Fusarium concolor X4, was applied to treat poplar CTMP for exploring the effects of biotreatment on brightness and light-induced yellowing of the pulp. The results indicated that the biotreatment with Fusarium concolor X4 could improve the brightness of poplar CTMP and inhibit light-induced yellowing of the pulp. The yellowing inhibition mechanism was explored by the analysis of enzyme production regularity during biotreatment, changes in chemical components, and the UV-Vis absorption spectra and FTIR-ATR spectra of pulps before and after biotreatment.High-yield pulps (HYPs), such as CTMP , are attractive due to their low cost and high wood utilization. However, their drawback of rapid brightness reversion (yellowing) limits wide use of the HYPs. In this study, a fungus,  The increasing forest resource consumption and environmental pollution are the two most serious problems for the pulp and paper industry. Researchers have done intensive studies for these problems . EfficieHowever, the well-known “brightness reversion ” of HYPs is a major drawback in HYPs application . GeneralAccording to previous researches, there are many methods to suppress the pulp yellowing. For example, UV absorbers are efficient substances to prevent brightness reversion. It had been reported that thiols or sodium hypophosphite were effective for preventing the light-induced yellowing , 10. 1-oE. grandis wood was treated with fungus C. subvermispora to produce bleached bioTMP  and found the bleached biotreated pulp had better thermal-stability than the control, while the photo-stability was similar for both pulps [In recent years, biotreatment is increasingly applied for pulping and bleaching processes in pulp industry because of its environmental friendliness and low cost. Many researchers had done lots of works in this field –14. For th pulps . Microorth pulps , 17. Theth pulps , 18, 19.Fusarium concolor X4 (F. concolor X4) is a fungus that has efficient delignification ability, which was screened and preserved in our laboratory. Our previous studies showed treatment with F. concolor X4 could effectively improve brightness and inhibit heat-induced yellowing of unbleached poplar CTMP and wheat straw semichemical pulp [F. concolor X4 pretreatment on light-induced yellowing of poplar CTMP, and the mechanism of yellowing inhibition was studied by investigating the changes on different types of enzymes produced by the strain and on chemical components of pulps, the UV-Vis absorption spectra, and FTIR-ATR spectra before and after the biotreatment.cal pulp –22. In tUnbleached Poplar CTMP was kindly provided by Hua tai Paper Co., Ltd. . The pulp was washed twice with tap water and preserved at 4°C.F. concolor X4 was screened from decayed wood and maintained on PDA slants in our laboratory [boratory .F. concolor X4. The strain was grown for about 3 days at 28°C at 150 rpm to inoculate the shake flask until the cell concentration reached 108 Colony-Forming Units per 1 mL (CFU/mL).The media (80 mL) contained 20 g/L glucose, and 5 g/L yeast extracted powder was used for precultivation of 4Cl 0.2 g, MgSO4·7H2O 0.5 g, KH2PO4 0.2 g, Na2HPO4 0.2 g, FeSO4·7H2O 0.007 g, MnSO4 0.035 g, CuSO4·5H2O 0.007 g dissolved in 1 L of distilled water) in flasks (3 L) and autoclaved at 115°C for 30 min. After cooling down to room temperature, the homogenized pregrown culture  was inoculated into the flasks. The pulps were incubated at 28°C for up to 10 days in an oscillation incubator with shaking at 150 rpm. The samples inoculated with inactivated pregrown culture by sterilization were used as control. For each sample, three replicates were made.Poplar CTMP (40 g oven dry weight) were adjusted to 5% consistency with solution . Flasks were shaken as far as possible to make the matrix evenly before sampling, and 2 g of pulp (oven dry weight) was taken out for each flask. The pulp slurry sampled were collected by centrifugation at 4°C at 8000 rpm for 10 min and then were washed twice in nylon cloth bags with 100 ml water for further experiments. The supernatants obtained by centrifugation were collected for the determination of enzymes activities.2SiO3 3%, MgSO4 0.5%, and H2O2 3% and pH of 10 to 11 for 2 h. After EDTA treatment and bleaching, the pulps were thoroughly washed to neutral with distilled water. The pH of pulp was adjusted by using H2SO4 2 mol/L and NaOH 1 mol/L. After bleaching, the test sheets were prepared using the bleached pulps by a Buchner funnel containing a cloth, compressed between filter papers, and dried at room temperature (25–27°C) in a dark environment.Biotreated pulps and the control were adjusted to 10% consistency, with pH 3.0 in polyethylene bags, and firstly treated with 0.5% EDTA at 80°C in water bath for 30 min and then were bleached under the conditions of pulp consistency 10%, 80°C, NaOH 1.5%, Naλ) of 295–350 nm, and λmax = 340 nm, were used for light-induced yellowing. The light intensities at the sample site were kept at 4.6 μW/cm2, which were determined by an ultraviolet irradiation meter. Temperatures in the aging box were kept at about 27°C by a cooling fan equipped in the box to minimize the heating effect of the light source. After 24 h irradiation, samples were conditioned in dark room for 4 h before analysis.Four UVB-340 lamps , which emit UV-Vis light with wavelength  with 1 mM 4-Nitrophenyl beta-D-xylopyranoside (pNPX) and suitable amount enzyme  and incubated for 10 min at 45°C. The reaction was stopped by 0.5 mL of Na2CO3 (1M). The released p-nitrophenol (pNP) was determined at 420 nm [μmol of sugar (or pNP) equivalents per minute under the assay conditions.Xylanase activity was determined by mixing 1.5 mL  of beech xylan solution prepared in 50 mM NaAc-HAc buffer of pH 4.8, and 0.5 mL of diluted enzyme, which was incubated at 50°C for 30 min. The reaction was stopped by adding 3 ml 3,5-dinitrosalicylic acid (DNS) reagent and boiled for 10 min . β-xylost 420 nm . Carboxyt 420 nm . One uniLaccase activity was determined by determining the rate of oxidation of 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) . LiP andR denotes ISO brightness of a thick pad at λ = 475 nm [ISO brightness of pulp samples were determined by brightness color tester . The degree of brightness reversion was expressed by post color number (PCN), which is calculated as follows:= 475 nm .4 as a background reference. The reflectance spectra were converted to K/S values according to the Kubelka–Munk equation [K/S vs. wavelength) were obtained by subtracting the reflectance spectrum (K/S vs. wavelength) of control sample  value was very small, and it could be considered as constant [K/S value was directly related to the amounts of chromophores and UV-active substances in the hand-sheet [K/S was approximately linear to the changes in chromophores or UV-active substances, which can be assumed according to the Kubelka–Munk theory.UV-Vis DR spectra of pulp samples were recorded on a UV-2550 spectrometer  equipped with an ISR-2200 integrating sphere, with BaSOequation . DiffereChemical compositions of pulps include the contents of acid-soluble lignin (ASL), acid-insoluble lignin (AIL), cellulose, and hemicellulose, were measured according to National Renewable Energy Laboratory  analytical methods . HexeneuF. concolor X4 minus that of samples without biotreatment (control).Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectra of pulps sheets were recorded with a Tensor 27 spectrophotometer  . The infP < 0.05 determined by using t-Student one-tail test.All of the FTIR-ATR spectra and UV-Vis DR spectra were representative of three independent experiments. The assays of pulp brightness, enzymes activities, and chemical components were performed in triplicate, and each experiment was repeated at least three times. The mean value and standard deviations were calculated using Microsoft Office 2010 Excell . The differences were considered statistically significant at F. concolor X4 treatment could improve pulp brightness. For example, the brightness of the biotreated pulp, respectively, increased by 5.0% and 12.2%  compared to the control after 2 and 6 days of F. concolor X4 treatment before light-induced yellowing. Subsequently, the increase of biotreated pulp brightness decreased, and only 3.7%  increased compared to the control after 8 days of treatment.The pulp brightness of biotreated pulps and the control before and after light-induced yellowing are shown in P < 0.01, n = 3) for pulps obtained in 2nd day, 6th day, and 8th day treatment with F. concolor X4 compared to the control, respectively.After light-induced yellowing, the increases on pulp brightness were 5.9%, 11.4%, and 1.6%  after treatment of 2 days. The MnP activity was 15.2 IU/L  at the 4th day of biotreatment. After 6 days treatment, laccase activity was detected (1 IU/L), and reached 1.2 IU/L at the 8th day. MnP and LiP activities peak appeared at the 10th and 8th day, and reached 29.8 IU/mL and 23.4 IU/L  respectively. The activities of CMCase and β-xylosidase were almost not detected during entry treatment period.During pulp treatment with detected . It was K/S) of biotreated pulps and control before and after light-induced yellowing. The pronounced signal peaks appeared at around 340 nm. And the strengths of signals at around 240 nm and 280 nm for controls were higher than that of biotreated pulps. For the biotreated pulp, the ▽K/S values were lower than that of control, especially for the pulps obtained by biotreatment of 6 days , respectively, after 2 days treatment of F. concolor X4, and by 3.1% and 12.6% , respectively, after 6 days of biotreatment. The HexA contents were also decreased by biotreatment. For example, decreased by about 6.8%  at the 2th day and 38.3%  at the 6th day, respectively.The chemical components of poplar CTMP before and after biotreatment of F. concolor X4 are shown in FTIR-ATR spectra of poplar CTMP and control before and after treatment of 2 days and 6 days with −1 related to vibrations of anomeric carbon (C1) in hemicellulose, and the absorptions at 1160 cm−1, 1103–1108 cm−1, and 1052–1053 cm−1 which related to vibrations of C-O-C, O-H, and C-O bands, respectively, in cellulose and hemicellulose were decreased. Absorption at 1031 cm−1 related to hemicellulose or G type lignin vibrations and absorption at 1231 cm−1 related to C-O (syringyl) bending vibrations were also decreased compared to the control  to chromophores  during light-induced yellowing [Lower ▽S values  of biotrS values  and PCN S values . This maS values  might beS values . That He Figures  suggesteellowing , 45, thuF. concolor X4 treatment, which was also proved by the decrease of hemicellulose contents shown in F. concolor X4 treatment and aromatic structure was not effectively destroyed. In the FTIR-ATR spectra of pulps, relative intensities of absorption bands related to hemicellulose were reduced, indicating that hemicellulose in pulp was degraded after F. concolor X4 increased the pulp brightness and inhibited light-induced yellowing of the pulp. It was found that different types of enzymes such as Lip, MnP, xylanase, laccase, a small amount of cellulase, etc. were produced in pulp during the F. concolor X4 treatment. The enzymes could degrade part light-active components such as lignin, hemicellulose, and HexA or change some UV-active structures in lignin, which may be some reasons for brightness improvement and the brightness stability enhancement.Biotreatment of poplar CTMP using


In this study, efforts were devoted to evaluate the benefits and drawbacks of O3–BAC–sand in a full-scale water treatment plant. The performance of the two configurations was compared in terms of particles, turbidity, CODMn and typical odorants and pesticides.To improve the microbial safety of drinking water, an arrangement of O3–BAC–sand yielded lower turbidity but higher CODMn (by approximately 7%) in the finished water than regular O3–BAC (sand–O3–BAC). Both systems removed odors in raw water; however, sand–O3–BAC was more effective on septic and musty odorants. The total pesticide removals by sand–O3–BAC and O3–BAC–sand were 78% and 72%, respectively; though the latter had shorter activated carbon durable years.The O3–BAC process in drinking water treatment.The re-location of the sand filter would sacrifice the BAC efficiency in removals of organic matter and micropollutants. This tradeoff is a result of the loss of the particulate organic matter removal by sand filters, because locating the sand filter behind BAC causes particle load increase on BAC; some measures of enhanced coagulation should be suggested to improve the turbidity and particle removal. The study will be helpful for improvement of the O Moreoveacterium , could pin China , giving cal risk  and has cal risk , 9. And Shanghai , Fuzhou Shanghai , Jinan [Shanghai  and JiaxShanghai .Kow > 2.5 tend to adsorb onto particles, resulting in higher removal efficiencies (> 80%) during sand filtration [Sand filtration is one key step in drinking water treatment to remove particles from raw water , 14, andltration , 16. Theltration . Consequltration , and thu3–BAC and O3–BAC–sand). Treatment performance was compared in the removal of chemical oxygen demand by KMnO4 titration (CODMn), the only parameter of comprehensive organic material concentration in the Chinese drinking water standard, and particles, turbidity, typical odorants as well as some typical pesticides.In this paper, the treatment performance for the rear sand filter arrangement is investigated and compared through experimental studies in a full-scale drinking water treatment plant with two parallel configurations . The sand–O3–BAC system, of 4 × 105 m3/days production capacity, consists of pre-ozonation, coagulation, sedimentation, sand filtration, post-ozonation and BAC in succession. The O3–BAC–sand system, of 3 × 105 m3/days capacity, varies in the location of sand filtration: pre-ozonation, coagulation, sedimentation, post-ozonation, BAC and sand filtration in succession. The sand–O3–BAC and O3–BAC–sand systems have operated for 47 and 33 months, respectively.The water treatment plant of 7 × 102O3 concentration) dose, 105-min sedimentation time, ozone dose of 0.5 mg/L for pre-ozonation (5 min) and 1.0 mg/L for post-ozonation (15 min), sand filtration velocity of 7.9 m/h, the empty bed contact time and filtration velocity of the BAC filter were 16 min and 8.3 m/h, respectively.During the four-month study period, typical operation parameters were aluminum sulfate at 40 mg/L . CODMn was measured on a HACH Model DR2800 spectrophotometer . Soluble CODMn (SCODMn) was obtained by measuring the samples after 0.45-μm membrane  filtration. Insoluble CODMn (inSCODMn) equals Total CODMn (TCODMn) subtracting SCODMn.The COMConcentrations of 25 pesticides  were meaFinally, particle size distribution was characterized on a Hybrid Particle Counter ZVL , in grab sample mode. The method measures both turbidity and particle count in 9 size ranges .Mn from 4.50 to 6.84 mg/L, counts for particles of 1–2 µm, 2–5 µm, 5–15 µm and > 15 µm from 268,836.00 to 731,408.25, from 32,278.64 to 294,281.25, from 18,726.58 to 47,688.50, and from 61.25 to 1783.08,respectively. It is also known for its septic/swampy odor occurrence and the presence of a variety of micropollutants because of pollution in source water [During the study period, the raw water turbidity ranged from 18.8 to 46.0 NTU, CODce water .3–BAC vs. 0.04 NTU in O3–BAC–sand (p = 0.020). The day-to-day monitoring data of the finished water also exhibited similar results  following ozonation was in contrast to an increase for the larger particles (> 2 µm). This observation apparently agrees with previous reports [3–BAC–sand system were 331.45, 50.06 and 3.14 count/mL, respectively, in comparison with 460.4, 109.8, and 14.0 count/mL, respectively, in sand–O3–BAC system. results . For thezonation  may be tzonation . Changes reports  that ozoMn along the treatment processes are shown in Mn value of finished water in sand–O3–BAC system was approximately 7% lower than that in O3–BAC-sand system. The statistically significant difference shows the better CODMn removal in sand–O3–BAC. Similarly, day-to-day monitoring data of the finished water  for ozone by i nSCODMn, and secondly, interference by accumulating particles in BAC to its biodegradation functions. The net result is lower CODMn removal efficiency of the rear sand filtration process . The total removals for sand–O3–BAC and O 3–BAC–sand were 78.43% and 70.03%, respectively.As shown in 3–BAC–sand might be attributed to the competition for ozone by the particulate organic matters (inSCODMn). Because of sand filtration upstream of ozonation, sand–O3–BAC benefited from the sand filter in removal of pesticides (14.70%) that are probably adsorbed in the particles. However, for both systems, BAC showed a relatively high removal rate for the pesticides that are mostly biorefractory, mainly because of the carbon adsorption [3–BAC than O3–BAC–sand system, suggesting that the process offers better efficiency in the rem oval of micropollutants like pesticides. The difference in pesticide removal can be also potentially attributable to the performance of BAC unit process. Prior to the comparative treatability study, the sand–O3–BAC system had already been operated by 14 months more than O3–BAC–sand system. Such BAC condition may still retain some adsorption capacity as suggested [The lower pesticide removal by ozonation in Osorption , 37. Reluggested  in a sepuggested .3–BAC and the O3–BAC–sand process in terms of their performance in the removal of particles, organic matter and micropollutants. On average, the results showed turbidity of 0.12 NTU in sand–O3–BAC vs. 0.06 NTU in O3–BAC–sand, and the average counts for particles of 2–5 µm, 5–15 µm and > 15 µm of O3–BAC–sand were 331.45, 50.06 and 3.14 count/mL, respectively, compared to 460.39, 109.80, and 14.02 count/mL, respectively, in sand–O3–BAC. However, the relocation of the sand filter might sacrifice efficiency in the removals of organic matter (by about 7%) and micropollutants. Thus, for the O3–BAC–sand application, some measures of enhanced coagulation should be suggested to improve the turbidity and particle removal, which would be helpful for enhancing the organic matter removal.A comparison was conducted for the sand–O


Chronic fatigue syndrome (CFS) is one of the most common chronic diseases in modern society and affects patients' quality of life to a certain extent. To date, the etiology and pathogenesis of CFS are still not completely clear. Various therapies have been developed, but there is still a lack of specific drugs or treatments. As a kind of adjuvant therapy, traditional Chinese medicine (TCM) has aroused widespread concern about the improvement of CFS. Although a large number of clinical randomized controlled trials have confirmed the therapeutic effect of TCM on CFS, the exact efficacy is still controversial. This article summarizes the clinical research methods and efficacy of TCM in the treatment of CFS over the past five years from the perspectives of syndrome differentiation, external treatment, and combination therapy. Since researchers led by the US Centers for Disease Control and Prevention coined the term Chronic fatigue syndrome (CFS), the history of the disease can be traced back to approximately 30 years ago . In 2018chronic fatigue syndrome, Chinese medicine treatment, and clinical trials. The English search terms included chronic fatigue syndrome, diagnosis, and treatment. The combined searching of subject words and free words was used as the search strategy. There were no language restrictions for document retrieval.The PubMed (2010～2020.3), Web of Science (2010～2020.3), JAMA Network (2015～2020.3), CNKI (2015～2020.3), SinoMed (2015～2020.3), and WanFang Data (2015～2020.3) electronic databases were searched. References were retrieved from the included documents. The Chinese search terms included (1) Clinical research literature related to the treatment of chronic fatigue syndrome with traditional Chinese medicine, clear diagnostic criteria and evaluation criteria for efficacy, complete treatment processes, and affirmative efficacy was selected. Literature about the progress of traditional Chinese medicine in the treatment of chronic fatigue syndrome and literature related to the treatment of chronic fatigue syndrome was also reviewed. (2) Duplicates or literature abstracts, meeting minutes, guidelines, regulations, government reports, animal experiments, unpublished theses, and other document syndromes were removed from the documents retrieved from the above document source databases according to the prescribed search strategy. According to the title of the article and the title of the author, the reference documents retrieved were removed from duplicate documents.In total, 331 articles were initially detected, and the two authors independently read the titles and abstracts, excluding 195 articles that did not meet the inclusion criteria. They further read the full text, screened the relevant literature of the original study as a randomized controlled trial, and finally included 77 articles, with 20 in English and 57 in Chinese.To date, the etiology and pathogenesis of CFS are still not completely clear. Modern medical considerations of the etiology and pathogenesis of CFS may be related to various factors, such as the immune and adrenal systems, genetic predisposition and certain personality traits, the neuroendocrine system, physical and emotional stress, biopsychosocial models, viral infections, childhood stress trauma, female gender, sleep quality, and nutrition –14. TherThe Golden Chamber, which is thought to be constituted as follows [TCM is a good choice as a therapy for CFS because of its long clinical history and universal efficacy. Although no exact records can be found in books, descriptions of fatigue and similar symptoms are equal to the records of “fatigue,” “emaciated”, “emptiness”, “slackness”, “exhaustion of limbs”, and “not lifting limbs” discussed by TCM . Zhang  in the H follows : (1) ins follows  analyzed follows  found thAt present, most clinical research on TCM syndrome differentiation treatment of CFS is mainly conducted through RCT, which covers TCM decoctions, proprietary Chinese medicines, and experiential prescriptions issued by clinical Chinese physicians. There is also syndrome differentiation treatment for a specific population. According to the main symptoms, they can be classified into liver stagnation and spleen deficiency syndrome, both heart and spleen deficiency syndrome, and other syndromes.Guiding Principles for Clinical Research of New Chinese Medicines and Diagnosis of TCM Syndromes [The total effective rate mentioned in this section equals the markedly effective cases plus the effective cases divided by the sample size and then multiplied by one hundred percent. The modern medical diagnostic criteria are based on the CFS diagnostic criteria revised by the Centers for Disease Control (CDC) in 1994 . The diayndromes , 25. CliAstragalus 30 g, Bupleurum 15 g, Agrimony 25 g, Atractylodes macrocephala 20 g, Pinellia tuber 15 g, Poria cocos 20 g, Tulip 20 g, Platycodon 6 g, Tangerine Peel 15 g, Scutellaria baicalensis 12 g, Licorice 12 g, and Jujube 12 g) at 150 ml bid po. After 4 weeks, the total effective rate of the observation group (97.06%) was higher than that of the control group (85.29%). Liu et al. [Angelica sinensis 10 g, Radix paeoniae alba 10 g, Bupleurum 6 g, Poria cocos 20 g, Atractylodes macrocephala 10 g, Roasted Licorice 6 g, Peppermint 6 g, Codonopsis 15 g, and Chinese yam 20 g) at 150 ml bid po. After 3 weeks, the total effective rate of the observation group (89.74%) was higher than that of the control group (68.29%).Li  observedu et al.  observedu et al.  observedAstragalus 20 g, Longan 15 g, fried Jujube seed 15 g, Poria cocos 15 g, Atractylodes rhizome 10 g, Angelica 10 g, Rhizoma chuanxiong 10 g, White peony root 10 g, Bupleurum 10 g, Citrus aurantium 10 g, Turmeric 10 g, Radix aucklandiae 10 g, Polygala 10 g, Tangerine peel 10 g, Cohosh 10 g, and Licorice 6 g). After 8 weeks, the total effective rate of the observation group (95.35%) was higher than that of the control group (72.09%). Ding [Astragalus 30 g, Semen ziziphi spinosae 25 g, Tuckahoe with pine 15 g, Longan 15 g, Polygala 15 g, Angelica 15 g, Atractylodes15 g, Codonopsis 15 g, Radix aucklandiae 7 g, and Licorice 10 g. After 8 weeks, the total effective rate of the observation group (93.33%) was higher than that of the control group (70.00%).Yang et al.  observed%). Ding  also repCodonopsis 15 g, Poria cocos 9 g, Atractylodes macrocephala 9 g, Radix paeoniae rubra 9 g, Astragalus 12 g, Herba Epimedii 9 g, Polygalaceae 9 g, Alisma 9 g, Divaricate Saposhnikovia root 9 g, Achyranthes bidentata 9 g, Adenophora 9 g, Rehmannia 15 g, Jujube 3 g, and Ginger 3 g). After 4 weeks, the total effective rate of the observation group (94.0%) was higher than that of the control group (48.0%). Li and Wang [Sun et al.  observedand Wang  observedAngelica sinensis 10 g, Rehmannia glutinosa 15 g, White peony root 10 g, Rhizoma chuanxiong 10 g, Dodder 15 g, Epimedium 12 g, Psoralen 10 g, and Chinese wolfberry 10 g) at 150 ml bid po. After 6 weeks, the total effective rate of the observation group (90.74%) was higher than that of the control group (75.93%). Liu et al. [Astragalus 30 g, Panax notoginseng powder 5 g, Acorus gramineus soland 5 g, and Aster 10 g) at 100 ml tid po. After 4 weeks, the total effective rate of the observation group (90%) was higher than that of the control group (80%). Ma et al. [Morinda officinalis 10 g, Astragalus 30 g, Codonopsis 15 g, Angelica 10 g, Cohosh 6 g, Bupleurum 6 g, Cork 5 g, and Anemarrhena 10 g) at 120 ml bid po. After 8 weeks, the total effective rate of the observation group (90.0%) was higher than that of the control group (77.5%).Kang et al.  summarizu et al.  observeda et al.  observedAstragalus 150 g, Codonopsis 120 g, Atractylodes macrocephala 100 g, Angelica 100 g, Rhizoma chuanxiong 100 g, Poria cocos 150 g, Chinese yam 150 g, Achyranthes bidentata 100 g, Fried Jujube seed 150 g, Albizia acacia peel 100 g, Fleece flower vine 300 g, Longan 100 g, Red peony 100 g, White peony 100 g, Bupleurum 60 g, Turmeric 100 g, Woody 60 g, Tangerine peel 60 g, Chickens gizzard-membrane 100 g, Licorice 100 g, and Tortoise plastron glue 250 g) at 15 g bid po. After 12 weeks, the total effective rate of the observation group (92.5%) was higher than that of the control group (70.0%). Wang [µg, lasting 7 days), which should be changed every 7 days, and dydrogesterone 5 mg qd po. After 4 weeks, the symptoms and laboratory indicators of the observation group improved more than did those of the control group. In this study, the medication time was relatively short, and the number of cases was small, so the results obtained were limited; thus, further research is needed. In a short period of time, the E2 patch is simple and safe to use and has fewer adverse drug reactions. It can significantly improve CFS symptoms and improve the quality of life, providing another choice for women who are not suitable for oral medication.Liu et al.  observed%). Wang  reported%). Wang  observedThe external treatment methods of TCM mainly include moxibustion, acupuncture, tuina, cupping, and other external treatment methods. Due to the diverse characteristics of methods and schools of TCM external treatment, when designing controlled experiments, we can see control experiments not only with external treatment methods but also with internal medicines.Acupuncture and Moxibustion [Differential Diagnosis of TCM Syndrome [Encyclopedia of Chinese Tuina [The diagnostic criteria of TCM mentioned in this section refer to the relevant content of ibustion , DiffereSyndrome , and Encse Tuina . The clise Tuina .Atractylodes macrocephala, and Salvia miltiorrhiza, with an unknown dose) at back-Shu points. The control group received oral oryzanol 10 mg tid po, vitamin B1 10 mg tid po, and vitamin B6 10 mg tid po, and the observation group added this method on the basis of the control group. Both groups were treated for 30 min once a day. After 4 weeks, the total effective rate of the observation group (91.4%) was higher than that of the control group (77.1%). Tian et al. [Chen et al.  observedn et al.  observedn et al.  observedn et al.  observedn et al.  observedModern TCM methods for acupuncture of CFS can be divided into body acupuncture and other acupuncture methods, mainly including abdominal acupuncture, auricular acupuncture, and acupoint embedding.Wei et al.  observedWang et al.  observedShi  observedLi and Hou  observedThe combination therapy of TCM includes two or more external treatments combined or syndrome differentiation combined with external treatments. The treatment of CFS with TCM combination therapy can often complement the weaknesses, highlight the curative effects, and reduce the side effects caused by a single treatment method. The difficulty of clinical trials is often that the primary and secondary treatments are confused during the experiment.Li and Sun  evaluateWu  observedQi et al.  observedAstragalus, Ligustrum lucidum, Eclipta prostrata, Polygonum multiflorum, Schisandra, Stir-fried Jujube seed, Lily, Turmeric, Epimedium, and Anemarrhena, with the specific doses not provided) at 250 ml bid po. After 4 weeks, the total effective rate of the observation group (77.45%) was higher than that of the control group (47.50%).Lu et al.  observedCurrently, there are many methods for treating CFS with TCM, showing its advantages. On one hand, treatments according to syndrome differentiation are diverse and involve herbal decoctions and proprietary Chinese medicines. External treatments include moxibustion, acupuncture, tuina, cupping, and other treatments. Combination therapies include two or more external treatments combined and internal and external treatments combined, which are often better than monotherapies and can remedy deficiencies. However, the clinical manifestations of CFS are different. Although fatigue is the main manifestation, there are individual differences in the appearance of insomnia or muscle pain in some patients. Treatments according to syndrome differentiation can provide patients with individualized treatments. On the other hand, TCM often has the advantages of simplicity, effectiveness, convenience, and a lower cost.However, there are still many problems in the treatment of CFS with TCM. For example, diagnostic criteria of syndrome differentiation, treatment methods, and efficacy evaluations have not yet reached a consensus. Some therapies are quite cumbersome or rare, but the efficacy is almost the same. Sometimes, combination therapy is taken, but the focus on primary and secondary treatments is not clear. Therefore, it is difficult to compare which therapy is better, which reduces the credibility of the studies. Among the databases that we examined, RCT experiments accounted for a large proportion, and the total effective rates of observation groups were often over 90% and were even as high as 100%. However, there was a lack of repeated experiments. The level of evidence is not good enough, and high-quality clinical trials are still needed, which remains the main contradiction in the treatment of CFS with TCM. In the process of searching the papers, we also found some folk-specific therapies, such as a classic medicated diet porridge using herbs , which can improve symptoms such as fatigue by taking 500 ml once every other day . Another


To investigate the effects of coadministration of esaxerenone with amlodipine on the pharmacokinetics (PK) of each drug, and of esaxerenone on the PK of digoxin.N = 24); single doses of amlodipine 2.5 mg , with esaxerenone 5 mg/day (Days 8–25) ; or digoxin 0.25 mg/day (Days 1–15) with esaxerenone 5 mg/day (Days 11–15) . PK parameters and safety were assessed.In three open-label, single-sequence, crossover studies, healthy Japanese males received single oral doses of esaxerenone 2.5 mg , with amlodipine 10 mg/day (Days 8–18)  and time to Cmax were unaltered by amlodipine coadministration, but mean half-life was slightly prolonged from 18.5 to 20.9 h. Geometric least-squares mean (GLSM) ratios for Cmax, area under the plasma concentration–time curve (AUC) from zero to last measurable concentration and from zero to infinity for esaxerenone + amlodipine versus esaxerenone were 0.958, 1.154, and 1.173, respectively. Study 2: corresponding GLSM ratios for amlodipine + esaxerenone versus amlodipine were 1.099, 1.185, and 1.214. Study 3: esaxerenone did not markedly alter digoxin PK. GLSM ratios for Cmax, trough plasma concentration, and AUC during a dosing interval for digoxin versus esaxerenone + digoxin were 1.130, 1.088, and 1.072, respectively.Study 1: esaxerenone peak plasma concentration ; Study 3: JapicCTI-163443 (registered on 24 November 2016).Studies 1 and 2:  Hypertension is an important public health issue and cardiovascular risk factor –3. Effecmax) was 2.5–3.5 h and elimination half-life (t1/2) was 22.3–25.1 h. In a mass balance study, about one-third of the clearance of esaxerenone was found to be through oxidative metabolism by CYP3A [Calcium-channel blockers (CCBs) are the most widely used antihypertensive agents, particularly in Asia, because of their potent BP-lowering abilities . One of by CYP3A .in vivo inhibitor of CYP3A [max) and area under the plasma concentration–time curve (AUC) for midazolam, a CYP3A-index substrate [Amlodipine is mainly metabolised by CYP3A and is a weak of CYP3A , 22. In ubstrate , 24, werubstrate . Therefoin vitro [Digoxin is a P-glycoprotein (P-gp) substrate and P-gp plays a major role in both the absorption and elimination of digoxin; thus, P-gp inhibition is a known risk factor for increased digoxin exposure , 27. Altin vitro , the effin vitro , a DDI sin vitro , 31, andin vitro .Therefore, the aim of the study was to clarify DDIs between esaxerenone and amlodipine or digoxin, by investigating the effects of esaxerenone and amlodipine coadministration on the pharmacokinetics (PK) of esaxerenone (Study 1) and amlodipine (Study 2), and the effects of esaxerenone on the PK of digoxin (Study 3).All studies had a single-centre, open-label, single-sequence design Fig. . All subPeriod 1 (Days 1–7) comprised the esaxerenone alone single-dose administration phase of the study. A single esaxerenone 2.5-mg tablet  was administered orally to fasting subjects . Period 2 (Days 8–19) comprised the coadministration phase of the study, commencing 7 days after the first dose of esaxerenone. On Day 8, amlodipine 10 mg  was administered orally after breakfast once daily for 11 days. On Day 15, esaxerenone 2.5-mg and amlodipine 10-mg tablets were coadministered orally to subjects in the fasting state , a single 2.5-mg dose of amlodipine was administered orally after breakfast (Day 1). During Period 2 (Days 8–26), esaxerenone 5 mg was administered orally after breakfast once daily for 18 days. On Day 21, esaxerenone 5 mg and amlodipine 2.5 mg were coadministered orally after breakfast , a 0.25-mg tablet of digoxin  was administered orally once daily after breakfast for 10 days (Days 1–10). There was no washout, and Period 2 started immediately after completion of Period 1. In Period 2 (Days 11–18), a 0.25-mg dose of digoxin and 5-mg dose of esaxerenone were administered orally at the same time each day (in the fed state) for 5 days  of ≥18.5 to < 25.0 kg/mIn Study 1, blood samples (3 mL) were collected for drug concentration measurement of esaxerenone on Days 1 and 15, before and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 24, 48, 72, and 96 h after esaxerenone administration. In Study 2, blood samples (5 mL) were collected for drug concentration measurement of amlodipine on Days 1 and 21, before and at 2, 3, 4, 5, 6, 7, 8, 9, 12, 24, 48, 72, 96, and 120 h after amlodipine administration. In Study 3, blood samples (3 mL) for drug concentration measurement of digoxin were collected on Days 10 and 15, before and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h after digoxin administration.g for 10 min) and was subsequently frozen (− 20 °C or lower) until delivered to the laboratory for analysis.Plasma for assays of esaxerenone, amlodipine, and digoxin was obtained by centrifugation of the blood samples (at 4 °C and 1700×Drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methodology for chromatographic separation and determination of esaxerenone used in Studies 1 and 2 has been reported previously , 33. ForIn Study 3, methods for the determination of esaxerenone plasma concentrations were identical to those used in Studies 1 and 2 (described above). For digoxin, plasma samples were treated by solid phase extraction  and chromatographic separation was performed using a SunShell C18 column  with an internal diameter of 2.1 mm, a length of 50 mm, and a pore size of 2.6 μm. Detection was performed using Triple Quad 5500  tandem mass spectrometry with ESI in the positive ion mode; MRM of digoxin (m/z 798–651) and its internal standard  was conducted. For digoxin test samples of 0.05, 0.1, 1.0, and 20.0 ng/mL, the intra-study assay precision rates were 3.3, 5.7, 1.5, and 1.8%, respectively. Accuracy of the assay ranged from − 4.0 to 7.0%, with an LLOQ of 0.05 ng/mL.PK parameters were calculated by non-compartmental analysis, using Phoenix® WinNonlin® .max and AUC to the last quantifiable time (AUClast) and from time zero to infinity (AUCinf) for esaxerenone. Secondary endpoints in both studies included time to maximum esaxerenone concentration (tmax), esaxerenone half-life (t1/2), and apparent total body clearance (CL/F).For Studies 1 and 2, the primary endpoints were Cmax, trough plasma concentration (Ctrough), and AUC during a dosing interval (AUCtau). Secondary endpoints included t1/2, tmax, and apparent total body clearance at steady state (CLss/F).For Study 3, the primary endpoints were CSafety was evaluated through the assessment of adverse events (AEs), laboratory tests, vital signs , and 12-lead electrocardiogram. AEs were coded using Medical Dictionary for Regulatory Activities  System Organ Class and Preferred Terms.max and AUC of 20 and 10%, respectively, based on previous studies [max and AUC were ≤ 1.05, when ratios were estimated after a single oral dose of test drug  and concomitant drug administration, a sample size of 18 subjects would provide ≥80% statistical power with two-sided 90% confidence intervals (CIs) for GLSM ratios of Cmax and AUC to detect the CIs within 0.80–1.25. To allow for unexpected circumstances, such as subject withdrawals, the number of subjects was specified as 24 in Study 1 and as 20 in Studies 2 and 3.The sample size was calculated assuming within-subject variations in C studies –35. AssuIn all studies, the PK analysis sets included subjects who received test drugs , and for whom data were available for at least one primary endpoint in Periods 1 and 2. The safety analysis sets included all subjects who agreed to participate in the study and who received at least a dose of drug . Differences in PK parameters between treatment groups were calculated by ratios of GLSM and their 90% CIs. No apparent DDI was concluded if the GLSM ratio was contained within the bounds (0.80–1.25) of 90% CIs. In all statistical analyses, SAS  was used.Baseline characteristics for subjects in all three studies are shown in Table max and tmax did not differ but AUClast and AUCinf were slightly increased when coadministered with amlodipine , 1.154 (1.118–1.190), and 1.173 (1.136–1.212), respectively , when amlodipine was coadministered with esaxerenone , 1.185 (1.132–1.240), and 1.214 (1.157–1.273), respectively  of digoxin reached steady state after Day 6  for Cmax, Ctrough, and AUCtau for digoxin alone versus esaxerenone plus digoxin were 1.130 (0.998–1.280), 1.088 (1.033–1.145), and 1.072 (1.015–1.133), respectively , including decreased appetite and gastroenteritis , and muscle spasms and increased creatine phosphokinase levels . Both events were of mild severity and resolved without treatment, but the subject with gastroenteritis discontinued study medication. No TEAEs were considered by the investigators to have a causal relationship with treatment. In Study 2, only one AE occurred (a case of tonsillitis 2 days after the single dose of amlodipine). This was of moderate severity, resolved with drug therapy, and was considered unrelated to study treatment. In Study 3, one subject had nasopharyngitis and one had increased levels of alanine aminotransferase and aspartate aminotransferase during treatment with digoxin alone; the AEs were mild in severity and resolved without treatment. No TEAEs occurred during coadministration of digoxin and esaxerenone.A summary of AEs in all three studies is provided in Additional file 2Data from these three studies indicate no clinically relevant DDIs or safety concerns associated with concurrent dosing of esaxerenone with either amlodipine or digoxin.max and AUC of esaxerenone with amlodipine versus esaxerenone alone were within the range 0.80–1.25, indicating that esaxerenone PK parameters were not affected by amlodipine. In contrast, evaluation of the effect of esaxerenone on amlodipine PK revealed increases of approximately 20% in the AUC for amlodipine. Given that an AUC increase of 60% was observed when amlodipine was coadministered with a moderate CYP3A4 inhibitor (diltiazem) [Regarding a potential effect of amlodipine on esaxerenone PK, 90% CI values for GLSM ratios for Cltiazem) , and theltiazem) , the AUCmax increased by approximately 13%. Although prior in vitro data revealed that esaxerenone had inhibitory activity against P-gp [in vivo DDI study with a P-gp probe substrate was not deemed necessary, based on available guidance for DDI studies [max increased slightly when digoxin was coadministered with esaxerenone, other parameters (including AUCtau) were within predefined ranges. Digoxin prescribing information states that dose adjustment is recommended when an increase in AUC is ≥14% [When digoxin was coadministered with esaxerenone, the digoxin Cnst P-gp , the inh studies . In this is ≥14% . TherefoThere were no safety issues when a single dose of esaxerenone 2.5 mg was coadministered with multiple doses of amlodipine 10 mg/day, a single dose of amlodipine 2.5 mg was coadministered with multiple doses of esaxerenone 5 mg/day, or when esaxerenone 5 mg was coadministered with digoxin 0.25 mg/day.The main limitation of these studies was that they were designed to evaluate PK parameters in healthy subjects and the efficacy and safety of the treatment combinations were not evaluated in patients. Although no notable safety concerns were raised in our analyses, assessment of long-term administration is warranted to confirm the detailed safety profile associated with concurrent dosing.max were observed during coadministration of esaxerenone, these alterations were not considered clinically relevant. No safety concerns were seen when amlodipine or digoxin was coadministered with esaxerenone. These findings indicate that, from a PK standpoint, no significant dosage adjustment is necessary for amlodipine or digoxin when administered with esaxerenone in hypertensive patients requiring combination therapy.The PK of esaxerenone were unaffected by coadministration of amlodipine. Although slight increases in amlodipine and digoxin CAdditional file 1. Additional methods describing details on treatments and exclusion criteria.Additional file 2: Table S1. Summary of adverse events.


Placobdelloides siamensis is a glossiphoniid leech with a short life cycle. In a laboratory setting, ten mature P. siamensis left their host (a turtle) after feeding for approximately three days and initiated copulation. The adults spent 3–4 days gestating before depositing eggs . The eggs then changed from a creamy white to a creamy brownish color before hatching. Hatching occurred after incubation on the parent's ventral surface for 5–7 days. The transparent brood, with a single pair of red eyes, spent a couple of weeks under the venters of their parents. After this period, they left their parents and grew to maturity in 10–15 days; leeches were considered mature when their color was similar to that of their parents and they performed their first copulation. In addition, the mature leeches survived for 163 days on one feeding. Hirudo medicinalis Linnaeus, 1758) [de novo in this family [Historically, glossiphoniid leeches have been favored over European medicinal leeches s, 1758) –4. Gloss8 –4. Glos family . During Glossiphonia complanata Linnaeus, 1758 [Helobdella stagnalis Linnaeus, 1758 [Helobdella triserialis Blanchard, 1849 [Theromyzon tessulatum Müller, 1774 [According to Kutschera and Wirtz 2001) , the Glo , the Gler, 1774 ) show a er, 1774 ,12.Placobdelloides siamensis Oka, 1917 [Cuora amboinensis Daudin, 1802 [Cyclemys oldhamii Gray, 1863 [Hieremys annandalii Boulenger, 1903 [Heosemys grandis Gray, 1860 [Malayemys khoratensis Ihlow et al., 2016 [Malayemys macrocephala Gray, 1859 [Malayemys subtrijuga Schlegel & Müller, 1845 [Siebenrockiella crassicollis [P. siamensis is adaptable to various freshwater conditions; however, the life cycle of this species, and the Placobdelloides genera, is still unknown. Therefore, to improve our understanding of the reproductive status of P. siamensis, the life cycle and the duration of each growth stage of this species were examined in a laboratory.Siamese shield leech ka, 1917 ) is a coin, 1802 , Cyclemyay, 1863 , Hieremyer, 1903 , Heosemy  is a cosicollis –24. PrevMalayemys macrocephala) was collected by hand from a pond at Kasetsart University, Bangkok, Thailand (13° 50'53.6" N 100° 33'47.3" E) on 24 July 2019, and delivered to a laboratory in the Department of Zoology, Faculty of Science   . The tur3) three-fourths full of water with an oxygen pump at room temperature. A single leech species, Placobdelloides siamensis, was identified following Chiangkul et al. (2018) [P. siamensis individuals with an average body length of 30.3±5.9 mm  were randomly transferred to the turtle container. The observation and recording of water temperature began on 25 July 2019.All the leeches, totaling 213 individuals, were carefully removed from the external surface of the turtle using forceps and kept separately in a glass container (30x20x20 cm. (2018) . Ten mat3) that was filled one-third of the way with water; a pot was placed above the water level for basking and an oxygen pump was placed in the water. The weight and carapace length of the turtle were recorded both pre- and poststudy, and Mazuri Aquatic Turtle Diet was provided once a day. Once a week, half the water was drained from the turtle container and fresh water was added until it reached the former level; this process was performed once a month for the leech container. Ten P. siamensis individuals were transferred into the turtle container for blood feeding, and their life cycles were observed. Four periods were recorded: the copulation period , incubation period (from the first egg deposition to the first hatching), parental care period (from the last hatching through the first juveniles leaving their parent), and maturation period (from the last juvenile leaving its parent for its first copulation). The water temperature in both containers was measured every six hours  every day.The turtle was maintained at room temperature in a glass container ; 1 hour for each concentration) using a critical point dryer  to reach a critical dry point. The specimens were then mounted on a stub using carbon tape and sputter-coated with gold particles. All of the coated specimens were examined with a scanning electron microscope.This research was approved by the Institutional Animal Care and Use Committee, Faculty of Science, Kasetsart University, Thailand, under project number ACKU61-SCI-028.Placobdelloides siamensis individuals had fed on the snail-eating turtle (in the turtle container), with blood filling their crop ceca, repeated copulations were observed at the bottom of the container for several hours. On separation, after the last copulation, each P. siamensis moved toward the substrate near the oxygen pump, where they attached and remained motionless. The gestation of creamy white eggs in both ovisacs occurred one or two days after copulation (th and 7th day of the observation period), the leeches deposited clutches of creamy white and roundish eggs on the substrates  near the oxygen pump and covered them with their bellies  , the average water temperature in the turtle container was 30.1±1.4°C (26.5–34.0°C). On the third day (27 July 2019), after the ten pulation . On the  venter) . There w venter) . In addi venter) .th to 14th day). Then, the eggs turned a creamy brownish color, signifying their maturation and readiness to hatch ; movement began in the second week, but the offspring remained on the ventral surface , the broods moved out from under their parents and began feeding on the turtle. At this stage, the offspring are considered "juveniles”. Eventually, in the fourth week after hatching , when the offspring had coloration similar to that of their parents and an average body length of 10.0±2.1 mm , they initiated copulation  with increased carapace length (17.1 cm) and weight (945 g).The turtle was released at the collection site on the 44th day) and reached 53% in the fourth month , the mean water temperature in the leech container (starting from 28.9±2.1°C) was as follows: 30.2±1.6°C (28.0–34.0°C) from 25–31 July 2019, 30.1±1.4°C (26.5–33.0°C) from 1–31 August 2019, 30.0±1.4°C from 1–30 September 2019, 28.9±1.0°C (22.0–33.0°C) from 1–31 October 2019, 28.5±1.4°C (26.0–31.0°C) from 1–30 November 2019, 26.9±2.9°C (24.0–31.0°C) from 1–31 December 2019, and 27.1±0.8°C (26.0–28.0°C) from 1–3 January 2020.Placobdelloides siamensis and the Placobdelloides genera. This genus is characteristic of the Glossiphoniinae subfamily due to its egg deposition on substrate. In contrast to what was described for this genus in Oka (1917) [Placobdelloides are described as brooding the eggs and juveniles on the ventral surface of the parent and the placement of eggs such as P. stellapapillosa Govedich et al. 2002 [This is the first report of the life cycle of a (1917)  and Sawya (1917) , Placobdal. 2002 , and cocPlacobdelloides siamensis has a 1-day copulation period, 3- to 4-day gestation period, 5- to 7-day incubation period, 14-day parental care period (from hatching to the juvenile stage), and 10- to 15-day maturation period (from the juvenile stage to the mature adult stage), when individuals are able to reproduce , H. stagnalis Linnaeus, 1758 [H. robusta Shankland et al., 1992 [T. tessulatum (6–10 days), and T. rude (5–10 days); shorter parental care period than G. complanata Linnaeus, 1758 [H. stagnalis (21–28 days), and H. conifera Moore, 1993 [A. polypompholyx (112–140 days) and H. conifera (35–76 days); and larger clutch size than A. polypompholyx (30–50 eggs/clutch), G. complanata (60 eggs/clutch), H. stagnalis (50 eggs/clutch), H. conifera (29–192 eggs/clutch), and P. sirikanchanae Click here for additional data file.


Faecalibacterium and Agathobacter were the most abundant populations in M2 soil and were not found in other soils. Pearson correlation analysis showed that soil moisture content, pH, AN, AP, AK and soil enzyme activity were significantly related to bacterial numbers, diversity and community distribution.The diversity and community distribution of soil bacteria in different land use types in Yangtze River Basin, Chongqing Municipality were studied by using Illumina MiSeq analysis methods. Soil physical and chemical properties were determined, and correlation analyses were performed to identify the key factors affecting bacterial numbers and α-diversity in these soils. The results showed that the soil physical and chemical properties of different land use types decrease in the order: mixed forest (M2) > pure forest (P1) > grassland (G3) > bare land (B4). There were significant differences in bacterial diversity and communities of different land use types. The diversity of different land use types showed the same sequence with the soil physical and chemical properties. The abundance and diversity of bacterial in M2 and P1 soils was significantly higher than that in G3 and B4 soils. At phylum level, G3 and B4 soils were rich in only Proteobacteria and Actinobacteria, whereas M2 and P1 soils were rich in Proteobacteria, Actinobacteria and Firmicutes. At genus level,  Soil is an important part of the ecosystem and plays an important role in maintaining the stability, material circulation and energy transformation of the terrestrial ecosystem . Soil miSoil microbial community and diversity, which are important dynamic indicators of soil quality, can be affected by land-use conversions. Environmental conditions such as soil types, soil physical and chemical properties, and land-use patterns are major factors that influence soil microbial community diversity . The comIn recent years, studies on biodiversity have attempted to describe soil microbiomes in different ecosystems  to understand the dynamics of microbial interactions with the environment . It was The Yangtze River Basin in Chongqing is an important area of diverse land-use types in China. It has long been affected by natural and high-intensity human activities, and extreme soil erosion has made its ecosystem quite fragile. The ecological environment of this basin has gradually attracted attention . Much ofThe selected study sites belong to the Yangtze River Basin of Chongqing Municipality, with a mild climate and a subtropical monsoon humid climate. The annual mean temperature in the region is 16−18 °C and the normal year annual rainfall is 1,000–1,350 mm, more than 70% of which occurs from May to September. The types of landforms in the Yangtze River Basin are complex and diverse. The main soil types are purple soily, ellow loam, paddy soil, red soil, new soil, and mountain meadow soil. The area is part of the key environmental protection areas of the Yangtze River Basin because of the complex natural landforms, abundant precipitation, and erosive soils.′24.54″, E106°31′35.55″) in September 2018. Soil type in the research area is “red soil”. A total of 12 soil samples (three random samples per environment) were taken at four locations from pure forest , mixed forest , grassland (G3), and bare land (B4). In each plot, the mixed soil samples (removing the surface litter layer) were collected from a depth of 0–20 cm by five-point sampling method and placed in individual sterile brown paper bags and stored at 4 °C for further studies. The fresh soil samples were divided into two parts, one part was passed through a 40 mesh sieve to determine the soil culturable bacterial number and diversity, while the other was naturally air dried to determine the soil nutrient.Based on, the characteristics of the Yangtze River Basin and the principle of vegetation division, the land types in the whole basin are divided into pure forest, mixed forest, grass, and bare land. The soil samples were collected randomly from different land-use types in the study area , available phosphorus (AP) and available potassium (AK) were determined as described. Briefly, soil water content was estimated by drying the sample at 105 °C for 24 h . Soil pHe method . Soil ave method . Soil ene method . CatalasThe culturable bacterial counts were determined by plate counting method . BrieflyDNA from all soil samples was extracted and purified using the E. Z. N. A.™ Mag-Bind Soil DNA Kit  using 0.2 g of soil as per the manufacturer’s instructions and stored at −80 °C until further analysis. For bacterial 16S rRNA, PCR amplification was performed using the primers 16S 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 16S 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with genomic DNA as template. The PCR reaction conditions were as follows: initial denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 20 s, extension at 72 °C for 60 s, for 32 cycles; and a final extension at 72 °C for 10 min. PCR products were stored at −20 °C and analyzedon 1% gel using DL2000 marker for quality examination.http://cutadapt.readthedocs.io/en/stable/) (http://github.com/torognes/vsearch/) (http://www.drive5.com/uparse/)  , and thesearch/)  to deletsearch/) . Clean ruparse/) . The OTUuparse/) . After op < 0.05.Statistical analysis of data was carried out using Origin 8.0 software . ANOVA with least-significant-difference (LSD) tests, cluster analysis, correlation analysis and principal component analysis were carried out using the SPSS 19.0 software . For all analyses, the results were considered to be significant at p < 0.05). Significant differences in soil nutrients were observed in different land-use types. The soil available P (47.00 mg/kg), available K (112.61 mg/kg), available N (94.83 mg/kg) contents in M2 were significantly higher than those in P1, G3 and B4 soils. The soil catalase and sucrase activities were similar in the four samples, but the soil urease activity varied greatly, ranging from 0.7816 to 3.0474 mg/g (p < 0.05). The M2 soil sample had higher urease activity when compared with P1, G3 and B4.The basic physicochemical characteristics of soil samples from different land-use types are shown in 5, 85. 3 ×105, 51. 3 ×105 and 10. 0 ×105 cfu/g respectively. The highest number of culturable bacteria was detected in M2 sample, while the lowest was found in B4.The number of culturable soil bacteria varied among different land-use types . The culSequences with 97% or more similarity were classified as an operational classification unit . As shown in The Good’ s coverage of P1, M2, G3 and B4 samples reached 99.1%, 98.7%, 99.1% and 99.0% respectively, that captured the majority of microbial diversity . The ChaNine bacterial phyla, Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Gemmatimonadetes, Chloroflexi, Fusobacteria and Tenericutes were identified in different land-use types analyzed . Among tDongia, Aeromonas, Candidatus_Bacilloplasma, Cetobacterium, Lysobacter and Agromyces were the predominant genera in P1 samples. In M2 samples, majority of the bacterial community belonged to eight genera, Romboutsia, Blautia, Fusicatenibacter, Colwellia, Faecalibacterium, Agathobacter, Reyranella and Bifidobacterium. Haliangium and Gaiella were the predominant genera in G3 samples, while Acidibacter and Polycyclovorans were the predominant genera in B4 samples. Other major genera were Bryobacter, Streptococcus, Streptomyces, Marmoricola, Pseudomonas, lamia, Sphingomonas and Bacteroides. Dongia was less abundant in M2, G3 and B4, and was found to be higher in P1 samples. Faecalibacterium and Agathobacter were highly abundant in M2 samples but were rarely detected in P1, G3 and B4 samples. The bacterial microbial community thus varied greatly, and the abundance of each genus varied among soil samples from different land-use types.At the genus level, the diversity of the bacterial microbiota in P1, M2, G3 and B4 samples was higher, especially in the M2 samples , 3. TwenPCoA and UPGMA analyses were performed to evaluate similarities in the bacterial communities of P1, M2, G3 and B4. One weighted  and another weighted  PCoA of bacterial communities was performed to analyze the community structure similarity . The UPGp < 0.01). The canonical correlation of the eight soil properties with the culturable bacterial numbers and α-diversity of the P1 soil are listed in P < 0.05), with correlation coefficients of 0.823 and 0.993 respectively. The Shannon index was negatively correlated (−0.806) and Simpson index was positively correlated (0.992) with pH. The Chao1 index and ACE index were significantly positively correlated with catalase, with correlation coefficients of 1.000 and 0.999 respectively (P < 0.01). The data in To further estimate the effects of environmental factors on microbial quantity and diversity, soil environmental factors such as moisture content, pH, available P, available N, available K, catalase, urease and sucrase in the samples were analyzed by correlation analysis using SPSS 19.0. These analyses demonstrated that environmental factors were strongly correlated with the microbial quantity and diversity (5 cfu/g) were significantly lower than those in P1, M2, G3 samples. We found that coverage by plants was a key factor influencing the number of soil bacteria, plant residues are considered to be important organic components of soil microbial growth (Previous studies have demonstrated that bacterial abundance and diversity can be influenced by factors such as soil type, organic matter content, soil properties and plant species . It is cl growth .Land usage has a significant impact on soil properties including moisture content, pH, soil nutrients and soil enzyme activity . Qi et aChang in land use not only affects soil physical and chemical properties but also affects soil microbe populations. Faecalibacterium and Agathobacter were especially dominant, and were rarely detected in P1, G3 and B4 samples. This finding is in accordance with that of Different land-use types had also had an important impact on soil bacterial community composition, which may change the soil function and ecological processes . PreviouFaecalibacterium and Agathobacter were dominant in M2 soil and different when compared with P1, G3 and B4 soils. Our study not only lays the foundation for understanding the relationships between microbes, plants and soil, but also provides a theoretical basis for land management.Our data highlight that land use has strong effects on soil properties, soil bacterial abundance, diversity and community composition. Correlation analysis showed that pH, water content, AN, AP, AK and soil enzymes were significantly related to bacterial count and diversity. The bacterial numbers were higher in M2 soil than in other land-use soils. The highest bacterial diversity was observed in M2 soil and that in B4 soil was lowest because of poor nutrient availability. Furthermore, the results revealed that 10.7717/peerj.9386/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9386/supp-2Supplemental Information 2Click here for additional data file.


Motivated by their fear of disapproval, individuals with social anxiety continually monitor their own behaviour during social interactions hoping to prevent dreaded negative outcomes. Ironically, they do evoke less positive reactions from others. This study investigated whether lower engagement in the interpersonal process of joint action by socially anxious individuals leads them to attract less positive ratings by their conversation partners.N = 36) and low socially anxious individuals  had separate conversations with a naïve conversation partner (N = 36). Conversations were filmed and analysed for joint action using the conventional manual way and a more exploratory automated way. Conversation partners rated the quality of the interaction and the person they talked to.High socially anxious (HSA; Conversation partners rated HSA participants less positively than LSA participants. The conventional manual method showed less joint action in conversations with LSA participants and crucially, joint action served as mediator between social anxiety status and general impression, quality of interaction and desire to meet again. These results were not replicated with the automated method.The study used an analogue sample and future research should investigate whether these findings also apply to a clinical sample. Future studies should further explore the utility of automated techniques to measure joint action.Reduced joint action may explain why socially anxious individuals tend to be perceived less positively by others. The findings emphasise the importance of taking an interpersonal and holistic approach to understanding aspects of social anxiety disorder. •Conversation partners perceive socially anxious participants less positively.•Manually rated joint action is lower in socially anxious individuals.•Joint action mediates relationship between social anxiety and positive ratings.•Movement synchrony as measured by motion energy is associated with positive ratings.•Movement synchrony as measured by motion energy is not related to social anxiety. AccordiStudies that have investigated the way in which socially anxious individuals are perceived by others during conversations have established two separate effects. First, socially anxious individuals tend to underestimate their performance compared to ratings made by their conversation partners . Second,In the past decades, social skills deficits have been considered a likely explanation for the fact that socially anxious individuals tend to be rated less positively by others in conversations and other social interactions . HoweverOne interpersonal process that might suffer from such depleted resources due to increased self-focused attention is the coordination between two individuals' actions, which serves as the basis of social connectedness  and is iThe present study is a further exploration of joint action in social anxiety. In contrast to 22.1High and low socially anxious individuals separately had a conversation with a third person (the conversation partner). Conversations were videotaped and conversation partners subsequently rated the conversation, the person they talked to, and their own feelings during the conversation. The order of conversations with low and high socially anxious participants was counter-balanced. Two different methods were applied to analyse the same visual recordings with regards to joint action: manual behavioural analyses by independent assessors and automated analyses using the Motion Energy Analysis (MEA) Software. Neither the conversation partner nor the independent assessors were informed about the level of social anxiety of the other person in the conversations.2.2Participants were recruited at the University of Oxford and in the general population using both online and offline advertisements . Exclusion criteria were a) age below 18 or above 65 years, b) severe levels of depression , c) suicidal ideation (a score of ≥2 on item 9 of the BDI); and d) currently receiving treatment for a psychiatric disorder or drug-related problem.Using the Social Interaction Anxiety Scale (SIAS) as a screening instrument, forty volunteers who scored 19 or below on the SIAS  took par2 = 4.44, p = .11, age, F = 0.22, p = .80, first language, χ2 = 2.37, p = .31, or level of education, χ2 = 13.91, p = .31, where level of education was tested with maximum likelihood ratio Chi-square as assumptions of Pearson Chi-square were violated , which hence was excluded from further analyses. Additionally, three triples were excluded as the third participant  in each did not show up on the day of testing, resulting in 36 triples  that were included in the analyses. The descriptives of the three groups are provided in violated .Table 1C2.32.3.1Beck Depression Inventory (BDI) is a well-established 21-items self-report measure of symptom severity of depression . The inv2.3.2Not at all characteristic of me) to 4  was used as a screening tool to assess fear in interaction and conversation settings. The questionnaire consists of 20 items which are rated on a 5-point Likert scale ranging from 0  to 4  was used to assess the fear of negative evaluation , which i2.3.4None) to 3 (Severe) for fear and from 0  to 3 . An overall score was obtained by summing the total scores of fear and avoidance. In the present study, internal consistency of the total scale was excellent (α = 0.96).This 24-item scale was used to measure fear and avoidance of social interactional (11 items) and public performance (13 items) situations . The par2.3.5Not at all) to 8 (Very much). The total sum score of the QI was used for further analyses. In the present study, only the QI filled out by the conversation partners were of relevance. The internal consistency was very good (α = 0.92).Quality of Interaction Scale (QI) was used to assess how the participants evaluated the conversations they just had. For instance, to what extent participants thought that the interaction was smooth, strained, and pleasant . The sca2.3.6Very few people would) to 81–100% (Almost everyone would). The total sum score of the DFIS was used for further analyses. In the present study, the internal consistency was good (α = 0.86).Desire for Future Interaction Scale (DFIS) measures the willingness to engage in future interactions with someone  to 100 (Very well). To reduce social desirability bias on the part of the conversation partners, a third-party perspective was taken . The average score of the GI was used for further analyses. In the present study, internal consistency was excellent (α = 0.95).Global impression scale (GI) was developed for the present study and includes three questions and can be rated on a visual analogue scale from 0  to 5 . In the present study, internal consistency for both the positive and negative scales was good .Positive and Negative Affect Scale (PANAS) comprises 10 items that assess positive affect and 10 items that assess negative affect. Each item is rated on a 5-point Likert scale from 1  had a 5-min conversation with the low socially anxious participant (LSA) or high socially anxious participant (HSA) separately, each in turn. Order of the conversations (HSA vs. LSA) was counterbalanced across triples. Conversation partners were told from the beginning that they will have two brief interactions with two individuals.To start the first conversation, the CP and the first participant  were seated facing each other. The experimenter said that she needed to leave the room to set up further equipment in another room before beginning the experiment properly. The experimenter encouraged the participants to ‘talk among yourselves for a moment’ and assured them she will be back in 5 min to start the experiment, during which the participants actually were filmed already. This instruction was intended to help participants feel less conscious despite the video equipment, so that they behaved in the same way they would in an ordinary conversation outside of the lab. Cameras recorded whole body shots. After 5 min the experimenter came back into the room and told the participants that there was a problem with the equipment and that they would skip this part of the experiment but would try to solve it before the second conversation. Essentially the same procedure was repeated with the third participant . After each of the conversations, CPs rated their partner's social performance on the QI, DFIS and QI. These three measures together represented likeability with Cronbach's α = 0.74, indicating that they do overlap but also that there is no redundancy. Additionally, CPs completed a measure of their own positive and negative affect . The testing session ended with the LSA and HSA participants watching the video footage of their conversations, while their eye-movements were recorded using an eye-tracking device. Afterwards, they rated their own social performance on the QI, DFIS, and GI. These last two parts of the testing session were unrelated to the topic of the current paper and will be reported elsewhere. Finally, participants were reimbursed and fully debriefed.2.5Two independent assessors, blinded for the condition , viewed and coded the videos of the conversations. The first assessor rated all videos during the entire 5 min of each conversation. The second assessor rated a third of all videos, which were randomly selected. The videos were muted to facilitate rating movement and form.2.5.1Not at all) to 9 (Completely), assessors were rated three items:The assessors used Bernieri's rating scheme (1988), which has been shown to be a valid method of measuring movement synchrony during social interactions . On a 9-Simultaneous movement. Simultaneous movement reflects the degree and quantity of changes in movement that begin and stop, change speed or direction at the same exact moment. For instance, when the narrator starts to turn her or his head at the precise instant as the listener raises her or his face  = -7.54, p < .001, , d = 1.82, as well as on the LSAS-SR, t(69) = -8.11, p < .001, , d = 1.92. CP scored lower than HSA, t(69) = -6.17, p < .001, , d = 1.47, and similarly to the LSA on the LSAS-SR, t(70) = 1.47, p = .15, , d = 0.35. On the BFNE-II, CP scored slightly higher than the LSA, t(70) = 2.31, p < .05, , d = 0.55, but lower than the HSA, t(69) = -4.68, p < .001, , d = 1.11.As can be seen from 3.2Interrater reliability between the two assessors for the three subscales of joint action, namely, simultaneous movement, tempo similarity and gestalt-like smoothness, were α = 0.62, 0.37, and 0.86, respectively. Given the low interrater reliability of tempo similarity, we decided to exclude the ratings of this subscore from further analyses. The global joint action score therefore from now onwards was the average of the score of simultaneous movement and gestalt-like smoothness. This resulted in an interrater reliability of the global joint action score of α = 72. which is comparable to what was found in earlier studies . Ratings3.3F = 2.77, p = .025; Wilk's Λ = 0.83, Ƞp2 = 0.17. Univariate tests showed that CP rated HSA lower than the LSA on the quality of the interaction, their general impression of the person they were talking with, and their desire to meet that person again (see t(70) = -1.59, p = .116, , for the QI t(70) = -1.95, p = .056, , and for the GI t(70) = -1.19, p = .239, , respectively.A multivariate analysis of variance (MANOVA) was used to investigate whether high social anxiety individuals are less liked or elicit less positive reactions from their conversation partners, as assessed by the DFIS, QI, GI and PANAS. As expected, there was a significant multivariate effect of social anxiety group (HSA vs. LSA), gain see .3 Effectgain see . Howevergain see . There w3.4A one-way Analysis of Variance (ANOVA) was used to analyse the degree of joint action during each interaction. Joint action was lower in conversations with socially anxious participants than in conversations with non-anxious participants according to the manual behavioural analyses see .3.5b = 0.12, t(69) = 0.184, 95% CI , p = .855, and negative items, b = -.25, t(69) = -1.12, 95% CI , p = .265. Mediation analyses were conducted with PROCESS macro . PANAS was associated neither with social anxiety , nor joiSS macro .3.5.1b = -2.53, t(70) = -2.51, 95% CI , p = .014, R2 = 0.08. Social anxiety was also a significant predictor of joint action, b = -.64, t(70) = -2.13, 95% CI , p = .041, R2 = 0.06. In turn, joint action, was a significant predictor of desire of future interaction, b = 0.85, t(69) = 2.23, 95% CI , p = .03. When the indirect effect of social anxiety on DFIS via joint action was taken into account, the direct effect of social anxiety on DFIS was no longer significant, b = -1.98, t(69) =-1.96, 95% CI , p = .054. These results provided support for partial mediation (R2 = 0.145). The bootstrap estimation approach with 5000 samples revealed that the indirect effect of joint action was significant, b = -.55, 95% CI . Thus, being socially anxious was associated with 0.55 points lower scores on DFIS as mediated by joint action.The mediation analysis revealed that social anxiety was a significant predictor of the DFIS score, ediation . Approxi3.5.2b = -3.42, t(70) = -2.08, 95% CI , p = .041, R2 = 0.06, and joint action, b = -.64, t(70) = -2.09, 95% CI , p = .041, R2 = 0.06. Furthermore, joint action predicted QI, b = 1.70, t(69) = 2.77, 95% CI , p = .007. More importantly, social anxiety no longer predicted QI when the indirect effect via joint action was taken into account, b = -2.33, t(69) =-1.44, 95% CI , p = .154. Therefore, joint action partially mediated the relationship between social anxiety and QI. Approximately 15% of the variance in QI was accounted for by both predictors (R2 = 0.153). The bootstrap estimation approach with 5000 samples revealed that the indirect effect of joint action was significant, b = -1.08, 95% CI . This means that, being socially anxious was associated with 1.08 points lower scores on QI as mediated by joint action.The mediation analysis with QI as dependent variable revealed similar results. Social anxiety predicted QI, 3.5.3b = -7.97, t(70) = -2.32, 95% , p = .023, R2 = 0.07, and joint action, b = -.64, t(70) = -2.09, , p = .041, R2 = 0.06. Joint action also predicted GI, b = 2.97, t(69) = 2.28, , p = .026. When the mediator (joint action) was taken into account, social anxiety no longer predicted GI, b = -6.08, t(69) = -1.77, , p = .081. Hence, joint action partially mediated the relationship between social anxiety and GI. Approximately 14% of the variance in GI was accounted for by both predictors (R2 = 0.136). The bootstrap estimation approach with 5000 samples revealed that the indirect effect of joint action was significant; b = -1.89, 95% CI . Therefore, being socially anxious was associated with 1.89 points lower scores on GI as mediated by joint action.The third mediation analysis revealed that social anxiety predicted GI, 3.6F = 0.07, 95% CI , p = .789. In fact, the mean difference of observed movement synchrony differs only about 0.001 between the high  and low socially anxious participants . However, movement synchrony appeared to have an effect on evoking positive reactions by their conversation partner. That is, movement synchrony was positively associated with DFIS, b = 70.94, t(69) = 2.90, 95% CI , p = .005, and with GI, b = 243.05, t(69) = 2.93, 95% CI , p = .005, but not with QI, b = 67.55, t(69) = 1.65, 95% CI , p = .103. Lastly, similar to the manually rated joint action, there was no effect of movement synchrony on the PANAS, neither the positive items, b = -32.21, t(69) = -0.80, 95% CI , p = .427, nor for the negative items, b = -18.22, t(69) = -1.27, 95% CI , p = .209.Interestingly, no evidence was found for an association between social anxiety and movement synchrony as measured by frame-differencing, 4Socially anxious individuals have consistently been shown to be less liked by others e.g., . In the On the contrary, findings from the Motion Energy Analysis suggest otherwise. Movement synchrony defined as differences in consecutive video-frames in the ROIs  was found not to be associated with level of social anxiety. It was positively related, however, to desire for future interaction and general impression of the person the CP just talked to. In other words, the more movement synchrony the person engaged in, the more positively did the CP rate the HSA or LSA individual. This lends further support to previous research that found that movement synchrony appears to be essential for rapport and good relationship quality in various contexts e.g. . Yet, wiIn both analyses conversation partner did not experience an effect on their affectivity after talking to LSA or HSA individuals. The PANAS was the only measure used that did not ask about the conversation or the (non-) anxious individual directly but exclusively about how the CP her- or himself felt. Possibly, it takes more to significantly change the CP's mood and feelings than a short 5-min conversation with someone they just met. The same applies to movement synchrony and joint action not being associated with the PANAS.The opposing results of an association between social anxiety and joint action or movement synchrony raise questions about the optional ways of measuring joint action. The MEA software belongs to “frame-differencing methods”, which have been argued to be the most promising non-verbal synchrony techniques , as it iA considerable body of research see  shows thAfter many inconsistent findings in studies of specific behavioural differences between high and low anxious participants regarding intrapersonal processes . Moreover, there was substantial overlap between the CP and LSA groups based on the SIAS cut-off scores. We cannot therefore exclude that CPs may have rated LSA participants more positively because of the similarity in this regard  and poorer joint action as egocentric focus has not been directly addressed in the present study.Conclusion: The goal of the present study was to shed more light on interpersonal processes that contribute to the reduced likeability of individuals with social anxiety. Poor engagement in joint action mediated the relationship between social anxiety and desire for future interaction, quality of interaction, as well as general impression. These results are in line with cognitive behavioural models of social anxiety emphasising the increased self-focused attention, which leaves anxious individuals with fewer cognitive resources to commit oneself to-the ongoing conversation and thereby, increasing the likelihood of evoking negative reactions from others . The funding source had no involvement in study design; the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.This research was supported by a grant