Background: Forty-four strains isolated from a cohort of cutaneous leishmaniasis (CL) patients who did or did not respond to one course of treatment with meglumine antimoniate were investigated to explore genetic polymorphisms in parasite kinetoplast DNA minicircles. Leishmania (Viannia) braziliensis strains isolated from responder (R) and non-responder (NR) patients who acquired infection in Rio de Janeiro or in other Brazilian states were studied using low-stringency single-specific primer polymerase chain reaction (LSSP-PCR) to identify genetic polymorphisms. Results: Polymorphisms were observed in parasites recovered from patient lesions. No association was found between a specific genotype and R or NR patients. Phenetic analysis grouped the genotypes into three main clusters, with similarity indices varying from 0.72 to 1.00. Although no specific genotype association was detected, at least one group of L. (V.) braziliensis genotypes that circulates in Rio de Janeiro was discriminated in clusters I and III, showing phenotypes of good and poor responses to treatment, respectively. Cluster I comprised parasite profiles recovered from R patients from Rio de Janeiro and in cluster III, NR samples were prevalent. Cluster II comprised 24 isolates, with 21 from Rio de Janeiro and three from other states, equally distributed between R and NR patients. Additionally, we found that parasites sharing all common genetic characteristics acted differently in response to treatment. Conclusions: These results are of clinical-epidemiological importance since they demonstrate that populations of L. (V.) braziliensis that exhibit high levels of genetic similarity also display different phenotypes associated with meglumine antimoniate responses in cutaneous leishmaniasis patients. Leishmania. The diseases occur in different environments and are endemic in 98 countries and three territories on five continents, affecting more than 12 million people worldwide. Tegumentary leishmaniasis (TL) is widely distributed, with approximately 0.7 million to 1.2 million TL cases diagnosed annually , lesion-evolution time in days, Montenegro skin test (MST) diameter of induration , ELISA , and IIF (cut-off point considered positive ≥1/40).The populations being studied were classified by therapeutic outcome. The R group included patients who presented clinical healing defined as complete epithelization of the cutaneous lesions up to 3 months after the conclusion of one course of treatment, and subsequent total regression of crusts, desquamation, infiltration, and erythema, without any sign of reactivation and absence of mucosal lesions over a 1 year observation period. The NR group included patients experiencing therapeutic failure (absence of complete skin-lesion epithelization up to 3 months after the end of one treatment) or lesion recurrence  requiring new courses of treatment.Leishmania promastigotes isolated from biopsies of cutaneous lesions were immediately stored in liquid nitrogen and subsequently recovered after two passages in Schneider's Drosophila medium  supplemented with 10% fetal bovine serum  at 26 °C. Parasites in the stationary phase were harvested by centrifugation at 4000 rpm for 10 min at 4 °C and washed several times with sterilized phosphate-buffered saline (pH 7.2). The L. (V.) braziliensis reference strain MHOM/BR/75/M2903 was used as a control in all experiments.Leishmania (V.) braziliensis by multi-locus enzyme electrophoresis (MLEE) analyses according to previously defined protocols  of the continuous variables. The association between categorical variables with clusters and therapeutic outcome was verified by Pearson's chi-squared test, Fisher's exact test, and the Shapiro-Wilk normality test. The Mann-Whitney 5+/kg/day, and three others received meglumine antimoniate 20 mg Sb5+/kg/day. Twenty-three patients (52.3%) were R to one course of treatment, and 21 (47.7%) were NR. However, all 21 patients healed after one to three additional (median = 1) courses of treatment. Seventeen of these patients showed lesion healing after further courses of 5 mg Sb5+/kg/day, including two patients who did not respond to 20 mg Sb5+/kg/day. Four other NR patients were cured after additional courses of amphotericin B (three) or pentamidine (one) (p = 0.048), and the NR group was more reactive to IIF as compared with the R group (p = 0.001).Forty-four patient samples were included in this study, with 26 (59.1%) from men and 18 (40.9%) from women [aged 11–59 years (34.25 ± 12.25)]. Patients harbored from 1 to 15 (median = 1) cutaneous lesions; in 23 patients (52.3%), lesions were localized in the lower extremities, in 13 (29.5%) on the upper limbs, in nine (20.5%) in the torso, and in six (13.6%) in the cervical-facial region (head), with evolution time ranging from 20 to 270 days (median = 60 days). Forty-one patients underwent MST testing and presented reactions ranging from 5 to 60 mm (median = 16 mm), with only one patient (2.3%) classified as a non-reactor (4 mm). The ELISA test was positive for 82.5% (33/40) of patients, and IIF was positive for 66.7% (26/39) of patients. Thirty-five patients (79.5%) acquired the infection in Rio de Janeiro and nine (20.5%) in other states of Brazil . Forty-one patients were treated with meglumine antimoniate 5 mg Sbne (one) . Table 1L. braziliensis complex, the diagnostic band of 750 bp was present in 100% of the L. (V.) braziliensis isolates (n = 44), as expected. Intra-population genetic variability was revealed by the polymorphic minicircle profiles obtained by the LSSP-PCR technique. Genetic profiles with different degrees of complexity were detected. L. (V.) braziliensis reference strain (MHOM/BR/75/M2903) was used as an internal control sample. In these experiments, as well as in others from our group [After specific PCR for the ur group , the repL. (V.) braziliensis reference (MHOM/BR/75/M2903), of which 10 were found presenting a similarity index of 1.00. Most samples were grouped by treatment-response profile, as well as by the site of infection. Moreover, nine of 13 NR samples were grouped in cluster III, which included the majority of patient samples (six) from other states in Brazil, with all six isolates sharing 100% similarity, and all of them were grouped based on treatment response and infection locality. In total, 20 samples displayed 100% similarity, with samples 310, 1127, and 946 the most genetically differentiated and presenting similarity indices of 0.72, 0.76, and 0.76, respectively.The isolates showed coefficients of similarity varying from 0.72 to 1.00 and were grouped into three main clusters  through phenetic analysis . ClusterPatients with samples grouped in cluster II were 9 years older than those in cluster III and presented a lower frequency of lesions on the upper limbs as compared with those with samples grouped in cluster I. Cluster III included a higher number of patients reactive to the IIF test as compared with cluster I. All samples in cluster I were isolated from patients who reported infection in Rio de Janeiro and responded well to the first treatment course. Cluster II included more patient samples from Rio de Janeiro than cluster III, but these clusters did not differ regarding frequency of responders or even the number of treatments . StatistPentavalent antimonials have been used to treat all clinical forms of leishmaniasis for the previous 70 years. Despite their efficacy, variability in responses has been reported, from clinical cure to the occurrence of therapeutic failures and relapses . StudiesThis study provided information on genetic polymorphisms in mitochondrial genomes (kDNA minicircles) from a panel of samples isolated from CL patients found to be R or NR to one course of treatment with meglumine antimoniate in Rio de Janeiro and other states in Brazil.Leishmania parasites can be used to determine possible correlations between different clinical forms, evolutionary patterns of the disease, and therapeutic responses [L. (V.) braziliensis parasites recovered from cutaneous lesions was evident, and three phenetic clusters were revealed. A previous study reported that switches in kDNA-minicircle dominance indicate that factors other than the amplified chromosomal DNA cause minicircle switching [Genetic polymorphisms in esponses ,12,14. Iwitching . Despitewitching .Leishmania(Leishmania) amazonensis selected in vitro for drug resistance have been reported [Extra-nuclear genomes evolve faster than nuclear genes, making kDNA a good target for evaluating intraspecies differences and discriminating species that have diverged more recently . The advreported . The domreported . Polymorreported . Such miL. (V.) braziliensis genotypes that circulates in Rio de Janeiro was discriminated in clusters I and III, showing phenotypes of good and poor responses to treatment, respectively. Therefore, we also established that parasites sharing all common genetic characteristics and belonging to the same phenetic cluster might be involved in different responses to treatment, as was the case in two patients from Rio de Janeiro (554 and 345). NR patients were prevalent in cluster III, given that most of them were isolated from Rio de Janeiro. Such results can be considered of clinical importance, because they allow genetic discrimination of distinct populations of L. (V.) braziliensis circulating in Rio de Janeiro and displaying different phenotypes.Although no specific genotype could be associated with response or non-response to treatment, at least one group of L. (V.) braziliensis [Leishmania spp. circulate, and greater genetic variability in L. (V.) braziliensis occurs [5+/kg/day. However, the NR group from Rio de Janeiro and other states included patients who required additional treatment with amphotericin B and pentamidine. Briefly, 81% of the NR patients (17 of 21) had their lesion healed after the second or third additional treatment with the low-dose regimen of meglumine antimoniate.Good response to antimony treatment in Rio de Janeiro might be explained by the genetic homogeneity observed in iliensis ,12. In oiliensis ,24. Howes occurs ,7,25. HoPatient age and lesion location(s) are among the factors that can interfere with the clinical course of the patient. Interestingly, we observed that the youngest group of patients placed in cluster III included a higher frequency of NR patients. Young age is considered to be the most solid predictor of treatment failure . Howeverp = 0.013), especially when compared with cluster I, where all were R patients. A strong immune response to cutaneous leishmaniasis that favors ultimate full recovery is characterized by the activation of T helper (Th)1 response, which stimulates the production of interferon-γ, tumor necrosis factor-α and interleukin (IL)-2 cytokines, and induces low production of antibodies [2 response is characterized by the secretion of IL-4, IL-10, and IL-13, which stimulate production of antibodies by B cells. This response is not efficient for infection control, and can cause susceptibility and persistence [It is also worth mentioning that patients presenting one cutaneous lesion, as observed in cluster I, might have influenced the good therapeutic response observed in this cluster . Additiotibodies . Howeversistence .It is important to highlight that the results obtained by LSSP-PCR can be considered to have clinical and epidemiological relevance. Further studies related to in vitro sensitivity of promastigotes and amastigotes to pentavalent antimonials are necessary to corroborate these findings.


This study has attempted to assess the effectiveness of quantitative faecal immunochemical tests (FIT) for triage of people presenting with lower abdominal symptoms, where a referral to secondary care for investigation of suspected colorectal cancer (CRC) is being considered, particularly when the 2-week criteria are not met.We conducted a systematic review following published guidelines for systematic reviews of diagnostic tests. Twenty-one resources were searched up until March 2016. Summary estimates were calculated using a bivariate model or a random-effects logistic regression model.Nine studies are included in this review. One additional study, included in our systematic review, was provided as ‘academic in confidence’ and cannot be described herein.n = 4091 participants, 176 with CRC), and the only study of HM-JACKarc to assess the 10 μg Hb/g faeces cut-off  reported a sensitivity of 100% (95% CI 71.5–100%). The corresponding specificity estimates were 85.8% (95% CI 78.3–91.0%) and 76.6% (95% CI 72.6–80.3%), respectively.When FIT was based on a single faecal sample and a cut-off of 10 μg Hb/g faeces, sensitivity estimates indicated that a negative result using either the OC-Sensor or HM-JACKarc may be adequate to rule out nearly all CRC; the summary estimate of sensitivity for the OC-Sensor was 92.1% , based on four studies  as well as CRC, the rule-out performance of both FIT assays was reduced.There is evidence to suggest that triage using FIT at a cut-off around 10 μg Hb/g faeces has the potential to correctly rule out CRC and avoid colonoscopy in 75–80% of symptomatic patients.PROSPERO 42016037723The online version of this article (doi:10.1186/s12916-017-0944-z) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is the third most common cancer in the UK population overall and in people aged 50 years and over, after breast and lung cancer for females and prostate and lung cancer for males. The Office for National Statistics (ONS) cancer registration data for 2013 showed approximately 35,000 new cases of CRC in England  [The UK has established bowel screening programmes with colonoscopy being offered following a positive faecal occult blood test using a guaiac test or qualitative faecal immunochemical test (FIT). Screening is offered to people between the ages of 60 and 74 years in England, Wales and Northern Ireland, and between the ages of 50 and 74 years in Scotland. Older people can opt to continue screening. Despite efforts to promote screening, the 2015 National Bowel Cancer Audit Report stated that, of all patients diagnosed with CRC in 2014, 55% were diagnosed following a referral by a general practitioner (GP), 9% (20% of those in the eligible age range for screening) were diagnosed through the National Health Service (NHS) Bowel Cancer Screening Programme and 20% were only diagnosed following an emergency presentation   have sugIn addition to the 2-week wait referral criteria, the 2015 version of the National Institute for Health and Care Excellence (NICE) guideline ‘Suspected cancer: recognition and referral’ NG12) recommended tests for occult blood in faeces in adults without rectal bleeding who are aged 50 years and over and have unexplained abdominal pain or weight loss; are aged under 60 years and have changes in their bowel habit or iron deficiency anaemia; or are aged 60 years and over and have anaemia in the absence of iron deficiency . These r recommenIt has been suggested that using quantitative FIT to select patients for referral has the potential to reduce unnecessary colonoscopies and provide more accurate classification of patients than traditional, symptoms-based guidelines . FIT is This systematic review analysed the clinical effectiveness of FIT for triaging referrals in people with lower abdominal symptoms, particularly those who would be considered to be at low risk of having CRC. The review was undertaken as part of a diagnostic appraisal to inform the development of new NICE diagnostics guidance (DG30) . The appWe conducted a systematic review with the main aim of summarising the evidence about the effectiveness of quantitative FIT for triage of people presenting with lower abdominal symptoms, where a referral to secondary care for investigation of suspected CRC is being considered, but the 2-week criteria are not met. Systematic review methods followed the principles outlined in the Handbook for Diagnostic Test Accuracy Reviews , the CenThe following databases were searched from inception to March 2016: MEDLINE; MEDLINE In-Process Citations and Daily Update; MEDLINE Epub Ahead of Print; Embase; Cochrane Database of Systematic Reviews (CDSR); Cochrane Central Register of Controlled Trials (CENTRAL); Database of Abstracts of Reviews of Effects (DARE); Health Technology Assessment (HTA) Database; NHS Economic Evaluation Database (NHS EED); International Network of Agencies for Health Technology Assessment (INAHTA); National Institute for Health Research (NIHR) Health Technology Assessment Programme; Aggressive Research Intelligence Facility (ARIF); PROSPERO. We also searched clinical trials registers  ClinicalTrials.gov, European Union (EU) Clinical Trials Register and World Health Organization (WHO) International Clinical Trials Registry Platform) and conference proceedings , Annual Meeting of the American Association for Clinical Chemistry and Laboratory Medicine (AACC), British Society of Gastroenterology (BSG) Annual Meeting, United European Gastroenterology Week (UEGW) and the European Congress of Clinical Chemistry and Laboratory Medicine (IFCC-EFLM), 2011–2015). Furthermore, we contacted experts in the field, with the aim of identifying any unpublished studies. Search strategies were based on index test (FIT assays) and target condition (CRC) and did not include any study design terms or filters [Diagnostic cohort studies, which assessed the accuracy of quantitative FIT assays in people with lower abdominal symptoms who were being investigated for suspected CRC, were eligible for inclusion.We included studies where the participant selection criteria were unclear, but where the population was described as symptomatic/suspected CRC and no asymptomatic participants were included. Where studies were conducted in mixed populations (both symptomatic and asymptomatic people included), study authors were contacted to request separate data for the symptomatic people sub-group. Studies conducted in people with pre-existing gastrointestinal tract co-morbidities were excluded.Only clinical evaluations of the following quantitative FIT assays, which are commercially available in the UK, were included: OC-Sensor ; HM-JACKarc ; FOB Gold ; Ridascreen .Included studies were required to confirm diagnosis using colonoscopy as the reference standard and to report sufficient data to determine the numbers of true positive (TP), false positive (FP), false negative (FN) and true negative (TN) test results. Where studies reported FIT uptake rates or test accuracy data for other target conditions, in addition to CRC , we also included these data in our review.Studies were screened for relevance independently by two reviewers, and full text articles of studies considered potentially relevant were assessed for inclusion by one reviewer and checked by a second. Disagreements, at either stage of study selection, were resolved through discussion and consensus, or by consultation with a third reviewer.One reviewer extracted data using a pre-study piloted data extraction form, and the extractions were checked by a second reviewer; any disagreements were resolved through discussion and consensus or by consultation with a third reviewer. Data were extracted on the following: study details, inclusion and exclusion criteria, participant characteristics , target condition , other significant bowel disease outcomes (as reported)), details of the FIT test , sampling procedure, detection method), details of the reference standard, definitions of the target conditions, test performance outcome measures  and proportion of study participants who returned a FIT sample (extracted as an indicator of acceptability).The methodological quality of included studies was assessed using Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) , which umetandi command. For analyses that would not run in Stata, we used Meta-DiSc [Sensitivity and specificity, with 95% confidence interval (CI), were calculated for each set of 2 × 2 data. The bivariate/hierarchical summary receiver operating characteristic (HSROC) model was used to estimate summary sensitivity and specificity with 95% CI and prediction regions around the summary points and to derive HSROC curves for meta-analyses involving four or more studies –25. Thiseta-DiSc .Studies were grouped by FIT assay type, by target condition and by cut-off f-Hb. Stratified results tables and ROC space plots are presented to illustrate the variation of test performance by cut-off f-Hb, and flow charts are provided to illustrate the progress of a hypothetical cohort of patients through a diagnostic work-up that includes triage using FIT at the optimal cut-off.The searches identified 5782 references; nine studies, reported in 26 publications –53, wereFive studies reported accuracy data for the OC-Sensor FIT assay ; one used the io analyser , one useAll studies included in this systematic review were diagnostic cohort studies (i.e. studies conducted in a group of patients in whom the presence of the target condition is suspected and which are therefore representative of the setting in which the test would be used in practice); diagnostic case-control studies  were excluded. The methodological quality of these studies was assessed using the QUADAS-2 tool . Two stu> 10 mm in diameter or adenomas with villous architecture or high grade dysplasia [All five studies that evaluated the OC-Sensor assay reported accuracy data, where CRC was the specified target condition , 32, 35.ysplasia , 31. Thrysplasia , 32, 35.The optimal test performance (maximising both sensitivity and specificity) appeared to occur with f-Hb cut-offs of 10 or 15 μg Hb/g faeces, with most data being available for the 10 μg Hb/g faeces cut-off. The summary estimates of sensitivity and specificity, using the 10 μg Hb/g faeces cut-off, were 92.1% (95% CI 86.9–95.3%) and 85.8% (95% CI 78.3–91.0%), respectively, based on data from four studies , 32, 35.Three studies reported separate accuracy data, using the 10 μg Hb/g faeces cut-off, for both CRC and the composite target condition AN , 29, 32.p = 0.041), iron deficiency anaemia (OR 2.99 (95% CI 1.27–7.03), p = 0.012) and f-Hb ≥ 10 μg Hb/g faeces (OR 86.60 (95% CI 11.70–641.16), p < 0.001) as independent predictors [p < 0.001), age (OR 1.36 (95% CI 1.13–1.63), p < 0.001) and f-Hb ≥ 10 μg Hb/g faeces (OR 7.54 (95% CI 5.03–11.28), p < 0.001) as independent predictors; age was treated as a categorical variable in this model  [One of the three studies described above also conducted multivariable analysis, using forward conditional logistic regression modelling, with the aim of identifying independent predictors of CRC and AN . The CRCedictors . The AN 0 years) . The res0 years) . The sco0 years) . The mod0 years) . In the 0 years) . We iden0 years) , which w0 years) . The log0 years) . The val0 years) , 33, 50,0 years) , 32, 33,Four studies reported information about uptake rates in participants invited to provide a sample for FIT , 31, 32.Two of the three studies that evaluated the HM-JACKarc assay reported accuracy data, where CRC was the specified target condition , 52. TheThe optimal test performance (maximising both sensitivity and specificity) appeared to occur at the f-Hb cut-off of 10 μg Hb/g faeces. The estimates of sensitivity and specificity at this cut-off, derived from a single study, were 100% (95% CI 71.5–100%) and 76.6% (95% CI 72.6–80.3%), respectively . Using aTwo studies reported information about uptake rates in participants invited to provide a sample for FIT , 52. TheOne study, reported in a conference abstract, assessed the performance of the FOB Gold FIT assay in symptomatic patients . This stAll studies included in our systematic review were diagnostic cohort studies reporting accuracy data. When FIT was based on a single faecal sample and an f-Hb cut-off of 10 μg Hb/g faeces, sensitivity estimates indicated that a negative result using either the OC-Sensor or HM-JACKarc may be considered adequate to rule out most CRC. The summary estimate of sensitivity for the OC-Sensor was 92.1% (95% CI 86.9–95.3%), based on four studies , 32, 35,Our study followed rigorous systematic review methodology, and our findings have informed the development of up-to-date guidance. The new NICE DG30 diagnostic guidance, now published, states: ‘The OC Sensor, HM-JACKarc and FOB Gold quantitative faecal immunochemical tests are recommended for adoption in primary care to guide referral for suspected colorectal cancer in people without rectal bleeding who have unexplained symptoms but do not meet the criteria for a suspected cancer pathway referral’ . LimitatSystematic reviews have previously been conducted which assessed the performance of various FIT assays in screening settings , 57. HowPopulation data indicate that f-Hb varies with age and sex, being higher in men and the elderly , 60. FurThe effects on FIT performance of using multiple faecal samples per patient remain unclear. One study  includedThe scope of our systematic review did not include evaluation of the performance characteristics of FIT when used in combination with other biomarkers. Two of the studies in our systematic review did compare FIT and calprotectin assays for detection of significant bowel disease and concluded that FIT alone had better rule-out performance , 52. ComThere is evidence to suggest that triage using OC-Sensor or HM-JACKarc FIT, at an f-Hb cut-off of 10 μg Hb/g faeces, has the potential to correctly rule out CRC and avoid colonoscopy in 75–80% of symptomatic patients. The relatively high proportion of FIT false positive results observed when the target condition is CRC may be mitigated by the potential to detect other bowel pathology in these patients. However, the importance of clinical judgement cannot be overemphasised. All of the studies included in this review were conducted in symptomatic populations selected on the basis of a GP’s intention to refer rather than on the presence of a specific set of symptoms alone; overuse of FIT or blanket referral following a positive result has the potential to overwhelm colonoscopy services. There are currently no data on the comparative performance of different FIT assays in this population. Given the trade-off between ease of use/simplicity and diagnostic performance, the clinical value of using additional variables (e.g. symptoms and further diagnostic tests) to develop risk scores for CRC and/or other significant bowel disease is likely to require further investigation.Additional file 1: Material S1.Full search strategies for MEDLINE and Embase. (DOCX 17 kb)Additional file 2: Table S1.Details of included studies and related publications. (DOCX 18 kb)Additional file 3: Table S2.Details of excluded studies with rationale for exclusion. (DOCX 37 kb)Additional file 4: Table S3.Data extraction tables. (DOCX 33 kb)Additional file 5: Table S4.PROBAST results for studies reporting the development and validation of risk scores that included FIT (DOCX 16 kb)Additional file 6: Table S5.Sub-group data for HM-JACKarc. (DOCX 19 kb)


Exposure to Formaldehyde (FA) results in many pathophysiological symptoms, however the underlying mechanisms are not well understood. Given the complicated modulatory role of intestinal microbiota on human health, we hypothesized that interactions between FA and the gut microbiome may account for FA’s toxicity. Balb/c mice were allocated randomly to three groups: a control group, a methanol group (0.1 and 0.3 ng/mL MeOH subgroups), and an FA group (1 and 3 ng/mL FA subgroups). Groups of either three or six mice were used for the control or experiment. We applied high-throughput sequencing of 16S ribosomal RNA (rRNA) gene approaches and investigated possible alterations in the composition of mouse gut microbiota induced by FA. Changes in bacterial genera induced by FA exposure were identified. By analyzing KEGG metabolic pathways predicted by PICRUSt software, we also explored the potential metabolic changes, such as alpha-Linolenic acid metabolism and pathways in cancer, associated with FA exposure in mice. To the best of our knowledge, this preliminary study is the first to identify changes in the mouse gut microbiome after FA exposure, and to analyze the relevant potential metabolisms. The limitation of this study: this study is relatively small and needs to be further confirmed through a larger study. The diverse populations of bacteria harbored in the human gastrointestinal tract are collectively referred to as the gut microbiome . At birtLactobacillus [The composition of the gut microbiota remains stable in the absence of any significant interference after evolving for several years after birth. However, some external factors such as antibiotics and toxic environmental chemicals can perturb the gut microbial community and induce dysbiosis ,17,18,19bacillus , arsenicbacillus , manganebacillus  and arsebacillus . However3, exceeding the national standard of 0.1 mg/m3 [Formaldehyde (FA), a toxic environmental chemical, is ubiquitously used in many industrial and consumer products. In recent years, the use of FA in China has surged dramatically, leading to severe environmental pollution. The average level of urban indoor air formaldehyde has reached 0.238 mg/m.1 mg/m3 . Many to.1 mg/m3 , neuroto.1 mg/m3 , genotox.1 mg/m3 , hepatic.1 mg/m3  toxicity.1 mg/m3 , and lat.1 mg/m3 . In view.1 mg/m3 ,28. TherA host of studies have been undertaken to elucidate the mechanisms underlying FA-induced cancers and other diseases, such as oxidative stress, DNA damage, chromosomal aberrations and apoptosis ,29,30. Ghttp://www.genome.jp/kegg/) and PICRUSt (http://picrust.github.io/picrust/) software programs [To test the hypothesis, we applied high-throughput sequencing of 16S ribosomal RNA (rRNA) genes to an experimental mouse model to investigate whether FA exposure can potentially alter the composition of the gut microbiota, and which specific types or classes or families of the microbiome are observed in those FA-caused landscape changes. Applying bioinformatic analyses with KEGG  with a 12 h light-dark cycle, and 55–75% humidity. To avoid coprophagia and other cross-contamination, each single mouse was housed in a separate cage. All experiments were approved by the Institutional Animal Care and Use Committee of Central China Normal University on 24 January 2016 (Ethics Ratification ID: CCNU-IACUC-2016-003). Balb/c mice were allocated randomly to three groups: unexposed control group , MeOH group , and FA group . Since this study is in the preliminary research stage and animal research ethics were also a cause of concern, groups of either three or six mice were used for the control or experimental groups [Balb/c mice  from the same parents were purchased from Hubei Research Center of Laboratory Animals . Before and throughout the experimental period, mice were provided with a standard pelleted rodent diet, filtered water Total DNA from the collected fecal pellets was extracted using the E.Z.N.A. DNA kit  according to the manufacturer’s instructions. All DNA samples were amplified by PCR using 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC to target the V3–V4 regions of the 16S rRNA gene of the gut microbiota. The DNA library was constructed via barcoding the resultant DNA according to Illumina standard protocol (see metadata). Sequencing was performed using an Illumina MiSeq platform  to generate 2X300bp pair-ended reads in the School of Life Science facilities of Central China Normal University.FLASH  was usedFor Alpha diversity and Beta diversity assays, the OTUs were rarefied to 29,276 reads per sample. Alpha diversities (chao1 richness and phylogenetic metrics), Beta diversities (Bray-Curtis matrix) and Principal Coordinate Analyses (PCoA) were analyzed using the QIIME and Vegan package in R program . Furthert-test, Two-sided) to reveal differences between the groups [Metagenomic content of the samples was predicted from the 16S rRNA profiles, and subsequently the KEGG pathways were categorized using the phylogenetic information by reconstructing the whole microbial community  . The mete groups .After FA treatment (at 1 and 3 ng/mL), the mice exhibited some signs of toxicity, such as weight loss and fatigue (data not shown).From the high-throughput sequencing of 16S rRNA gene, the mouse feces microbiome composition was profiled. Bacteroidetes  and Firmicutes  dominated the microbe taxa at the phylum level (Actinobacteria (0.3%), TM7 (0.3%), Cyanobacteria (0.1%), Deferribacteres (0.1%), and Tenericutes (0.1%). This result is similar to that of Dimova et al. [Clostridiales order (24.5%). The relative abundance of a bacterial genus (Turicibacter) was greatly reduced in the MeOH or FA treated samples.Distribution and alteration of the microbe taxa in the different groups of mice  were further analyzed. Regardless of the fecal sample considered, um level , followermicutes .7%, bluep-value = 0.02, permutations 999) (Beta-diversity measured by Bray–Curtis distance metrics showed significant differences between the treatments (ANOSIM test ons 999) , suggestProteobacteria and Actinobacteria) and decrease in the relative abundance of phylum Cyanobacteria were observed when mice were subjected to treatment of 3.0 ng/mL FA. Since decrease in the relative abundance of Cyanobacteria was also observed when mice were subjected to treatment of 1.0 ng/mL FA, it implied that Cyanobacteria were affected by the presence of FA in the mouse gut. An increase in the relative abundance of Proteobacteria was observed in the mice treated with 3.0 ng/mL FA when compared to the mice treated with 1.0 ng/mL FA.According to their relative abundance, members of the bacterial community can be categorized as: rare members (<0.1% in relative abundance), common members (between 0.1% and 1%), and abundant members (>1% in relative abundance)  Figure B. No sigShifts of fecal microbial composition were then examined at the genus level. Among the 55 genera detected in this study, 12 dominant genera exhibited average relative abundance over 1% , Panel APrevotella, Bacteroidales) associated with MeOH treatment, it was observed that there were significant increases in the abundance of thirteen genera and decreases in the abundance of four genera when comparing both FA treated samples (from 1 ng/mL FA to 3 ng/mL FA treated mice) with untreated samples. Among genera with increasing relative abundance, Prevotella is one of the dominant genera. Five genera  and an unidentified member of the Erysipelotrichaceae family are common members. Four genera  and two unidentified genera (from the Bacilliaceae family and the Mogibacteriaceae family) are rare members. The four genera with decreasing abundance upon FA treatment are Bacteroides and three unidentified genera . These genera include one dominant member (Bacteroides), one common member (from YS2 order), and two rare members .The shift of genera upon FA or MeOH treatment was analyzed by STAMP software . Figure The relative richness of the fecal samples from the FA or MeOH treated mice was further analyzed using heatmap illustrations . The taxActinobacteria, TM7, Firmicutes, Proteobacteria, and some unassigned bacteria. Firmicutes is the dominant phylum in this cluster. The other cluster includes Bacteroidetes, Tenericutes, Cyanobacteria, Deferribacteres. Bacteroidetes is the dominant phylum in this cluster. The families could be divided into four major clusters. The genera could be divided into seven major clusters. Based on the clustering analysis, we found that the MeOH or FA treated samples share higher similarity than those from untreated samples at the phylum, family and genus levels. Samples from MeOH groups shared higher similarity than samples from FA groups.The phyla could be divided into two major clusters. One cluster includes The functional capacity of the gut microbiome after FA or MeOH treatment was evaluated with PICRUSt software  based onAfter eliminating pathways that may be associated with MeOH treatment , it was Increases in the abundance of eight KEGG pathways and decreases in the abundance of five KEGG pathways were detected when comparing the 3.0 ng/mL of FA treated samples with the 1.0 ng/mL of FA treated samples or with the untreated samples. Although six KEGG pathways were detected when comparing untreated sample with FA treated sample, changes in seven KEGG pathways were observed between 3.0 ng/mL and 1.0 ng/mL FA treated samples. We suspected that these pathways were also associated with FA treatment.Associations among human diseases, gut microbiota and exposures to environmental pollutants (such as FA) have been previously reported ,11,26,27Although the physiological and anatomical structures of humans and mice are very similar, and the mechanisms of interactions between microbial communities and hosts are shared among most of these species, there are some key differences between mouse models and humans. For example, although 85% of the bacterial species found in mice do not exist in humans , a similFirmicutes and Bacteroidetes phyla dominated the fecal bacterial communities, which is consistent with previous studies [In this study, the  studies ,47. Alth studies .Although there have been many reports of FA inhalation and resultant symptoms, FA ingestion has only rarely been described in the literature. Hawley and Harsch  reportedPrevotella is one of the dominant genera in the mouse gut. Changes in Prevotella are related to the development of osteomyelitis. [Erysipelotrichaceae is observed to be associated with Major Depressive Disorder [Desulfovibrio is a genus of sulfate-reducing bacteria that stimulate gut immune responses and contribute to inflammation in experimental colitis [Dorea was reported with a remarkable change of increases in the relative abundance in stool microbiomes during a course of exclusive enteral nutrition in paediatric Crohn’s Disease [Adlercreutzia is capable of metabolizing isoflavones to equol [Anaeroplasma and Coprococcus are two genera occasionally reported in gut mirobiota [We found that there was an increased abundance of thirteen genera. Among these genera, yelitis.  ErysipelDisorder . Desulfo colitis . Dorea w Disease . Adlercrto equol . Anaeropirobiota . Candidatus Arthromitus is a group of morphologically distinct bacteria found almost exclusively in terrestrial arthropods as filamentous bacterial forms associated often with the gut wall [Delftia is a genus of Comamonas bacteria. There have been reports of isolation of the organism from serious infections like central venous catheter associated bacteremia, corneal ulcers and otitis media [Lactococcus is a genus of lactic acid bacteria that are able to produce lactic acid, the main, or only, product of glucose fermentation. Serratia belongs to the Enterobacteriaceae family. The bacterium is an opportunistic human pathogen and responsible for about 2% of nosocomial infections of the bloodstream, lower respiratory tract, urinary tract, surgical wounds, and skin and soft tissues in adult patients [gut wall . Delftiais media . Lactocopatients .Bacteroides are members of the human gut microbiota that confer myriad benefits on their hosts. These bacteria are involved in polysaccharide metabolism and utilization [Coriobacteriia are associated with human oral infections as well as severe blood bacteraemia and ulcerative colitis [Among bacteria with decreased relative abundance upon FA treatment, the lization . The Cor colitis ,68,69.The metabolic properties of gut microbiota were inferred from the PICRUSt predicted KEGG pathways. Increases and decreases have been observed in the abundance of many KEGG pathways, which may present a general response of gut microbiomes to environmental stress. Alpha-Linolenic acid (ALA) is an n-3 fatty acid. It is one of two essential fatty acids (the other being linoleic acid), so called because they are necessary for health but cannot be produced within the human body. It is interesting to understand the role of “alpha-Linolenic acid metabolism” in resistance to changes caused by FA exposure. Similarly, some cofactors and vitamins might be affected by treating mice with FA. Therefore, the metabolism of cofactors and vitamins might also be affected. Inositol phosphates are a group of mono- to poly-phosphorylated inositols. They play crucial roles in diverse cellular functions, such as cell growth, apoptosis, cell migration, endocytosis, and cell differentiation. Increases of the two KEGG pathways “Inositol phosphate metabolism” and “Transcription related proteins”, suggest that metabolisms relating to specific proteins might be improved. Naphthalene and Seleno compounds are both toxic. The increase in “Naphthalene degradation” and “Seleno compound metabolism” pathways implies that a detoxification pathway might be activated. The increase of “Ion channels” and “Other ion-coupled transporters” pathways implies there might be increased communication between cells and environments after FA treatment.Helicobacter. A decrease in “Antigen processing and presentation”, “Linoleic acid metabolism”, and “Lysine biosynthesis” pathways might be related to FA influencing normal metabolisms.A decrease in several KEGG pathways was also observed. A decrease in the pathway “Epithelial cell signaling in Helicobacter pylori infection” might be related to a decrease of richness of Disorders of the KEGG pathways colorectal cancer, “p53 signaling pathway”, “pathways in cancer”, “small cell lung cancer”, and “sulfur relay system” might be related to the carcinogenic mechanism of FA. Disorders of the KEGG pathways “hypertrophic cardiomyopathy (HCM)”, “Iifluenza A”, “viral myocarditis”, “african trypanosomiasis”, “toxoplasmosis”, “chagas disease (American trypanosomiasis)”, “insulin signaling pathway”, and “renin-angiotensin system” might explain the serious adverse health effects caused by FA exposure. Disorders of the KEGG metabolism pathways “biosynthesis of unsaturated fatty acids”, “D-alanine metabolism”, “ether lipid metabolism”, “protein folding and associated processing”, “steroid biosynthesis”, “arachidonic acid metabolism”, and “drug metabolism—other enzymes” indicated that FA treatment might affect a variety of metabolisms and result in complicated damage to the host. More investigations are needed to address the disorders of “progesterone-mediated oocyte maturation”, “transcription machinery” and “photosynthesis—antenna proteins” pathways.The limitation of this study: the low number of animals makes statistical analysis less robust, except in the case of phylum level variations. We need further confirmation from a larger study.This study explored FA-induced alterations to the mouse gut microbiome using the high-throughput sequencing of 16S rRNA gene and metagenomics approaches. Alterations were observed in bacterial richness and diversity of the fecal microbiota after exposure. However, the relative richness was significantly affected in the abundance of several bacterial genera and families as well as several PICRUSt predicted KEGG pathways. The affected genera included abundant members as well as rare members in the bacterial community. A number of KEGG pathways including detoxification and signaling pathways might be affected by FA exposure.


Minimally invasive pancreatoduodenectomy (MIPD) has been gradually attempted. However, whether MIPD is superior, equal or inferior to its conventional open pancreatoduodenectomy (OPD) is not clear.Studies published up to May 2017 were searched in PubMed, Embase, Cochrane Library, and Web of Science. Main outcomes were comprehensively reviewed and measured including conversion to open approach, operation time (OP), estimated blood loss (EBL), transfusion, length of hospital stay (LOS), overall complications, postoperative pancreatic fistula (POPF), delayed gastric emptying (DGE), post-pancreatectomy hemorrhage (PPH), readmission, reoperation and reasons of preoperative death, number of retrieved lymph nodes (RLN), surgical margins, recurrence, and survival. The software of Review Manage version 5.1 was used for meta-analysis.P < 0.01), lower transfusion rate , shorter LOS . There was no significant difference in time to oral intake, postoperative complications, POPF, reoperation, readmission, perioperative mortality and number of retrieved lymph nodes.One hundred studies were included for systematic review and 26 out of them  were included for meta-analysis. In the early years, most articles were case reports or non-control case series studies, while in the last 6 years high-volume and comparative researches were increasing gradually. Systematic review revealed conversion rates of MIPD to OPD ranged from 0% to 40%. The mean or median OP of MIPD ranged from 276 to 657 min. The total POPF rates vary between 3.8% and 50% observed in all systematic reviewed studies. Meta-analysis demonstrated MIPD had longer OP , lower blood loss  contains supplementary material, which is available to authorized users. Gagner & Pomp  describeSystematic searches of PubMed, Embase, Cochrane Library, and Web of Science were performed to identify articles published up to May 2017. Strategies included the terms “minimally invasive”, “laparoscopic”, “laparoscopy”, “robot”, “robotic”, “Da Vinci”, “pancreaticoduodenectomy”, “Whipple”, “PD” and “pancreatic resection” and “pancreatic surgery”. The language of the publications was confined to English. References were searched for studies not found through the initial search.The inclusion criteria for systematic review were prospective or retrospective case series studies assessing surgical outcomes of MIPD, and comparative studies of MIPD and OPD, but case reports were excluded. Abstracts, letters, comments, conference proceedings, reviews were also excluded. Studies included more than 20 cases of MIPD were used for summarizing perioperative the relevant information. Studies included in the meta-analysis fulfilled the following criteria: (1) compared the outcomes of MIPD and OPD; (2) reported on at least one of the outcomes of interest mentioned below; (3) had adequate raw data reported such that we could extract the number or percentage of events for categorical outcomes; (4) continuous variables should both present means and standard deviations (SDs). We did not estimate the means and SDs from medians and ranges or inter-quartile ranges as the method of Hozo et al. , becauseFirst, two investigators (Chen K and Pan Y) independently tabulated the data. A double-check procedure was performed to make sure of the accuracy of the data extracted. Then, a manager inputted the extracted data into a spreadsheet. The measured outcomes of all eligible publications can be divided into four categories: ① Study baseline: author, geographical region, facility, journal, publishing year, study period, sample volume, study subject, indications and surgical procedure; ② Intraoperative effects: conversion to open approach, operation time (OP), estimated blood loss (EBL), transfusion; ③ Postoperative recovery: time to first flatus or restart oral intake, length of hospital stay (LOS), morbidity and mortality ; ④ Oncologic outcomes: retrieved lymph nodes (RLN), surgical margins, recurrence, and survival. Relied on the complication reporting system of Memorial Sloan-Kettering Cancer Center , postopeP < 0.05 was deemed as statistically important.The risk ratio (RR) was utilized to analyze the dichotomous variables and the weighted mean difference (WMD) was utilized to assess the continuous variables. Based on Der Simonian and Laird approach, the random-effects model was utilized so as to account for clinical heterogeneity due to both sampling variability and other factors such as differences in surgical skill and the numbers of procedures carried out by a surgeon. According to the overall POPF rate, the bias of potential publication was determined by carrying out informal visual inspection of funnel plots. The software of Review Manage version 5.1 (RevMan 5.1) that was downloaded from Cochrane Library was used to conduct data analysis. n = 3) [n = 54), followed by Japan (n = 20), China (n = 19) . The relevant literature was very scarce prior to 2004 (n = 3) . After tThe majority of papers were single center research based on their initial experience with short-term surgical outcomes. Thirteen studies reported mid- or long-term postoperative results , 96, 107Of these 37 cohort studies, which have compared MIPD and OPD with perioperative outcomes, four studies were excluded from the meta-analysis due to overlap patient cohorts , 85, 107A total of 3402 patients were included in the analysis with 1064 undergoing MIPD 31.3%) and 2338 undergoing OPD 68.7%). Total sample size for included studies ranged from 16 to 1239, with patient numbers in the MIPD arms ranging from 8 to 211. They represented an international experience . Because MIPD was still much less performed than OPD in many centers, approximately, half of the included studies used case-match comparative analysis [.3% and 2.7%. TotaThe number of surgeons who performed robot-assisted surgery was reported in 80.8% of the included studies 21/26). Most of the included studies represented the experience of single surgeon or term     Fig. . Another01) Fig. . The mea01) Fig. . The met01) Fig. . Another01) Fig. . Transfu02) Fig. . The intP < 0.01). Two studies reported the times of analgesic injection to evaluate the postoperative pain, and the pooled-data showed less analgesic use of MIPD than OPD . Besides, Wang et al. [P = 0.08). Tan et al. [P = 0.01). Other two studies only revealed analgesic usage tended to favor the MIPD group but was not statistically significant [P < 0.01) and Chen et al. [P < 0.01) after LPD. As for quantitative meta-analysis, only three studies with data available reported the postoperative time to restart oral intake. Analysis of the pooled data revealed early time to oral intake in MIPD group, but the difference did not reach statistically significant . Forty-nine of systematic reviewed studies reported on mean or median length of hospital stay, which ranged from 6 to 25 days, respectively, and the recorded length ranged from 3 to 118 days . We summarized the specific postoperative surgical and medical complications based on all reviewed researched in Table P = 0.25) , which was defined as grade B and above according to the ISGPF. The meta-analysis also did not find any statistically significant differences between groups regarding other complications, such as DGE and PPH. Fourteen studies reported reoperation cases, and there was no significant difference in reoperation rate . The specific reoperation reasons observed in the meta-analysis included studies are summarized in Additional file P = 0.13). The overall perioperative mortality rates range from 0%–6.9% in all systematic reviewed studies . The specific death reasons in the meta-analysis studies are summarized in Additional file The majority of the included studies reported on adverse events with a range of 18.2%-87.5% for MIPD and 8.0%–92.9% for OPD. According to the results of the meta-analysis, though the incidence of postoperative complications tended to favor the MIPD group, but there was not statistically significant difference  Fig. , as was P = 0.04). Survival status is the most critical outcome for evaluating surgical interventions in oncological therapy. However, limited long-term survival outcomes are available from this review. We summarized the information available about long-term survival outcomes in Table P = 0.06). Five studies reported comparable survival time or rate between two groups [The mean or median number of retrieved lymph nodes ranged from 6 to 32 observed in systematic review studies , with significant heterogeneity mainly due to different methods of EBL. Therefore, the results should be interpreted prudently and need further study to confirm.The substantial risk of perioperative mortality and morbidity remains a major concern to limit the development of MIPD. Although surgical safety had been improved gradually mainly related to centralization, the conventional OPD was still associated with postoperative morbidity rates of 23% to 66% and mortality rates of 3% to 5% even in high-volume centers –114. TheAs for postoperative recovery, the pooled data demonstrates mixed outcomes with less time spent in ICU and less usage of analgesic in MIPD group than OPD group, while only moderate earlier time to oral intake in the MIPD group without significant difference, indicating MIPD likely enhanced the recovery of intestinal function. Reduced use of analgesic drugs, shortened time of abdominal cavity exposure, and earlier postoperative activities are considered to be the main reasons for earlier gastrointestinal recovery from MIPD.As the removal of a sufficient amount of lymph nodes can enhance the accuracy of staging and regional disease control, appropriate lymphadenectomy is one of the most crucial steps in radical pancreatic surgery. The mean or median number of retrieved lymph nodes ranged from 6 to 32 observed in systematic review studies and the quantitative meta-analysis showed no significant difference between MIPD and OPD groups. It is well-documented that only about 20% eligibility rate for surgery among patients with pancreatic cancer when diagnosed. It has also been suggested that even patients who are eligible for surgery preoperatively cannot be offered potentially curative resection mainly due to vascular invasion , 121. BeOur researches have some limitations as below: (1) No RCTs were included: All the evidence was derived from observational studies with either concurrent or historical controls, and the use of some historical control groups that may be inadequately matched to the intervention group and because of poor or selective outcome reporting. (2) Selection bias: Most hospitals are still in the learning curve now and during that phase only the most ideal patients are selected. (3) Clinical heterogeneity: The variety of postoperative management, discharge criteria and application of fast track protocol et al. will lead to clinical heterogeneity and a sequence of biases. (4) Lacking long-term outcomes: large studies on the long-term outcomes, such as oncological efficiency, especially for long-term survival and quality of life (QOL) are scarce.Our systematic review and meta-analysis demonstrates that MIPD is technically feasible and safety, especially in experienced hands. MIPD alleviates surgical traumas and enhanced postoperative recovery. Account for the inherent limitations of our study, our findings should also be interpreted with cautions. Robust prospective comparative studies and randomized clinical trials are awaited to bring more high quality evidences.Additional file 1:Number of original publications concerning MIPD by year according to the study design . (TIFF 56 kb)Additional file 2:Number of total publications concerning MIPD by year according to the place of origin . (TIFF 67 kb)Additional file 3:Published articles of MIPD. (DOCX 52 kb)Additional file 4:Baseline and quality assessment based on the NOS for meta-analysis included studies. (DOCX 30 kb)Additional file 5:Summary of indications and laparoscopic technical details in the meta-analysis included studies. (DOCX 26 kb)Additional file 6:Summary of perioperative outcomes in reviewed studies with more than twenty cases of MIPD. (DOCX 37 kb)Additional file 7:Summary of reasons for conversion. (DOCX 22 kb)Additional file 8:Summary of perioperative outcomes in studies without quantitative data presenting mean and standard deviation. (DOCX 24 kb)Additional file 9:Summary of the specific reoperation reasons. (DOCX 22 kb)Additional file 10:Summary of the specific perioperative death reasons. (DOCX 23 kb)


Importantly, acupuncture reversed the downregulation of PKA-α and p-CREB. The expression of PKA-α was upregulated by fluoxetine, but not p-CREB. No significant difference was found between Acu and FLX groups on the expression of PKA-α and p-CREB. Interestingly, H89 inhibited the effects of acupuncture or fluoxetine on upregulating the expression of p-CREB, but not PKA-α. There was no significant difference in expression of CREB among the groups. Conclusively, our findings further support the hypothesis that acupuncture could ameliorate depressive-like behaviors by regulating PKA/CREB signaling pathway, which might be mainly mediated by regulating the phosphorylation level of CREB.Protein kinase A (PKA)/cAMP response element-binding (CREB) protein signaling pathway, contributing to impaired neurogenesis parallel to depressive-like behaviors, has been identified as the crucial factor involved in the antidepressant response of acupuncture. However, the molecular mechanisms associated with antidepressant response of acupuncture, neurogenesis, and depressive-like behaviors ameliorating remain unexplored. The objective was to identify the mechanisms underlying the antidepressant response of acupuncture through PKA signaling pathway in depression rats by employing the PKA signaling pathway inhibitor H89 in in vivo experiments. Our results indicated that the expression of hippocampal PKA- Depressive disorder is a common mental disorder which has been affecting millions of people worldwide . The maiPresently, antidepressant drugs  are the major treatment for depression and have been widely used for the treatment of depression in the clinic. Fluoxetine, one of the antidepressant drugs characterized by a selective serotonin (5-hydroxytryptamine (5-HT)) reuptake inhibitor (SSRI), has been approved by the Food and Drug Administration (FDA) to treat stress-related disorders (including depression and anxiety) in patients . AntidepThe precise contributing factors and mechanisms of depression are still unknown. The precise pathogenesis and etiology of depression has been a challenging issue recently. Nowadays, the involvement of alterations concerning immune response and inflammatory response in the pathophysiology of depression and stress has been evidenced by various studies –16. NumeDuring our previous studies, we have been focusing on investigating the clinical effects and mechanisms of acupuncture on depression. We found distinct abnormalities in regional brain activity , 28. Morα, CREB, and p-CREB were assessed. We aimed to elucidate the molecular mechanisms underlying the antidepressant response of acupuncture and shed new light on conceptual frameworks of prospects for new therapies in the treatment of depression.Here, we established a rat model of depression induced by CUMS and assessed the difference in antidepressant effect between acupuncture and fluoxetine. Importantly, H89, a moderately specific inhibitor for PKA , 32, wasAdult 6-week-old male Sprague-Dawley (SD) rats, weighing 220 ± 20 g, were obtained from Weitong Lihua Experimental Animal Center of Beijing, China. Rats were housed in a quiet room with a controlled environment of 23°C–26°C and 50% ± 10% humidity. The rats subjected to CUMS were housed separately in different cages for social isolation, and 5 animals per cage were housed for rats in the control group. All experimental procedures were in full observance of the Bioethical Committee of the Institute of Animal Care Committee, Beijing University of Chinese Medicine, Beijing, China (permit no. Kj-dw-32-20150612).μM, 5 μl) were administered in Acu + H89 and FLX + H89 groups 60 minutes before the CUMS procedure, once every other day, and then, acupuncture stimulation and intragastric administration of fluoxetine were conducted, respectively (The body weight (BW), sucrose preference test (SPT), and open-field test (OFT) were investigated to guarantee the consistency of baseline characteristics before the experimental procedure was conducted. Five rats were excluded due to the inconsistent baseline characteristics. Then, a total of 60 rats under the circumstance of similar baseline characteristics of BW, SPT, and OFT were assigned into control, model, model + acupuncture (Acu), model + fluoxetine (FLX), model + acupuncture + H89 (Acu + H89), and model + fluoxetine + H89 (FLX + H89) groups at random, with 10 rats in each group. All rats were exposed to social isolation and CUMS for 21 days excluding rats in the control group. Thirty minutes before CUMS procedure, the rats in the Acu group were acupunctured at Baihui (GV 20) and Yintang (EX-HN 3); the rats in the FLX group were administered with fluoxetine (0.18 mg/ml) by gavage (1 ml/100 g). Intracerebroventricular injections of the PKA signaling pathway inhibitor H89  cannulae implanted 7 days before the experimental design as described by previous studies , 35. RatμM), 5 μl, was infused bilaterally into the encephalocele at a rate of 0.25 μl/min for 2 min, once every two days. Then, the infusion probes were left in place for an additional 2 min to allow solutions to diffuse away from the probe tips. After redisinfection with 75% alcohol, the ICV cannulae were closed with a dummy cannula.In the microinfusion experiments, the ICV cannulae were strictly disinfected with 75% alcohol. Microinfusion probes were gently inserted through the guide cannulae. The solution of PKA inhibitor H89  were inserted transversely (keeping the angle between the needle and the skin surface at 15°) into Baihui (GV 20) and Yintang (EX-HN 3)  was injected into the lateral ventricle. After being exposed to free activity for 4 h, rats were deeply anesthetized with 10% chloral hydrate  and then perfused intracardially with 100 ml of 0.9% sodium chloride solution followed by 200 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Then, rats were decapitated, and the brains were removed and placed into 4% paraformaldehyde in 0.1 M PBS for 72 h at 4°C. The brains were transferred to 20% and 30% sucrose solutions for dehydration at 4°C. The morphology of lateral ventricle was detected and visualized under a laser confocal scanning microscope  through frozen sections (8 μm) .To verify the effectiveness and validity of ICV injection of H89 in rats in the Acu + H89 and FLX + H89 groups, another 6 rats were subjected to ICV catheterization and ICV injection of 1% Evans blue. Briefly, rats that received posttraining infusions of 0.9% sodium chloride solution after successful ICV catheterization were returned to the holding cage for better recovery for 7 days from surgery. Then, 0.1% Evans blue , sucrose preference test (SPT), and open-field test (OFT) were investigated at least 12 hours after the stress stimulation at the end of experimental period. Mood states, quality of feces, and appetite of rats were observed.The changes in body weight gain in comparison to the baseline were calculated to evaluate the states of food preference and nutrition status. Body weight was detected on day 0 and day 21 for each rat throughout the experimental procedures.Locomotor activity of each rat was detected through open-field test (OFT) as illuminated by previous studies , 39. TheReferring to investigations of recent studies , 33, sucα , p-CREB , CREB , and GAPDH  at 4°C overnight. Following incubation with the primary antibody, the membranes were incubated with goat anti-rabbit HRP-conjugated IgG  at room temperature for 60 min. The bound antibodies were visualized using an enhanced chemiluminescence reagent by ECL kit  and quantified densitometrically using Gel-image analyzing system . The experiments were performed in triplicates with triplicate samples. The mean optical density value of each protein band relative to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band from the same sample was calculated.Following the last experimental procedure, the rats were sacrificed and decapitated. The brains were removed quickly, and the hippocampus was isolated and stored at −80°C for the next process. The samples were homogenized with RIPA lysis buffer, containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.5% Na deoxycholate and protease inhibitor cocktail (or phosphatase inhibitor cocktail for phosphorylated protein) for protein extraction. And then, the supernatant was collected following centrifugation at 13,000 rpm at 4°C for 20 minutes. The total protein content was determined by using bicinchoninic acid (BCA) assay. Following the quantitative determination of the total protein content, the proteins of each sample were denatured at 100°C for 5 min and fractionated through 10% SDS polyacrylamide gel electrophoresis (PAGE). The proteins of samples were electrotransferred onto polyvinylidene difluoride membranes with voltage at 80 V for 60 min. The membranes were blocked with 5% bull serum albumin- (BSA-) TBST for 1 h at room temperature. Protein expression was subsequently detected by incubation with rabbit polyclonal primary antibodies against PKA-α, CREB, and p-CRE were analyzed by a one-way analysis of variance (ANOVA) test. Differences between individual means were tested for significance using Fisher's least significant difference (LSD) or Tamhane's T2 procedure. The horizontal and vertical motion scores were analyzed by the Kruskal-Wallis H test. Probability values less than 0.05 were considered greatly significant.All data were statistically analyzed by SPSS 22.0 software  and expressed as the mean ± standard deviation     Tables . Both acP < 0.01; P < 0.01), which might be due to the anesthetization during ICV catheterization surgery disturbing the regular diet order. After 7 days of recovery from surgery, the baseline characteristic of behaviors of rats in the Acu + H89 and FLX + H89 groups kept pace with the others. Then, the BW, SPT, and OFT of pre-experiment versus postexperiment were observed. The results showed that, compared with the Acu group, the gain in body weight and the sucrose intake were significantly reduced , and the times of horizontal and vertical motion scores were notably reduced in the Acu + H89 group, all with statistical significance (P < 0.05 and P < 0.01)   (α induced by CUMS. No significant difference was found between acupuncture and fluoxetine groups (P > 0.05). Similarly, no significant differences were found between the Acu group versus Acu + H89 group and the FLX group versus FLX + H89 group .The results showed that, compared with control group, the expression of hippocampal PKA- < 0.01) . Followi < 0.05) . Both acP < 0.01)  (P < 0.01). While the expression of p-CREB in the fluoxetine group presented a trend of escalation in comparison with the model group (P > 0.05). No significant difference was found between the acupuncture and fluoxetine groups. The expression of p-CREB was notably downregulated between the Acu group versus Acu + H89 group and the FLX group versus FLX + H89 group  . Similar < 0.01) . By cont89 group .The present study aimed to identify molecular and neurobiological mechanisms responsible for antidepressant response following the treatment of acupuncture. A rat model of depressive disorder induced by CUMS was established. Open-field test (OFT), sucrose consumption, and body weight were employed to evaluate the depression-relevant behaviors, including the ability to adapt to new environments, the sensitivity to reward stimulation and pleasure, or mainly the state of anhedonia. Our findings indicated that the rats subjected to CUMS were observed to exhibit obviously poor appetite and significantly slow increase in body weight, the adaptive regression to new environments, the stagnancy to reward stimulation and pleasure, or altered mood switching from irascibility to low spirits throughout the CUMS procedure. The result of the present study is consistent with the current study illustrated that CUMS could induce depressive-like behaviors, effectively imitating the symptoms of depression in the patients , 41.α and p-CREB, induced by CUMS, were reversed by acupuncture. What is more, the upregulated expression level of p-CREB, but not PKA and CREB, was inhibited by H89, indicating that the increased p-CREB expression (phosphorylation of CREB) might be partly attributable to the increased activation of PKA. Although no significant difference was found in acupuncture versus fluoxetine, concerning inhibiting the phosphorylation of CREB (expression of p-CREB) by the PKA inhibitor H89, it is notable that the antidepressive effects of acupuncture on alleviating sucrose intake level and the horizontal motion scores were more compelling than fluoxetine following the administration of H89. All these results might indicate that the antidepressive response of acupuncture is not just dependent on PKA/CREB signaling, which is identical with our previous studies [Acupuncture showed compelling antidepressant effects on ameliorating depression-related behaviors. Importantly, PKA, the CREB upstream regulator, and the phosphorylation of CREB on Ser133 by PKA, p-CREB, also showed a strain-dependent expression pattern. Although PKA and p-CREB expression levels were upregulated following the treatment of acupuncture and fluoxetine, inhibition of PKA-CREB signaling by H89 reversed the upregulation of p-CREB expression level (but not PKA and CREB) and the antidepressant effects on ameliorating depression-related behaviors of acupuncture and fluoxetine, suggesting that both acupuncture and fluoxetine could achieve the antidepressant effects by promoting the phosphorylation of CREB on Ser133 by PKA-CREB signaling. Other studies concerning the CREB signaling have shed light on the potentially promoting effects on neurogenesis implicated in cognitive behaviors or synaptic plasticity function involved in antidepression –44. In t studies .Acupuncture, one of the conventional therapies in traditional Chinese medicine (TCM), contributes to therapeutic effects by regulating the nervous, endocrine, and immune systems  and playThe present study has been evidenced that the activation of phosphorylation of CREB through the strain-dependent PKA/CREB signaling exhibits compelling antidepressant responses to acupuncture. Further studies will focus on the expression of BDNF and the downstream effector of PKA/CREB signaling and other signaling commonly responsible for individual differences in antidepressant responses between antidepressants and acupuncture, which in turn will elucidate the underlying mechanisms concerning the antidepressant response of acupuncture and explore new prospects of integrated medicine in the treatment of depression.The authors declare that they have no competing interests.


The triglyceride glucose (TyG) index has been suggested as a simple surrogate marker of insulin resistance. However, there are limited data regarding the association between the TyG index and arterial stiffness in adults. Therefore, we evaluated the relationship between the TyG index and arterial stiffness as measured based on brachial ankle pulse wave velocity (baPWV) in Korean adults.A total of 3587 subjects were enrolled in this study. Anthropometric and cardiovascular risk factors were measured. The TyG index was calculated as ln[fasting triglycerides(mg/dl) × fasting glucose(mg/dl)/2], and the insulin resistance index of homeostasis model assessment (HOMA-IR) was estimated. Arterial stiffness was determined by measuring baPWV.The subjects were stratified into four groups based on the TyG index. There were significant differences in cardiovascular parameters among the groups; the mean baPWV increased significantly with increasing TyG index. According to the logistic regression analysis after adjusting for multiple risk factors, the odds ratio (95% CI) for increased baPWV (> 75th percentile) for the highest and lowest quartiles of the TyG index was 2.92 (1.92–4.44) in men and 1.84 (1.15–2.96) in women, and the odds ratio for increased baPWV for the highest and lowest quartiles of the HOMA-IR was 1.80 (1.17–2.78) in men and 1.46 (1.06–2.47) in women, respectively.The TyG index is more independently associated with increased arterial stiffness than HOMA-IR in Korean adults. Cardiovascular disease (CVD) is a leading cause of death worldwide . In partInsulin resistance (IR) is one of the most important risk factors for CVD . IR is aMany recent studies have shown that the triglyceride glucose (TyG) index is associated with IR, as assessed by hyperinsulinemic euglycemic clamp testing and HOMA-IR. Thus, the TyG index has been proposed as a reliable and simple surrogate marker of IR –10.Consistent with these data, there is growing evidence to suggest that the TyG index is associated with cardiovascular disease –13. HoweThe study population consisted of 5989 Korean subjects who participated in a comprehensive health examination as part of a self-referred health checkup program at the Gangnam Severance Hospital Health Promotion Center from January 2008 to February 2015. The exclusion criteria of this study were: subjects with elevated triglyceride levels (> 400 mg/dl) and subjects with any history of malignancy, acute inflammatory disease, infectious disease, or renal disease. We also excluded subjects with a history of hypertension, angina, myocardial infarction, cerebrovascular accident, or an ankle–brachial index (ABI) < 0.90. Subjects taking statins or triglyceride-lowering medications (fenofibrate or omega-3) were also excluded. In addition, we excluded men and women with a history of alcohol consumption in excess of 30 and 20 g/day, respectively. After applying the exclusion criteria, a total of 3587 subjects were enrolled in our final analysis. The study protocol was approved by the Institutional Review Board of Yonsei University College of Medicine.2). Lifestyle information, personal medical history of acute and chronic illnesses, and medication history were collected using a standard questionnaire. Systolic and diastolic blood pressures  were measured by experienced technicians using an automated blood pressure (BP) monitor  after a 5-min rest period with the patient’s arm placed at heart level. The diagnosis of diabetes mellitus was based on a previous history of diabetes or the ADA’s diagnostic guidelines. Hypertension was defined as systolic and/or diastolic blood pressures > 140/90 mmHg or the current use of an antihypertensive medication. Current smokers were defined as having smoked cigarettes regularly over the previous 6 months.We measured the height and weight of each study participant and calculated the body mass index , total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were determined using enzymatic methods with a Hitachi 7600–120 automated chemistry analyzer . Low-density lipoprotein cholesterol (LDL-C) was calculated according to the Friedewald formula. The TyG index was calculated as ln[fasting triglycerides (mg/dl) × fasting glucose (mg/dl)/2] [Arterial stiffness was measured using an automatic plethysmographic instrument  as previously described . BrieflyContinuous variables with normal distributions were expressed as the mean ± standard deviation (SD), whereas continuous variables with skewed distributions were expressed as the medians with interquartile ranges and were log transformed for analysis. Intergroup comparisons were performed using ANOVA. Chi square tests were used to compare categorical variables with percentages. Age-adjusted baPWV means and standard errors were calculated using analysis of covariance (ANCOVA) according to TyG quartiles. The relationships between the baPWV and various clinical parameters were examined using Pearson’s correlation. The odds ratios (OR) and corresponding 95% confidence intervals (CI) for high PWVs were estimated by multivariate logistic regression analysis after adjusting for confounding variables across TyG index and HOMA-IR quartiles. Covariates in the multivariable model, which were chosen for their clinical importance, as well as statistical significance, were age, SBP, BMI, LDL-C, HDL-C, diabetes mellitus, and menopause in women. Because an absolute cut-off value of normal baPWV was not available, high baPWV was arbitrarily defined as a value greater than the cut-off level between the third and fourth quartiles (> 75th percentile), which was 1437.5 cm/s for men and 1411.5 cm/s for women. All statistical analyses were performed using SPSS for Windows 23.0 . P values less than 0.05 were considered statistically significant.Table We found that baPWV was positively associated with increasing TyG quartiles. Figure Pearson’s correlation analysis was used to examine the relationship between the baPWV and clinical variables. The baPWV was significantly correlated with age SBP, DBP, BMI, TC, LDL-C, HDL-C, TG/HDL-C, LDL-C/HDL-C, non HDL-C HOMA-IR, and the TyG index Table .Table 2CThe association between the TyG index and high baPWV was further explored by categorizing the TyG index levels into quartiles and using the first quartile as a reference. Based on age-adjusted multivariable logistic regression analysis, with Q1 set as the reference, the TyG index levels for Q2, Q3, and Q4 were associated with increased ORs for high baPWV in all subjects. Both in men and in women, this relationship remained statistically significant after adjusting for confounding variables. In addition, higher HOMA-IR quartiles were significantly associated with increasing baPWV, and this relationship remained significant even after adjusting for metabolic variables. After adjusting for multiple risk factors, the OR (95% CI) for increased baPWV in the highest HOMA-IR quartile was 1.80 (1.17–2.78) in men and 1.46 (1.06–2.47) in women, whereas the OR of the TyG index in the highest quartile was 2.92 (1.92–4.44) in men and 1.84 (1.15–2.96) in women, respectively (Table In this study, we identified a significant association between the TyG index and baPWV as a marker of arterial stiffness. To the best of our knowledge, this is the first study to demonstrate such a relationship between the TyG index and arterial stiffness in the general population. Furthermore, our results showed that the TyG index is independently and more strongly associated with increased arterial stiffness compared to HOMA-IR after adjusting for metabolic risk factors.The TyG index which is a surrogate marker of IR, is known to be associated with metabolic parameters and CVD , 13. FurA few reports have investigated whether the TyG index can serve as an independent predictor of subclinical atherosclerosis in both the general population as well as in diabetic subjects , 14, 15.Although the mechanism underlying the relationship between TyG index and arterial stiffness has not been fully elucidated, it may be linked to IR. Indeed, previous studies have demonstrated that HOMA-IR, which reflects insulin resistance, is related to PWV –24. BothOur study had several limitations. First, this was a cross-sectional observational study. Thus, a causal relationship cannot be established based on the results of this study. Second, the study population consisted of Korean men and women enrolled at a single institution, and the proportions of young adults and pre-menopausal women were relatively small. Thus, the generalizability of the results may be limited. Lastly, we were unable to obtain histories of alcohol consumption and smoking in a quantitative manner, and we could not adjust for nutritional and exercise habits, which can affect blood triglyceride levels.We demonstrated a significant association between the TyG index and baPWV as a marker of arterial stiffness in both sexes. Moreover, the TyG index was found to be independently associated with increased arterial stiffness to a greater degree than was HOMA-IR. As a marker of insulin resistance, the TyG index is a simple to calculate, and it appears to be a useful marker of arterial stiffness and thus reflective of cardiovascular risk. Further prospective large-scale studies will be needed to elucidate the exact mechanism of the relationship between the TyG index and arterial stiffness.


Dracaena plants, and has been widely used as a traditional medicine in East and Southeast Asia. The major components of dragon’s blood are flavonoids. Owing to a lack of Dracaena plants genomic information, the flavonoids biosynthesis and regulation in Dracaena plants remain unknown. In this study, three cDNA libraries were constructed from the stems of D. cambodiana after injecting the inducer. Approximately 266.57 million raw sequencing reads were de novo assembled into 198,204 unigenes, of which 34,873 unique sequences were annotated in public protein databases. Many candidate genes involved in flavonoid accumulation were identified. Differential expression analysis identified 20 genes involved in flavonoid biosynthesis, 27 unigenes involved in flavonoid modification and 68 genes involved in flavonoid transport that were up-regulated in the stems of D. cambodiana after injecting the inducer, consistent with the accumulation of flavonoids. Furthermore, we have revealed the differential expression of transcripts encoding for transcription factors  involved in flavonoid metabolism. These de novo transcriptome data sets provide insights on pathways and molecular regulation of flavonoid biosynthesis and transport, and improve our understanding of molecular mechanisms of dragon’s blood formation in D. cambodiana.Dragon’s blood is a red resin mainly extracted from  Dracaena plants, which has been used as a famous traditional medicine since ancient times1Dracaena cochinchinensis and Dracaena cambodiana were the mainly plant resources of dragon’s bloodDracaena plants grow very slowly, and only trees with 30–50-years-old have the possibility to produce a small amount of dragon’s blood. In addition, natural Dracaena resources have been destroyed severely owing to overexploitation4D. Cambodiana78910Dracaena plants genomic information, the molecular basis of specific flavonoids biosynthesis and the regulation of formation in Dracaena plants remain unknown.Dragon’s blood is a red resin excreted by a part of the genus D. cambodiana. However, the lack of the transcriptomic and genomic data has made it difficult to investigate the mechanisms of flavonoid accumulation and dragon’s blood formation in D. cambodiana.The flavonoids metabolism pathway  has been well characterized in model plants at the genetic, biochemical and molecular levels12Dioscorea alataPrunus aviumOlea europaeaGentiana stramineaD. cambodiana using RNA-Seq complemented with HPLC flavonoid profiling to identify potential candidate genes involved in flavonoids accumulation. This study, therefore, may serve as a basis for the future discoveries on pathways and molecular regulation of flavonoid biosynthesis and transport in D. cambodiana.Next generation sequencing platforms provide highly efficient tools to identify novel genes associated with biosynthesis of various secondary metabolites in non-model plant speciesD. cambodiana changed from light yellow to brown after injecting the inducer  and constructed. By sequencing on the platform of Illumina Hiseq 2000, total raw reads of 266.57 M were generated and trimmed to exclude low-quality reads , the Swiss-Prot protein database, the KEGG database, and the COG database. A total of 34,873 (17.5%) unique sequences could be matched to known genes in the public databases . The proExpression levels of unigenes were determined by aligning the RNA-Seq reads from each library to the assembly. As a result, a total of 148,875 genes were expressed in all three samples, and 2,724, 1,698, 2,155 unigenes were specifically expressed in 0d, 3d and 6d respectively . The difWe did functional categorize to the differential expression unigenes by using gene ontology (GO) terms. The GO terms allow for the definition and standardization of the properties of gene products in any organism. The 3d against 0d libraries, 1,297 different genes were annotated. Of the three main sub ontologies, molecular function was the highly represented, with 1,129 unigenes followed by biological process with 795 unigenes and finally, cellular component with 584 unigenes. Besides this, 1,711 and 1,606 unigenes were annotated in other groups . GO enriD. cambodiana’s transcriptome, where flavonoids, flavone and flavonols, isoquinoline alkaloids, stilbenoid, diarylheptanoid and gingerol biosynthesis were the most relevant pathways by KEGG enrichment (The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways represent collections of manually drawn pathway maps and that are helpful for the understanding of the biological functions and interactions of genes. By mapping Enzyme Commission (EC) numbers to the reference canonical pathways, a total of 998, 1,296, 1,419 distinct genes matched the metabolic pathways in the KEGG pathway database at 3d/0d, 6d /0d, 6d /3d, respectively. Phenylpropanoid biosynthesis, flavonoid biosynthesis and flavone and flavonol biosynthesis were the most prominent classes . From thrichment .D. cambodiana are flavonoids, especially flavanes, with 7,4′- dihydroxy flavane as basic skeleton. Up to now, the biosynthetic pathway of flavanes has not been reported in other plants. A brief schematic of flavonoid biosynthesis is shown in D. cambodiana. Flavonoids are synthesized via the phenylpropanoid pathway and are converted from phenylalanine to chalcone by the enzymes phenylalanine ammonia-lyase , cinnamate 4-hydroxylase , 4-coumarate CoA ligase  and chalcone synthase . Chalcone isomerase  catalyses the isomerisation of chalcones into flavanone. Flavanone can be converted either to flavonols through the subsequent action of flavanone 3-hydroxylase  and flavonol synthase  or to flavane through the action of dihydroflavonol 4-reductase  and leucoanthocyanidin reductase . However, no unigene coding for flavone synthase  was detected by RNA-sequencing. The reason may be that FNS genes were short fragments without sequence similarity.The main components of dragon’s blood of It is known that most genes involved in flavonoid biosynthesis are coordinately expressed, consistent with flavonoid accumulation. We compared the differences in gene expression profile of those flavonoid biosynthesis genes to identify putative genes co-expressed with flavonoid accumulation. Among the above described genes involved in the flavonoid biosynthesis, five 4CL unigenes, six CHS unigenes, two CHI unigenes, seven DFR unigenes, one LAR unigenes, two F3H unigenes and two FLS unigenes were significantly up-regulated after injecting the inducer, consistent with flavonoid accumulation . Those gD. Cambodiana , UDP-glycosyltransferase (UGT) and O-methyltransferase (OMT) have been shown to play roles in the modification of flavonoidsmbodiana .+-ATPases and H+-PPases have been claimed to play roles in sequestration of flavonoids into the vacuole22D. Cambodiana transcriptome, 88 unigenes encoding MRP/ABCG, 34 unigenes encoding MATE, 20 unigenes encoding GST, 4 unigenes encoding VSR, 12 unigenes encoding SNARE, 74 unigenes encoding H+-ATPases and 13 unigenes encoding H+-PPases were found , 33 bHLH unigenes (15 up-regulated and 18 down-regulated), and 12 WD40 unigenes (5 up-regulated and 7 down-regulated) in the stem of D. cambodiana after injecting the inducer (D. cambodiana.It is well known that transcription factors (TFs) play an essential role in regulating flavonoid biosynthesis and transport. In most species, flavonoid biosynthesis and transport is controlled by a ternary complex of MYB-bHLH-WD40, which generally regulate expression of many structural genes inducer . These dD. cambodiana.To confirm the unigenes obtained from sequencing and to further analyze the reliability of RNA-seq data in present study, 15 DEGs involved in flavonoid biosynthesis, transport and regulation were chosen for real-time quantitative PCR assay. The expression profiles of these unigenes are shown in D. cambodiana is an important medicinal plant used as a source of dragon’s blood. The results of the analyses of chemical constituents indicate that the main components of dragon’s blood are flavonoids78910D. cambodiana. In the present study, we firstly provideed the assembly transcriptome sequence for D. cambodiana. Based on the sequencing results, 198,204 unigenes were generated, 34,783 of which were similar to known proteins. All sequences involved in flavonoid biosynthesis, modification, transport and regulation were identified by searching these transcripts against sequence databases using the BLASTx search; and their expression levels were monitored by comparing them in the stems of D. cambodiana after injecting the inducer characterized by different flavonoid accumulation.D. cambodiana was activated obviously after injecting the inducer, while the upstream of which, phenylpropanoid metabolic pathway had no significantly change.Flavonoids are synthesized by the phenylpropanoid metabolic pathway catalyzed by PAL, C4H and 4CL, which also serves as a starting point for the production of many other important compounds, such as coumarins, stilbenes, aurones and lignans2728+-ATPases (and H+-PPases in the tonoplast) has been proposed to be the main driving force for the transport of some flavonoids31323334D. cambodiana transcriptome, 18 MATE, 2 VSR, 4 SNARE, 18 H+-ATPase and 2 H+-PPase DEGs were found to be significantly up-regulated after injecting the inducer. Additionally, GSTs could act as flavonoid binding proteins, have been described as participating in vesicle uploading or vacuolar transpor37D. cambodiana; transport of flavonoids may be a multifactorial process, involving different strategies and the contribution of several proteins. These findings provide insights into the research of flavonoid transport mechanisms in D. cambodiana. However, in recent years, significant progress in flavonoid transport mechanisms has been focused on anthocyanin, transport of other flavonoids such as flavanes has been limited. Further research is needed to confirm whether there are three similar transport mechanisms in D. cambodiana and which mechanism is prevalent in transport of flavanes.Controlled transcription of biosynthetic genes is one major mechanism regulating secondary metabolite production in plant cells25TT12 to control the flavonoids transportLcGST4 was activated by LcMYB1, a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in litchiSbMYB2 in Scutellaria baicalensis, which was identified as regulators in the control of flavonoid biosynthesisMdMYB1, which was also involved in anthocyanin biosynthesis and transport in appleGL3, which has been reported to positively regulate anthocyanin synthesisD. cambodiana. Further studies are still needed to determine whether the flavonoid metabolism in D. cambodiana is regulated by these candidate genes, and which is independently regulated by a single TF, or controlled by combinations of TF complexes.The transport of flavonoids from the cytosolic side of the endoplasmic reticulum (ER) to the vacuole may occur through three basic mechanisms: membrane transporter-, glutathione S-transferase (GST)-, or vesicle trafficking-mediated transport22D. cambodiana are still unknown. In this work, we performed transciptome analysis of three samples with different flavonoid accumulation to identify putative genes for flavonoid metabolism in D. cambodiana. As a kind of plant secondary metabolite, the formation of dragon’s blood was considered as a result of plant defense response to biotic and abiotic responses42+ sensors, playing critical roles in Ca2+-mediated signaling in plant responses to environmental stresses by binding and regulating downstream effectors472+ sensors  significantly up-regulated were found from the DEGs , indicating that Ca2+-mediated signaling may be involved in the formation of dragon’s bloodinduced by the inducer. Increasing evidence indicates that calcium plays an import role in flavonoid biosynthesis. It has been reported that the calcium can regulate anthocyanin accumulation, possibly by activating flavonoid pathway genes49502+, calmodulin, and protein kinases have been shown to be involved in regulating the anthocyanin level5152D. cambodiana  will send out to extracellular in response to a variety of stress. Future studies will focus on verifying this proposed model by identifying putative genes involved in flavonoid accumulation.The molecular mechanisms of flavonoid biosynthesis, transport and regulation have been well characterized genetically and biochemically in model plants. Many genes involved in the flavonoid biosynthetic pathway have been cloned and characterized. However, the mechanisms of flavonoid biosynthesis and accumulation in mbodiana . In thisDe novo characterization of transcriptome of D. cambodiana was firstly identified and genes expression was analyzed during formation of dragon’s blood on transcriptome level. A total of 198,204 unigenes were identified from the three cDNA libraries, which will contribute significantly to further research of this specie and other related species. Many candidate genes involved in flavonoid biosynthesis, modification, transport and regulation were identified, which are worthy of further functional research. The results provide insights on pathways and molecular regulation of flavonoid biosynthesis and transport, and improve our understanding of molecular mechanisms of dragon’s blood formation.In summary, Dracaena cambodiana Pierre ex Gagnep were planted in Hainan Province of China. The stems were injected with the inducter (37.5 g/L NaCl and 1.25 ml/L acetic acid), and samples were taken from 6 cm above the inject site at 3 days and 6 days after transfusing. The stems cut from healthy trees were used to generate material for the 0 day library. All samples were collected, immediately frozen in liquid nitrogen and stored at −80 °C prior to RNA extraction.Three-year old Total RNAs from stems of three replicates were extracted using the RNA easy plant Mini Kit (QIAGEN) and treated with DNase I (Thermo) according to manufacturer’s instructions. RNA quality was examined using 1% agarose gel and the concentration was determined using Nanodrap (Thermo).D. cambodiana were refluxed with 95% EtOH. The solvent was evaporated under reduced pressure to yield the 95% EtOH extract. The flavonoids in the extract was chromatographed and detected with an Agilent 1260 Infinity HPLC system equipped with a diode array detector  and a C18 Analytical HPLC Column  as the stationary phase. The mobile phase consisted of acetonitrile (A) and 0.5% (v/v) formic acid aqueous solution (B), with a gradient elution program, 0–40 min: 10% to 25% A; 40 to 110 min: 25–45% A at a flow rate of 1.0 mL∙min−1. The HPLC chromatogram was monitored at 279 nm and the column temperature was set at 30 °C. The compounds were determined by comparing to standards isolated in our previous work and their structures were identified by 1HNMR, 13CNMR and MS.The dried stems of TM RNA sample prep kit (Illumina) according to the manufacturer’s recommendations. Briefly, mRNA was isolated from 0.5 mg of total RNA using oligo (dT) magnetic beads. mRNA was cut into short fragments by adding fragmentation buffer. First-strand cDNA was synthesized using random hexamer-primers, taking these short fragments as templates. RNaseH, buffer, dNTPs, and DNA polymerase I were used to synthesize second-strand cDNA (NEBNext® mRNA library prep master mix set for Illumina®). Short fragments were purified with Takara’s PCR extraction kit . Sequencing adapters were ligated to short fragments and resolved by agarose gel electrophoresis. Proper fragments were selected and purified and subsequently PCR amplified to create the final cDNA library template.Paired-end Illumina mRNA libraries were generated from 4 mg of total RNA following the manufacturer’s instructions for mRNASeq sample preparation . Library quality was assessed with the 2100 Bioanalyzer . The samples for transcriptome analysis were prepared using a TruseqTM 2000. Four fluorescently labeled nucleotides and a specialized polymerase were used to determine the clusters base by base in parallel. The size of the library was approximately 100 bp and both ends of the library were sequenced. The 100 bp raw pairedend reads were generated on the Illumina sequencing platform. Image deconvolution and quality value calculations were performed using Illumina GA pipeline v1.6. The raw reads were cleaned by removing adaptor sequences, empty reads, and low quality sequences (reads with unknown sequences ‘N’ or less than 25 bp). The clean reads were assembled into non-redundant transcripts using the Trinity, which has been developed specifically for the de novo assembly of transcriptomes using short reads.The transcriptome was sequenced using the Illumina HiSeqwww.ncbi.nlm.nih.gov) and the Swiss-Prot protein database (http://www.ebi.ac.uk/uniprot) with E-value cut-off of 1e-5. The annotations of the best hits were recorded. Gene Ontology (GO) (http://www.geneontology.org/) were further used to category the function of the unigenes by Blast2GO, and the unigenes were assigned to biological functions on the macro levels of “biological process”, “cellular component” and “molecular function”. The Kyoto Encyclopedia of Genes and Genome (KEGG) pathways database (http://www.genome.jp/kegg/) were assigned to unigenes by KEGG Automatic Annotation Server (KAAS).Unigenes were used as query sequences to search against the non-redundant protein (NR) database at NCBI  values during the assembly and clustering process. False discovery rate (FDR) < 0.001 and an estimated absolute log2 fold-change (log2 FC) ≥ 1 were used as threshold for identifying differentially expressed genes (DEGs).D. cambodiana actin gene was used as an internal control. The sequences of primers used in this study are provided in To verify the RNA-Seq results, quantitative RT-PCR was conducted using SYBR-green  and Stratagene Mx3005 P Real Time Thermal Cycler  with the following program: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, and then annealing at 65 °C–95 °C for 30 s. We used the Primer Premier v. 5.0 and Vector NIT v.11.0 software (Applied Biosystems) to design primers based on the sequences of key genes of interest identified in our library. The How to cite this article: Zhu, J.-H. et al. De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon’s blood. Sci. Rep.6, 38315; doi: 10.1038/srep38315 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.