The Cornales is the basal lineage of the asterids, the largest angiosperm clade. Phylogenetic relationships within the order were previously not fully resolved. Fifteen plastid genomes representing 14 species, ten genera and seven families of Cornales were newly sequenced for comparative analyses of genome features, evolution, and phylogenomics based on different partitioning schemes and filtering strategies.rpl22 and ycf15) contained premature stop codons in seven and five species respectively. The phylogenetic relationships among all sampled species were fully resolved with maximum support. Different filtering strategies  of sequence alignment did not have an effect on these relationships. The topology recovered from coding and noncoding data sets was the same as for the whole plastome, regardless of filtering strategy. Moreover, mutational hotspots and highly informative regions were identified.All plastomes of the 14 Cornales species had the typical quadripartite structure with a genome size ranging from 156,567 bp to 158,715 bp, which included two inverted repeats  separated by a large single-copy region  and a small single-copy region  region. These plastomes encoded the same set of 114 unique genes including 31 transfer RNA, 4 ribosomal RNA and 79 coding genes, with an identical gene order across all examined Cornales species. Two genes  contains supplementary material, which is available to authorized users. Cornus), Nyssaceae , Curtisiaceae (Curtisia), Grubbiaceae (Grubbia), Hydrostachyaceae (Hydrostachys), Alangiaceae (Alangium), Davidiaceae (Davidia), and Mastixiaceae  [The Cornales is a relatively small but diverse group, representing the basal lineage of the largest angiosperm clade, the Asterids –4. It coastixia) , 5–8. CoThe circumscription and phylogenetic relationships of the order have been investigated by a number of phylogenetic analyses, mostly based on plastid DNA, beginning from the early twentieth century such as Olmstead et al.  and ChasIntegrating genomic data into plant phylogenetic investigations is developing rapidly due to the availability of new methods of sampling genomes  facilitated by next-generation sequencing (NGS) technologies –14. CompPlastid genomes of angiosperms generally contain 110 to 130 distinct genes, and range in size from 120 to 160 kb. They usually show a typical quadripartite circular structure of two copies of large inverted repeat (IR) separated by the small single-copy (SSC) and the large single-copy (LSC) regions . AlthougIn the present study, a total of 15 complete plastid genomes of 14 species representing 10 genera and seven families of Cornales were obtained. The main objectives of this study were to 1) characterize and compare the structure and gene organizations of plastid genomes in Cornales; 2) explore the potential effects of different partitioning schemes and alignment strategies on phylogenetic inferences; and 3) assess the application of the complete plastid genome sequences in Cornales, and provide genetic resources for future research.http://www.theplantlist.org/) . A total of 15 individuals representing 14 species of 10 genera from 7 families in Cornales mainly occurring in China were sampled. Samples of three families  could not be obtained for this study. The sampled species hence represented four out of the five major lineages suggested by Xiang et al. [Cornus capitata were sequenced here to investigate the intraspecific variability within plastid genome. As outgroups, the plastid genome of Fouquieria diguetii of Ericales was newly sequenced, and the plastomes of three species within Caryophyllales  were obtained from another parallel work (unpublished data). These two orders are phylogenetically closest to Cornales [The circumscription of families of Cornales followed Xiang et al. , and taxCamptotheca acuminata, Cornus controversa, Mastixia caudatilimba and Hydrangea heteromalla. These were sequenced instead from total DNA using Illumina Hiseq4000 after short-insert (500 bp) libraries constructed following the manufacturer’s protocol (Illumina HiSeq 4000) and 143 bp paired-end reads for Hydrangea heteromalla and 90 bp paired-end reads for the other three species, which were generated at BGI Shenzhen, China.Total genomic DNA was isolated from about 100 mg fresh leaf material with a modified CTAB method  in whichCamellia sinensis (NC_020019.1) was used as a reference, and contigs of each individual sample were aligned to it using local BLAST, from which the contigs of plastid genome can be selected. For Cornus controversa and Hydrangea heteromalla, this process produced two and three long plastid contigs respectively, which were easily assembled into a complete genome by overlaps using Geneious v 8.1 [The raw sequence reads were assembled using following steps. First, all reads were de novo assembled into contigs with CLC Genomics Workbench 8.0.2 (CLC Bio) under a word size of 60 bp, minimum contig length of 500 bp and map reads back to contigs with default settings. Second, a closely related genome of us v 8.1 . Among tVerification of the assembly was performed in three ways: 1) by mapping the reads to the assembled plastid genome sequences, 2) by comparing the 14 manually assembled genomes with two easily assembled ones, and 3) by obtaining the four boundary regions using newly designed primers under Sanger sequencing, which were showed in Additional file The complete genome sequences were annotated using the online program DOGMA  to prediCornus controversa as reference. Plastomes of Cornales were also aligned using MAFFT [The 15 whole plastid genomes were aligned with Mauve v 2.3.1  plugin ing MAFFT  and manung MAFFT : the prong MAFFT .rpl22 is disabled or not, the ratio of nonsynonymous and synonymous  of rpl22 for different branches was calculated in PAML v4.7 [To test whether the abnormal gene of AML v4.7  using thThe whole plastid genomes of the 15 individuals of Cornales and the four outgroup species were aligned using the program MAFFT v 7.22 with default settings. Three primary data sets were generated for phylogenetic inference. The first data set comprised coding regions, i.e. exons of protein-coding genes, tRNAs and rRNAs; the second comprised all noncoding regions, i.e. intergenic regions and introns; the third comprised the entire plastid genome. Each gene and intergenic or intron was realigned using MAFFT v 7.22 with G-INS-i algorithm plugin in Geneious v 8.1. One of the IR regions was removed for all data sets to reduce overrepresentation of duplicated sequences.Some regions in the whole plastome data set are highly variable and poorly aligned. So, in order to assess the effect of alignment quality on phylogeny, we compared the results from three different analysis strategies. First, the unfiltered alignment included all sequence positions of the plastomes in the alignment. Second, the lightly filtered alignment was created using the program Gblocks  to removhttp://www.phylo.org/), to estimate the optimal model of molecular evolution with the Akaike Information Criterion (AIC). Maximum likelihood (ML) analyses were conducted using RAxML v8.1.11 [For the unfiltered, lightly filtered and strictly filtered alignments of coding, noncoding and complete plastome data sets, jModeltest v2.1.6  was used v8.1.11  as imple v8.1.11  as impleTo investigate the issues of data partitioning for the plastid phylogenomic analysis, an algorithmic method for estimating an optimal partitioning scheme was conducted for the complete unfiltered data set. It was partitioned into the maximum possible number of data blocks based on genomic composition. We divided the whole plastid genome into 174 subsets: each gene, intergenic region or intron was regarded as a distinct subset, while subsets of less than 200 bp, or regions that only contained invariable nucleotide sites, were combined into large data subsets according to their function   to 2757× (Camptotheca acuminata). Henceforth, all text describing plastid genomes refers only to Cornales unless stated otherwise. The size of the 15 Cornales plastid genomes ranged from 156,567 bp in Nyssa sinensis to 158,715 bp in Diplopanax stachyanthus, and both individuals of Cornus capitata examined had the same plastid genome size  (Table ycf3 and clpP) contained two introns. The length and GC content of coding, noncoding and complete plastid genome data sets are shown in Table Fouquieria diguetii of Ericales , resulting in the presence of a part of the rps19 gene in the IRb. In Hydrangea davidii and Deutzia crassifolia, the junction was located in the rps19-rpl2 spacer. The IRb/SSC boundary positions in all species were located in the ycf1 gene, with part of this gene duplicated from 972 to 1246 bp. The ndhF gene in seven species was completely located in the SSC region, whereas in the others it extended fractionally into the IRa region , but onThe plastid genomes within Cornales showed high sequence similarities with identities of only a few regions below 90%  were identified from the plastid genomes, which might be undergoing more rapid nucleotide substitution. These comprised 2 gene regions and 7 intergenic regions: rpl22 and ycf15 were interrupted by internal stop codons in seven and five Cornales species respectively. Both of them were further verified by Sanger sequencing using newly designed primers  . Despite this, the gene still exhibited nearly 80% nucleotide identities between species, with no big difference between those species with and those without internal stop codons. Furthermore, the ratio of nonsynonymous and synonymous  of rpl22 for different branches showed similar values in both the Cornaceae-Alangiaceae-Curtisiaceae clade (ω = 0.34569) and Mastixiaceae clade (ω = 0.35594), and no significant difference with background  was found. This indicated that those genes containing stop codons have not accumulated mutations at an increased rate, and hence may not have lost their functions.For all species from Cornaceae, Alangiaceae and Curtisiaceae, a frameshift mutation generated premature termination codons within GA) Fig. . Furtherdon Fig. . rpl22 wycf15 varied from 102 bp to 249 bp among the 15 sequenced individuals of Cornales. For two species of Alangium, ycf15 contained a large deletion (84 bp) at the 5′ end and a 10-bp deletion near the 3′ end, potentially causing a loss of function. Additional 4-bp and 5-bp deletions within ycf15 led to internal stop codons in Davidia involucrata and Hydrangea aspera, respectively. Furthermore, in Deutzia crassifolia, a single substitution (G to A) within ycf15 likewise resulted in an internal stop codon (TAG). In the remaining nine Cornales species, ycf15 did not contain stop codons, and there was no evidence of loss of function   for all nodes Fig. . NyssaceLikewise, using lightly filtered and strictly filtered data sets, both the topology and support values were almost identical . Topology and support values obtained from this analysis were consistent with unpartitioned analysis, except for a decrease in BS support value from 100% to 95% for the sister relationship of Curtisiaceae to Cornaceae-Alangiaceae in ML analysis  and an intervening region of about 250 bp in some other taxa  that renders it as a pseudogene [ycf15 transcripts in taxa with or without the insertion suggested that this gene may not be a protein-coding gene even when it is intact [ycf15 were detected in some taxa like Camellia, it may have been removed from the pre-mRNA after transcription in order to activate the function of other genes, thus ycf15 is possibly an intergenic sequence without function [ycf15 hypothesis to some extent is supported by data from Cornales, within which four independent mutation events within ycf15 either inserted stop codons  or deleted parts of the gene (Alangium). The evolutionary patterns of ycf15 showed that they evolved in a discontinuous fashion across angiosperms [Alangium, Davidia and Deutzia. These three genera belong to distinct clades, implying separate and independent alterations in each case; hence ycf15 might not provide phylogenetic implication  on each of these three data sets. Phylogenetic inference from BI and ML analyses based on all data sets provided the same topology. All data sets supported the sister relationship between Curtisiaceae and Cornaceae-Alangiaceae, support for this clade from the coding region and its filtered data subsets was relatively low, e.g. 81%/1.0 for unfiltered  and a broader taxon sampling, recovered five major clades: Cornaceae-Alangiaceae, Curtisiaceae-Grubbiaceae, Mastixiaceae-Nyssaceae-Davidiaceae, Hydrostachyaceae, and Hydrangeaceae-Loasaceae; relationships between these clades were well supported. However, cpDNA-based relationships were contradicted by 26S rDNA data in the study, and relationships within some families  were not fully recovered [matK, ndhF) and seven regions trnK-rps16, rpoB-trnC, trnT-trnE, petA-psbJ, psbE-petL, rpl32-trnL, and rps15-ycf1 that are mutational hotspots, and are hence recommended as phylogenetic markers within Cornales, and perhaps beyond it.Preliminary phylogenetic frameworks for Cornales have previously been provided based on a few molecular markers, but relationships among families of the order tended to be poorly resolved , 7, 72. ecovered . Therefoecovered , 73, 74.rpl22 and ycf15) contained premature stop codons in seven and five species respectively. Plastid genomes showed strong potential for resolving phylogenetic relationships within Cornales, both for the interfamily and intrafamily relationships, with very strong support. Different partitioning schemes and filtering strategies  of sequence data sets have no effect on phylogenetic relationships. The topology recovered from coding and noncoding data sets was likewise identical to that for the whole plastome. However, the coding data set provided lower support values than the latter two data sets. Mutational hotspots and highly informative regions of Cornales were identified. All data presented here are fundamental to phylogenomic analyses of Cornales, and will be a useful genomic resource for future studies of evolutionary biology.Phylogenomic data have rapidly accumulated and been broadly used for resolving phylogenetic relationships in the last few years. In the present study, fifteen full plastid genomes of 14 Cornales species were sequenced to investigate the phylogenetic relationships and plastome evolution of Cornales. Comparative analysis of the plastid genomes revealed that plastomes of the order have a conserved collinear structure with identical gene content and order. Two genes . Table S2. Molecular models selected for all the data sets of the three alignment strategies. Table S3. Model selected for each data partition identified by software PartitionFinder for unfiltered complete plastid genomes. Table S4. Gene category and gene contained in plastid genomes of Cornales. (DOC 147 kb)The primers newly designed in this study for four junctions and two genes A percent identity plot showing the overall sequence similarity of the fourteen Cornales plastid genomes.


Scutellaria baicalensis and heartwoods of Acacia catechu containing free-B-ring flavonoids and flavans, respectively, were combined into a proprietary blend called UP326. A similar bioflavonoid composition, UP446, has been reported with modulation of pathways related to systemic inflammation. To test the effect of UP326 on memory and learning, a radial arm water maze (RAWM) and contextual fear conditioning (CF) were utilized in aged F344 rats fed with UP326 at doses of 3, 7, and 34 mg/kg for 11 weeks. The 7 and 34 mg/kg dosage groups had significantly fewer errors than aged vehicle control animals and their performance was equivalent to young animal controls. In a separate human clinical trial, test subjects orally given 300 mg of UP326 BID for 30 days showed marked improvement in speed and accuracy of processing complex information in computer tasks and reduced their standard deviation of performance compared to baseline and the placebo group. This data suggest that UP326 may help maintain memory, sustain speed of processing, and reduce the number or memory errors as we age.Dementia and cognitive impairment have become the major concerns worldwide due to a significantly aging population, increasing life span and lack of effective pharmacotherapy. In light of limited pharmaceutical drug choices and the socioeconomic implications of these conditions, the search for safe and effective alternatives from natural sources has gained many attractions within the medical food and dietary supplement industry. Two polyphenol extracts derived from roots of There is a growing interest in alternative medicine and use of dietary or nutritional supplements that have a variety of putative beneficial activities, including weight loss, athletic performance, general health, and cognition. Of particular interest are traditional homeopathic products that can stabilize or enhance cognitive function. Among these conditions, Alzheimer's, disease (AD), a slow neurodegenerative disorder of the brain characterized by progressive cognitive dysfunction and memory impairment, is a major public health concern , 2.α, MIP1-α, and IL-1β expression), and the pathogenesis of AD leading to loss in memory and speed of information processing in specific animal models  during the initial RAWM testing without delay. When a 4 h delay was inserted between trials 3 and 4, there was a clear effect of age  . Following the baseline training, the aged animals were assigned to treatment groups  in a counterbalanced manner based on their performance on trial 4 of the 4 h delay baseline test. After 11 weeks of treatment, the rats were tested again on the 4 h delay version of the task  = 103.6, p < 0.0001]. All groups decreased the number of errors from trials 1–3. There was significant group effect on trial 4. The aged controls had significantly more errors on trial 4 than the young controls. The UP326-treated animals were not significantly different from young controls. Two of the UP326-treated groups (3 and 34 mg/kg) had significantly fewer errors than the aged controls (p < 0.005).During the baseline training, all animals learned the basic underlying principles of the task as evidenced by the rapid decrease in the number of total errors across trials . There w< 0.002] . Post hothe task . There wF = 7.941, p < 0.001]. The aged control group froze significantly less than the young group (p < 0.0001). The 7 and 34 mg/kg UP326-treated groups froze significantly higher than the aged controls (p's < 0.0005)  .Elevated plus maze and open filed studies were also carried out to assess the anxiolytic effect of UP326 on young (6-month-old) animals. No behavioral changes were observed as a result of UP326 for all the dosages (data not shown).All study compounds were well tolerated with no reports of serious or unexpected treatment related adverse effects. All subjects completed the study.p = 0.1314); however, there were significant improvement across weeks of testing (p < 0.0001) and a significant group × week interaction (p = 0.05). Across the weeks of testing, there was a decrease in the RT median from baseline to week 4, indicating a faster rate of responding. For the week by group interaction, subjects in group B showed the largest improvement  (p < 0.05) during the weeks 2 and 3 of treatment. These results suggest that the composition can increase cognitive processing (decision-making) speed without impairing choice accuracy, improving the rate of responding to cognitively demanding or complex choice situations.The reaction time median (RT med) scores, a measure of executive function, show the most promising results for this study . The maip = 0.3962); however, there was significant improvement across weeks of testing (p < 0.0002) but there was no significant group × week interaction (p = 0.303). Across the weeks of testing, there was a decrease in the standard deviation from baseline to week 4. For the weekly between-group differences, the significant improvement  was for group B at week 3 of treatment. These results suggest that the composition may increase sustained attention, improving the consistency of responding to cognitive demanding or complex choice situations. Reaction standard deviation (RTSD) also suggests some promising findings complementing the working memory speed . RTSD is Scutellaria baicalensis and Acacia catechu for short term memory in aged rats and for speed and accuracy of processing complex information in humans.Due to significantly aging population, increasing life span, and lack of effective pharmacotherapy options, AD and vascular dementia have become the major concerns worldwide. In spite of advances in therapeutic discoveries, there is no cure for AD. Significant bodies of evidence have been documented detailing the pathogenesis of AD in relation to inflammation , oxidati in vitro and in vivo. For example, 26-week repeated oral toxicity study in rats [ In vitro, no mutagenicity or drug interactions were seen by AMES and cytochrome P450 enzyme inhibition, respectively [While the nonsteroidal anti-inflammatory drugs seem to possess activities suggestive of benefit in neurodegenerative diseases, their chronic consumption is hindered due to their gastrointestinal, renal, and cardiovascular side effects. To achieve a meaningful therapeutic effect, patients with AD or dementia have to be in a long term treatment schedule. This condition will expose the patients to deleterious pharmacotherapy adverse effects. Hence, there is always a need for safe and efficacious options from natural sources to help maintain cognitive health. Besides its beneficial activities described here, rigorous safety evaluations have been carried out on the composition, UP326, in rats  and in b in rats , reprodu in rats –43, a 90 in rats , and tox in rats  have bee in rats . In vitr in vivo experiments found in the current study is that UP326 reversed age-related memory deficits in rats in two different learning and memory tasks. In the experimental design, studies conducted at the no delay condition demonstrate the animals' ability to perform the task and act as a control for differences in the ability to perform the task . The delay task introduces a 4 h delay between trials 3 and 4, making the task more difficult. It is under the delay condition that the age-related memory impairments were demonstrated. Treatment of aged F344 rats with middle and high doses of UP326 reversed an age-related impairment in spatial memory 11 weeks after treatment. In the contextual fear conditioning task, there was a significant age-related impairment in hippocampal-dependent memory, as demonstrated by the decreased freezing in the training context. This age-related memory impairment was ameliorated by treatment with middle and high doses of UP326.The overall strength of the preclinicalIn the pilot clinical study, performance for the working memory task was already high at baseline in this healthy population. The lack of significance between group differences for working memory speed performance and flexibility may be due to the study participants having normal cognitive function that is close to their maximal level of ability. Implying that ceiling effects were attained, this leaves no room for treatment related statistically significant improvements. Regardless, results from this study suggest that the composition may increase sustained attention and improve the consistency of responding to cognitive demanding or complex choice situations. The data also further warrant additional studies involving large numbers of participants targeting the right age group.μmole TE/g suggesting its capacity to reduce ROS [ cox-2, 45-fold for il-1β, 3.3-fold for tnf-α, 37-fold for il-6, and 2.2-fold for nfκb [μg/mL, while the mixed extract showed a value for 5-LOX enzyme activity of 25 μg/mL [Previously, UP326 has been reported to have a high ORAC value of 70,930 duce ROS . When infor nfκb . The 50%25 μg/mL , 45. Cle Scutellaria baicalensis and catechins from Acacia catechu extracts have demonstrated their cognitive deficit amelioration effect in multiple in vivo models. It has been postulated that these effects could be as a result of their anti-inflammatory and free radical scavenging activities. For instance, baicalein administered orally ameliorated memory impairments induced by chronic bilateral common carotid artery occlusion used as a model for vascular dementia [β-amyloid peptide infusion induced model for amnesia [ Acacia catechu, catechin, has also shown antiamnesic [Furthermore, augmented reports have been documented from various animal models describing the beneficial usage of flavonoids in attenuating the characteristic symptoms of AD. In particular, active components such as baicalin, wogonin, and Oroxylin A fromdementia  and β-am amnesia . In addi amnesia , 50. In  amnesia . Similar amnesia  and scop amnesia  suggestiiamnesic  and antiiamnesic  properti Scutellaria baicalensis and Acacia catechu, to the best of our knowledge, there is no report for the combined use of two standardized extracts in maintaining cognitive health. Acknowledging the complexity of aging process as well as global inflammation and generation of oxidative stress in relation to cognitive impairment, we believe that combining active fractions from these two medicinal plants together could be a preferred approach for mitigation of cognitive decline in healthy individuals. In summary, animal and human clinical results with UP326 suggest that there is significant attenuation of memory deficits in animal models and increases in speed and accuracy of processing in healthy human subjects, respectively. These results may be due to a reduction of eicosanoid inflammation and ROS effects on NFκB-induced inflammatory pathways as a result of administration of UP326. The pathologic lesions associated with AD develop many years before these clinical manifestations; however, early diagnosis is still a major challenge for the prevention of the disease. Therefore, it could be inferred that regular consumption of a bioflavonoid supplement containing UP326 starting at a relatively early age in life could possibly prevent or delay the clinical manifestations of dementia.While these individual reports in animal neurodegenerative models suggest the beneficial use of active components isolated from


The objective of this study is to investigate the association between the incidence of pancreatic fistula after pancreaticoduodenectomy (PD) and the degree of pancreatic fibrosis.Between January 2013 and December 2016, the analysis of the clinical data of 529 cases of pancreaticoduodenectomy patients of our hospital was performed in a retrospective fashion. The univariate analysis and multivariate analysis were done using the Pearson chi-squared test and binary logistic regression analysis model; correlations were analyzed by Spearman rank correlation analysis. The value of the degree of pancreatic fibrosis to predict the incidence of pancreatic fistula after pancreaticoduodenectomy was evaluated by the area under the receiver operating characteristic (ROC) curve.2, pancreatic duct size ≤ 3 mm, pancreatic CT value< 30, the soft texture of the pancreas (judged during the operation), and the percent of fibrosis of pancreatic lobule ≤ 25% are prognostic factors of pancreatic fistula after pancreaticoduodenectomy (P < 0.05); the pancreatic CT value and the percent of fibrosis of pancreatic lobule in pancreatic fistula group were both lower than those in non-pancreatic fistula group (P < 0.05). Results indicated that there is a negative correlation between the severity of pancreatic fistula and the pancreatic CT value or the percent of fibrosis of pancreatic lobule . The areas under the ROC curve of the percent of fibrosis of pancreatic lobule and the pancreatic CT value were 0.756 and 0.728, respectively.The total incidence of pancreatic fistula after pancreaticoduodenectomy was 28.5% (151/529). Univariate analysis and multivariate analysis showed that BMI ≥ 25 kg/mThe degree of pancreatic fibrosis is a prognostic factor which can influence the pancreatic texture and the incidence of pancreatic fistula after pancreaticoduodenectomy. The pancreatic CT value can be used as a quantitative index of the degree of pancreatic fibrosis to predict the incidence of pancreatic fistula after pancreaticoduodenectomy. Their age ranged from 14 to 82 and the average age is 55.1. Among the above patients, 355 patients were male and 174 were female. Pathological diagnosis showed that there were 348 cases of pancreatic ductal adenocarcinoma (PDAC), 102 cases of cancer originated from gastrointestinal tract, 28 cases of neuroendocrine neoplasm, 25 cases of inflammatory change, 16 cases of pancreatic papilloma, eight cases of serous cystadenoma, and two cases of retroperitoneal liposarcoma (RPLS). All the operations utilized Child procedure to rebuild the digestive tract and were performed by doctors who at least have the title of associate professor.The diagnostic and including criteria of pancreatic fistula in this research adopted the definition of pancreatic fistula which is given by the International Study Group on Pancreatic Fistula (ISGPF)CT value is the attenuation value of X-ray after the absorption of the tissue it passed.All the pathological sections in this research were sampled from the remaining pancreatic tissue closing to the incision, and embedded in paraffin. The thickness of all the sections was about 5 μm. Stained by hematoxylin-eosin. Then, the sections were observed by electron microscope.P = 0.05.Data were statistically analyzed by SPSS 18.0. Univariate analysis was done using the Pearson chi-squared test. Then, the factors which are statistically significant were analyzed by binary logistic regression analysis. The correlation between pancreatic CT value and the percent of fibrosis of pancreatic lobule was analyzed by Spearman rank correlation analysis. We used the area under the ROC curve to evaluate the pancreatic CT value and the percentage of fibrosis of pancreatic lobule to predict the incidence of pancreatic fistula after PD. The area under the ROC curve higher than 0.9 indicates a high predictive value; the area under the ROC curve ranges from 0.7 to 0.9 indicates a moderate predictive value; the area under the ROC curve ranges from 0.5 to 0.7 indicates a low predictive value. Differences were considered as significant at the level of Within 1 month after PD, there were 151 cases of pancreatic fistula in 529 patients (28.5%), including 79 cases of grade A pancreatic fistula (14.9%), 60 cases of grade B (11.3%), and 12 cases of grade C pancreatic fistula (2.3%).P < 0.05); however, there is no correlation between the incidence of pancreatic fistula after PD and gender, age, diabetes history, hypertension history, history of abdominal surgery, hemoglobin, albumin, total bilirubin, ALT, AST, alkaline phosphatase, surgical method, intraoperative blood loss, or situation of vessels invaded by carcinoma (P > 0.05) , and the percent of fibrosis of pancreatic lobule (5) Table .Table 1R2, pancreatic duct size ≤ 3 mm, CT value < 40, hard texture of pancreas (judged during the operation), and percent of fibrosis of pancreatic lobule ≤ 25% are prognostic factors of pancreatic fistula after PD . The pancreatic CT values and percent of fibrosis of pancreatic lobule of mild pancreatic fistula patients are higher than those of heavy pancreatic fistula patients, and pancreatic CT value and percent of fibrosis of pancreatic lobule all had a rank correlation with the severity of pancreatic fistula (P < 0.01)  Table .Table 3Cr = − 0.297, P < 0.05; r = − 0.342, P < 0.05).We assigned non-pancreatic fistula, grade A, grade B, and grade C to 0, 1, 2, 3, respectively. Analyzed by linear correlation analysis, we found that there is a negative correlation between the severity of pancreatic fistula and pancreatic CT value or percent of fibrosis of pancreatic lobule (P = 0.026).Spearman rank correlation analysis suggested that there is a high correlation between pancreatic CT value and percent of fibrosis of pancreatic lobule  (Fig. Results showed that the areas under ROC curve are 0.756 and 0.728, respectively. The areas under ROC curve also indicated that preoperative pancreatic CT value and the postoperative percent of fibrosis of pancreatic lobule could be used to predict the incidence of pancreatic fistula after PD and both have a moderate predictive value (95% Pancreatic fistula is one of the main causes which lead to other complications and death.In 1984, Longmire18At present, most researchers determine whether the texture of the pancreas is soft or not depending on their subjective experience, lacking in objective uniform standards. Although some studies utilized the CT value to evaluate the degree of pancreatic fibrosis, the included cases were too scarce to form a scientific evaluation system.After analyzing the data of preoperative pancreatic CT value and postoperative percent of fibrosis of pancreatic lobule, linear regression equation was established, and correlations were examined as well. Results indicated that there is a linear correlation between the above two indexes, so these two indexes both can reflect the degree of pancreatic fibrosis and predict the incidence of pancreatic fistula after PD. In the meantime, results of ROC curve also suggested that preoperative pancreatic CT value and postoperative percent of fibrosis of pancreatic lobule can be used to predict the incidence of pancreatic fistula after PD. Based on the above results, we assumed that preoperative pancreatic CT value and postoperative percent of fibrosis of pancreatic lobule both can be used as objective indexes to reflect the degree of pancreatic fibrosis. Hence, they can be used to predict the incidence of pancreatic fistula after PD. From a practical standpoint, however, results of postoperative pathological examinations cannot instruct the formulation of preoperative therapeutic plan. On the contrary, preoperative pancreatic CT value has a great significance in instructing the formulation of perioperative therapeutic plan, as CT value can be obtained noninvasively and before operation.In conclusion, our research suggests that BMI, pancreatic duct size, pancreatic CT value, texture of the pancreas, and postoperative percent of fibrosis of pancreatic lobule are prognostic factors of pancreatic fistula after PD. There is a good linear correlation between pancreatic CT value and postoperative percent of fibrosis of pancreatic lobule, so they can be used as objective indexes to reflect the degree of pancreatic fibrosis. It can be more scientifically to predict the incidence of pancreatic fistula after PD if the above two indexes combine with subjective judgment. However, it still needs further research to understand whether there are differences in pancreatic CT value and postoperative percent of fibrosis of pancreatic lobule among cohorts and to determine how to judge the degree of pancreatic fibrosis preoperatively.


Fungia, along that cross-shelf environmental and water quality gradient. The main research questions were: (1) How do water quality and bacterial community composition change along a coastal shelf gradient? (2) Which water quality parameters influence bacterial community composition? (3) Is there a difference in bacterial community composition among the investigated habitats? For this purpose, a range of key water parameters were measured at eight stations in distances from 2 to 55 km from urban Makassar. This was supplemented by sampling of bacterial communities of important microbial habitats using 454 pyrosequencing. Findings revealed that the population center Makassar had a strong effect on the concentrations of Chlorophyll a, suspended particulate matter (SPM), and transparent exopolymer particles (TEP), which were all significantly elevated at the inshore compared the other seven sites. Shifts in the bacterial communities were specific to each sampled habitat. Two OTUs, belonging to the genera Escherichia/Shigella (Gammaproteobacteria) and Ralstonia (Betaproteobacteria), respectively, both dominated the bacterial community composition of the both size fractions of the water column and coral mucus. The sampled reef sediments were more diverse, and no single OTUs was dominant. There was no gradual shift in bacterial classes or OTUs within the sampled habitats. In addition, we observed very distinct communities between the investigated habitats. Our data show strong changes in the bacterial community composition at the inshore site for water column and sediment samples. Alarmingly, there was generally a high prevalence of potentially pathogenic bacteria across the entire gradient.Coastal eutrophication is a key driver of shifts in bacterial communities on coral reefs. With fringing and patch reefs at varying distances from the coast the Spermonde Archipelago in southern Sulawesi, Indonesia offers ideal conditions to study the effects of coastal eutrophication along a spatially defined gradient. The present study investigated bacterial community composition of three coral reef habitats: the water column, sediments, and mucus of the hard coral genus  Coastal coral reef systems in close vicinity to highly populated urban areas are often impacted by land-based activities. The Spermonde Archipelago, including its ~150 small islands, is located on a narrow, 60 km wide carbonate shelf platform in southern Sulawesi, Indonesia. The coral reefs fringing the islands are essential to sustain the livelihoods of thousands of fishermen in the archipelago as a source of income and building material for local construction . For the water quality analyses five replicate water samples were taken from 5 m water depth (which was ~1 m above the substrate) with a 5 l Niskin bottle . From each replicate subsamples were taken for measurement of the water quality parameters . Samples were stored at −20°C in the dark until analysis immediately after returning to the field station. The longest travel time was no more than 3 h. Inorganic nutrients  where measured spectro-photometrically with a Flowsys continuous flow analyser . For DOC, samples were filtered through 0.45 μm pore Whatman GF/F filters  and acidified with HCl (pH below 2). The measurement was completed via high-temperature oxic combustion (HTOC) using a TOC-VCPH TOC analyzer . Hansell artificial seawater standards  and ultrapure water blanks were used for calibration and quality control. To determine SPM mass, pre-combusted GF/F filter were weighed on a ME 36S balance  before and after filtration of known volume of sample water. Difference in weight was determined after filters were dried for 24 h at 40°C. TEP was quantified with an updated protocol  and chlorophyll l Engel,  of the sl Engel, . In shorWhatman, GE Healthcare, Pittsburgh, USA), to separate bacterioplankton in two selected size fractions representing “particle-attached” and “free-living” bacteria, respectively. DNA extraction for water column samples followed the protocol established by Boström et al.  with modification of two steps of the protocol: (1) we did not incubate for 5 min at 4°C but went straight for the centrifugation and (2) we used 50 μl of elution buffer instead of 100 μl. Extracted DNA samples were sequenced, after 16S rRNA amplification (PCR), Roche/454-tagging and preparation of the Pico Titer Plate, on a Genome Sequencer FLX System + Titanium  by LGC Genomics . 16S rRNA primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 1061R (5′-CRRCACGAGCTGACGAC-3′) were used, targeting the V3–V6 hypervariable region  before enumeration. Ten fields of view were counted at 1,000x magnification from triplicate slices for each sample.For cell enumeration staining with 4′6-diamidino-2-phenylindole (DAPI) for epifluorescence microscopy was conducted by incubating filter slices with 20 μl of a 1 μg mlSystat Software, Inc., San Jose, California, USA). Values are given as arithmetic mean ± standard deviation. All parameters failed the Shapiro-Wilk test for normal distribution, so alternative non-parametric Kruskal-Wallis ANOVA on ranks was performed. Whenever significant differences were detected, pair-wise comparisons were conducted using the implementation of the Tukey's HSD post-hoc test for non-parametric data in SigmaPlot. Principle component analysis (PCA), including all environmental and water quality parameters, was conducted using the PRIMER 6.16 software , q = 1 , and q = 2 (inverse Simpson index) were calculated based on 100 repeated subsampling runs to rarefy the data set to the minimum library size (191 sequences). Kruskal-Wallis and spearman correlations were conducted to test the effect of reef habitat and distance to Makassar on bacterial alpha diversity, respectively. Differences in bacterial community composition (beta diversity) among reef habitats and with increasing distance to Makassar were tested using PERMANOVA in combination with ANOSIM.Alpha diversity of the bacterial communities was assessed using Hill numbers , as revealed by Kruskal-Wallis ANOVA  across the surveyed gradient from the inshore site to the outer shelf break (Tables a exhibited significantly higher concentrations inshore (1.50 ± 0.21 μg l−1) compared to the furthest outer shelf site (0.14 ± 0.05 μg l−1), but overall correlation to distance from Makassar remained weak (Spearman's ρ −0.44). No significant differences were found in SPM concentrations although there was a weak general decreasing trend from inshore to the outher shelf (Spearman's ρ −0.28). There were significant differences between the highest concentrations of DOC (97.44 ± 17.76 μmol l−1) at the closest near-shore station and lowest concentrations, 78.46 ± 18.04 μmol l−1, at the inshore station. DOC concentrations between these two zones were also significantly different. Although cross-shelf concentrations of DOC were significantly different (P < 0.01), there was only a very low correlation to the distance from Makassar (Spearman's ρ −0.22). Overall chlorophyll a, TEP and SPM showed a very similar pattern with steeply declining concentrations between the inshore and the first near-shore site. Near-shore concentrations of TEP were significantly (P < 0.02) higher (301.38 ± 11.98 μg Xeq l−1) than the most distant outer shelf site (26.34 ± 1.43 μg Xeq l−1), and the correlation to distance from Makassar was strong (Spearman's ρ −0.83). The two near-shore sites showed intermediate concentrations while all following stations were in a comparable range to the outermost site.The individual islands differed significantly in many of the sampled environmental and water quality parameters, including chlorophyll VA Table . Howeverk Tables , 2. OnlyA PCA of the environmental and water quality data was able to capture 66.2% of the variation among sites on the first two principal components Figure . The ins−1 h−1) and the lowest at 11 km distance from Makassar (0.010 ± 0.008 mg l−1 h−1). For both surface and 3 cm deep sediments the highest oxygen uptake was measured at the 11 km site , while lowest BOD was measured at the outer shelf site at 55 km .Biological oxygen demand of the water column and sediments showed high variability and did not exhibit a clear pattern across the gradient Table . Within 6 ± 4.70 × 106 cells ml−1 in the outer shelf area to 7.94 × 106 ± 1.08 × 106 cells ml−1 at the inshore site , followed by the particle-attached fraction of the water column (from 3.68 ± 0.03 to 11.54 ± 0.09) and sediments (from 87.10 ± 1.37 to 156.48 ± 0.85). Bacterial communities in the reef sediments were generally more diverse  = 7.90, P < 0.001], but not with distance to Makassar . Subsequent pairwise tests showed that all habitats, except the Fungia mucus compared with both size fractions of the water column, were significantly different from each other .The composition of the bacterial communities in the sediment was distinct from the other habitats based on the clustering of Bray-Curtis dissimilarity coefficients Figure . This waEscherichia/Shigella (Gammaproteobacteria), Ralstonia (Betaproteobacteria), Stenotrophomonas (Gammaproteobacteria), and Phenylobacterium  at the mid-shelf site at 11 km, and decreased toward the outer shelf sites (8% at 19 km). Stenotrophomonas and Phenylobacterium showed no obvious trends across the gradient, and their relative contribution to the total community varied from <1 to 24 and 2 to 10%, respectively. Other potentially pathogenic bacterial groups that were found among the 10 most dominant OTUs were Mycobacterium, the causative agent of tuberculosis and leprosy, and Staphylococcus (Firmicutes). There was no overall trend in the abundance of the potentially pathogenic OTUs with distance from Makassar. While e.g., Escherichia/Shigella increased in abundance with distance, Mycobacterium (Actinobacteria) is most abundant at the very inshore site closest to Makassar.The free-living fraction of the water column bacterial communities showed variation among stations across the spatial gradient Figure . Three cEscherichia/Shigella and Ralstonia were, on average, also the dominant OTUs that were retained on 3.0 μm filters, and thus being defined as “particle-attached.” The highest relative abundance of Escherichia/Shigella was found at the 22 km site (44%), while the lowest abundance was observed at the 14 km site (6%). Ralstonia was generally found in lowest abundance at 14 km . Calculated Bray-Curtis dissimilarities were also often >50% , and unclassified members of the gammaproteobacterial JTB255 marine benthic group (2%) and the firmicute family Carnobacteriaceae (1%). Additionally, the relative contribution of the investigated potentially pathogenic OTUs to the overall community composition was much lower (Figure Overall the sediment communities were more diverse and even in their community composition and showed less variation across the shelf on the class level (Supplementary Table a, and TEP. Although SPM showed a very similar pattern, the differences were not significant. While there were no significant effects of the water quality parameters and distance from Makassar on the bacterial community composition we detected distinct and significant differences between the habitats. Overall, the sediment bacterial communities were more diverse and heterogeneous, comparable to previous descriptions of carbonate-based reef sediments (Hewson et al., Fungia mucus, Planctomycetes was the most abundant class in the reef sediments.With this study we can shed new light on shifts in bacterial communities of the water column, reef sediments and coral mucus in response to coastal eutrophication. Similar to other previous investigations we found strong changes in water quality parameters along the gradient (Sawall et al., a, TEP and SPM in the Spermonde Archipelago where within the range of other near-shore reef ecosystems of the western Pacific, e.g., the Great Barrier Reef, Australia (Alongi et al., a, previous studies of the same area (Sawall et al., a and the severity of eutrophication-related processes were highly correlated to water residence time (Delesalle and Sournia, Excluding the highly eutrophied inshore site, concentrations of chlorophyll a, SPM concentrations and DAPI cell counts in the water column. Additionally, TEP may also serve as a food source and habitat for bacteria (Passow, TEP concentrations can be used as a good integrative indicator for differences in the water quality among the sites as well as for a strong interaction between phytoplankton and the bacterial communities, which results in TEP production (Smith et al.,  Passow, , increas Passow, .a, TEP, and SPM. This could be explained through a combination of DOC rapidly being take up by heterotrophic bacteria in the water column and aggregation into larger particles (Passow and Alldredge, Concentrations of DOC in the Spermonde Archipelago are at the higher end described for tropical reef ecosystems (Dinsdale et al., Escherichia/Shigella was the dominant OTU. Bacteria of this genus are known human pathogens, as well as indicators for drinking water quality and fecal contamination (Baudišová, Mycobacterium and Staphylococcus, were found at highest relative abundances closer to the mainland. This is noteworthy, as they thrive well in eutrophic conditions (Jacobs et al., SAR11/Pelagibacter clade (Tout et al., Ralstonia as the main constituents of that class, are very common to freshwater (Lau et al., Ralstonia in free-living and particle-attached habitats.On the community level there were no corresponding shifts along the measured water quality gradients. In the present study, e.g., significantly higher bacterial cell counts in the water column were observed at the inshore site at 1 km distance to Makassar. This can be the result of an increased uptake of available DOC at the inshore site (Ferrier-Pagès et al., Escherichia/Shigella was again the single most common OTU in the mucus samples. This is an unusual observation, as members of the Escherichia/Shigella taxon are usually not present in mucus samples in such high relative abundances (Koren and Rosenberg, Fungia corals at the sampled islands and their fragile host-symbiont balance are already severely disturbed potentially enabling Escherichia/Shigella to colonize the coral mucus and capitalize on the available carbon sources. Findings concerning the Betaproteobacteria from the present study are supported by comparisons of bacterial communities in mucus of Fungia sp. from the Red Sea and aquaria by Kooperman et al. (Observations made on the community composition of the mucus samples were similar to those of the water column, which was reflected in the non-significant differences in overall community composition. Again, Gamma- and Betaproteobacteria were on average the most dominant classes. n et al. . They aln et al. .a, TEP, and SPM at near-shore sites, indicating a high productivity and anthropogenic impact at the site closest to shore, may support the high prevalence especially of Gammaproteobacteria at this site. As a result from high chlorophyll a and SPM concentrations, a constant supply of particles rich in organic matter to the sediments may favor fast-growing, heterotrophic bacteria.The highly diverse reef sediment community was, on the class level, dominated by Planctomycetes, Bacteroidetes, and Gammaproteobacteria, particularly at the inshore site closest to Makassar. Planctomycetes are common colonizers of marine sediments, and are commonly found in anaerobic, high-nutrient conditions and are effective metabolizers of DOC (Chipman et al., Fungia mucus, showed statistically significant differences in bacterial community composition. Although the two size fractions of the water column were quite similar in the general composition of the most dominant OTU, there were still significant differences in the overall community composition. This is a common observation, also in tropical waters, and often related to differences in food and habitat availability (Zhang et al., Synechococcus is almost exclusively found in the particle-attached fraction of the water column.Our results suggest that much of the observed variability in bacterial community composition in the Spermonde Archipelago is a result of differences between the sampled habitats. All three habitats, except between the water column and the Escherichia/Shigella, Ralstonia, and Mycobacterium in the present study, can indicate a failure of control mechanisms for mucus colonization due to stress.There is also a frequent exchange between bacterial communities of the water column and the coral mucus, usually controlled by bacteria inhabiting the mucus Ritchie, . Cell co2 and subsequently available electron acceptors after the first few mm (Rasheed et al., Reef sediments are a biogeochemically very different environment compared to habitats exposed to water column. As observed for the present study, they are also often more diverse than communities from the water column, and the full diversity is rarely assessed (Uthicke and McGuire, Escherichia/Shigella, Ralstonia, and Mycobacterium were among the most abundant taxa observed in the water column and coral mucus in this study, while there was no dominant OTU in the sediments. Unfortunately, our identification of potentially pathogenic OTUs was only based on high sequence similarity to known pathogenic strains, and we could not test for actual pathogenicity of these OTUs. Further, tests for the functional traits are required to confirm our assumption. Alarmingly, the prevalence of many of the observed, potentially pathogenic bacteria was much higher at the chronically impacted sampling sites closer to urban Makassar. If coastal development and waste water management remains unchanged this may have unpredictable consequences for the coastal and island populations that strongly depend on the natural resources taken from the Archipelago.This study contributes the first conclusive overview of changes in bacterial communities from the water column, sediments, and coral mucus in relation to changes in the water quality of the Spermonde Archipelago. There were significant differences between the investigated habitats, namely between the sediments, coral mucus, and the water column as well as between the two size fractions of the water column. The potentially pathogenic bacterial OTUs HK, AG, MT, CW, and ML developed the idea for this project. HK conducted the fieldwork with support from JP and AG and logistical support from ML. The statistical analysis was conducted by HK, CH, and JP. HK wrote the manuscript with input and critical revision from all co-authors.This project was part of a Junior Research Group Leader position granted to AG by the Leibniz Association.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Finally, the results of dewatering tests demonstrated that sludge dewaterability was greatly improved by adding the synthesized novel flocculants, and the sludge-specific resistance to filtration, filter cake moisture content and residual turbidity all reached a minimum  at 40 mg·L−1. The PAB flocs were large, compact, difficult to break, and easy to regrow. Furthermore, PAB was more effective in the removal of protein from soluble extracellular polymeric substances (SEPSs). In summary, this study provides a novel solution to synthesize cationic microblock polyacrylamide for improving sludge dewatering.Cationic polyacrylamides have been employed widely to improve sludge dewatering performance, but the cationic units are randomly distributed in the molecular chain, which restricts the further enhancement of dewaterability. Common template technology to prepare block copolymers requiring a huge number of templates reduces the polymer purity and molecular weight. Here, we adopted the surface-active monomer benzyl dimethyl 2-(methacryloyloxy)ethyl ammonium chloride (BDMDAC) to synthesize cationic microblocky polyacrylamide initiated by ultrasound. The reactivity ratio of monomers suggested that novel cationic monomer BDMDAC had higher homopolymerization ability, and was thus more prone to forming a microblock structure. The statistical analysis of sequence-length distribution indicated that the number and length of cationic segments increased in the PAB molecules. In addition, the characteristic results of Fourier transform infrared (FTIR), proton nuclear magnetic resonance ( The manl groups . The colA variety of chemical conditioning agents, such as Fenton’s reagents, surfactants, polyaluminum chloride and polyelectrolytes, are added to sludge in order to improve dewaterability . SpecifiIn recent years, the influence of the chemical structure, especially the block structure, of the polymer on dewatering performance has also been investigated. The polymer molecular chain with a random cation distribution often adopts a flatter adsorption on the surface of the particles, which reduces the opportunity for bridging interactions with other sludge particles and squanders the advantage of the long linear molecular chain. Inversely, the microblock structure limits the adsorption sites for the CPAM, which then adopts a more extended adsorbed configuration, resulting in effective bridging flocculation . TemplatSurface-active monomers (surfmers) containing amphiphilic structure and polymerizable vinyl double bonds display unique physicochemical properties . They caFree radical copolymerization in aqueous solution can be initiated by heat, rays and ultraviolet. The above-mentioned initiation processes have disadvantages, for instance, elevated temperature is used for breaking apart the chemical bonds and a radiation source which requires specific safety precautions is employed by photochemical initiation . Ultraso1H NMR spectroscopy, and differential scanning calorimetry and thermogravimetric analysis (DSC–TGA). Finally, the sludge dewatering tests were carried out in terms of residual turbidity of the supernatant (RTS), filter cake moisture content (FCMC), specific resistance to filtration (SRF), flocs morphological properties and extracellular polymeric substance (EPS).In this study, the surfmer, benzyl dimethyl 2-(methacryloyloxy)ethyl ammonium chloride (BDMDAC), reported as forming micelles in aqueous solution, was adopted to prepare the P (AM-BDMDAC) (PAB) initiated by ultrasound with potassium persulfate as the initiator. Another monomer, methacrylatoethyl trimethyl ammonium chloride (DMC), was used to prepare the copolymer (P (AM-DMC) (PAD)) under the same synthetic conditions in that the DMC is similar to BDMDAC in structure but without self-assembly properties. In order to elucidate the effect of surfmer on cationic microblock structures, a comparison between PAB and PAD in this study was conducted. The reactivity ratios of monomers were calculated, and the sequence distribution of the molecules was statistically analyzed. The properties of polymers were characterized by Fourier transform infrared (FTIR), w/w) was obtained from Lanjie Tap Water Company ; BDMDAC (80 wt % in water) was procured from Zibo Jinyin Chemical Co., Ltd. ; and DMC (80 wt % in water) was provided by Nanjing Jingrui Jiuan Biotechnology Co., Ltd. . Analytical grade urea (CO(NH2)2), hydrochloric acid (HCl), sodium hydroxide (NaOH) and potassium persulfate (K2S2O8) were purchased from Aladdin Co. Ltd. . All aqueous and standard solutions were prepared with deionized water supplied by a Milli-Q Plus water purification system .The acrylamide (AM), BDMDAC, and DMC used in this experiment were all of technical grade and were used without further purification. The monomer AM  ultrasonic generator  operating at a frequency of 22 kHz controlled by standard power source was used as an ultrasonic source to initiate the polymerization. The monomer solution was prepared by adding predetermined amount of AM (155.93 mmol) and BDMDAC (17.33 mmol) in distilled water to reach a monomer mass ratio of 40%, and it was then transferred to the reactor. Then, urea  was added to increase the solubility and the pH of the reaction solution was adjusted to 4 (±0.2) by HCl and NaOH solution. Prior to the addition of the photoinitiator, the reaction vessel was purged with nitrogen gas (99.99%) for half an hour. Finally, the irradiation time under ultrasonics was set to 30 min . After irradiation, a milk-white semitransparent solid was obtained which indicated the formation of copolymer. The products were purified with acetone and ethanol for several times and then dried in a vacuum oven at 60 °C for 2 days. PAD was prepared by the same synthesis process. The possible reaction mechanisms of PAB and PAD are shown in r), namely, the ratio of chain radical homopolymerization rate constant to the copolymerization rate constant reflects the relative polymerization activity [k11 and k12 are the rate constants of homopolymerization and copolymerization, respectively. When r is more than 1, the monomer tends to homopolymerize with the same monomers; otherwise, the monomer tends to copolymerize with other monomers. Besides, the larger the value is, the higher the relative activity will be. The monomer reactivity ratio is a crucial parameter to control polymer composition and structure in binary copolymerization [The monomer reactivity ratio FSequence-length distribution is referred to the monomer unit amount in its continuous chain segment and is important to characterize the block structure of the polymer ,20,21. I1H NMR spectra were obtained in deuterium oxide (D2O) with an AVANCE 500 NMR spectrometer . The thermal decomposition property of copolymer was determined by thermal gravimetric analysis (TGA) at a heating rate of 10 °C·min−1 under nitrogen flow of 20 mL·min−1 over a temperature range from 20 °C, up to 600 °C on a DTG-60H synchronal thermal analyzer .The intrinsic viscosity of copolymer was measured by an Ubbelohde viscosity meter  equipped with a thermostatic water container . Fourier transform infrared (FTIR) spectra of the dried copolymer samples were recorded with KBr pellets by a 550Series II infrared spectrometer . The The sludge samples were collected from secondary sedimentation tanks of the Jiguanshi Wastewater Treatment Plant in Chongqing, China. The initial sludge characteristics are listed in The conditioned sludge was poured into Buchner funnel and filtrated under a constant pressure of 0.07 MPa. The volume of filtrate was measured by graduated flask which was used to calculate SRF. The resulting filter cakes were transferred to a crucible and dried at a thermostatic drying oven. FCMC and SRF were obtained according to the calculation method described in 50 (50% of the flocs were of the sizes in the range 0–d50) was adopted to represent the average floc size. Floc strength and recovery factors were widely used to compare the degree of breakage and re-growth and were calculated as follows [A, B and C are the average flocs sizes in the steady phase before breakage, after the breakage period and after the re-growth to new steady phase, respectively.Dynamic sludge flocs size was monitored using a laser diffraction analyzer  during the whole formation, breakage and re-growth process. d follows :(4)streThe sludge cake after vacuum filtration was sampled and pretreated with spray gold after vacuum freeze drying. Then, the morphology of sludge was observed with a MIRA 3 LMU scanning electron microscopy (SEM) system .g for 15 min. The supernatant was carefully collected as SEPS of which the chemical oxygen demand (COD) was determined using HACH DR2800 spectrophotometer  with 2 h of digestion. The polysaccharides and proteins from SEPS were measured by Anthrone method [On the basis of previous work, the extraction and analysis method was designed described below . A totale method  and Lowre method , respectrAM and rDMC were both lower than 1, which indicated the monomer copolymerization ability was stronger than that of homopolymerization [rBDMDAC was greater than 1 in PAB system, which was attributed to stronger self-assembly ability of BDMDAC. Before polymerization reaction, BDMDAC could form micellar aggregation in the aqueous phase whereas DMC just assumed the dispersed state. The concentration of surfmers in micellar aggregation was similar to bulk polymerization, facilitating homopolymerization. When encountering a growing AM macroradical headgroup, they were embedded in polymer in the form of aggregation, of which the structure was regarded as a micro block. Accordingly, it was deduced that there were more and longer cationic blocks in the molecular chain of PAB than in PAD. Whereas, owing that AM reactivity ratio was less than 1, the PAB copolymer was composed of long BDMDAC segments embedded in short AM chain segments [The reactivity ratios of AM and cationic monomers calculated according to three methods are shown in rization . Therefosegments . The abof2 (DMC−PAD) = 0.38, i.e., the feed molar ratio of DMC was higher than that in copolymers when beyond 0.38, otherwise it was the opposite. Moreover, we noticed that the curve of BDMDAC-PAB without intersecting the diagonal had no azeotropic point, which was in agreement with block copolymerization. The molar ratio of BDMDAC was invariably higher than the feed ratio, demonstrating a high utilization of cationic monomer. Furthermore, PAB would possess greater CD than PAD at same feed ratio since the PAB curve was always above PAD. Based on the above discussion, the new monomer not only enhanced the charge effect but also improved conversion.The composition equations of the polymers were obtained on the basis of monomer reactivity ratios and feed ratios which are depicted in f2 = 0.2 accounted for 28.22% (30.71%), 20.26% (21.28%), and 14.54% (14.74%), respectively. Correspondingly, the DMC (BDMDAC) segments with the length of 1, 2, 3 in PAD (PAB) accounted for 90.97% (72.49%), 8.21% (19.94%), and 0.7% (5.49%), respectively. This illustrated that the sequence distribution of cationic monomers was narrower with shorter chain segments, whereas AM sequence distribution was wider and longer under this condition. When the initial molar ratio of reactants was increased to 0.4 or 0.6, monomers in the two polymers tended to an alternate distribution along the molecular chain. Sequentially increased to 0.8, the situation of the AM and DMC (BDMDAC) segments was just the contrary to f2 = 0.2. Our results suggested that monomers sequence distributions were mainly dependent on f2, namely, feed ratio.The sequence distributions of the monomer segments were in terms of microscopic perspective to evaluate molecular arrangements in comparison with macroscopical polymer composition equations. The results are summarized in In addition, there were more long cationic chain segments in PAB than those in PAD under the same feed ratio. Besides, the length advantage became more and more obvious with the increase of feed ratio. This observation may be explained by the fact that BDMDAC monomers are inclined to form larger micelle aggregates at higher dosages, which contributed to longer cationic chain segments being embedded on encountering growing levels of macroradicals. The results are in agreement with the analysis of reactivity ratios and composition equations of polymers. In short, PAB had more and longer cationic blocks in the molecular chain than PAD.−1, which were ascribed to the strong stretching vibrations of the –NH and –C=O in the AM unit, respectively [−1 were attributed to –CH3 bending vibrations of –N+(CH3)2− in the BDMDAC unit, whereas the absorption peaks at 2940 and 2785 cm−1 were from the –CH3 and –CH2 asymmetric stretching vibrations, respectively [−1 of PAB were due to the benzene skeleton vibration in the BDMDAC unit [−1 were assigned to quaternary ammonium groups from DMC and BDMDAC, respectively. The spectral structures in −1 in PAD proved the lack of benzene ring, which was consistent with its theoretical chemical structure. Overall, the FTIR characterization results showed the formation of both copolymer PAB and PAD.The FTIR spectra of (a) PAM; (b) PAD and (c) PAB are shown in ectively . The chaDAC unit . The cha1H NMR spectra of poly(benzyl dimethyl 2-(methacryloyloxy)ethyl ammonium chloride) (PBDMDAC), PAB, poly(methacrylatoethyl trimethyl ammonium chloride) (PDMC), and PAD, respectively, for convenient comparison and research. It could be seen from 1) and methine groups (b1), respectively. Protons in the two sequential methylene groups  connected to the ammonium group of BDMDAC were observed at δ = 4.64 and 3.79 ppm. The sharp peak at δ = 3.15 ppm was attributed to the protons of the two equivalent methyl (e1) at quaternary ammonium groups. The peaks at δ = 7.60 and 4.61 ppm were assigned to protons of the phenyl group (g1) and methylene (f1) connected to the phenyl group. The comparison between PBDMDAC and PAB revealed that most protons of cationic monomer appeared in PAB, which confirmed the successful copolymerization of BMDAC and AM. Moreover, the chemical shifts of adsorption peaks were very slight, indicating that the chemical environments of cationic monmer in PAB and homopolymer were almost identical. On the other hand, there are many split peaks marked with arrows in PAD compared with PDMC shown in DSC−TGA analysis was often performed to estimate the thermal stability of products. In recent years, many researchers identified polymer microstructures using this method ,32. FiguThe above characterization results of two copolymers corroborate that there is a blocky distribution of cationic units in PAB and a random distribution in PAD. Therefore, to accurately evaluate the impact of cationic sequence distribution on sludge dewatering efficiency, four copolymers detailed in The zeta potential of the supernatant was a reasonable parameter in terms of interpreting flocculation mechanism . As show−1) was depicted in Fs) of sludge flocs shown in F) of PAB was 34.15%, whereas the RFs of PAD, CPAD and CPAA were 12.36%, 13.84% and 7.59%, respectively, which demonstrated re-growth capability except when PAB was quite low.The dynamic sludge floc size during flocculation, breakage and re-flocculation stage of PAB, PAD, CPAD and CPAA at optimal dosage  chemical properties ,44,45. T1H NMR, and TGA provided evidence for the synthesis of cationic microblock copolymer.The reactivity ratio of monomers suggested that novel cationic monomer BDMDAC had higher homopolymerization ability, and thus are more prone to forming microblocks. The statistical analysis of sequence-length distribution indicated that the number and length of cationic segments were increased in the PAB molecules. In addition, the characteristic results of FTIR, 12 m·kg−1, 2.6 NTU, respectively) at 40 mg·L−1. The excellent performance was associated with the combined effect of surfmers and ultrasound.Sludge dewaterability was greatly improved by adding the synthesized novel flocculant and the sludge-specific resistance to filtration, filter cake moisture content and residual turbidity all reached a minimum (68.7%, 5.4 × 10The bigger and more compact sludge flocs conditioned by PAB were not easy to break when exposed to high shear force. Furthermore, their re-growth capability was relatively high, benefiting from patch re-flocculation.PAB with cationic microblock structure was more effective in removal of PN in SEPS because of enhanced electrical neutralization and adsorption bridging ability, which was conducive to reducing the sludge viscosity and compressibility.In this study, a novel flocculant PAB with a cationic microblock structure was successfully synthesized by the ultrasound-initiated polymerization technique, which significantly improved the sludge dewatering performance. Based on the investigations, the main conclusions can be drawn as follows:


Here, we propose a novel in situ advanced restoration-friendly program for the endangered epiphytic orchid species Dendrobium devonianum, in which a series of in situ symbiotic seed germination trials base on conspecific fungal isolates were conducted at two sites in Yunnan Province, China. We found that percentage germination varied among treatments and locations; control treatments (no inoculum) did not germinate at both sites. We found that the optimal treatment, having the highest in situ seed germination rate (0.94-1.44%) with no significant variation among sites, supported a warm, moist and fixed site that allowed for light penetration. When accounting for seed density, percentage germination was highest (2.78-2.35%) at low densities and did not vary among locations for the treatment that supported optimal conditions. Similarly for the same treatment, seed germination ranged from 0.24 to 5.87% among seasons but also did vary among sites. This study reports on the cultivation and restoration of an endangered epiphytic orchid species by in situ symbiotic seed germination and is likely to have broad application to the horticulture and conservation of the Orchidaceae.Due to increasing demand for medicinal and horticultural uses, the Orchidaceae is in urgent need of innovative and novel propagation techniques that address both market demand and conservation. Traditionally, restoration techniques have been centered on  These include: the generation of clones  Do seeds sown in situ with symbiotic mycorrhizal fungus have higher germination rates compared to seeds sown without? (2) Does seed germination rate vary among microclimatic conditions and location? (3) Does seed density at the time of sowing influence seed germination rate?, and 4) Does the season in which seeds are sown influence germination rate? Ultimately, we hope to determine if in situ symbiotic seed germination can be used to conserve the endangered species D. devonianum by concomitantly rebuilding populations in the species’ natural range and meeting the demand of industry for cultivated medicinal orchid materials. This study is the first to report on the cultivation and restoration of an endangered epiphytic orchid species by in situ symbiotic seed germination, and is likely to have broad application to the horticulture and conservation of orchids.To address the needs of the Chinese medicinal orchid industry as well as the needs of orchid conservation and restoration management, we assessed “D. devonianum typically resides as an epiphyte on tree trunks of Camellia spp., our study sites were centered on two traditional tea gardens that contained numerous mature specimens of Camellia sinensis var. assamica in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China. In this area, annual precipitation due to the tropical monsoon climate (rainy season occurs from May to October) ranges between 1,400 and 1,800 mm/yr. The rainy season is followed by a misty cool season from November to February, both March and April are the dry hot season. Annual average temperature and average relative humidity at both sites range between 18–20°C and 86–89%, respectively. Longpa traditional tea garden in Jinghong city  is contiguous with tropical montane evergreen broad-leaved forests. Most of the tea trees had been cultivated for more than 200 years and they have an average height of 2 m. The remaining native shade trees include Schima wallichii, Nephelium chryseum, Castanopsis echidnocarpa, Machilus gamblei, Alseodaphne andersonii, Meliosma arnottiana, and Macaranga indica. Yiwu traditional tea garden is located in Mengla county  and was established in the 1970s. The main shade trees are Lauraceae sp. and Ehretia thyrsiflora. Tea trees at the Longpa site were on average larger than those at Yiwu site, and the two sites are 90 km apart.Given that D. devonianum, is an epiphytic orchid widely distributed in the subtropical and tropical areas of SE Asia, including southwest China at altitudes from 1100 to 1900 m. In Xishuangbanna, it flowers from April to May and seeds mature in the following March  was isolated from each of the five protocorms. We experimentally tested that the fungus was capable of significantly promoting seed germination and subsequent seedling development . The strain was deposited in Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences (HMAS1: accession number CGMCC No. 9551). The ITS sequence was submitted to GenBank database in the National Center for Biotechnology Information (NCBI: accession number KM226996.1).The study species, ng March . In JulyD. devonianum formed by natural pollination were collected in April 2014 in Tengchong county, Yunnan Province, within the natural range of the species. Seed capsules were sterilized in 75% (v/v) ethanol for 2 min and dried at room temperature before being opened with a scalpel under sterile conditions. The extracted seeds were transferred to airtight glass containers with calcium chloride anhydrous. After 4 days, the seeds were stored in a glass vial at -20°C for long-term preservation. Seed viability was 79.6% based on an assessment of approximately 300 seeds using a tetrazolium test (TTC).Mature, undehisced seed capsules of in situ seed germination rates observed in this study are the result of the Tulasnella sp. inoculum that was isolated and applied in this study (as opposed to in situ contamination), in vitro symbiotic seed germination trials were conducted confirming the compatibility, efficacy and specification for promoting seed germination in D. devonianum (unpublished data). Subsequently the fungus was cultured in 10-50 ml conical flasks (500 ml) with 100 ml sterilized potato dextrose broth (PDB) placed on a shaker  at 150 rpm and 25°C for 7 days. The fermented mycelia were filtered with medical gauze and washed with sterile distilled water to avoid dextrose residue. One gram of fresh mycelium was homogenized in 50 ml sterile ddH2O by a sterile homogenizer. Seeds were added to this mixture and shaken to obtain a homogeneous liquid suspension. Prior to each round of seed sowing, seed density/ml of suspension was calculated by placing 200 μl of suspension on a glass slide and counting the total number of seeds/slide. This procedure was replicated three times per suspension and the mean was calculated.To ensure that Camellia assamica trees: in each case, 4 ml of liquid seed suspension was applied using a medical syringe. Because orchid seeds are easily flushed by rain due to their small size and require a moist, fertile inoculated substrate for seed germination, one of the goals of our study was to evaluate a number of plausible scenarios that might influence epiphytic in situ seed germination. The treatments were (1) Sphagnum: seed suspension applied between the trunk and a sphagnum wrap; (2) Trunk: seed suspension applied directly on the trunk; (3) Dung: seed suspension mixed with cow dung and applied directly to the trunk; (4) Plastic Wrap: seed suspension applied between the tree trunk and a plastic wrap; (5) Plastic Wrap + packet: seed suspension sealed in nylon packets attached to trunks using Plastic Wrap; (6) Control: seed suspensions containing seeds mixed in 0.1% sterile agar suspension without fungal inoculum. To ensure the liquid seed suspension remained fresh over time and was applied across a wide range of microhabitats on each tree, we randomly applied each treatment (∼8.5 treatments/tree) to approximately 200 trees that were randomly selected at the Longpa site (N = 1748). To evaluate the effect of location for treatments that showed the highest germination rates at the Longpa site, percentage germination was also assessed at the Yiwu site for treatments (4)–(6). Each treatment was also randomly applied to approximately 200 randomly selected trees .Once the liquid suspension was prepared, seeds were sown on the trunks of After 3 months, the number of seedlings for each treatment was counted and the percentage germination was calculated as (the number of seedlings/the number of viable seeds sown) × 100. Because percentage germination data (arcsine square root transformed) did not meet the assumptions of normality associated with the residuals of a one-way analysis of variance (ANOVA) model, we conducted non-parametric comparisons for each pair of treatments using Wilcoxon/Kruskal–Wallis tests (Rank Sums) among all six treatments. A number of sources of variation were evaluated. These include: (1) treatment, (2) seed density at time of sowing and (3) sowing season  location. Given a number of practical limitations associated with our study, such as two sites spanning multiple seasons, our design did not facilitate the testing of interaction terms among all treatments, densities, and seasons ; not all terms were represented in all categories. A one-way Chi-square approximation analysis for location as a source of variation was restricted to treatments (4)–(6); all three treatments were represented at both sites. In addition, and for the same reasons, we restricted our analysis of density of seeds sowed and sowing season as sources of variation to treatments (4) and (5). Here, we used a Wilcoxon/Kruskal-Wallis (Rank Sums) non-parametric comparison for each pair of terms in the model that included a treatment × density and a treatment × season analysis. For treatment (4) and (5), we analyzed three seed densities . All statistical analyses were conducted using JMP 10.Dendrobium devonianum. Within sites, mean germination rate was 0.59 ± 0.09% (mean ± SE) and 0.51 ± 0.10% at Longpa (N = 1748) and Yiwu sites (N = 1215), respectively.Overall, seed germination rates for the 2963 replicates conducted in this study across both sites varied from 0 to 83.01% (mean ± SE: 0.56 ± 0.10%) in X2 = 90.11; DF = 5, p < 0.0001): the treatment (5) Plastic Wrap + packet  had significantly higher percentage germination than the treatment (4) Plastic Wrap , and the treatment (1) Sphagnum . The treatment (2) Trunk , treatment (3) Dung , and treatment (6) Control  had significantly lower percentages of germination than all other treatments . Similar significant differences among the three treatments that were tested at that Yiwu site were found : the treatment (5) Plastic Wrap + packet  had significantly higher percentage germination than the treatment (4) Plastic Wrap  . Similar to the results found at the Longpa site, both of treatment (4) Plastic Wrap and (5) Plastic Wrap + packet at the Yiwu site had significantly higher germination than treatment (6) Control .Significant differences among the treatments were found at the Longpa site . No difference in germination rates between the Longpa and Yiwu sites was found for treatment (5) Plastic Wrap + packet .When the effect of location was compared directly, germination rate was significantly higher at Longpa site compared to the Yiwu site for treatment (4) Plastic Wrap (SE = 0.08%) to 0.52% (SE = 0.11%) which did not differ significantly from each other . Seed germination for treatment (4) at the Yiwu site, was significantly higher for the medium seed densities (0.15 ± 0.03%) compared to seeds sown at lower (0.01 ± 0.01%) and higher (0.08 ± 0.03%) densities which were also significantly different from each other . For treatment (5) Plastic Wrap + packet, seed germination at the Longpa site did not differ significantly among low (2.78 ± 1.06%), intermediate (0.30 ± 0.06%) and high (0.20 ± 0.04%) seed densities . Seed germination at the Yiwu site, was significantly higher for the lowest (2.35 ± 0.94%) and intermediate (0.08 ± 0.03%) seed densities compared to seeds sown at the highest (mean = 0) density but did not differ significantly from each other .Percentage germination differed significantly for seed density at time of sowing for the two treatments analyzed in each site. For treatment (4) Plastic Wrap, seed germination at the Longpa site was significantly lower for the highest seed density (0.24 ± 0.04%) compared to seeds sown at lower densities that ranged from 0.29%  and high  densities compared to the Yiwu site. Germination did not differ between sites for medium densities  . When the effect of location for treatment (5) Plastic Wrap + packet was compared directly, germination rate was significantly higher at the Longpa site compared to Yiwu site for the highest density  but the two sites did not differ significantly from each other at the lowest  and intermediate  densities .Significant differences among sowing densities were found for the two treatments analyzed in each site. For treatment (4) Plastic Wrap, seed germination at the Longpa site was significantly higher at low . Seed germination at the Yiwu site, was significantly higher for the misty cool season (0.83 ± 0.22%) compared to seeds sown during the rainy season (0.16 ± 0.02%) and the dry hot season (mean = 0) which were significantly different from each other . For treatment (5) Plastic Wrap + packet, seed germination at the Longpa site did not differ significantly among the rainy (0.24 ± 0.03%), misty cool (5.87 ± 2.24%), and dry hot (1.97 ± 0.98%) seasons . Seed germination at the Yiwu site, was significantly lower for the rainy season (0.147 ± 0.02%) when compared to the misty cool season (1.74 ± 1.65%), although having the highest seed germination rates the dry hot season (2.55 ± 1.13%) was not significantly from either .Percentage germination differed significantly for sowing seasons for the two treatments analyzed in each site. For treatment (4) Plastic Wrap, seed germination at the Longpa site was significantly lower for the rainy season (0.25 ± 0.04%) compared to seeds sown at misty cool season (0.85 ± 0.24%) but neither differed significantly from the dry hot season (0.85 ± 0.23%)  and the dry hot season  compared to the Yiwu site. Germination rates did not differ significantly between sites for the rainy season  . When the effect of location for treatment (5) Plastic Wrap + packet was compared directly, germination rate did not differ significantly among sites for the rainy season , misty cool season , and dry hot season  .Significant differences among the two sites were found for the sowing seasons analyzed in each site. For treatment (4) Plastic Wrap, seed germination at the Longpa site was significantly higher during the misty cool season  suggesting that protocorms or seedlings can be formed only with the presence of symbiotic fungi . Critical to seed germination and seedling success, compatible fungi provide carbohydrates, vitamins, and growth factors that appear, based on our results, imperative for the culture of orchids in situ.It is well known that the seeds of orchid species require compatible mycorrhizal fungi for germination to occur in nature, and symbiotic fungi have widely been shown to promote seed germination and facilitate seedling establishment in both terrestrial and epiphytic orchids . For somex situ . Althougex situ . As a reotential . In thisin situ symbiotic propagation and conservation of orchid species , indicating that use of the fungal symbiont isolated from orchids in the natural population may lead to successful germination and development of seedling in the in situ restored population. Interestingly, Aa achalensis to promote seed germination and found that seed germination depended on the strains used. Varying results have also been shown in Gavilea australis where percentage seed germination varied depending on the origin of the mycorrhizal fungi isolated from the host (roots or protocorm) or conspecific vs. heterospecific species . The water retention capabilities of this treatment, coupled with the fact that our seeds were not washed or blown away over time, seems a likely cause for the success of this treatment. Many cultivation parameters, however, such as microclimatic conditions, seed density at time of sowing, and planting season, to name a few, may also be affecting seed germination of D. devonianum in field. Although our research is limited to in situ seed germination of this wild Chinese species, below we discuss some of the main sources of variation that may be contributing to our results and may have broader application to the Orchidaceae.Historically, attempts to initiate germination in epiphytic orchids ccessful . Sphagnuccessful . To evalCaladenia arenicola by in situ symbiotic seed germination but none of the seedlings survived summer dormancy, perhaps due in part to the lack of favorable microclimatic conditions necessary for the maintenance of seedlings and fungal vitality. Although the extant natural distribution of D. devonianum includes both our study sites and thus both locations were suitable for seed germination and growth, seed germination for our Plastic Wrap + packet treatment was less affected by microhabitat variation.Germination for the two plastic treatments occurred at both sites but significant variation in germination among sites only occurred for the Plastic Wrap treatment and not for the Plastic Wrap + packet treatment. Thus it would appear that the conditions promoted by the Plastic Wrap + packet treatment buffers microclimatic variation among sites whereas our Plastic Wrap treatment seems to be more sensitive to unfavorable conditions. Previous studies have shown that seed germination may be influenced by locations that have distinct microhabitats which reflect differences in light, temperature, and humidity; all of which are probably indirectly related to seed germination rates by affecting seed viability or by influencing fungal growth or survival. For example, Table 2 and Figure 2). Although we are not aware of any previous studies on density-dependent processes associated with in situ seed germination in epiphytic orchids, symbiotic seed germination trials on OMA medium showed that the seeds of D. devonianum could not initiate germination with >1500 seeds in a 9 cm petri dish (unpublished data). In vitro symbiotic seed germination usually has <200 seeds per petri dish , a result that is concordant with most ex situ studies. Although the exact mechanism driving this threshold remains elusive, there is some evidence to suggest that seed germination rate is partly dependent on the seed density in suspension prior to application of our treatments because of increased carbon acquisitions driven by increasing volume of fungi in suspension. Additionally, the effect of seed density varied among sites for only the Plastic Wrap treatment, indicating that a density by microhabitat interaction may be also important, although given the statistical power of our dataset we were unable to test this interaction directly. It would appear that when microclimatic conditions promote a stable, moist, warm substrate (Plastic Wrap + packet treatment) seed germination is not only better but more consistent regardless of seed density at the time of sowing.We found that seed germination was significantly higher at low seed densities compared to germination at higher densities . Although we were unable to directly test the season × density interaction  due to lost degrees of freedom, the most likely abiotic factor influencing in situ seed germination across seasons is the humidity level of the microhabitat, which can in turn affect germination success, subsequent protocorm development and survivorship. Because plantlets can not survive for more than several days without humidity around their roots, the absolute number of seedlings formed during the rainy season was much higher and the seedlings looked more fit compared to results found for other seasons . Additionally, the effect of season on seed germination varied among sites for only the Plastic Wrap treatments, indicating that a season and microhabitat interaction may be an important factor driving our results, although we were also unable to test this directly due to lack of statistical power in our dataset. It would appear that regardless of season, microclimatic conditions favoring a more stable moist and warm substrate (the Plastic Wrap + packet treatment) promote more consistent and better seed germination. Given that natural seed formation occurs from May to March and protocorms that are used for fungal isolation are typically found in July, we speculate that the ideal season for in situ seed sowing as an advanced cultivation-friendly technique at our sites is from November to January (the misty cool season).Seed germination rate varied among seasons and trends varied among sites. Most surprising was that the highest seed germination was during the misty cool season followed by the dry hot season, both in stark contrast to the lowest rates which were generally found during the rainy season  will have high germination rates regardless of variation in microhabitat conditions or location, and variation associated with seed density, or the timing of planting (season), although geographic origin and potential ecological risks remain to be tested  for use in the medicinal plant trade, produced through the cultivation techniques outlined in this paper, will have the same quality as their wild counterparts. Our results have enormous potential for the production and sale of locally produced materials while simultaneously easing collection pressures on native plant populations. Although our research is the only based on in situ seed germination of Chinese wild species, our study has broad application for the both commercial and conservation initiatives for the Orchidaceae.Advanced restoration-friendly cultivation of orchids through e tested . We hopeSCS and JYG designed experiments. SCS, QL, XLF, HH, and JYG conducted experiments. SCS, KB, JCS, and JYG wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.