. At both farms water and feed were supplied ad libitum but the farms had a different feed supplier and received broilers from different commercial hatcheries. Diets on both farms were wheat based, combined with feeding of whole wheat at later ages. In addition to soybean meal, sunflower seed meal and rapeseed meal were added at maximum 6.5% inclusion.Data for this study were obtained from two broiler farms in The Netherlands, with Ross 308 broilers, low historic use of antibiotics, and good production performance. No antibiotics were used. Coccidiostatic drugs were applied as standard in the feed of both flocks. Both farms used a combination of nicarbazin and narasin  from days 0–3, 20 h/d for days 4–6 and 18 h/d for days 7–35. Temperature was set to gradually decrease from 34 °C at day 0 to 20 °C at day 35. Farm 1 used wood shavings and Farm 2 peat as litter material. During one production cycle both farms were visited on day 0 , 2, 7, 14 and 35. One farm was visited in August 2016, and the second farm was visited in June 2017. From each poultry house, five broilers were randomly selected, one from each corner and in the middle of the poultry house. The start of the sampling of broilers took place at least 30 min after the end of a dark period, to reduce potential effects of fasting on the amount of content in the intestinal tract and microbiota composition.During each visit samples were taken from the five broilers per house before, i.e., cloacal swabs, and after euthanization, i.e., cecal content and, ileal content, and from the poultry house, i.e., cecal droppings from special feces collection boxes, and boot socks. The broilers were collected from the broiler house and immediately a cloacal swab  was taken. The swab was placed in a 2 mL sterile cryotube  and cut with a sterile scissor to fit in the tube. Each tube with swab was placed on dry ice before being stored in a freezer (−80 °C) for future processing. Subsequently, the broilers were euthanized by cervical dislocation, immediately followed by removal of the gastrointestinal tract. This procedure was carried out in as sterile a way as possible, and between broilers sterile gloves were changed and the table, scissors, and tweezers were cleaned with 70% ethanol to prevent cross contamination between broilers. The distal end of the cecum was cut to collect cecal content. Ileal content was collected distal and close to the Meckel’s diverticulum. The intestinal content was gently squeezed into a 2 mL sterile cryotube and snap frozen on dry ice and stored at −80 °C for microbial genomic DNA extraction. The time between euthanization and placing the samples on dry ice was between 3–5 min.In each poultry house five fecal collection boxes of wood with iron netting (70 cm × 50 cm × 5 cm) were placed on the litter, with one in each corner and one in the middle of the poultry house . At the Per broiler house, at each sampling day, five boot socks  were used to collect litter samples at the same locations where the fecal collection boxes were placed . Boot soIn total, 50 cecal and 50 ileal content samples, 47 cecal droppings, 50 cloacal swabs and 50 boot socks were analyzed. DNA was extracted from 0.25 g cecal and ileal content and cecal droppings using 700 μL of Stool Transport and Recovery (STAR) buffer . All samples were transferred to a sterile screw-capped 2 mL tube  containing 0.5 g of zirconium beads  and 5 glass beads  was used.The cloacal swab samples were directly transferred to a sterile screw-capped 2 mL tube (BIOplastics BV) containing also 0.5 g of zirconium beads  and 5 glass beads  but 300 μL of Stool Transport and Recovery STAR buffer. g for 5 min at 4 °C). The supernatant was removed and of the 10 mL tube again 1.5 mL boot sock content was added and centrifuged . The pellet was resuspended in STAR buffer. All samples were treated in a bead beater  at a speed of 5.5 m/s for 3 × 1 min, followed by incubation at 95 °C with agitation (15 min and 300 rpm). The lysis tube was centrifuged , and the supernatant was transferred to a 2 mL microcentrifuge tube. Thereafter, the aforementioned process was repeated with 300 μL STAR buffer for the cecal, cecal droppings and ileal content samples, and with 200 μL STAR buffer for the swab and boot sock samples.Before DNA was extracted from a boot sock sample, the boot sock was cut in half with a sterile scalpel and put into two different 50 mL Falcon tubes. Then 35 mL phosphate-buffered saline (PBS) was added to both 50 mL tubes and the tubes were vortexed for 20 s (s) at maximum speed. Thereafter the tubes were placed for 30 min in a carousel  to soak the feces from the boot sock (room temperature). From both tubes, 5 mL was pipetted in a 10 mL sterile tube. Next the tubes were vortexed for 5 s at maximum speed to mix and 1.5 mL of boot sock content was transferred to a sterile 2 mL microcentrifuge tube and centrifuged . The remainder of the extraction protocol was then carried out in the Maxwell® 16 Instrument  according to the manufacturer’s instructions. The DNA concentrations were measured with a NanoDrop ND-1000 spectrophotometer , and the DNA samples were stored at −20 °C until further use.An aliquot (250 μL) of the combined supernatants from the sample lysis was then transferred into the custom Maxwell−1 in nuclease free H2O. All polymerase chain reaction (PCR) plastics were ultraviolet (UV) radiated for 15 min before use. For 16S rRNA gene-based microbial composition profiling, barcoded amplicons covering the variable regions V5-V6 of the bacterial 16S rRNA gene were generated by PCR using the 784F and 1064R primers as described in Ramiro-Garcia et al. [Extracted DNA was diluted to 20 ng μLa et al. .Each sample was amplified in duplicate using Phusion hot start II high fidelity polymerase  checked for correct size and concentration on a 1% agarose gel and subsequently combined and purified using CleanNA magnetic beads . A detailed description of the PCR reactions was described previously .Positive and negative controls were added to the data set to ensure high quality sequencing data. As positive controls we used synthetic communities of known composition , and as To compare sample types we applied widely used diversity measures and univariate and multivariate statistical analyses. Alpha diversity (within sample) was determined using Faiths phylogenetic diversity  which noBacillaceae, Burkholderiaceae, Halomonadaceae, Micrococcaceae, or Shewanellaceac. The ileal content and boot sock samples of day 0 were excluded because these contained a large number of families associated with the negative control samples, and therefore did not pass our quality control standards  associated with an unknown domain or the order Mitochondria were removed from all sequenced samples. About 90% of the families in the negative controls were associated with five families: tandards .2 = 10.6, p = 0.001 and χ2 = 18.5, p < 0.001). The cecal content was not significantly different from cecal droppings and boot socks based on phylogenetic diversity . Alpha diversities Chao 1 and Shannon, that do not take into account the phylogenetic relatedness, showed one different result of the comparisons. According to Chao 1 and Shannon measures, the ileal content samples were not significantly different when compared to cloacal swab samples . No age effect was found for ileal content samples  and Jaccard (3.6%) , Table 1Clostridiaceae , Corynebacterium (14–28% points) and Staphylococcus (11–18% points) showed a higher relative abundance in the boot socks compared to cecal content samples  in the cloacal swab samples . Only in the boot socks was it also possible to observe a higher relative abundance in Bacteroides in Farm 2 (p = 0.029). No difference in genera between farms could be detected in cecal droppings, cloacal droppings, or ileal content samples.Concerning differences between farms, within the cecal content samples the only difference observed was a higher relative abundance of the genus The aim of this study was to evaluate different types of samples with regard to characterization of the intestinal microbiota of individual broilers and flocks from the first week of life onwards until the end of the production cycle. In particular, we compared invasively collected cecal and ileal content samples to non-invasive samples, such as cloacal swabs, cecal droppings and boot sock samples collected from the litter. In addition, the invasive and non-invasive sample types were evaluated on the ability to distinguish between ages and among farms. The outcomes of this study suggest that cecal droppings and boot socks are useful non-invasive alternatives for cecal content samples to determine intestinal microbiota longitudinally in broilers and broiler flocks.In our study, the cecal microbiota had the highest diversity and showed less variation over time compared to the microbiota of the ileal content, which is consistent with other studies ,24,25. ANo differences between the farms were observed in cecal content based on α diversity and β diversity taking phylogenetic relatedness into account. This is in contrast with another study where differences in microbiota composition of cecal content were found between broiler flocks . Our resLactobacillus compared to cecal content. These results add new information to previous results described in literature, which only showed that cecal droppings reflected cecal content microbiota of broilers at the end of the production cycle [When comparing non-invasive sample types to the cecal content, the cecal droppings represented the cecal microbiota of the five individual birds across different time points quite well, even though we collected those samples in pools from the litter in the poultry house. Only on day 2 of age did cecal droppings show a higher relative abundance of the genus on cycle . Cecal don cycle . NeverthSalmonella [Campylobacter in the environment [Lactobacillus, Escherichia-Shigella and Enterococcus were lower in relative abundance in the boot socks. However, on day 35, 20 genera were different between the boot socks and the cecal content, which indicated that boot socks were less useful to reflect the cecal content microbiota near the end of the production cycle. When comparing our results to the literature, it must be pointed out that Corynebacterium, Sphingobacterium, and Lactobacillus were observed to be highly abundant in litter samples on day 35 [Another surprising potentially useful non-invasive sample type to monitor the cecal microbiota at flock level are boot socks samples. This method is commonly used in poultry houses to monitor the presence of lmonella ,41, and ironment . To our n day 35 , which in day 35 ,59.Lactobacillus, Streptococcus and Clostridium [It was expected that cecal and ileal content samples would be distinct from cloacal swabs, because that has been described before ,21,28. Tstridium ,60,61, cstridium ,24,25,62Consistent with our present findings, previous research also showed that age explained part of the individual variation in ileal content microbiota of broilers . NeverthCurrently, it is unclear how the intestinal microbiota can be manipulated to optimize the robustness of broiler flocks, for example, to improve the alertness of the immune system while maintaining production efficiency . KnowledIn conclusion, this study shows that the value of non-invasive sample types varies at different ages and depends on the goal of the microbiota characterization. We have shown that cecal droppings and boot socks, collected from a broiler house, are useful alternatives for cecal samples collected during post-mortem, to determine intestinal microbiota composition longitudinally in broiler flocks and in an experimental setting. These sample types may be a useful expansion of the current toolbox for microbiota studies. Further studies should be done to validate the use of those microbiota sample types as a diagnostic tool early in the production cycle, e.g., by studying broiler flocks with differences in health and productivity status in further detail. Non-invasive longitudinal sampling to monitor the development of the intestinal microbiota will facilitate the development of new and better interventions to improve the health and performance of broiler chickens.