Secondary hyperparathyroidism (SHPT) is a common complication of chronic kidney disease (CKD), and as the disease progresses SHPT is associated with systemic consequences, termed CKD-mineral and bone disorder. Currently, cinacalcet is indicated for the treatment of SHPT; however, cinacalcet is associated with upper gastrointestinal adverse events. Evocalcet has been developed to address these issues, but the long-term safety and efficacy of evocalcet need to be evaluated. To more accurately reflect clinical practice, this phase 3, multicenter, open-label study was specifically designed without a cinacalcet washout period, and focused on those patients who switched from cinacalcet to evocalcet. A total of 137 SHPT patients undergoing hemodialysis were enrolled, of whom 113 switched from cinacalcet to evocalcet. The most frequent type of adverse drug reaction was decreased adjusted calcium. The incidence of gastrointestinal-related adverse events did not increase in a dose-dependent manner as the dose of evocalcet was increased. The percentage of patients achieving the target intact parathyroid hormone concentration increased from 40.9% to 72.3% with 52-week treatment. The corrected serum calcium and phosphorus levels remained largely unchanged throughout the study. The long-term safety and efficacy of evocalcet was confirmed using a clinically relevant intra-subject dose-adjustment strategy in SHPT patients undergoing hemodialysis. SHPT is a maladaptive response triggered by hypocalcemia, hyperphosphatemia, and active vitamin D deficiency, which in turn causes parathyroid cells to overproduce parathyroid hormone (PTH)2. Excess levels of PTH are associated with systemic toxicity, known as CKD-mineral and bone disorder, which represents the cardiovascular and bone diseases3. SHPT has typically been treated with the active form of vitamin D to reduce PTH levels; however, this type of treatment has been associated with elevations in serum calcium levels, causing arterial calcification2.Secondary hyperparathyroidism (SHPT) is a major and common complication that develops in chronic kidney disease (CKD) patients undergoing hemodialysis (HD)6. In addition to controlling PTH secretion, cinacalcet has been demonstrated to be useful for improving mortality risk and cardiovascular outcomes in clinical research8. While cinacalcet is an effective agent for the treatment of HD patients with SHPT, it is commonly associated with gastrointestinal (GI) symptoms that cause poor drug adherence and treatment discontinuation10. Moreover, even in patients who continue therapy, these adverse events can result in inadequate dosages of cinacalcet being administered.Cinacalcet, a type of calcimimetic agent, was developed to interact with calcium-sensing receptors (CaSR) as a positive allosteric modulator to suppress PTH secretion11. Previous clinical trials have highlighted the dose-response relationship of evocalcet (from 1 to 12 mg) and safety in HD patients13. More recently, a 30-week phase 3 study indicated that 1–8 mg of evocalcet was non-inferior to cinacalcet, with approximately 70% of patients showing improvement in intact PTH (iPTH) levels while also reporting fewer GI adverse events14. These results were not inferior to the guidelines set by The Japanese Society for Dialysis Therapy (JSDT) for the management of SHPT in HD patients15.Evocalcet is a novel calcimimetic developed as an equally efficacious, but more tolerable, alternative to the currently available calcimimetics for SHPT patients. In pre-clinical studies, evocalcet demonstrated efficacy in suppressing serum levels of PTH and with less-marked effects on the GI tract in animalsThe current 52-week phase 3 study examined the long-term safety and efficacy of once-daily oral administration of 1–12 mg evocalcet in SHPT patients undergoing HD. Additionally, patients who were currently receiving cinacalcet were switched to evocalcet so as to accurately reflect the clinical setting.This study was reviewed and approved by the institutional review board of each participating site (Supplementary Text S1) and conducted in compliance with the Declaration of Helsinki, Good Clinical Practice, and applicable regulations. This study was registered on ClinicalTrials.gov  and JAPIC . All patients provided written informed consent.Patients were enrolled according to the following key inclusion criteria: iPTH >240 pg/mL only for patients who had not previously been treated with cinacalcet, or cinacalcet treatment for at least 2 weeks prior to screening; corrected calcium serum levels ≥8.4 mg/dL; provision of written consent; aged ≥20 years at the time of consent; and undergoing thrice-weekly HD for at least 12 weeks prior to screening.Prior treatment with cinacalcet was permitted until the day before initiation of evocalcet treatment, and no washout period was required, although patients who commenced or had a change in cinacalcet administration 2 weeks prior to screening were excluded. Patients who had previously been treated with cinacalcet were exempt from the iPTH eligibility criterion of >240 pg/mL, but still underwent screening without a washout period to determine evocalcet starting dose.Patients who received treatment with bisphosphonates, denosumab, or teriparatide within the 24 weeks before the screening; parathyroidectomy within 24 weeks before screening; or peritoneal dialysis within 12 weeks before screening, were excluded. Patients who changed the dose or dosing regimen of an activated vitamin D drug or its derivative, phosphate binder, or calcium preparation from 2 weeks prior to screening were excluded, while patients who changed dialysis conditions  from 2 weeks prior to screening were also excluded.15. The starting dose of evocalcet was nominally set at 1 mg, or 2 mg if patients had an iPTH concentration of ≥500 pg/mL and a corrected serum calcium concentration of ≥9.0 mg/dL at screening, regardless of pre-treatment cinacalcet dose. Treatment with cinacalcet, bisphosphonates, denosumab, or teriparatide; parathyroidectomy; and peritoneal dialysis were prohibited during the study. Changing or beginning an activated vitamin D drug or its derivative, phosphate binder, or calcium preparation was permitted throughout the study period. Further details on study design and exclusion criteria are summarized in Supplementary Text S2.This was a phase 3, multicenter, open-label, intra-patient dose-adjustment study conducted in 15 participating centers (Supplementary Text S1) in Japan from September 2015 to December 2016. The study design is shown in Fig. All clinical examinations were performed in centralized laboratories at LSI Medience Corporation. iPTH was measured using electro-chemiluminescence immunoassay . Intact fibroblast growth factor 23 (FGF23) was measured using enzyme-linked immunosorbent assay .Safety was primarily evaluated based on adverse events, adverse drug reactions, clinical laboratory values, vital signs, body weight, body mass index (BMI), 12-lead electrocardiogram (ECG), and ophthalmological examinations. Adverse drug reactions were defined as adverse events that were judged by an investigator to be causally related to evocalcet. Ophthalmological examinations and measurement of 12-lead ECG were performed before the administration of evocalcet and prior to HD at week 0, and then at any time needed thereafter. All other measures were performed before HD and evocalcet treatment after the maximum interdialytic interval. Adverse events and adverse drug reactions were coded by MedDRA ver. 19.0. Decreased adjusted calcium adverse events were reported when corrected serum calcium concentrations were ≤7.5 mg/dL or as judged by an investigator.The efficacy endpoints included the proportion of patients with an iPTH concentration of 60–240 pg/mL, the percentage change in iPTH concentration from baseline, and the percentage of patients who achieved ≥30% decrease in iPTH concentration from baseline.2/dL2) were also evaluated. Parathyroid volume and blood flow were assessed by carotid ultrasound.The median (interquartile range [IQR]) iPTH (pg/mL), whole PTH (pg/mL), intact FGF23 concentration (pg/mL), and bone turnover markers ; and the mean (standard deviation [SD]) corrected serum calcium concentration (mg/mL), ionized calcium concentration (mEq/L), serum phosphorus concentration (mg/dL), and corrected serum calcium-phosphorus product . The efficacy analysis was carried out using the full analysis set (FAS), which included all patients enrolled in the study who were administered evocalcet at least once and had iPTH levels measured at least once after the study began. The safety analysis was carried out using the safety analysis set, which included all patients enrolled in the study who were administered evocalcet at least once.No statistical adjustment was made to missing data for continuous variables in the efficacy analysis of the present study. Missing data were treated as patients who did not achieve the target iPTH concentration of 60–240 pg/mL and, in order to show robustness, two different versions of the data (with and without missing data) are presented.Adhoc analyses were conducted to evaluate the efficacy and dose of evocalcet in patients stratified by pre-treatment cinacalcet dose. The changes from baseline in iPTH, whole PTH, intact FGF23 concentration, and bone turnover markers were tested using Wilcoxon signed-rank tests. The percent change from baseline in iPTH and the changes from baseline in corrected serum calcium, ionized calcium, serum phosphorus, and corrected serum calcium-phosphorus concentration were assessed using t-tests. The number of patients who achieved target iPTH and patients who achieved a ≥30% decrease in iPTH from baseline were assessed using chi-squared tests.Written informed consent was obtained from 152 Japanese HD patients with SHPT. Of these, 15 were judged to be ineligible for enrollment, and a total of 137 patients were enrolled in the present study to receive oral administration of evocalcet Fig. . The demn = 11), 25 mg (n = 46), 37.5 mg (n = 4), 50 mg (n = 28), 75 mg (n = 18), and 100 mg (n = 6) cinacalcet. The remaining 24 patients did not receive the study drug on day −1. Concomitant therapy with activated vitamin D, calcium-based phosphate binders and calcium preparation is shown in Table Of the 137 patients, 113 patients had the following cinacalcet treatment on the day before beginning evocalcet treatment (day −1): 12.5 mg (Adverse events occurred in 136 of the 137 patients (99.3%), and those judged to be moderate or severe in severity occurred in 29 patients and 14 patients, respectively  and 22 (16.1%) of the 137 patients, respectively. The types of events were decreased adjusted calcium (the most frequent type), occurring in 10 patients (7.3%); decreased blood calcium, in four patients (2.9%); constipation and diarrhea, each in two patients (1.5%); and erosive gastritis, intestinal obstruction, nausea, vomiting, chest discomfort, and decreased blood parathyroid hormone, occurring in one patient each (0.7%).Upper GI-related adverse events that occurred in ≥5% of patients included nausea (11.7%), abdominal discomfort (8.8%), and vomiting (8.8%) Table . Upper GNo adverse events resulted in death, and there were no clinically significant changes in the clinical laboratory values, body weight, vital signs, ophthalmological measures, and 12-lead ECG for all patients throughout the 52-week study period.The incidence of GI-related adverse events did not increase in a dose-dependent manner as the dose was increased from 3 mg to ≥8 mg evocalcet in 1-mg increments Fig. . No new n = 24), the proportion of patients that achieved the target iPTH concentration at week 52 was 87.6% (99/113 patients). The target achievement rate also increased in the cinacalcet pre-treatment group from 49.6% (56/113 patients) at baseline to 70.8% (80/113 patients) at week 52  of patients achieving an iPTH concentration of 60–240 pg/mL was 56 patients (40.9%) at baseline and 99 patients (72.3%) at week 52  in iPTH concentration from baseline was −64.9% (17.3%), and the number (%) of patients who achieved a decrease in iPTH concentration of ≥30% from baseline was 19 (79.2%) (Table The mean percent change (SD) in iPTH concentration from baseline was −15% (82%) at week 52. The number (%) of patients who achieved a decrease in iPTH concentration of ≥30% from baseline was 62 45.3%). The results of the 5.3%. The%) Table . The numThe mean (SD) corrected serum calcium concentration was 9.14 (0.57) mg/dL at baseline and 8.90 (0.57) mg/dL at week 52. Although the serum calcium concentration initially fluctuated, it remained almost unchanged throughout the study Fig. . The meaThe mean ionized calcium, corrected serum calcium-phosphorus product levels, and the median intact FGF23 also remained relatively unchanged Fig. . The medad hoc analysis show that the mean (SD) doses at week 51 in each subgroup were 1.7 (1.0) mg, 2.9 (2.6) mg, 4.8 (2.8) mg, 6.2 (3.9) mg, 5.4 (4.4) mg, and 10.3 (2.9) mg, respectively.The median (IQR) evocalcet dose of all the patients increased from a baseline dose of 1.0 (0.0) mg to a final dose of 2.0 4.0) mg with >50% of patients treated with ≤2 mg evocalcet , the evocalcet doses required to return the mean percent change in iPTH concentration to 0% were 1.09 mg, 1.82 mg, 1.75 mg, 5.52 mg, 4.50 mg, and 10.33 mg, respectively. Therefore, the ratios of pre-treatment cinacalcet dose divided by evocalcet dose were 11.5, 13.8, 21.4, 9.1, 16.7, and 9.7, respectively. Moreover, in the 40 patients who achieved an iPTH concentration of 60–240 pg/mL at both week 0 and 52, the mean ratio was 10.5. These ratios are consistent with a previous clinical trial, which showed that the efficacy of 2 mg evocalcet was nearly equivalent to that of 25 mg cinacalcet (ratio = 12.5)18. Because switching from cinacalcet to evocalcet was unique to the design of the present study, and the safety of evocalcet in Japanese hemodialysis patients with SHPT has been already elucidated in another head-to-head phase 3 study14, this discussion focuses on the secondary endpoints of the present study.This was the first study to evaluate the long-term efficacy and safety of evocalcet in patients with SHPT undergoing HD, including patients that had switched from cinacalcet. Japanese patients have a longer duration of dialysis compared with patients in the US and EU; therefore, the development of a suitable therapy for Japanese patients with SHPT is needed23.In this study, the number of patients who reached the target range (iPTH concentration of 60–240 pg/mL) progressively increased with continued administration of evocalcet over the 52-week study period. The majority of patients (82.5%) were switched from cinacalcet to evocalcet; therefore, it was assumed that the baseline parameter values would have been influenced by the prior treatment with cinacalcet. As such, patients pre-treated with cinacalcet had a median iPTH concentration at baseline of 210 pg/mL, whereas in patients who were not pre-treated with cinacalcet, the median iPTH concentration was 386 pg/mL. This indicates that there was some level of improvement already accomplished by cinacalcet prior to this study. However, in patients who had been previously treated with cinacalcet, the iPTH concentration decreased further after switching to evocalcet. As a result, in these patients, the proportion of patients who achieved the target iPTH range increased from 49.6% (56/113 patients) at baseline to 70.8% (80/113 patients) at week 52. In patients who had not been previously treated with cinacalcet, the percentage change in iPTH concentration from baseline was −64.9%, and the number (%) of patients who achieved a decrease in iPTH concentration of ≥30% from baseline was 19 (79.2%). These iPTH target achievement results are comparable to those achieved with other calcimimetic agentsad hoc analyses to evaluate the changes in these parameters according to pre-treatment cinacalcet dose. This analysis showed that patients pre-treated with a higher dose of cinacalcet had temporary increases in the efficacy parameters; however, these were then subsequently controlled with evocalcet dose adjustment. Additionally, the doses of activated vitamin D, calcium-based phosphate binders and calcium preparation tended to be increased throughout the study period to recover serum calcium levels that had decreased owing to the dose escalation of evocalcet. Overall, in all patient groups stratified by pre-treatment cinacalcet dose, the target iPTH achievement rate improved.Although iPTH, calcium, and phosphorus levels temporarily increased after switching from cinacalcet, these fluctuations may be attributed to the pre-treatment cinacalcet dose as well as the relatively low evocalcet dose (1 or 2 mg) these patients were initially administered. To evaluate the impact of pre-treatment cinacalcet dose, we performed The data on FGF23, the bone turnover markers, and the results of the parathyroid volume analyses were inconclusive. However, this is believed to be due to the influence of pre-treatment with cinacalcet.Evocalcet was generally well tolerated at the doses administered in this long-term study, particularly in regards to GI-related adverse events. Furthermore, there were no differences in the safety profiles between patients who were pre-treated with cinacalcet and those patients who were not. Most significant adverse events were deemed to be mild. Serious adverse drug reactions included congestive heart failure and cardiomyopathy; however, these occurred in only one patient each, and they either improved or were resolved with or without treatment. No significant events occurred due to a dose increase in evocalcet, which allows for dose escalation, further improving drug efficacy. Previous treatment with cinacalcet posed no safety concerns for patients in this study, and the incidence rates of adverse events were not affected by patients’ history of cinacalcet treatment.Overall, the present study demonstrated that the safety profile of evocalcet supports the long-term administration with clinically relevant dosages. In some patients, a target iPTH level was only achieved after receiving an escalated evocalcet dose of 12 mg, which corresponds to a ratio that exceeds that of the previously reported 12.5. In these patients, the evocalcet dose was escalated to 12 mg because it was considered that these patients could not increase their pre-treatment cinacalcet dose sufficiently to achieve the necessary target iPTH. Additionally, baseline iPTH levels, intact FGF23 levels, parathyroid gland volume, and pre-treatment cinacalcet dose in these patients were higher than those reported for the overall average.This study is limited by the unknown disease management status or compliance with cinacalcet treatment in those patients who had been treated with cinacalcet prior to the start of this study. Moreover, mineral and bone disorder markers might also be affected by concomitant therapy with activated vitamin D, calcium-based phosphate binders and calcium preparation. This study was also limited by not being placebo controlled, having a small number of patients, the possibility of attrition bias, and including only Japanese patients, which limits the generalizability of the results.This study demonstrated the long-term safety and efficacy of evocalcet in HD patients with SHPT over a 52-week period. The iPTH concentrations and the percentage of patients achieving the target range improved from baseline throughout the study after switching from cinacalcet. Evocalcet was well tolerated and the incidence of GI complications was low. Patients who had been pre-treated with cinacalcet were able to switch to evocalcet with no serious safety concerns observed.Supplementary Information


Asarum spp. are important medicinal plants that have the potential for use in treating various types of fevers. Aristolochic acid is one of the main toxic compounds present in these plants. To improve our understanding of the biosynthetic pathway of aristolochic acid, we sequenced the transcriptome of the root and leaf tissues of Asarum heterotropoides and performed de novo sequence assembly. The data were stitched together to produce 468,357 transcripts with an N50 of 611 bp. The data were annotated with various databases  and were annotated. There were 205,165 transcripts (43.81%) of differentially expressed genes in the roots and leaves, which were shown to be involved in biosynthesis, transport, and catabolism, and 100 genes in defence mechanisms. Three candidate transcripts  were discovered in these differential genes. TyrDC may be a key enzyme in the biosynthesis pathway of aristolochic acid identified using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography (HPLC). The transcriptome data and analysis presented here lay the foundation for further research into these important medicinal plants. Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. is a medicinal plant that dispels chills associated with exposure of the body to cold produced in vivo, and it has analgesic and antitussive effects. It is an important medicine for treating wind chill, headache, and cough. Liu et al.1 and Zeng et al.2 identified various components from the volatile oils of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and Asarum siedboldii Miq. var. seoulense Nakai., including methyleugenol, safrole, elemicin, and demethylcoclaurine. L-sesamin and aristolochic acids A, B, C, and D were identified in non-volatile oils3. The current edition of the Chinese Pharmacopoeia4 lists medicinal Asarum-based plant materials as the dried roots and rhizomes of A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., A. siedboldii Miq. var. seoulense Nakai., and A. siedboldii Miq. Asarum has a wide geographical distribution; A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., and A. siedboldii Miq. var. seoulense Nakai. grow as undergrowth or on mountainous wetland4. The main producing areas are Jilin, Liaoning, and Heilongjiang in China. A. siedboldii Miq. grows in damp places near creeks or rocky areas of Shanxi, Gansu, Zhejiang and other places in China5.8. To effectively use A. heterotropoides and other aristolochic acid-containing medicinal herbs, various traditional Chinese medicine formulation have been studied, mostly with a focus on reducing their toxicity by preparing slices from the medicinal plant parts. Traditional breeding can also remove plant toxicity. The traditional Chinese medicine involves the use of concoctions (Paozhi) and solvents or diluents such as wine, vinegar, salt, and honey are used to treat slices of traditional Chinese medicine formulations. Only slices of traditional Chinese medicine preparations can be used to treat diseases. Traditional Chinese medicine methods such as concocting and cooking show reasonable compatibility and can remove aristolochic acid, but completely eliminating its toxicity is difficult. Genetically removing or reducing the aristolochic acid content in A. heterotropoides is the most effective method. Although this has not been previously performed, it is possible to elucidate the synthesis pathway of aristolochic acid in plants. Therefore, in this study, we identified genes with a strong correlation in the synthesis pathway of aristolochic acid using high-throughput sequencing and transcriptome data analysis. Reduction or blockade of the gene expression using gene silencing could reduce the toxicity of aristolochic acid-containing plants to facilitate their safe use and breeding of new plant varieties with high efficacy and low toxicity.Many Chinese herbal medicine formulations containing aristolochic acid I are banned or restrictedAristolochiaceae, there are reports in the literature that Aristolochia L. plants Aristolochia fimbriata and Aristolochia tagala have available expressed sequence tags (ESTs) and transcriptome data, respectively10, while Saruma henryi Oliv. has transcriptome information11. The chloroplast genome of A. heterotropoides was sequenced in Asarum L. in 201712. Presently, there is no literature report of the sequencing of whole genomes or transcriptomes of A. heterotropoides. In this experiment, the roots and leaves of A. heterotropoides were collected on 3 September, 2016, and a cDNA library was established. Transcriptome data were obtained using high-throughput sequencing. An analysis of the transcriptome data determined that the tyrosine decarboxylase (TyrDC) family of enzymes is involved in the biosynthesis pathway of aristolochic acid in A. heterotropoides, and the full-length gene sequences of three enzymes were identified.Among the plants of A. heterotropoides were obtained, and 268,492,920 raw readings were generated with a read length of 100 bp. After trimming to remove adaptors, primer sequences, polyA tails, and short and low-mass sequences, 265,808,030 (99.0%) high-quality reads were recovered  database and used the Basic Local Alignment Search Tool (BLAST) to search data in several public databases.GO is a biological information network initiative with a three-tier system that defines and describes the function of genes and proteins as well as other gene products. The GO database consists of three major categories: molecular functions, cell components, and biological processes. Based on the differential transcript annotations to GO, we focused on the three categories that matched the molecular functional domain the most: “binding”, “catalytic activity”, and “structural molecule activity” . The three most abundant categories under cellular components were “cell part”, “organelle”, and “organelle part” . The three most common categories of biological processes were “metabolic process”, “cellular process”, and “single-organism process” . After comparison and analysis, we mapped the GO functional classification annotation  UniGene sequences were annotated. They consisted of 201,141 (42.95%), 120,029 (25.63%), 102,165 (21.81%), and 43,684 (9.33%) in Nr, Swiss-Prot, COG, and Kyoto Encyclopedia of Genes and Genomes (KEGG) in the total UniGenes list, respectively. The remaining 56.19% of transcripts were not annotated, indicating that there was no significant protein match in the database. Furthermore, there were unreported or multifunctional proteins in the The KEGG database simulates biological reactions through computational analysis and calculates and predicts metabolic pathways and functions of cellular gene products. It can provide integrated metabolic pathways and relationships between pathways, and comprehensively annotate enzymes that catalyse each step of the reaction. From this analysis, we found that 43,684 transcripts could be assigned to 311 pathways in six major categories. The six main categories were “organismal systems” “metabolism,” “genetic information processing”, “human diseases”, “environmental information processing”, and “cellular process”. The highest numbers of UniGene sequence transcripts included in each category were as follows: “endocrine system”, 4,575 (10.47%); “carbohydrate metabolism”, 6,433 (14.73%); “translation”, 6,915 (15.83%); “infectious diseases: viral”, 4,848 (11.10%); “signal transduction”, 11,136 (25.49%); and “transport and catabolism”, 3,510 (8.03%), as shown in Fig. The COG/EuKaryotic Orthologous Groups (KOG) database is based on the phylogenetic relationship of encoded proteins of the complete genomes of bacteria, algae, and eukaryotes. The alignment could annotate a specific protein sequence to a specific COG, and each cluster of the COG is composed of orthologous sequences, which enables the identification of sequence functions. This analysis identified 102,165 UniGene sequences that were annotated  to map high-quality reads from individual samples onto the The KEGG enrichment scatter plot is a graphical representation of the KEGG enrichment analysis results and as shown in Fig. In the COG/KOG annotation analysis of differentially expressed genes, we focused on the biosynthesis, transport, and catabolism of secondary metabolites 739); amino acid transport and metabolism (633); and defence mechanisms (100) as shown in Fig. ; amino aet al.13 reported the formation of radioactive aristolochic acid I when -tyrosine was added to Aristolochia sipho. The synthesis pathway of aristolochic acid I is speculated to be tyrosine → tyramine → 4-hydroxy-phenyl-acetaldehyde → (s)-nor-amethoxine → (S)-Nor Laudanine → orientaliue → stephanine → aristolochic acid I, adapted from the literature published by Comer (1969)14 14 Fig. . SchutteA. heterotropoides has a high content of active ingredients and a small amount of aristolochic acid I in the three basic plant species, as stipulated by the Chinese Pharmacopoeia, which may be why it is difficult to enrich the terminal enzyme gene in the entire plant metabolic network in transcripts.Combining the results of the analysis of the putative aristolochic acid synthesis plant metabolic network and transcriptome data, it was determined that possible precursor compounds for the synthesis of aristolochic acid were in KEGG00950 and 00350. Differentially expressed genes from these plant metabolic networks in the transcriptome data were screened, and 34 genes with strong correlation were obtained . The results of the qRT-PCR in the transcriptome data were compared to verify that the transcriptome results were authentic and reliable. Based on the analysis of the expression of tyrosine decarboxylase in A. heterotropoides and leaves and the content of aristolochic acid I, the results confirmed that tyrosine decarboxylase had a strong positive relationship with the biosynthesis of aristolochic acid I  of three genes based on predicted genes in the transcriptome data. This finding demonstrated the existence of the gene in A. heterotropoides. Furthermore, information on these three genes was submitted to GenBank . The phylogenetic tree was based on TyrDC sequences of 13 species  Kitag.] seedlings from the Jilin Agricultural University of China Medicinal Materials College at the on 3 September 2016. The growth conditions were as follows: in the early stage of cultivation, we built an awning shed and manually weeded around the plants regularly during the growing process, and the rest were untreated. The seedlings had underground roots and leaves, and the dark green leaves that were free from worms were stored at −80 °C.We collected 6-year-old Total RNA from the roots and leaves of three plants was extracted and triplicate samples were processed separately. The quality of the RNA samples was evaluated using a bioanalyzer . The mRNA was captured using the NEBNext Poly(A) mRNA Magnetic Isolation Module, and the cDNA libraries were constructed using the NEBNext Ultra RNA Library Prep kit for Illumina and purified using Beckman Agencourt AMPure XP beads, followed by detection and quantification using the Agilent bioanalyzer 2100 and Qubit. cBOT auto-clustering was performed using the TruSeq PE cluster kit v4. The clustered library was processed using the TruSeq SBS kit v4-HS and then sequenced using the Illumina HiSeq 2000 platform using the two-terminal sequencing method.16 (v2.17.1.14) software and FastQC (v0.10.1) were used to analyse the initial sequence data quality to obtain the pass filter data. The raw data read from six RNA-sequences (RNA-seqs) were subjected to quality trimming using the second-generation sequencing data quality statistics software, Cutadapt17 (version 1.9.1), to sequence the pass filter data to remove the linker and low-quality sequences to obtain clean data for subsequent information analysis. With the UniGene sequence file as a reference gene file, RSEM18 (v1.2.6) estimated gene and isoform expression levels from the pair-end clean data. Paired-end sequencing used 2 × 150 bp sequenced arm. The Cutadapt procedure was as follows: the adaptor sequence and bases whose masses at the 5′ or 3′ end were lower than 20 were removed, the reads containing more than 10% of N were removed, and reads less than 75 bp long were removed after trim removal.The bcl2fastqA. heterotropoides, it was necessary to splice the clean data. Trinity19 (v2.2.0) was used to assemble the sample data from scratch. The assembly results were further sequenced and de-redundantly processed using sequence clustering to obtain long non-redundant UniGene sequences.Because there is no report on the genome or transcriptome data of Assembly was performed by Trinity, which represents a novel method for the efficient and robust de novo reconstruction of transcriptomes from RNA-seq data. Trinity combines three independent software modules: Inchworm, Chrysalis, and Butterfly, applied sequentially to process large volumes of RNA-seq reads. Duplicated contigs were removed by cd-hit to obtain the UniGene sequence file.Open reading frames were predicted using the TransDecoder, and the data were annotated using the Nr, PFAM, and Swiss-Prot databases using the BLASTX program. The KEGG automatic annotation server 63 was used to map the transcript pathways in the non-redundant UniGene collection.20 (2.1.0) software with default parameters was used to compare short reads to quantify the genes.In the absence of a reference genome, UniGene was used to concatenate the Trinity data, the clean and UniGene data were compared, and then Bowtie221 (v1.18.0) Bioconductor package, a model based on the negative binomial distribution. After adjustments using Benjamini and Hochberg’s approach for controlling the false discovery rate, P-values of the genes were set at <0.05 to detect differentially expressed genes . The entire FPKM26 density distribution reflected the UniGene expression data of the roots and leaves of A. heterotropoides.Differential expression analysis used the DESeq2−ΔΔCT method30.The qRT-PCR was performed using the ABI StepOne system and the Brilliant II SYBR Green qRT-PCR Master Mix kit One-Step. β-Actin was used as the reference gene, and the specific primer sequences are shown in Table A. heterotropoides plant material were pulverised with liquid nitrogen, and 0.5 g of the powdered mixture was placed in a labelled 10 mL centrifuge tube, which was vortexed with 5 mL fresh 70% methanol (chromatography grade) for 30 s, kept aside for 1 h, and then weighed. After ultrasonication for 1 h, the tube was removed and then weighed again after cooling. The weight loss was compensated by adding 70% methanol, and 1.5 mL of the extract was centrifuged at 12,000 rpm for 1 min. The supernatant was filtered through a 0.22 μm organic membrane into a vial and stored airtight at 4 °C in the refrigerator.The roots and leaves of the The fresh leaves and roots were placed in a 40 °C oven and dried to a constant weight. The extraction was performed using the extraction step described above.32.High-performance liquid chromatography (HPLC) detection was performed using a Shimadzu LC-10ATvp system with a ZORBAX SB-C18 column . Chromatographic conditions for determining aristolochic acid I  content were as follows: mobile phase, methanol–0.1% aqueous acetic acid (72:28); column temperature, 27 °C; flow rate, 1.0 mL/min; detection wavelength, 315 nm; and injection volume, 20 μL. The retention time was determined using standard samples, and the aristolochic acid I content of each tissue sample was determined using a linear regression equation. Each sample was analysed in triplicatehttp://www.snapgene.com/) was used to detect the cleavage sites and positions of TyrDC1, TyrDC2, and TyrDC3 gene sequences. Primer 5  was used to determine the CG content and Tm value of the primers, according to the predicted gene screening of restriction sites and the principles of primer design. An OMEGA gel extraction kit  was used to recover the PCR products from the gel, which were then examined using the NanoDrop 2000 instrument (Thermo Fisher Scientific)33.SnapGene Viewer  to generate the FASTA format, and the data were analysed using DNAman8 (https://www.lynnon.com/index.html). Phylogenetic trees (ML) were constructed using MEGA7 (https://www.megasoftware.net/)37.The obtained Te datasets of raw read sequences from each tissue were deposited in the NCBI Short Read Archive  database under the BioProject accession number PRJNA477885.Supplementary InformationDataset 1


In light of rampant childhood diarrhoea, this study investigated bacterial pathogens from human and non-human sources in an urban informal settlement.n = 85), river water (n = 64), and diarrheic stool (n = 66) were collected between September 2015 and May 2016. A duplex real-time PCR, gel-based PCR, and CHROMagar™STEC were used to screen Tryptic Soy Broth (TSB) for diarrheic E. coli. Standard methods were used to screen for other selected food and waterborne bacterial pathogens.Meat from informal abattoirs , Plesiomonas shigelloides , Aeromonas sobria , Campylobacter jejuni , Shigella flexneri , Vibrio vulnificus , and diarrheic E. coli  respectively. All the isolates were resistant to trimethoprim–sulphamethoxazole.Pathogens isolated from stool, meat, and surface water included There was a high burden of drug resistant diarrheal pathogens in the stool, surface water and meat from informal slaughter. Integrated control measures are needed to ensure food safety and to prevent the spread of drug resistant pathogens in similar settings. Diarrheal disease is a major cause of morbidity in the Western Cape province and the city of Cape Town in South Africa . Infant Public primary health care practice in South Africa relies on the syndromic management of diarrheal disease . The SouSupranational surveillance projects such as the Global Enteric Multicenter Study (GEMS)  have pro2. Eighty-one percent of households have access to communal flush toilets connected to a sewer system, while 11% rely on bucket toilets; and 3% report no access to toilet facilities. Only 53.5% of households have piped water inside their dwellings [The City of Cape Town is temperate with moderately wet winters and dry, warm summers. The Nyanga urban informal settlement surveyed is comprised of ~ 16,000 dwellings with ~ 58,000 residents. The population density is estimated at 19,000/kmwellings . The NyaThe caregivers of children (under 12 years of age) with diarrhoea, attending the local Community Health Center (CDC) on Monday, Wednesday, and Friday of every week between October 2015 and April 2016, were approached at random for enrolment after informed consent was obtained. Clinical and epidemiological data was collected from consenting caregivers. Freshly passed stool was collected using a screw-capped stool collection container  from the diaper (or a triple tissue layer). Meat samples  from all road-side stalls in the community were collected randomly in sterile stomacher bags  on the first two Fridays of every month using the guidelines for integrated surveillance of foodborne antimicrobial resistance . SurfaceThis non-interventional survey study was approved by the Human Subjects Research Ethics Committee of the University of Cape Town (HREC 2015/140) and the City of Cape Town Health Department, Klipfontein Health sub-district .Informed consent: “Informed consent was obtained from all individual participants included in this study.Shigella and Salmonella, and Cefsulodin -Irgasan-Novobiocin  for Aeromonas and Yersinia with overnight incubation at 35 °C. For Campylobacter, samples  were directly streaked onto Charcoal Cefoperazone Deoxycholate modified Agar (CCDA) and incubated under microaerophilic conditions for 72 h at 35 °C. To culture Vibrio, 10 ml of water or 1-10 ml of stool was inoculated in 90 ml of Alkaline Peptone Water and incubated at 42 °C for eight hours. Subsequently, a loopful of the surface pellicle was sub-cultured onto a Thiosulfate – Citrate – Bile salts – Sucrose (TCBS) agar and incubated overnight at 35 °C. For P. shigelloides, a loopful of TSB was used to inoculate MacConkey agar with crystal violet (McA) and Cefsulodin – Irgasan - Novobiocin (CIN) agar and incubated at 35 °C for 24 h. Non- lactose fermenting colonies on McA and opaque colonies with a pink center on CIN, were then tested for oxidase activity before confirmatory identification. A maximum of five mauve colonies from each CHROMagar™STEC plate were sub-cultured separately on McA agar with crystal violet and sorbitol MacConkey agar plates. Suspect colonies from the different selective media were picked for confirmatory identification and characterisation. All isolates were identified, and their minimum inhibitory concentration (MIC) values determined using VITEK 2 . E. coli isolates from CHROMagar™STEC were tested for the presence of stx using real-time PCR on a LightCycler®480 II Instrument  using LightCycler® 480 Probes Master mastermix and primers as previously described. , and the filtrate (on a 0.45 μm membrane filter) of 100 ml of surface water were inoculated in 90 ml, 225 ml, and 90 ml of TSB  respectively and incubated at 35 °C overnight as previously described , 16. FrodaaC) for diffusely adherent E. coli (DAEC), the anti-aggregation protein transporter gene (aat) for enteroaggregative E. coli (EAggEC) heat-stable (ST) and heat-labile (LT) enterotoxin genes for enterotoxigenic E. coli (ETEC), the invasive plasmid antigen (ipa) gene for enteroinvasive E. coli (EIEC), the bundle-forming pili (bfp) gene for typical enteropathogenic E. coli (EPEC) and the intimin coding gene (eae) for EPEC were determined using end-point PCR on an Applied Biosystems™ 2720 Thermal Cycler platform using the QIAGEN PCR-multiplex kit  followed by agarose gel detection as previously described [The presence of the fimbrial adhesion gene :):E. colSimilarly, sample TSB enrichment broths from all sources were subjected to nucleic acid extraction using the MagNApure bacterial/fungal DNA extraction kit on a MagNApure LC instrument . PCR testing was done as described above .Shigella were serotyped using the Wellcolex* colour Shigella Rapid latex agglutination test , while Salmonella and E. coli (O-typing) [-typing)  were serSix ten-fold serial dilutions of 10 ml of canalized water in 90 ml of sterile water were filtered through 0.45 μM nitrocellulose membranes  under vacuum suction and incubated on modified mTEC agar  at 35 °C for two hours and later at 44 °C for 22 h as previously described, followed by manual counting of colonies .p <  0.05 considered significant. MIC data were analyzed using the WHONET version 5.6 software .Data on clinical and epidemiological characteristics were analyzed using Epi Info 7™ . Comparison of proportions was done using a two-tailed Chi-square test or Fischer’s exact test, with , 5% (3/66), 20% (13/66), and 17% (11/33) reported bloody stool, vomiting, and fever respectively , 85 meat samples and 64 community canal water samples. Thirty-nine percent of children had received rotavirus immunisation. This study targeted patients three years and younger that presented with diarrhoea to the local centre during the study period. Among the children that presented with diarrhoeaShigella flexneri and STEC-one patient, Shigella flexneri and Campylobacter jejuni-one patient, Aeromonas sobria and DAEC-one patient, Shigella flexneri and DAEC-one patient, DAEC and EAEC-one patient, DAEC and EAggEC-one patient). There was a high prevalence of S. flexneri (Table A. sobria (1/2), and DEC (1/14) were isolated. Notably, none (0/11) of the S. flexneri cases presented with bloody stool.Overall, an enteric bacterial pathogen was isolated from 55% 36/66) of the cases; in 17% (6/36) of these, more than one bacterial pathogen was isolated  that were tested carried various bacterial pathogens which included diarrheic ng Table .E. coli pathotypes isolated on CHROMagar™STEC. This could be explained by the fact that not all diarrheic E. coli are tellurite resistant and so would not form colonies on CHROMagar™STEC.For all the pathotypes, more of the virulence marker genes were detected Fig.  in TSB estx1  in TSB enrichment broths of these samples. For raw beef, S. enterica, P. shigelloides, A. sobria, and EPEC were isolated. For raw pork, only DAEC was isolated while S. enterica and C. jejuni were isolated from raw chicken  was the most abundant enteric bacterial pathogen in water; others included S. flexneri, V. vulnificus, A. sobria, EAggEC, STEC, and DAEC. The samples, however, had a high prevalence of daaC  and stx1 .Surface water from the community canal  yielded high numbers of faecal coliforms (mean CFU/ml =2.74E+ 05–1.11E+ 06) with the highest levels seen in February (p = < 0.001), which was also the driest (zero precipitation) and hottest month; precipitation in March 2016 coincided with the lowest measured surface water contamination level , there was resistance to the following antibiotics: ampicillin, amoxicillin-clavulanate, cefoxitin, cefuroxime, cefotaxime, cefepime, ciprofloxacin, trimethoprim-sulfamethoxazole, and nitrofurantoin (Table All the pathogens from the different sources were resistant to trimethoprimin Table .Table 8Plesiomonas (from water and stool) and Aeromonas (from meat and stool) were resistant to ampicillin while all Salmonella enterica Idikan and Salmonella enterica non – typeable were resistant to ciprofloxacin (Table Only three isolates were multi-drug resistant and were isolated from meat (one isolate) and stool (two isolates). All in Table .Table 9This study revealed a high prevalence of food and waterborne, as well as sanitation associated bacterial pathogens in meat, surface water and diarrheal stool from a low-income informal settlement setting in Cape Town, South Africa. Assessment of non-human sources for diarrheic bacterial pathogens is important because environmental modifications of potential reservoirs would aid the prevention of infections with drug-resistant pathogens., 5% (3/66), 20% (13/66), and 17% (11/33) reported bloody stool, vomiting, and fever respectively. Among the bacterial pathogens detected, S. flexneri, C. jejuni, STEC, and EIEC have been commonly reported to cause invasive disease. The associations between the presence of these bacterial pathogens and the different clinical presentations could not be determined due to small sample size. Notably, none (0/11) of the S. flexneri cases presented with bloody stool - a finding consistent with previous studies that reported the poor sensitivity of this clinical sign for the recovery of Shigellae [S. flexneri was the second most prevalent pathogen (the highest was DAEC) in this setting; it was the most prevalent among systemic shigellosis cases diagnosed between 2003 and 2009 in South Africa [Shigella and other dysentery-causing infections may be underappreciated in primary health care in South Africa. Appropriate laboratory testing and surveillance to support the differential diagnosis and targeted treatment of enteric infections in vulnerable communities are urgently needed to aid patient care and reduce secondary transmission in the community. At least seven outbreaks of shigellosis or bacillary dysentery have been reported from South Africa since 1994. These have been associated with poor water and sanitation infrastructure. Our study community experienced an outbreak of bacillary dysentery in 1999 that affected 82 people and resulted in 3 deaths in children under two years of age [Among the children that presented with diarrhoeahigellae , 10. S. h Africa . Shigells of age . WithoutCampylobacter (compared to 5% in this study) in the stool in the Venda region, Limpopo, South Africa [E. coli that were detected in stool in this setting, DAEC were the most common  and EIEC were the least common . An earlier report in Egypt, 2013, established presence of DAEC associated with diarrhoea in human and non-human sources [E. coli that were detected, more of the virulence marker genes were detected by these methods, compared to the isolates on CHROMagar™STEC from the same sample. Infection with multiple organisms was common . All co-infected patients were infected with at least one of S. flexneri or DAEC. We did not test for infection or co-infection with viruses or intestinal parasites. Consistent with the reported inadequate and low second rotavirus vaccine coverage in the Cape Town metro [Similar studies in South Africa, albeit conducted in non – informal settlement settings, have reported a prevalence of 10% for h Africa . Among t sources . Our stuS. flexneri from open surface water in the community is alarming as Shigellae are particularly evanescent outside the host, suggesting a substantial presence. P. shigelloides and V. vulnificus were abundant in canal water raising concern for those accessing the canal for recreation, washing, food extraction or drinking; and to those executing monitoring activities. The prevalence of stx1 genes in water (30% of samples) was higher than the 15% previously detected in the Berg River system [rfbE and wbdl in the water highlights it as a potential reservoir of STEC O157: H7 and STEC O111. This is of clinical and epidemiological significance because these are highly pathogenic STEC serotypes [Most of the cases (79% - 52/66) used the communal standpipe as the primary source of water. According to the City of Cape Town Water and Sanitation Department, the communal standpipe nozzles in the informal settlements often were contaminated . Lack ofr system . The preerotypes .C. jejuni and S. enterica Idikan were most common pathogens identified. Of concern, A. sobria and P. shigelloides, typically found in aquatic habitats, were recovered from meats, raising concern over vendor access to safe water and sanitation infrastructure. S. enterica Idikan was isolated from 5% (4/85) of the meat samples. It has previously been isolated from food-producing animals; animal feeds, meat from abattoirs, water, and associated environments in South Africa at a prevalence of 6% (between 2012 and 2014) [C. jejuni in South Africa is commonly isolated from poultry and is increasingly becoming resistant to antibiotics [Salmonella, Shigella or E. coli of any quantity as unacceptable [Raw meat samples collected from informal street-side vendors showed nd 2014) . C. jejuibiotics –37. The ceptable .E. coli as sentinels for surveillance of foodborne antimicrobial resistance in this setting, these pathogens showed resistance to antibiotics usually used as first-line options for treatment of infections caused by Gram negative bacilli, such as ampicillin, amoxicillin-clavulanate, and extended-spectrum cephalosporins.Considering diarrheic Pathogen-specific enrichment methods would have been more sensitive for isolate recovery. Viral and parasitic causes of diarrhoea were not targeted in this study.S. flexneri spreads from person-to-person as a result of poor sanitation and inadequate quantities of water for personal hygiene, highlighting the need to urgently address inadequate water and waste provision in communities such as this one.We showed that antimicrobial resistance to a number of antimicrobials is present in diarrheal pathogens from human, food, and environmental sources in an informal settlement in Cape Town.Globally, informal settlements present unique public health challenges due to dense human populations, poor residential infrastructure, and poor hygiene practices. Informal abattoirs do not follow recommended slaughter practice and often contaminate the environment. In a setting where diarrhoea is often empirically managed, knowledge of the sources, prevalence and antibiotic susceptibility patterns of bacteria causing enteric infections is critical.


Proteus mirabilis is the third common species responsible for UTIs that are mostly treated with fluoroquinolones or aminoglycosides. Treatment of the UTI caused by P. mirabilis is problematic due to the ability to form biofilm on the urinary catheters. The aim of the study was to evaluate the influence of ascorbic acid in combination with antibiotics on P. mirabilis abilities to form biofilm. The susceptibility of P. mirabilis reference strain ATCC® 29906™ and four clinical strains isolated from the urine samples of patients with urinary catheter were evaluated according to EUCAST recommendations. The influence of ascorbic acid (0.4 mg × mL−1) in combination with antibiotics on biofilm formation was evaluated spectrophotometrically. Aminoglycosides at sub-inhibitory concentrations more successfully limited biofilm formation by P. mirabilis strains without ascorbic acid addition. Inhibition rate differences at the lowest concentrations of gentamicin and amikacin were statistically significant (p ≤ 0.05). Ascorbic acid addition to the culture medium limited the inhibitory effect of fluoroquinolones, facilitating biofilm formation by P. mirabilis strains. The addition of ascorbic acid during aminoglycosides therapy may disturb treatment of urinary tract infections related to the presence of P. mirabilis biofilm.Vitamin C has antimicrobial activity and is often used as an oral supplement accompanying antibiotic treatment in urinary tract infections (UTI).  Vitamin C  was first identified in the 1920s by a Hungarian biochemist, Albert Szent-Györgyi . The uriVitamin C has antioxidant properties, therefore any effects of its presence in the human body may be most prominent under conditions of the enhanced oxidative stress, i.e., during the process of inflammation . Viral, Escherichia coli and Klebsiella pneumoniae) is inhibited at the vitamin C concentration of 10 mg × mL−1 and 20 mg × mL−1 . Thech lower , therefoE. coli reference strains growth on the surface of the urinary catheter and they noted 1000-fold (105 to 108) increase in the number of both bacteria strains after application of one vitamin C dosage to the culture medium for 24 h. El-Gebaly et al. (2012) [−1 of vitamin C were used, respectively. The concentrations of AA applied by them [P. mirabilis strains examined in our study, so it is very probably that concentrations higher than MIC values are able to inhibit biofilm formation. Habash et al. (1999) [E. coli and Enterococcus faecalis adhesion to biomaterials that the catheters are made from. This tendency was not observed for other urinary tract pathogens [Similar results were obtained by Carlsson et al. 2005) . They ex . They e. (1999)  showed tathogens .P. mirabilis biofilm. Vitamin C is commonly prescribed with antibiotics during UTI treatment [P. mirabilis strains biofilm formation. Similar results for the planktonic cells were obtained by Masadeh et al. (2012) [E. coli cells with 10 mM AA inhibits ciprofloxacin-induced hydrogen peroxide generation and reverse its antibacterial activity. Moreover, Aiassa et al. (2012) [P. mirabilis strains when applied simultaneously with 10 mM of vitamin C. The observed decreased sensitivity to ciprofloxacin was explained by antioxidant activity of vitamin C, influencing the ciprofloxacin mode of action on bacteria cells, based on oxidative stress induction. The increase of MIC value for another fluoroquinolone (pefloxacin) was also detected for S. aureus in the presence of vitamin C in the study conducted by Awofisayo et al. (2012) [The strategy of our own study presented was to evaluate the influence of AA addition on antibiotics activity against reatment . In our . (2012)  for cipr. (2012)  investig. (2012) .−1) applied on biofilm examination by El-Gebaly et al. (2012) [P. mirabilis. The planktonic forms of both strains examined by them [Bacillus subtilis biofilms synthesis may led to the increase of bacteria susceptible to vitamin C-induced oxidative stress compounds or antimicrobials. Yassein (2004) [The conflicting conclusions were obtained for higher concentrations of vitamin C (80 and 100 mg × mL. (2012) . They de by them  were res by them  demonstrn (2004)  explainsn (2004) .−1 of AA against multidrug resistance P. aeruginosa strains, but they did not find any interactions between tobramycin and the same concentration of AA. Goswami et al. (2007) [E. coli strain to streptomycin in a manner similar to fluoroquinolones. Andrade et al. (2014) [Acinetobacter baumannii and methicillin-resistant S. aureus (MRSA). They do not observed this effect for any of the examined aminoglycosides. In this study we demonstrated that AA strongly decreases the BIR value. Moreover, we observed the enhancement biofilm formation at the lowest sub-inhibitory concentrations of amikacin or gentamicin in the presence of AA while in the medium supplemented only with the antimicrobials—the inhibition of biofilm formation was demonstrated. Similar results were also proven by Wasfi et al. (2012) [The influence of antioxidants on aminoglycosides remains unknown. However, Cursino et al. (2005)  demonstr. (2007)  observed. (2014)  study ob. (2014)  also obs. (2012)  for ceftIn the available literature, very few information on the influence of vitamin C supplementation on the CAUTIs treatment can be found. Thus, further studies of that issue are necessary, especially in terms of bacterial biofilm production.Proteus spp. strains were isolated from patients of the Antoni Jurasz University Hospital No 1 in Bydgoszcz, Poland. The examination included only strains obtained from urine samples collected using bladder catheters. Strains were identified during microbiological investigation at the Department of Microbiology.Proteus spp. strains was examined using BD Phoenix™ system (Becton Dickinson) and interpreted according to EUCAST recommendation [® 29906™) were included into the study  recommendation (2012) [−1 was evaluated. Escherichia coli strain ATCC® 25922™ was used for the control assays for antimicrobials. The MIC value was read-out visually by the presence of turbidity of the suspension in the wells of microtiter plates at a given concentration of antibiotic or AA. Investigation was done in three independent replications.The MIC value for gentamicin (GEN), amikacin (AMK), ciprofloxacin (CIP), norfloxacin (NOR), levofloxacin (LEV), and ascorbic acid (AA)  of n (2012) . The groThe ability to form biofilm by the studied strains was examined using 0.1% 2,3,5 triphenyltetrazolium chloride  by the microtiter methodology that was described previously . That coP. mirabilis were cultivated on the Mueller-Hinton Agar II  and cultured at 37 °C for 18 h. Next, the single colonies of each strain were inoculated into Tryptic Soy Bullion . After 18 h at 37 °C, each culture was centrifuged for 15 min at 4000 rpm and the supernatants was discarded. The remaining pellet was rinsed with 3 mL of phosphate buffered saline solution (pH = 7.2) . Next, the bacterial suspension was centrifuged at 4000 rpm for 10 min and the pellet was used to make the suspension of 0.5 McFarland turbidity in Mueller-Hinton Broth . Then, 100 μL of every suspension was placed in the wells of polystyrene 96-well plate, three repetitions each. The wells were filled with: (a) 100 μL of sterile MHB , (b) 100 μL of AA  in MHB (AA impact assessment). The culture was incubated in a humid chamber at 35 ± 2 °C for 24 h. After incubation, the solutions were removed and the wells rinsed tree times with sterile PBS. Next, 100 μL of TSB and 100 µL of 0.1% TTC were added to each well. The plates were placed on a shaker at 250 rpm for 5 min at room temperature. Next, the plates were placed at 37 °C. After 3-h incubation, the TTC was removed and plates were rinsed three times with sterile water. Finally, 200 μL of 96% methanol (Avantor) were added to each well and left on a shaker at 400 rpm for 5 min at room temperature. Results were conducted with a spectrophotometer as described above.The examined strains of Based on MIC values results, the sub-inhibitory concentration of the studied antibiotics were prepared. The antibiotic concentrations were equivalent to 0.064, 0.125, 0.25, 0.5, and 1.0 of MIC values for particular antimicrobial. The preparation of examined strains inoculum was based on methods described in the previous section. Bacteria suspension (100 μL) was placed into the wells of polystyrene 96-well plate. The wells were filled with: (a) 100 μL of an antimicrobial at the concentration in final medium in range 0.064–1 MIC, diluted in MHB (sub-inhibitory antibiotic concentration impact assessment), (b) 100 μL of MHB only . The culture of biofilm was conducted under conditions described above. Results were given as absorbance values.−1) in MHB (antibiotic + AA impact estimation). The culture of biofilm was conducted under conditions described above. Results were given as absorbance values.Preparation of the examined strains inoculum based on the methods was described in the previous section. Bacteria suspension (100 μL) was placed in the wells of polystyrene 96-well plate. Then, the wells were filled with: (a) 50 μL of an antibiotic at concentration in the final medium in the range 0.064–1 MIC, diluted in MHB, and 50 μL of sterile MHB , (b) 50 μL of an antibiotic at the same concentration as in the previous point, diluted in MHB, and 50 μL of AA  stands for the absorbance value of positive control, and (Ay) for the absorbance value of the strain subjected defined concentration of antibiotics or/and ascorbic acid.We introduced value described as Biofilm Inhibition Rate (BIR) to express our final results. It normalized the impact of TTC to formazan metabolism measurement time, regardless of the examined strain properties. BIR is based on absorbance values and is calculated according to the formula:Statistical analysis was performed using Statistica 12.5 PL (StatSoft) software. The confidence interval for all the tests applied was α = 0.05. The obtained results normality was evaluated with the Shapiro-Wilk test. Bonferroni test was applied to compare the impact of various concentrations of the studied antibiotics on biofilm formation , and U M


This paper aims to investigate the correlations between the concentrations of nine heavy metals in moss and atmospheric deposition within ecological land classes covering Europe. Additionally, it is examined to what extent the statistical relations are affected by the land use around the moss sampling sites. Based on moss data collected in 2010/2011 throughout Europe and data on total atmospheric deposition modelled by two chemical transport models , correlation coefficients between concentrations of heavy metals in moss and in modelled atmospheric deposition were specified for spatial subsamples defined by ecological land classes of Europe (ELCE) as a spatial reference system. Linear discriminant analysis (LDA) and logistic regression (LR) were then used to separate moss sampling sites regarding their contribution to the strength of correlation considering the areal percentage of urban, agricultural and forestry land use around the sampling location. After verification LDA models by LR, LDA models were used to transform spatial information on the land use to maps of potential correlation levels, applicable for future network planning in the European Moss Survey.Correlations between concentrations of heavy metals in moss and in modelled atmospheric deposition were found to be specific for elements and ELCE units. Land use around the sampling sites mainly influences the correlation level. Small radiuses around the sampling sites examined (5 km) are more relevant for Cd, Cu, Ni, and Zn, while the areal percentage of urban and agricultural land use within large radiuses (75–100 km) is more relevant for As, Cr, Hg, Pb, and V. Most valid LDA models pattern with error rates of < 40% were found for As, Cr, Cu, Hg, Pb, and V. Land use-dependent predictions of spatial patterns split up Europe into investigation areas revealing potentially high (= above-average) or low (= below-average) correlation coefficients.LDA is an eligible method identifying and ranking boundary conditions of correlations between atmospheric deposition and respective concentrations of heavy metals in moss and related mapping considering the influence of the land use around moss sampling sites.The online version of this article (10.1186/s12302-018-0183-8) contains supplementary material, which is available to authorized users. The United Nations Economic Commission for Europe (UNECE) Convention on Long-range Transboundary Air Pollution (CLRTAP) of 1979 and its eight protocols are aimed at limiting and reducing air pollutants. Under the LRTAP convention, the European monitoring and evaluation programme (EMEP) gathers information on emission from its parties, collects data on air and precipitation quality and models atmospheric transport and deposition of air pollutants –4 to detBased on EMS data from 2005, atmospheric deposition has been identified as the main factor determining the spatial variation of concentrations of cadmium (Cd) and lead (Pb) in moss specimens collected throughout Europe –8. HarmeCorrelation analysis Examination of correlations between concentrations of HM in moss from the EMS 2010/2011 and respective atmospheric deposition as modelled by use of the CTMs EMEP MSC-East and LOTOS EUROS (LE), and to which extent the correlations are specific for ecological land classes of Europe (ELCE) [e (ELCE) .Statistical modelling Calculation, to which extent the amount of ELCE-specific correlation coefficients is affected by the areal percentage of land use around the sampling sites potentially indicating influences of local emission sources as for instance agricultural and urban land use or point sources of air pollutants. Hence, the reason for different ELCE-specific correlation coefficients was investigated.Predictive mapping Land use-dependent predictions and mapping of correlation patterns across Europe (site-related/area-related) and, finally, aggregation of predicted spatial patterns for decision support (e.g. moss survey network planning).The above-mentioned findings were verified in the investigation presented in this paper using data collected in the EMS 2010. The present study addresses the following objectives.For this investigation, data on atmospheric deposition of HM derived from the EMEP MSC-East  were supData on element concentration in moss were correlated with respective modelled atmospheric deposition specifically for ecological land classes of Europe (ELCE) and major land use categories around the sampling sites derived from CORINE land cover 2006 and Global land cover 2000 , 18 (TabPleurozium schreberi (Brid.) Mitt., Hylocomium splendens (Hedw.) Schimp., Hypnum cupressiforme Hedw. s.str. and Pseudoscleropodium purum (Hedw.) M. Fleisch (synonym Scleropodium purum Hedw. Limpr.) [In 2010/2011, moss specimens were collected at 4499 sample sites in 26 countries across Europe following a standardized experimental protocol . FurtherStatistical relations between element concentrations in moss and atmospheric deposition of HM derived from the numeric chemical transport models (CTMs) LOTOS-EUROS (LE) , 21 and The EMEP deposition data were supplied by the meteorological synthesizing centres MSC-East (Moscow) of EMEP operating under the LRTAP convention. Travnikov and Ilyin  used emiFollowing Harmens et al. , in this40) to 200 (ELCE200) classes identified by 48 geo-data layers on potential natural vegetation [40 and ELCE200 were calculated and mapped by means of classification and regression trees [40 was used, whereby ELCE units occurring sporadically and with a total spatial extent below 4.2% were summarized to one class (“others”).The data on element concentrations in moss and atmospheric deposition were spatially joined to the map of ecological land classes of Europe (ELCE)  for quantifying the relation between concentrations in mosses and modelled atmospheric deposition of HM and N  within participating European countries were used as coding variable for calculating ELCE-specific correlations. Due to a non-normal distribution in most of the subsamples, Spearman rank correlation coefficients (rs) were determined. The correlation coefficients were classified according to Brosius [The statistical design comprises the calculation of Spearman rank correlation coefficients  and respective coefficients . The aim is to separate groups of data in a scatterplot so that the variation in data within each group is minimized [The second step is to investigate the reason for the difference of ELCE-specific correlations. LDA models were used to find linear separation lines as best discriminate of sampling sites in ELCE regions revealing high or low element-specific correlation coefficients. LDA attempts to find a multivariate discriminant function inimized –33 and einimized  were exainimized . OverallLogistic regression (LR) is similar to LDA, as it also explains a categorical variable by the values of continuous independent variables. LR is preferable in applications where the independent variables are not normally distributed. Since LR is less concrete, LDA in the present study was used for model building and LR for verification of LDA results.LDA models were firstly applied on the Europe-wide dataset of moss sampling sites with information on land use density around the sampling sites. Model-specific error rates (%) were calculated by means of confusion matrix values . Charts for the linear discriminant functions were used for plausibility checks. Logistic regression models were built using the same predictors from the LDA models. Confusion matrices and error rates (%) specified for each LR model were calculated and compared with the statistical characteristics of the LDA models.To verify to which extent the models really separate sampling sites showing high or low correlations between element concentrations in moss and respective atmospheric deposition, bivariate Spearman coefficients for the correlations between element concentrations in moss and atmospheric deposition were again calculated for the following subsamples: sampling sites located within all ELCE classes, ELCE classes showing correlations above and below the element-specific class boundaries between high and low correlation levels defined in Table Geographic information on the spatial density of agricultural, forestry, and urban land use within a 1, 5, 10, 25, 50, 75, and 100 km radius around the sampling sites available with blanket coverage of Europe was taken as predictors for estimating categories of correlations  between atmospheric deposition of nine HM in Europe using LDA models and to transform spatial information on the land use to spatial correlation patterns across Europe. Finally, spatial patterns estimated by the best LDA models were aggregated by calculating the number of element-specific A classifications  of the sampled area in the countries participating in the EMS .All analyses with HM concentration in moss were based on a reasonably large sample size of at least 3274 (As) out of 3965 (Zn) sample points. The minimum sample sizes for elements and ELCEr et al. , 29. As rs) ranging from 0.6 to 0.8 were achieved for 7% (EMEP) up to 10.5% (LE) of the area of ELCE40 coverage of all countries participating in the EMS 2010 together  are located in Poland, Switzerland and Austria . Moderate rs values occurred for ELCE40 units covering 47.0–49.6% of the landmass. Moss data from each 5 ELCE40 (for LE in parts other than for EMEP) are weakly correlated with the modelled Cd deposition . 7 (EMEP) up to 9 (LE) out of 27 ELCE40 units reveal non-significant or very weak correlations (23.5–31.1%).Strong correlations between element concentrations measured in moss and modelled deposition  with coefficients   and 7  ELCE units show Spearman’s rank coefficients between 0.2 and 0.4. For another 10 ELCE categories (LE) and, respectively, 11 ELCE categories (EMEP), correlation coefficients came out to be between 0.4 and 0.6. The area comprises 36.6–43.5% of the total area covered by moss samples mainly located in Finland, Sweden and France  were found for max. 5 ELCE classes: D_13 (only EMEP), S_0 (only LE), B_2, C_0, F1_1, “others” (both EMEP and LE) (15.6–16.8% of the landmass) predominately distributed in Norway. With regard to the samplings across Europe, Spearman’s rank coefficients are 0.64 (LE) and 0.7 (EMEP).For Pb, in 4 (EMEP) up to 6 (LE) out of 27 ELCErs values were not significant, below 0.02 or even negative . It is clear that the correlation between moss data from the EMS 2010 and EMEP modelled deposition is very low . 14 out of 27 ELCE40 units reveal non-significant correlations within 41.1% of the sampled ELCE40 area . Three ELCE40 classes reveal significant weak correlations with rs values between 0.2 and 0.4 (13.1%). Merely 4 ELCE40 units show moderate coefficients between 0.4 and 0.6 . The ELCE40 unit with the highest correlation was U_1  comprising dispersed small areas within the participating countries (1%)  were found rs = 0.. 14 out rs = 0.03, Table 40 units, the rs values were not significant (49.5% of area of ELCE units covered by moss sampling sites), and for 37.3%, the rs values were below 0.02 or even negative  could be shown for land class D_17, covering 2.4% of the analysed area, located in Sweden, Finland and Russia. D_22 (5.4%) as a part of Sweden reveals at least moderate correlations (rs = 0.43). The remaining surface showing low correlations is allocated to ELCE unit D_7, which covers 5.4% of the landmass.Of all elements examined, Cr reveals the weakest Europe-wide correlation between concentrations in moss and total deposition modelled by LE (rs) between 0.2 and 0.4. Moderately strong correlations were found for 6 out of 27 ELCE40 units  sparsely distributed in almost every participating country and comprising 15.6% of the ELCE units. In comparison, Europe as a whole is also characterized by an intermediately strong correlation (rs = 0.5). All other 10 out of 27 ELCE40 units reveal non-significant correlations within 35.5% of the sampled ELCE40 area.The largest area covered by moss sampling sites (48.9%) is allocated to low correlations  or non-significant values (53.8%). Significant positive correlations in ELCE40 classes were found for D_17 (0.2 > rs > 0.4), D_18, G2_0 (0.4 > rs > 0.6) and J_2 (0.6 > rs > 0.8)  reveal significant positive, low Spearman’s rank coefficients . They cover 25.7% of the sampled area and can be primarily found in Fennoscandia, northern Spain and France . The 7 out of 26 ELCE40 classes with at least low correlations were the following: D_18, F1_1, F2_5, F3_1, G1_0, U_1, and “others”, located in eastern and northern parts Europe. The coefficients for the remaining ELCE units are very low or non-significant (29.5% and 45.6% of the landmass).On the European level, the correlation between modelled Zn deposition (LE) and concentrations in moss is significantly low with The frequency of the predictors used as discriminants in the 11 LDA models ranges between 1 and 3, which means that none of the factors in particular stands out Fig. . MoreoveLDA models with the highest quality corresponding to error rates ≤ 30% were found for Cr and V followed by As, Cu, Hg, and Pb (only LE) with error rates ≤ 40% , As, Cr, Cu, Hg, and V were selected as the most valid models further pursued for predictive mapping of correlations in “The application of LDA models for As, Cr, Cu, Hg, Pb (LE), and V on the whole European dataset with information on density of land use around the sampling sites led to typical correlation patterns as depicted in Fig. An aggregated view on LDA predictions for As, Cr, Cu, Hg, Pb, and V feasible within countries supplying sufficient information on LDA predictors with blanket coverage derived from CLC 2006 is shown in Fig. Areas with 5–6 out of 6 LDA models revealing potential high correlations are located in Fennoscandia, Scotland, Austria and parts of the Baltics, South-eastern Europe, Turkey, France, and Spain . Information about the elements being relevant at specific locations is given in Fig. n > 10. This is in line with similar findings for Cd, Hg and Pb based on data from 2005 [For all investigated HM , the correlations between concentrations in moss and the modelled total atmospheric deposition  were land class specific and element specific. Significant positive correlations were found for about 30% (As), 78–81% (Cd), 15% (Cr), 63% (Cu), 44% (Hg), 15% (Ni), 78–89% (Pb), 19% (V), and 44% (Zn) of 27 ELCE units being represented by moss sample sizes rom 2005  and to srom 2005 , 39. Parrom 2005  probablyrom 2005 , 13, butrom 2005 –43. Furtrom 2005 . Also prrom 2005 . Also, oThe use of LDA enables a separation of moss sampling sites regarding their contribution to the correlation level in dependency on urban, agricultural and forestry land use around the sampling location. LDA and LR help explaining spatial patterns of ELCE-specific correlation levels by identifying most relevant land uses and radiuses around the sampling sites. A very valuable indicator for this was that moss sampling sites classified by LDA as A within ELCE B units defined by below element-specific average correlations show stronger correlations than the whole set of sampling sites within ELCE B units . Vice versa, category B sites from ELCE A units reveal lower correlations than the whole subsample from ELCE A units . This means that the ELCE-specific correlation levels are more strongly dependent on land use within the specified radii around the sampling sites and less on the spatial assignment to specific ELCE units, which per se have high or low correlations. For example: Each random subsample within regions showing low correlations between Hg concentrations in moss and respective atmospheric deposition is expected to reveal low correlations. If LDA selects a subsample indicating high correlations within regions showing low correlations, the importance of the predictors for Hg can be graded as high. Best LDA models with error rates < 40% were found for As, Cr, Cu, Hg, Pb, and V. Similar error rates were found by means of LR, confirming the validity of LDA models, which are more graphic and less difficult to interpret. This supports our approach to use LDA in conjunction with a confirmation by LR, which is in any case recommended for multivariate statistics, especially since predictors are not normally distributed. Likewise, relevant factors for predicting potential correlation levels are element specific. While density of forestry land use might characterize a seclusion of sampling locations, urban and agricultural land use around the sampling sites might indicate pronounced influences of local emission sources. For Cd, Cu, and Ni, small radiuses are relevant. For Cd and Cu, the relevance of agricultural land use within a small range of 0–5 km could be caused by fertilizers (Cd) and copper-containing pesticides . For Cd LDA modelling in combination with LR is an eligible method for predicting and mapping spatial patterns of correlations in dependency of the influence of the environmental factors. Based on error rates and spatial patterns analysis, the explanatory power of 6 out of 11 LDA models with error rates < 40%  was found to be sufficient. However, error rates of 26–39% imply strong influences of other factors, i.e. the predictions are connected with high uncertainties and should be regarded as indications only.The predictive mapping provides spatial indication on the presence of other factors for element concentration in moss than those considered in the modelling of background atmospheric deposition, e.g. local emission sources or mineral windblown dust from soil . The LDAAdditional file 1: Figure S1. Map of Ecological Land Classes of Europe [Table S1. Legend of the map on Ecological Land Classes of Europe [Table S2. Description of Chemical Transport Models (CTM) used as data source. Figure S2. Predicted correlation patterns for Cd, Ni, Pb, and Zn  at site level as classified according to their surrounding land use with above element-specific average (= A) or below e.-s. average (= B) correlations.f Europe . Table Sf Europe . Table S


After propensity score matching (PSM), 26 pairs of well-matched HCC patients were selected; patients in the TACE–SBRT group showed better overall response rates in trend compared to those in the TACE–sorafenib group. The hazard ratio (HR) of OS to PFS for the TACE–SBRT approach and the TACE–sorafenib approach was 0.36  and 0.35 , respectively. For HCC patients with macrovascular invasion, TACE plus SBRT could provide improved OS and PFS compared to TACE–sorafenib therapy.Stereotactic body radiotherapy (SBRT) has shown promising results in the control of macroscopic vascular invasion in patients with hepatocellular carcinoma (HCC); however, its efficacy in comparison to sorafenib when combined with transarterial chemoembolization (TACE) remains to be determined. Between 2009 and 2017, 77 HCC patients with macroscopic vascular invasion receiving TACE–SBRT or TACE–sorafenib combination therapies were enrolled. The best treatment responses, overall survival (OS), and progression-free survival (PFS) of the two treatment arms were compared. Of the patients enrolled, 26 patients (33.8%) received TACE–SBRT treatment, and 51 (66.2%) received TACE–sorafenib treatment. The patients in the TACE–SBRT group were more frequently classified as elder in age ( The macroscopic vascular invasion of hepatocellular carcinoma (HCC), including portal vein tumor thrombosis (PVTT) or inferior vena cava tumor thrombosis (IVCTT), corresponds to the Barcelona Clinic Liver Cancer (BCLC) stage C disease and remains a daunting challenge for treatment ,2. The cTransarterial chemoembolization (TACE) is the most popular palliative treatment for patients with unresectable HCC. In the past decade, the strategy of combining conventional TACE and sorafenib to treat HCC patients with macroscopic invasion had been proposed ,6,7 and In recent years, the advances in radiotherapy techniques, including three-dimensional conformal radiotherapy (3D-CRT), have facilitated the safe and effective delivery of a high dose of radiation to the tumor while preserving the surrounding tissue . A recenTherefore, the goal of this study was to retrospectively compare the OS and PFS in advanced HCC patients with macroscopic vascular invasion who are undergoing combined TACE–SBRT treatment versus combined TACE–sorafenib treatment.p = 0.012), having recurrent disease (p = 0.026), and having single intrahepatic lesions (p = 0.005) than patients in the TACE–sorafenib group. There were no significant differences in the distribution of gender, Child–Pugh class, cause of disease, tumor size, alpha-fetoprotein (AFP) level, type of PVTT, and hepatic vein/inferior venae cava (HV/IVC) invasion between the two groups.Of the 77 patients enrolled, 26 (33.8%) were in the TACE–SBRT group and 51 (66.2%) were in the TACE–sorafenib group. p = 0.001; p = 0.076; The mean follow-up time for the TACE–SBRT group and TACE–sorafenib group were 12.2 months  and 10.4 months , respectively. Among the 26 patients in the TACE–SBRT, seven were still alive, nine had died, and 10 were lost to follow-up by the end of this study. Among the 51 patients who received TACE–sorafenib, two were still alive, 39 had died, and 10 were lost to follow-up. The median OS time was 24.2 months for the TACE–SBRT group and 12.9 months for the TACE–sorafenib group. The one-, three-, and five-year OS rates were 81.8%, 49.4%, and 35.3% in the TACE–SBRT group, and 52.1%, 11.4%, and 0.0% in the TACE–sorafenib group, respectively. The TACE–SBRT group had significantly higher OS rates than the TACE–sorafenib group  who underwent TACE–SBRT and 43 of the patients who underwent TACE–sorafenib (84.3%) during follow-up. The median PFS time were 10.1 months for TACE–SBRT groups and 3.6 months for the TACE–sorafenib group. The one-, three-, and five-year PFS rates were 44.7%, 26.2%, and 9.4% in the TACE–SBRT group, and 13.4%, 2.2%, and 0.0% in the TACE–sorafenib group, respectively. Patients in the TACE–SBRT group had significantly higher PFS rates compared with those in the TACE–sorafenib group (p = 0.139).By utilizing one-to-one PSM, 26 patients from each group were selected, with well-matched confounding factors . The resp < 0.001) and PFS rates (p < 0.001) than patients in the TACE–sorafenib group (p = 0.003) and 0.31 , respectively.The median OS and PFS time in the TACE–SBRT group were 24.2 and 10.0 months, and in the TACE–sorafenib group these were 8.4 and 3.5 months, respectively. The one-, three-, and five-year OS rates were 81.8%, 49.5%, and 35.3% in the TACE–SBRT group, and 46.9%, 7.8%, and 0.0% in the TACE–sorafenib group, respectively. The one-, three-, and five-year PFS rates were 44.7%, 26.2%, and 9.4% in the TACE–SBRT group, and 12.5%, 0.0%, and 0.0% in the TACE–sorafenib group, respectively. Patients in the TACE–SBRT group had significantly higher OS  is mandatory and fundamental to ensure precision. The key to safe SBRT is to match the target in the planning CT to the target itself on the cone-beam CT before each fraction . Since tThis study had several limitations. First, it is a retrospective study. Second, the sample size of patients enrolled in each group was relatively small, which impeded further stratified analysis. Third, all TACE procedures were performed in our cancer center, which may cause bias as TACE is operator dependent. For these reasons, a multi-institutional clinical trial is needed in the future.From January 2009 to January 2017, a consecutive series of 746 HCC patients with macroscopic vascular invasion were retrospectively reviewed. This study was approved by the SYSUCC Hospital Ethics Committee (No. B2018-156-01), which waived the need for written informed consent because of the retrospective nature of the study. The inclusion criteria were: (a) initially diagnosed HCC with macroscopic vascular invasion or recurrent HCC after surgical resection with macroscopic vascular invasion; (b) received TACE as initial treatment and achieved technical success; (c) potential candidates for SBRT of vascular tumor thrombi evaluated by two radiation oncologists with more than 10 years’ experience; (d) received SBRT of vascular invasion within 3 months after first TACE or started sorafenib treatment within 1 week after first TACE as combination treatment; and (e) Child–Pugh A or B. The exclusion criteria were: (a) presence of extrahepatic metastasis detected on abdominal CT/MRI scan or chest CT scan/digital chest radiograph; (b) recurrent tumors with no intrahepatic lesions; (c) received other treatment including ablative therapies, surgical resection, transplantation within the first 6 months after initial TACE; and (d) other malignancies. A total of 77 patients were enrolled in this study with 26 in the TACE–SBRT group and 51 in the TACE–sorafenib group .In this study, each case was fully evaluated by a multidisciplinary team before the initiating treatment, including interventional radiologists, oncologists, pathologists, radiologists, and radiation oncologists. Each patient was informed of the advantages and disadvantages of the two treatment plans before initial TACE, including costs, treatment-related morbidities, and treatment outcomes. The decision regarding the treatment plan was made jointly by the patient and doctors.For the TACE procedure, based on the tumor size, location, number, and vascular supply, a super-selective microcatheter was inserted into the supplying artery of the tumor. Then a combination of lipiodol (5–15 mL), lobaplatin (30–50 mg) and pirarubicin (30–50 mg) was introduced into the tumor. Technical success was defined as full embolization of the tumor feeding artery and with no tumor staining observed by angiogram at the end of procedure. Transarterial chemoembolization was repeated every 4–5 weeks thereafter, and was discontinued if the patients could not tolerate additional procedures due to adverse effects or when they refused further treatment. For the initial 6 months, the mean repeats of TACE for patients in the TACE–SBRT group was 1.50  before SBRT and 0.3  after SBRT ; the meaIn the TACE–SBRT group, SBRT was scheduled after one to two administrations of TACE and completed within 3 months after the initial TACE. All patients underwent four-dimensional computed tomography (4DCT) scans at 2.5- or 3-mm slice thickness during free breathing. The tumor thrombosis visualized on the CT/MRI images and the contiguous HCC were defined as gross tumor volume (GTV) for all patients; the intrahepatic metastases of HCC were not included in GTV. The internal target volume (ITV) was defined as the combined volume of GTVs in the multiple 4DCT phases. To take interfractional motion variability and daily setup errors into account, the planning target volume (PTV) was acquired by adding a 6-mm margin to the ITV.TM, Stockholm, Sweden), was utilized to perform SBRT. Planning of VMAT was conducted utilizing Monaco TPS  and consisted of a single arc [Volumetric modulated arc therapy (VMAT) techniques, which typically was planned for a 6 MV photons Elekta accelerator  to PTV in six fractions, 3 days per week. The total radiation dose was determined on the basis of tumor size, tumor location, and patients’ Child–Pugh Class. The dose constraints for organs at risk were defined as follows: mean dose of normal liver <18 Gy, mean dose of kidneys <15 Gy, maximum dose to spinal cord <27 Gy, and maximum dose to >0.5 cc of esophagus, stomach, or small intestine <30 Gy. Daily image guidance was performed using cone-beam CT for alignment of liver anatomy prior to each fraction.For patients in the TACE–sorafenib group, sorafenib was continuously administered orally at a dosage of 400 mg twice a day (800 mg/day). Dosage reductions and termination of treatment were permitted if unacceptable treatment-related toxicity occurred or disease progressed.Patients in both groups were followed up and evaluated for response every 4–5 weeks during sessions of treatment and then every 3 months until death through contrast-enhanced CT or MR scans. The primary endpoint was overall survival (OS), which was calculated from the date of the initial treatment to death by any causes. The secondary endpoint was progression-free survival (PFS), which was calculated from the date of the initial treatment to disease progression or death.Tumor response assessments were made by two independent radiologists using the modified Response Evaluation Criteria in Solid Tumors (mRECIST), who were blinded to each other’s opinion . Accordi2 test or Fisher’s exact test. Ordinal variables between groups were compared using Wilcoxon rank sum test. OS and PFS were estimated by Kaplan–Meier method and the differences between the groups were compared using the log-rank test. Multivariate Cox regression model in backward conditioned method was utilized to determine the independent prognostic factors for survival.Categorical variables between treatment groups were compared using Pearson χp < 0.05 was considered statistically significant.Propensity score matching analysis was performed to reduce the potential bias in patient selection. The selected variables entered into the propensity model included age, gender, disease onset, Child–Pugh Class, AFP level, liver tumor size, number of tumors, type of PVTT and HV/IVC invasion. One-to-one matching between the groups was accomplished using the nearest-neighbor method, with the caliper of 0.25. Following propensity score matching, the adjusted comparisons were based on data from 26 patients in each treatment arm. Statistical analyses were performed using SPSS 21.0 or R 3.3.2 . The value www.researchdata.org.cn), with the approval RDD number as RDDA2018000943.The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform (This retrospective study provided encouraging evidence that the combination therapy of TACE–SBRT can significantly enhance the control of tumor thrombus and prolong the OS and PFS in HCC patients with macroscopic vascular invasion when compared to TACE–sorafenib therapy. Further randomized control trials are needed to confirm our findings.