GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y‐shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y‐shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases ( The majority of Firmicutes members including lignocellulolytic degraders were Clostridium, Anaerobacter, Caloramator, Caldicellulosiruptor, and Thermoanaerobacter , located in Nevada (77–85°C)   is located in Perak, Malaysia. Previously, we conducted microbial diversity analysis of the main water source of the Sungai Klah (SK) hot spring  using American Public Health Association standard protocols. At least 20 pieces of submerged foliage with no apparent biofilm were collected with a sampling dipper and transferred to polypropylene Ziploc bags using a tweezer. Submerged foliage with green biofilm was collected and stored separately in 1 L Schott bottles. Degraded foliage was collected at the base of SK‐Y with green biofilm, and nondegraded plant litters were carefully removed in situ.Freshly picked foliage samples from trees growing along the SK‐Y were collected and stored in sterile polyethylene bags. The lignin, cellulose, and hemicellulose content of these foliage samples were analyzed at the Malaysian Agricultural Research and Development Institute (MARDI), a local service provider of acid detergent lignin (ADL), acid detergent fiber (ADF), and neutral detergent fiber (NDF) analysis using a modified protocol based on Van Soest and Wine .2.2Unless specified, all samples were kept at 4°C. DNA extraction was completed within 2 days after sample collection. To study the microbial diversity of the SK‐Y, total community DNA extraction of the following samples was performed: (1) pooled water of four sites with an equal volume ratio, (2) submerged foliage with no apparent biofilm, (3) submerged foliage with green biofilm, and (4) degraded foliage collected at the base of the SK‐Y.g for 2 min at 4°C. The supernatant was discarded and the pellet was resuspended using Tris‐EDTA buffer (10 mmol/L Tris‐HCl (pH 7.5), 1 mmol/L EDTA) and modified cetyltrimethylammonium bromide (CTAB) lysis buffer  . Then, the membrane was placed in 10 ml of autoclaved 1 ×  concentration phosphate‐buffered saline  containing sterile glass beads and shaken vigorously for 5 min. Next, the membrane was removed and the leftover liquid was centrifuged at 17,000g for 10 min at 4°C. The pellet was subjected to the aforementioned conventional total community DNA extraction.Approximately 100 g of foliage samples were separately placed according to sample type  inside a 500 ml autoclaved glass bottle containing sterile PBS with 0.05% Tween 20 , and sonicated  for 1 min at 25°C. The preparations were then hand‐shaken vigorously for 30 s, and leaf debris was discarded, and the remaining liquid was aliquoted into a 50 ml tube and centrifuged at 14,800™ 1000 spectrophotometer , and a Qubit® 2.0 Fluorometer .To improve the purity of the extracted total community DNA, inhibitors such as humic acids were removed using the Agencourt AMPure XP System . Quality and yield of the purified total community DNA were examined using 1% w/v agarose gel electrophoresis, a Nanodrop2.3TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG‐3′; (2) 16S rRNA V3 and V4 bacterial amplicon PCR reverse primer 5′‐GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C‐3′; (3) targeted archaeal amplicon PCR forward primer 5′‐TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CMG CCG CGG TAA‐3′; and (4) targeted archaeal amplicon PCR reverse primer 5′‐GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTA CNV GGG TAT CTA ATC C‐3′. The underlined and nonunderlined sequences refer to the Illumina adapter overhang nucleotide sequences and locus‐specific primers for the regions to be targeted, respectively. The amplicons were then subjected to a series of library quantification steps to accurately quantify the NGS (next‐generation sequencing) sample libraries. The amplicons were quantified with Qubit dsDNA HS Assay Kit (Invitrogen) on a Qubit® 2.0 Fluorometer, and the library size was selected based on Agilent Technologies 2100 Expert Bioanalyzer using Agilent High Sensitivity Kit . The number of amplifiable molecules in a library were quantified absolutely using Eco Real‐Time PCR System with KAPA Library Quantification Kit  prior to sequencing. Next, libraries were sequenced using paired‐end sequencing on an Illumina MiSeq sequencer  with MiSeq Reagent Kit V2 (2 × 250 base pairs) and V3 (2 × 300 base pairs) for the archaeal primers amplicons and bacterial primers amplicons, respectively.The Illumina 16S rRNA Metagenomic Sequencing Library Preparation Guide was followed for the preparation of the libraries. Two sets of primers were used to target bacterial and archaeal hypervariable 16S rRNA conserved regions . Adapter sequences were trimmed and reads were filtered to ensure an average Phred score of 20. Paired‐end reads were merged  in CLC Genomics Workbench 7.0. The assembled reads were then subjected to chimera filtering and microbial taxonomic classification using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline  to determine the number and types of GH families for these taxa.33.1More than a dozen hot spring sites are present in the Sungai Klah hot spring park. Previously, we performed a 16S rRNA amplicon and shotgun sequencing for samples obtained from one of the SK hot springs , ADF (cellulose and lignin), and ADL   were not found in the literatures to date, except as reported by Codron, Lee‐Thorp, Sponheimer, and Codron  SK‐Y water, 2) submerged foliage with no apparent biofilm (labeled as nondecay), (3) submerged foliage with green biofilm (labeled as green biofilm), and (4) degraded foliage (labeled as decay) , Chloroflexi (12.8%), Firmicutes (10.8%), and Cyanobacteria (8.4%) , Chlorobi (0.60%), Nitrospirae (0.70%), Spirochaetes (0.78%), and Synergistetes (0.78%) were also detected at lower percentages. The decay sample had higher percentages of Acidobacteria, Aquificae, and Thermotogae compared to the other three samples. Photosynthetic phyla, such as Chloroflexi and Cyanobacteria, were present in lower percentages in the decay sample than those in green biofilm. A broad range of Proteobacteria, including Alpha‐, Beta‐, Gamma‐, Delta‐, and Epsilonproteobacteria, was observed in all samples.Unless specified, the values shown in this subsection are the average number for the four samples. Generally, the taxonomic assignment of bacteria in the four samples could be classified into 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera. The 10 most abundant phyla contributed 75.5% of the total bacterial diversity, including Actinobacteria, Aquificae, Deinococci, Bacilli, Clostridia, Ignavibacteria, Planctomycetia, Alphaproteobacteria, Thermodesulfobacteria, Thermotogae, unclassified Acidobacteria, and other unclassified classes or blast hits. All these classes were also present in green biofilm, nondecay, and water samples, but occurred in different proportions. The main classes associated with green biofilm, but present in lower percentages in the decay sample, included Blastocatellia, Bacteroidia, Cytophagia, Caldilineae, Chloroflexia, Cyanobacteria, Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria.The following classes contributed to almost 69% of the total population in the degraded foliage microbiota: Thermaceae (4.5%), Caldilineaceae (3.8%), Chloroflexaceae (3.6%), and Roseiflexaceae (3.6%) representing the most abundant groups. In addition, Fervidobacteriaceae (5.0%) was found to be the predominant family in the decay sample. For bacterial genera, the four samples in SK‐Y shared most OTUs but with different abundances , Meiothermus (3.8%), Chloroflexus (3.6%), Roseiflexus (3.6%), Thermoanaerobaculum (3.1%), Melioribacter (2.4%), Geobacillus (2.2%), Desulfurobacterium (1.8%), Thermosipho (1.3%), Thermoanaerobacterium (1.0%), Fervidobacterium (1.0%), Acidimicrobium (1.0%), Chloracidobacterium (1.0%), and Thermoanaerobacter (0.9%).A total of 171 families were observed across all the samples, with s Figure b, domina3.6Thaumarchaeota (85.5%). In SK‐Y water, green biofilm, and decay samples, Euryarchaeota was the second‐most abundant archaeal phylum, followed by Crenarchaeote. Crenarchaeota was the second‐most dominant phylum in the nondecay sample, followed by Euryarchaeota. Within Thaumarchaeota, the most abundant class in all samples was Nitrososphaeria, with Nitrososphaera (84.0%) as the dominant genus. Furthermore, the next most abundant class in SK‐Y water, green biofilm, and decay samples was Methanomicrobia, mainly represented by Methanofollis. Thermoprotei was the second‐most dominant class in nondecay sample with Aeropyrum as the second abundant genus. Genus level assignment revealed that Methanocorpusculum (3.6%), Nitrosopumilus (1.5%), Methanogenium (0.4%), Ignicoccus (0.3%), Methanotorris (0.3%), Methanosphaerula (0.3%), Halapricum (0.2%), and Methanothermococcus (0.2%) genera were present in all the samples , Proteobacteria (14.4%), and Chloroflexi (13.1%) were the main three phyla that contributed to green biofilm communities. Cyanobacteria and Chloroflexi are chlorophyll‐based phototrophic bacteria. Their growth rates are strongly affected by temperature, pH, sulfide concentration, sunlight, and other factors  genus, a chemo‐organotroph that thrives in anaerobic habitats  were possibly present in SK‐Y. Based on CAZy classification of AA enzymes, genes for AA are lacking or missing from most of the thermophiles listed in Table Thermus OTUs were identified in SK‐Y as minority taxa. A thermostable and chloride‐tolerant laccase  from Thermus thermophilus SG0.5JP17‐16 was cloned and overexpressed , Amycolatopsis , Azoarcus , Pseudoxanthomonas (AA 3 and AA 6), and other AA enzymes present in Anoxybacillus, Thermodesulfobacteriaceae, Clostridium, Geobacter, Bacillus, and some other minority members. Despite being generally mesophilic, proteins from Bacillus spp. may be thermostable. For instance, laccase from Bacillus licheniformis ATCC 9945a had an optimum temperature of 90°C and a half‐life of 50 min at 70°C  enzymes originate from fungi (Levasseur, Drula, Lombard, Coutinho, & Henrissat, 5The work presented here describes the microbiota within a heated biomass degrading bioreactor hot spring. The microbial community within SK‐Y included 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera. Thus, SK‐Y represents a good source for isolation of efficient biomass degrading thermophiles and thermozymes. Biomass degradation at high temperatures may involve various community members. Abiotic factors such as temperature, dissolved oxygen level, and stage of degradation may affect the population of the microbiota members. The microbial profiling data demonstrated that at least 18 genera found in this natural ecosystem are potential candidates for efficient lignocellulosic enzymes. Analysis of these genera, based on existing sequenced genomes, revealed at least 61 GH enzymes that exemplify the important interplay between diverse microorganisms in communities that contribute to the enzyme repertoires required for the degradation of lignocelluloses. Further analyses using metatranscriptomic sequencing may provide more detailed insight into the gene expression level of GHs, and may possibly mine new AA enzymes.The authors declare that they have no competing interests linked to the data presented in this manuscript. All the authors consented to the publication of this work. Click here for additional data file.


Four members of the retinoic acid-related orphan receptor α (RORα) family  are transcription factors that regulate several processes including circadian rhythm, lipid metabolism, cerebellar development, immune function, and cancer. Only two isoforms, RORα1 and 4, are specifically co-expressed in the murine and human. In the present study, we identified a specific N-terminal domain (NTD) of RORα1 that potentiated the downregulation of target genes involved in tumor progression and proliferation, based on results from RORα-deficient mouse embryonic fibroblasts and prostate carcinoma tissues. The hyperactivation of proliferative target genes were observed in RORα-deficient embryonic fibroblasts, and reconstitution of RORα1 inhibited this activation by a NTD dependent manner. Downregulation of RORα1 and upregulation of Wnt/β-catenin target genes were correlated in prostate cancer patients. These findings revealed the control of invasive growth by NTD-mediated RORα1 signaling, suggesting advanced approaches for the development of therapeutic drugs. Shh, Slc1a6, N-myc, Cav3, Cpt1, Pcp2, Pcp4 and Crygf [Defining the molecular strategies by which distinct signaling pathways control downstream target genes coordinately is central for understanding development, disease, and homeostasis. The Orphan nuclear receptor (ONR) family perform vital roles in controlling subsets of target gene expression both positively and negatively in diverse signaling pathways in the regulation of disease, neurogenesis, and homeostasis ,2. ONRs nd Crygf ,10,11,12nd Crygf ,15,16,17The crosstalk between the nuclear receptor family and the Wnt/β-catenin pathway is emerging as a clinically and biologically significant issue ,19. GiveRORα-deficient mouse model, and in clinical samples of patients with prostate carcinomas.Canonical Wnt signaling causes β-catenin accumulation with T cell factor (TCF)/lymphoid enhancer factor (LEF) in the nucleus; which then regulates target gene expression ,28. In tp value < 0.001), whereas RORα4 + 1NTD permitted the transcriptional activation function comparable to that of RORα1  mice, which had a spontaneous loss-of-function mutation in the RORα gene [cyclin D1, c-jun, and c-myc, and quantitative RT-PCR analysis revealed that Wnt target genes in RORα-deficient fibroblasts were hyperactivated when compared with those in wild-type (+/+Rora) fibroblasts  prepared from ORα gene . LiCl inroblasts A. Increaanalyses B. These sg/sgRora MEFs. ChIP assay unveiled that recruitment of RORα1 to the cyclin D1 promoter with decreased Pol II levels on the target gene , Cyclin D1 , c-jun , c-myc , Lamin A/C , tubulin , RORα1 , and common RORα . The following commercially available antibodies were used: Anti-FLAG antibodies , anti-β-catenin , and anti-RNA Polymerase II antibodies . LiCl was purchased from Sigma and added at 10 mM for 24 h before cell harvest.staggerer mutation (C57BL/6J-RORsg/+) were purchased from the Jackson Laboratory and housed in the animal facility of the Sookmyung Women’s University according to standards of the Association for Assessment and Accreditation of Laboratory Animal Care . The animals were genotyped by PCR. Genomic DNA was extracted from tail biopsies and amplified in two sets of reactions, one for each allele. The staggerer allele primers (5′–3′) were: CGTTTGGCAAACTCCACC and GTATTGAAAGCTGACTCGTTCC. The + allele primers were: TCTCCCTTCTCAGTCCTGACA and TATATTCCACCACACGGCAA. The amplified fragments  were detected by electrophoresis on agarose gel. Homozygous sg/sgRora mice and their +/+Rora littermates were obtained by crossing heterozygous male and female breeders. The mice were weaned at four weeks of age. Male +/+Rora and sg/sgRora mice were used in the experiment. Mouse embryonic fibroblasts were generated from a litter of embryos on embryonic day 13.5. Briefly, embryos were taken out aseptically and the head and liver were carefully removed from the isolated fetus, minced and trypsinized for 30 min at 37 °C. Cells were harvested, resuspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and plated on a 10 cm dish for each embryo.A pair of the heterozygous mice having the To examine the effect of RORα constructs on the binding to β-catenin, we first prepared GST β-catenin bound to Glutathione-Sepharose beads. The beads were incubated with the isolated RORα proteins in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Nonidet P40, and 10% glycerol. After extensive washing, the bound materials were subjected to Western blot analysis.Tissue array  was stained as previously described . The metsg/sgRora MEFs as previously described [cyclin D1 promoter sense strand CCGGGCTTTGATCTTTGCTTA, antisense strand TCTGCTGCTCGCTGCTACTG.The ChIP was conducted in escribed ,41 usingCt method and normalized by using primers to detect β-actin or HPRT. All reactions were performed as triplicates. Primers (5′–3′) were: mCyclinD1, AACTACCTGGACCGCTTCCT and CCACTTGAGCTTGTTCACCA; mc-jun, TGAAAGCTGTGTCCCCTGTC and ATCACAGCACATGCCACTTC; mc-myc, TGAGCCCCTAGTGCTGCAT and AGCCCGACTCCGACCTCTT; mMcp-1, GGCTCAGCCAGATGCAGTTAAC and AGCCTACTCATTGGGATCATCTTG; mβ-actin, TAGCCATCCAGGCTGTGCTG and CAG GATCTTCATGAGGTAGTC; hRORα1, CGGTGCGCAGACAGAGCTAT and CCACAGATCTTGCATGGAATAATT; hCyclin D1, CTACTACCGCCTCACACGCTT and GGCTTGACTCCAGCAGGGCT; hc-Jun, GTCCACGGCCAACATGCTCA and TGTTTGCAACTGCTGCGTTAG; hc-Myc, CAGCTGCTTAGACGCTGGATT and GTAGAAATACGGCTGCACCGA; hHPRT, TGACACTGGCAAAACAATGCA and GGTCCTTTTCACCAGCAAGCT.The abundance of mRNA was detected by an ABI prism 7300 system with SYBR Green (molecular probes). Primer pairs were designed to amplify 90–150 bp mRNA specific fragments and confirmed as a unique products by melting curve analysis. The PCR conditions were 95 °C (5 min) and 40 cycles of 9595 °C (30 s), 5695 °C (30 s), and 7295 °C (30 s). The quantity of mRNA was calculated using ΔΔ4 cells each) were used in triplicates. Assay was performed as previously described [LNCaP, RORα1-LNCaP, and RORα4-LNCaP cells . Conditioned RPMI1640 medium containing 15% fetal bovine serum was added to the bottom chamber as a chemoattractant. After 22 h incubation, the cells that had migrated to the lower chamber of the filter were fixed with 100% methanol, stained with Giemsa, and quantified by counting the total number of cells in four different fields. All experimental studies were performed according to the manufacturer’s protocols. Values were expressed as means ± standard deviations for at least three independent experiments.LNCaP cells stably expressing RORα1, RORα4, or RORα4+1NTD were used in Matrigel invasion assays along with control cells. Matrigel invasion assay was conducted as previously described . Culture5) were placed in Iscove’s media containing 0.4% noble agar containing 10% FCS. Cells were allowed to grow for three weeks in 5% CO2, and the formation of colonies containing >50 cells was analyzed. Colonies were counted in ten different fields, and the total colony number/well was calculated. The representative image is shown for each group.Anchorage-independent growth of LNCaP, LNCaP-RORα1 and LNCaP-RORα4 was determined by analyzing cellular growth in semisolid medium. Cells . Tumors were measured weekly and the experiment was terminated when the largest tumor was about 10 mm in diameter. Tumors were excised and weighed. Statistical differences in tumor weights were determined by a Student’s t-test using the Statview package . These experiments were carried out with the approval of the Institutional Animal Care and Ethics Committee.For experiments examining tumor formation in vivo, a total of ten million cells with an equal volume of Matrigel  was injected subcutaneously at the left flank into three groups of 6-week-old athymic p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.Values were expressed as mean ± SD or SEM. Significance was analysed by a one-tailed, unpaired t-test or one-way ANOVA using GraphPad Prism software.


To explore a comparable method to Gd-contrast enhancement in the preoperative evaluation of anal fistula to evaluate its morphology changes.Forty-six patients with anal fistula were enrolled. Each patient acquired a 3.0T magnetic resonance imaging (MRI) routine sequence, diffusion-weighted imaging (DWI) sequence and fat suppression T1 weighted imaging (FS T1WI) contrast enhancement (CE) scanning. To record the morphology performances of the internal orifice and the fistulas on the transverse images of fat suppression T2 weighted imaging (FS T2WI), DWI, FS T2WI combined with DWI, FS T1WI Gd-CE, with the standard of the surgical pathology results. Two observers evaluated images in consensus. The conspicuity and the diagnostic performance rate were compared between the 4 imaging data sets.The consistencies of interobservers about the conspicuity scores and the diagnostic performance rates of the internal orifice and the fistula were good. The conspicuity of the internal orifice was higher for the set of FS T2WI, FS T2WI+DWI, and FS T1WI+CE than DWI. The diagnostic performance rate of the internal orifice was higher for the set of FS T2WI, FS T2WI+DWI, and FS T1WI+CE than DWI. The conspicuity of the fistula was higher for the set of FS T2WI+DWI and FS T1WI+CE than FS T2WI or DWI. There were no significantly differences between the 4 sets of FS T2WI, DWI, FS T2WI+DWI, and FS T1WI+CE in the diagnostic performance rate of the fistula.The set of FS T2WI combined with DWI was comparable to FS T1WI CE in evaluation of anal fistula morphology changes. It occurs to patients of any age and the average age is between 20 and 40. The incidence of anal fistula is high,3 while the clinical cure rate is relatively low, which is attributed to the hidden secondary fistula or small abscess being ignored.Anal fistula refers to the anomalous connection between the anus or rectum and the skin around the anus with the granulation tissue lining the anal fistula, It is demonstrated that the postoperative complications of anorectal fistula could be decreased by about 75% thanks to MRI examination.Magnetic resonance imaging (MRI) is currently recognized as the best method for detecting and evaluating the details of the anal fistula. fat suppression T2 weighted imaging (FS T2WI),8 and fat suppression T1 weighted imaging contrast-enhanced (FS T1WI+CE) sequences. However, the use of contrast agent is relatively contraindicated in patients with renal insufficiency result from the increased risk of renal systemic fibrosis. And multiple studies have shown that repeat administration of the gadolinium may result in the deposition in the deep nuclei of the brain.,12 It is critical for looking for a noninvasive method to assess anal fistula. In another hand, there was no report about the comparison between FS T2WI+DWI and either FS T2WI or DWI alone and the comparison between FS T2WI+DWI and FS T1WI+CE in the literature. Therefore, the purpose of this study is to compare the lesion conspicuity and diagnostic performance rate of four imaging data sets , FS T2WI combined with DWI[FS T2WI+DWI], FS T1WI+CE) in assessing perianal fistulas, so as to explore a comparable method to Gd-contrast enhancement in the preoperative evaluation of anal fistula.The great significance of the 3 imaging data sets has been emphasized in the literature including the T2 weighted imaging (T2WI),22.1From January 2017 to December 2018, 323 patients were enrolled in the study who had been clinically diagnosed perianal fistulas with detailed surgical and pathology reports. The inclusion criteria were as follows:(1)Patient who underwent magnetic resonance (MR) imaging before surgery and(2)Patient without operation history because of perianal fistula.A total of 52 patients were selected. Six patients were excluded for unavailable DWI (n = 3), FS-T2WI (n = 2) or FS T1WI+CE (n = 1). Thus, 46 patients  were confirmed in the final study, aged 18 to 80 years , complaining of the perianal swelling, fever or pain. There was a fistula or a hard knot around the anus. And sometimes purulent or bloody discharge could be seen. With the consent of the hospital ethics committee, all patients were informed of the condition before examination, and the informed consent was signed by the patient or legal guardian.2.2All the patients underwent MRI scanning within 1 week before surgical treatment, and were confirmed as anal fistula by the surgery and pathology. All patients were scanned with 3.0T Siemens MR scanner , covered with 18-channel body coil, in supine position with head first, with the symphysis pubis as the center. No intestinal preparation was required.2.The MRI protocols contained axial T1-weighted imaging turbo spin echo (TSE), coronal T2-weighted imaging, axial and sagittal T2-weighted imaging with fat suppression, followed by DWI. Finally, the volumetric interpolated breath-hold examination (VIBE) acquisition technology was used to conduct T1-weighted with fat suppression sequence before and after intravenous gadolinium administration . The main parameters were as follows: turbo spin echo (TSE) T1-weighted imaging (T1WI): axial; repetition time (TR)/echo time (TE), 600/18 ms; field of view (FOV), 280 mm × 280 mm; matrix, 314 × 448, slice thickness, 4 mm; and number of slices, 24. TSE T2-weighted imaging (T2WI) with spectral adiabatic inversion recovery (SPAIR) for fat suppression: axial; TR/TE, 3320/85 ms; FOV, 280 mm × 280 mm; matrix, 307 × 384; slice thickness, 4 mm; and number of slices, 24. T2-weighted imaging (FS-T2WI): sagittal; TR/TE, 5450/86 ms; FOV, 260mm × 260 mm; matrix, 320 × 240; slice thickness, 4 mm; and number of slices, 25. DWI : axial; TR/TE, 5800/76 ms; FOV, 320 mm × 320 mm; matrix, 118 × 168; slice thickness, 5 mm; number of slices, 25; NSA, 1; b = 0.800 s/mm2.3 the anal fistula could be divided into 4 types. According to 4-point scale in the references by Hori, and the definition, contour and margin of the internal orifice and fistula, and the relationship between them with the sphincters, scored each sequence on the conspicuity and diagnostic performance rate of the internal orifices and fistulas on a 4-point scale: 1, probably not a fistula or an internal orifice; 2, uncertain or indistinct; 3, possible a fistula or an internal orifice but obscure; and 4, definite and legible fistula or an internal orifice.After the scanning, the images were transmitted to the picture archiving and communication system (PACS). The surgical pathology results were taken as the standard. Two physicians with more than 8 years’ experience in MR diagnosis analyzed and evaluated the images independently. After the initial independent analyses, the results were discussed by the 2 observers and modified if necessary, applying a consensus reading method. Recording the detection of internal orifice, main fistula, secondary or branch fistula or abscess of anal fistula on the transverse images of FS T2WI, DWI, FS T2WI+DWI, FS T1WI+CE. According to the Parks classification,2.4P value of less than .05 was considered to indicate a statistically significant difference. Bonferroni correction (n = 4) was used for multiple comparisons, a P value of less than .008 ((2 × 0.05)/4 (4–1)) was considered to indicate a statistically significant difference.Statistical analysis was done with the software package SPSS 25.0. Kappa conformance test was performed on the 2 observers: k ≤ 0.4, poor consistency; 0.4 < k ≤ 0.6, medium consistency; 0.6 < k ≤ 0.8, good consistency; k > 0.8, very good consistency. The diagnostic performance rate of the anal fistula was compared with chi-square test; the conspicuity scores of the internal orifices and fistulas were compared with rank test. A 33.1In 46 cases of anal fistula, a total of 51 internal orifices, 62 primary fistulas and 34 abscesses were found in surgery, with 2 internal orifices found in 5 patients, and 3 horseshoe fistulas. According to the classification system of Parks, the perianal fistulas consisted of 22 intersphincteric fistulas (47.8%), 20 transsphincteric fistulas (43.5%), 3 suprasphincteric fistulas (6.5%) and 1 extrasphincteric fistula (2.2%).3.2The consistencies of interobservers about the conspicuity score of the internal orifice of anal fistula , the diagnostic performance rate of the internal orifice , and the conspicuity score of the fistula . T2WI, k = 0.824; DWI, k = 0.882; FS T1WI+CE, k = 0.718; FS T2WI+DWI, k = 0.851), the diagnostic performance rate of the fistula  were normal.3.3P = .000263, .000002, .000155, respectively). However, there was no significant differences between the sets of FS T2WI, FS T2WI+DWI, and FS T1WI+CE . However, there was no significant between the sets of FS T2WI, FS T2WI+DWI, and FS T1WI+CE  was slightly higher than that by FS T1WI CE (92%), while for observer 2, FS T2WI+DWI (88.2%) are slightly higher than FS T1WI+CE (84.3%). In general, there was no difference between T2WI and DWI (96%) and FS T1WI+CE (96%). Our results showed that FS T2WI+DWI (88.2%), FS T1WI+CE (84.3%), FS T2WI (82.4%), and DWI (54.9%). FS T2WI+DWI, FS T1WI +CE, and FS T2WI were superior to DWI, respectively, but there was no significant difference between the first three sets. Hori et al evaluated 20 fistulas in 13 patients by FS T2WI, DWI, and FS T1WI+CE, which demonstrated that there were no differences between DWI combined with FS T2WI (95%) and FS T1WI+CE combined with FS T2WI (95%) in the fistula display rate and both were superior to FS T2WI (90%). In theory, when the fistula directly extends to the mucous membrane of the anal canal, it is easy to confirm the location of internal orifice. However, the circumferential thickness of the anas and the mucosa of the internal sphincter are not uniform in normal people actually. Sometimes local thickening of the mucosa shows high signal on FS T2WI sequence. Small vessels around the anas can also show high signal like dot or strip, which are similar to the signal of internal orifice. It is often difficult to distinguish. Although contrast enhancement (CE) can improve the detection rate of the internal orifice, it is sometimes difficult to distinguish from the perianal blood vessels or mucosa. However, the combination of DWI sequence becomes essential to increase the diagnostic accuracy (Fig. It have been shown in the study that the detection rate of anal fistula by MRI is about 82.7% to 97%,WI+CE 84.%. In genOur study may have some limitations. First, our retrospective study had a relatively low number of samples. Therefore, we need to expand the samples for further prospective investigation, while the imaging findings and conclusions should be interpreted as preliminary. Second, in order to improve the reliability of scoring and enhance the accuracy of diagnosis, we performed the scanning by multiple sequences and multiple directions in the examination of anal fistula, so it is unavoidable to combine other sequences, and the double-blind method was not used strictly to evaluate the single sequence. Future studies should take care to this point.5The conspicuity and diagnostic performance rate of FS T2WI combined with DWI were comparable to that of FS T1WI CE. The combination of FS T2WI and DWI could be the best sequence for noninvasive evaluation of anal fistula morphology changes. MRI could assess the morphology changes of anal fistula accurately, which is of great guiding significance for the treatment of anal fistula.Conceptualization: Chao Gu, Yu Wang, Weiwei Han, Jiansheng Li, Haichang Xing, Chuanting Li, Keyun Bai.Data curation: Chao Gu, Yu Wang, Lixia Lai, Weiwei Han, Jiansheng Li, Haichang Xing.Formal analysis: Yu Wang, Keyun Bai.Investigation: Chao Gu, Yu Wang, Lixia Lai, Jiansheng Li, Keyun Bai.Methodology: Chao Gu, Yu Wang, Lixia Lai, Weiwei Han, Jiansheng Li, Haichang Xing, Yongjun Huo, Chuanting Li, Keyun Bai.Project administration: Chao Gu, Yu Wang, Haichang Xing, Yongjun Huo, Chuanting Li, Keyun Bai.Resources: Weiwei Han, Jiansheng Li, Haichang Xing, Yongjun Huo, Keyun Bai.Supervision: Chao Gu, Yu Wang, Lixia Lai, Weiwei Han, Haichang Xing, Yongjun Huo, Keyun Bai.Writing – original draft: Yu Wang, Lixia Lai, Weiwei Han, Haichang Xing, Keyun Bai.Writing – review & editing: Chao Gu, Chuanting Li, Keyun Bai.


Plastome mutants are ideal resources for elucidating the functions of plastid genes. Numerous studies have been conducted for the function of plastid genes in barley and tobacco; however, related information is limited in Chinese cabbage.cdm was cytoplasmically inherited. Physiological and ultrastructural analyses of cdm showed impaired photosynthesis and abnormal chloroplast development. Utilizing next generation sequencing, the complete plastomes of cdm and ‘FT’ were respectively re-mapped to the reference genome of Chinese cabbage, and an A-to-C base substitution with a mutation ratio higher than 99% was detected. The missense mutation of plastid ribosomal protein S4 led to valine substitution for glycine at residue 193. The expression level of rps4 was analyzed using quantitative real-time PCR and found lower in than in ‘FT’. RNA gel-blot assays showed that the abundance of mature 23S rRNA, 16S rRNA, 5S rRNA, and 4.5S rRNA significantly decreased and that the processing of 23S, 16S rRNA, and 4.5S rRNA was seriously impaired, affecting the ribosomal function in cdm.A chlorophyll-deficient mutant of Chinese cabbage that was derived by ethyl methanesulfonate treatment on isolated microspores showed uniformly pale green inner leaves and slow growth compared with that shown by the wild type “Fukuda 50′ (‘FT’). Genetic analysis revealed that cdm was a plastome mutant and that chlorophyll deficiency might be due to an A-to-C base substitution of the plastome-encoded rps4 that impaired the rRNA processing and affected the ribosomal function.These findings indicated that The online version of this article (10.1186/s12870-018-1353-y) contains supplementary material, which is available to authorized users. Mutant lines are ideal resources for discovering novel gene functions and deciphering unknown mechanisms , 2. In hArabidopsis thaliana [Chlorophyll-deficient mutants can be generally divided into three types: mutants controlled by nuclear genes, mutants controlled by cytoplasmic genes, and mutants controlled by nuclear-cytoplasmic interactions . Most chthaliana , rice [7thaliana , soybeanthaliana , carrot thaliana , and cabthaliana , are conthaliana . Only a thaliana .Mirabilis jalapa and Pelargonium zonale [Brassica campestris, plastids and mitochondria are maternally inherited, whereas in Brassica napus, mitochondria are not strictly maternally inherited [Chlorophyll-deficient mutants have been used as a genetic tool for the initial study of cytoplasmic inheritance , 15. Them zonale –16. Ris m zonale  as well m zonale  reportedm zonale . Cytoplam zonale . Of thesnherited , 22.A limited number of cytoplasmic inherited chlorophyll-deficient mutants with a non-chromosomal stripe phenotype are mitochondrial mutants generally caused by rearranging , 24. MosPlastome mutants are classified into three categories: those with mutations in genetic system genes, in photosynthesis-related genes, and other genes and conserved reading frames . NumerouA. thaliana. The rps2, rps4, rpl18, and rpl20 knockout mutants in tobacco show misshapen leaves, revealing the essential role of plastid proteins in leaf development [rps15 knockout mutant shows chlorophyll deficiency [prps1, rps5, prsp17, prpl11, and prpl24 mutants in A. thaliana show chlorophyll deficiency and reduced growth, and of these, prps17 and prpl24 show rRNA process impairment [Genetic system genes, including 62 plastome-encoded genes, 30 tRNAs, 4 rRNAs, and 21 ribosomal protein genes, constitute the largest group in the plastome, which is associated with plastid gene expression , 48. Plaelopment , whereasficiency . In addipairment –52. Therpairment . The idecdm and Fukuda 50  were respectively re-mapped to the cpDNA reference genome of Chinese cabbage [cdm compared with those in ‘FT’. Our results suggested that RPS4 is associated with chloroplast rRNA processing and chlorophyll deficiency in cdm.In this study, a maternal inherited mutation causing chlorophyll deficiency was identified in Chinese cabbage. The complete plastomes of  cabbage  using IlIn this study, a doubled haploid line rived from the Chinese cabbage variety ‘FT’ was used as a donor for isolated microspore culturing to create a mutant library. In August 2014, ‘FT’ seeds of a doubled haploid line derived from the Chinese cabbage variety Fukuda 50 (‘FT’) were vernalized at 4 °C for 20 d after accelerating germination and then, sown in an aperture disk on a seedbed. Seedlings were re-potted in late September, and the unopened flower buds were used for isolated microspore culturing.v/v) was filter-sterilized through a 0.22-μm filter membrane. Isolated microspores were washed by B5 medium and centrifuged twice at 120×g for 3 min. The precipitate acquired after the first centrifugation was suspended in B5 medium containing EMS  for 10 min. The microspores were re-suspended and cultured in NLN-13 medium [Procedures of microspore isolation, purification, and culture were performed as described previously , 55. Iso3 medium , incubat3 medium  and cultcdm and ‘FT’ were respectively selected at the seedling stage (4-week-old) and the flowering stage (8-week-old) and submerged in 80% acetone for 24 h in the dark. The absorbance of the supernatants was recorded at 645 nm and 663 nm using the DU 800 UV/Vis Spectrophotometer .Total chlorophyll was extracted as described by Inskeep and Bloom . In briecdm and ‘FT’ were selected at the seedling stage, cut into small pieces (approximately 1 mm2), and fixed in 4% (v/v) glutaraldehyde in a 0.1 M phosphate buffer solution  at 4 °C for 7 d. The fixed samples were post-fixed in 1% (w/v) aqueous osmium tetroxide for 4 h and rinsed 3 times in PBS for 5 min each time. The samples were gradually dehydrated with various concentrations of ethanol, and then, impregnated and embedded in Epon812. Ultrathin sections were made using the LKB2088 ultramicrotome  and double stained with uranyl acetate and lead citrate. Finally, the samples were observed under the H-7650 transmission electron microscope .The chloroplast and mitochondrial ultra-structures were characterized as described by Lichtenthaler et al.  and Huancdm and ‘FT’ plants with consistent growth were selected for measuring fluorescence kinetic parameters by applying A Fluor Cam Portable ChI/GFP Luminoscope  after 20 min of dark adaption using leaf clips. The optional maximal photochemical efficiency of PS II (Fv/Fm), the effective quantum yield of PSII (ФPSII), the photochemical quenching coefficient (QP), and the non-photochemical Chl fluorescence quenching (NQP) were calculated as described by Bilger and Bjorkman [Ten 4-week-old Bjorkman  and MaxwBjorkman . Each mecdm and ‘FT’ plants were placed at 4 °C for 48 h in the dark to eliminate chloroplast starch granules and reduce the plastid breakage. Fresh leaves collected from three plants were mixed, cleaned with double distilled water, dried on filter paper, and the main nerve was cut off. A 30-g sample was ground to less than 1 mm2 in liquid nitrogen. Total cpDNA was isolated using the column Plant chloroplast DNAout kit , according to the manufacturer’s instructions with minor modifications. This experiment was performed on ice, and all vessels and solution were precooled.At the seedling stage, well-grown cdm and ‘FT’ was used for constructing an NGS library using the NEBNext Ultra DNA Library Prep Kit for Illumina, according to the manufacturer’s instructions. In brief, 1 μg cpDNA was randomly fragmented to < 500-bp fragments by sonication and treated with End Prep Enzyme Mix, followed by adaptor additions to both ends. Fragments of approximately 410 bp with a 350-bp insert were purified using the AxyPrep Mag PCR Clean-up kit  and then, amplified using P5 and P7 primers. Purified PCR products were validated by the Agilent 2100 Bioanalyzer  and quantified by the Qubit2.0 Fluorometer . DNA libraries were multiplexed, loaded on an Illumina HiSeq instrument, and sequenced by a 2 × 150 paired-end (PE) configuration with the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) reading sequence information.Purified cpDNA from http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?opt=plastid&taxid=3398), cpDNA sequence reads were isolated from the raw sequence reads of mixed nucleus and mitochondrial DNA. The filtered cpDNA sequence reads from cdm and ‘FT’ were used for re-sequencing as described by [Based on the reference cpDNA genome of Chinese cabbage , introduced into the pGEM®-T Easy Vector , and transformed into the JM109 competent cell . The recombinant plasmids were sequenced by the Institute of Beijing Genomics, China, and the sequences were aligned using DNAMAN .Based on gene annotation information  and nuclcdm, ‘FT’ × cdm and cdm × ‘FT’ plants were used for RNA isolation using TRIZOL (Invitrogen). First-strand cDNA was synthesized with random hexamer primers. A 10-fold dilution of cDNA was used for quantitative real time PCR (qRT-PCR) by the Bio-Rad IQ5 Real Time PCR System  with gene-specific primer sets  formamide, 5× saline sodium citrate (SSC), 50 mM phosphate buffer, 0.1% N-lauroylsarcosine sodium salt, 7% (m/v) sodium dodecyl sulfate (SDS), and 2% (m/v) blocking reagent (Roche). The membrane was pre-hybridized at 50 °C for 1 h, and then hybridized at 55 °C for 24 h after adding the specific probe in a hybridization oven (UVP American). The membrane was washed in 2× SSC containing 0.1% SDS at 25 °C, and subsequently, washed twice in 0.1× SSC, containing 0.1% SDS, at 68 °C. A 1.5% blocking reagent (Roche) in maleic buffer was applied for immunological detection as described by Huang et al. [Total RNA was quantified by Infinite M200PRO . Then, 5-μg aliquots were denatured in RNA loading buffer with ethidium bromide for 15 min at 65 °C, separated in formaldehyde-containing 2% agarose gels, transferred onto Hybond N+ membranes  by capillary blotting using standard protocols, and fixed by applying a crosslinker . Digoxigenin-labeled plasmid DNA probes were amplified using the PCR DIG Probe Synthesis Kit  with gene-specific primers listed in Additional file g et al.  and the cdm)  were obtained from EMS mutagenesis on isolated microspores, and the variant plants were screened. All the plants were selfed to obtain M1 seeds. In M1 generation, all the new variant plants were also screened. A chlorophyll-deficient mutant (dm) Fig.  was idencdm plants were reciprocally crossed with ‘FT’ plants. The 376 F1 individuals were different, but consistent with the characteristics of female parent  rRNA, 16S (1.9 kb) rRNA, and 4.5S rRNA were over-accumulated in cdm  residues, which primarily induces C-to-T changes and ultimately leads to an amino acid change or deletion . The A-tthaliana , barley thaliana , 70, andenothera , 71. Theenothera . The plaenothera . Since Erps5, prps17, and prpl24 impairs rRNA processing in A. thaliana [rpl33 mutants in E. coli showed low-temperature tolerance [rpl33 mutants in tobacco showed decreased viability and growth under chilling stress [Escherichia coli [rps4 might affect the chloroplast ribosomal assembly process and impair plastid rRNA processing in cdm. By running denaturing agarose gels, we found that abundance of plastid rRNAs in cdm and cdm × ‘FT’ was decreased significantly than those in ‘FT’ and ‘FT’ × cdm. These results indicated that the impaired rRNA processing was maternal inherited. Northern blot data revealed a reduced abundance of mature 23S rRNA, 16S rRNA, and 4.5S rRNA, but over-accumulation of 23S rRNA, 16S rRNA, and 4.5S rRNA precursors in cdm. The missense mutation in RPS4 was the cause of aberrant rRNA processing in cdm that might be due to the reduced transcript level of RPS4 or the Val to Gly mutation in RPS4.Ribosomal proteins are essential components of the plastid ribosome and play different roles in rRNA processing and abundance. Mutations in plastid ribosomal proteins result in a range of developmental phenotypes, of which, some impair rRNA processing. Previous studies demonstrated that a mutation in thaliana , 72. Plaolerance , 74, wheg stress . RPS4 ishia coli  and is ehia coli ; thus, trRNA is stable only when integrated into ribosomal subunits; thus, rRNA abundance can serve as an indicator of the chloroplast ribosome content . The ribrps4 was associated with Chlorophyll deficiency and slow growth mutation in Chinese cabbage that impaired the rRNA processing and affected the ribosomal function and plastid translation.We first reported the identification of a homoplasmic plastome mutant in Chinese cabbage this study. The A-to-C point in the plastome-encoded Additional file 1:Table S1. Primers used in this study. (XLSX 11 kb)Additional file 2:Table S2. Mutation sites in the plastome of wild type ‘FT’ compared with the reference genome. (FREQ 10682 kb)Additional file 3:Table S3. Mutation sites in the plastome of cdm compared with the reference genome. (XLSX 9748 kb)Additional file 4:Figure S1. rRNA accumulation pattern of cdm, ‘FT’, ‘FT’ × cdm and cdm × ‘FT’ in ethidium bromide-stainde gel. (TIF 21348 kb)Additional file 5:Figure S2. Loading controls for RNA gel blotting. (TIF 16387 kb)


Cyrtotrachelus buqueti (bamboo snout beetle) to evaluate the efficiency of bamboo lignocellulose degradation.As an important biomass raw material, the lignocellulose in bamboo is of significant value in energy conversion. The conversion of bamboo lignocellulose into fermentable reducing sugar, i.e. the degradation of bamboo lignocellulose, is an important step in lignocellulose conversion. However, little research has focussed on excavating the enzymes and microbes that are related to the degradation of bamboo lignocellulose, which is important for its utilisation. This study used evgtrinloc27093t1 and evgtrinloc16407t0) and laccase (evgtrinloc15173t0 and evgtrinloc11252t0), were found to be upregulated after feeding. Faecal component analysis showed that the degradation efficiencies of cellulose, hemicellulose and lignin were 61.82%, 87.65% and 69.05%, respectively. After 6 days of co-culture with crude enzymes in vitro, the degradation efficiencies of cellulose, hemicellulose and lignin in bamboo shoot particles (BSPs) were 24.98%, 37.52% and 26.67%, respectively. These results indicated that lignocellulosic enzymes and related enzymes within the insect itself co-degraded bamboo lignocellulose. These finding can potentially be used for the pre-treatment and enzymatic hydrolysis of bamboo lignocellulose.RNA sequencing was conducted to sequence the transcriptome of the insect before and after feeding on bamboo shoots. The expression levels of genes encoding several carbohydrate-active enzymes, such as endoglucanase  genes were upregulated in the transcriptome, including those for cellulase, xylanase and ligninase genes. Therefore, we proposed a scheme for applying the lignocellulolytic enzymes from C. buqueti to degrade bamboo lignocellulose using genetic, enzymatic and fermentation engineering techniques to overexpress the lignocellulolytic enzymes genes in vitro and obtain large quantities of enzymes that could efficiently degrade bamboo lignocellulose and be used for lignocellulose bioconversion.Our results showed that intestinal digestive enzymes from The online version of this article (10.1186/s13068-019-1406-y) contains supplementary material, which is available to authorized users. It has a short growth cycle, high yield and high lignocellulose content which provide many advantages for bioenergy utilisation . In addiorldwide –6. AlthoCyrtotrachelus buqueti [Anoplophora glabripennis (Asian longhorn beetle) [Diacritical virgifera virgifera (Western corn rootworm) [Rhynchophorus ferrugineus  [Enzymatic hydrolysis is an important step in lignocellulose bioconversion. During this step, cellulose and hemicellulose are converted into their sugar constituents, which can be fermented to produce biofuels . However buqueti . Neverth buqueti , Anoplop beetle) , Diacritootworm) , and Rhy weevil) . HoweverC. buqueti to degrade bamboo lignocellulose in vivo and in vitro were investigated. The digestive systems of C. buqueti were extracted before and after feeding for total RNA isolation and high-throughput RNA sequencing. Transcriptional profiling revealed that the expression levels of CAZymes were increased after feeding. The crude enzymes were extracted and used to degrade bamboo shoot particles (BSPs) in vitro over 6 days, and we found that the lignocellulose was partially removed. This indicated that insect enzymes could degrade bamboo shoot lignocellulose both in vivo and in vitro, which could provide new enzyme resources for the biodegradation of bamboo lignocellulose.In this study, the abilities of adult C. buqueti libraries, respectively. All clean reads were assembled into transcripts using Trinity RNA-Seq [Clean reads were obtained from raw reads after removing reads that were of low quality or were ambiguous; adapters and duplicates were also removed. This resulted in 72,931,350 and 70,126,886 clean reads in the control and treatment  RNA-Seq . The resDendroctonus ponderosae (mountain pine beetle) (52.1%), Tribolium castaneum (red flour beetle) (25.9%), Oryctes borbonicus (scarab beetle) (1.9%), Homo sapiens (1.2%), and Lasius niger (0.6%) representing 82% of all annotated species.The assembled unigenes were annotated against several databases: non-redundant (nr) 14,212), conserved domains (CDD) , Swiss-Prot , protein families (PFAM) (9547), gene ontology (GO) , the Kyoto encyclopaedia of genes and genomes (KEGG) (8778), Translation from EMBL (TrEMBL)  and euKaryotic Orthologous Groups (KOG) (9088) , cellular component (CC) and molecular function (MF) , 53 GT families (184 GTs), 8 CE families (63 CEs), 1 PL family (1 PL), 7 AA families (52 AAs) and 14 CBM families (81 CBMs) , PLs, CEs, AAs and CBMs . The funamilies 10 GHs, 53The GH families were primarily represented by the families GH1, GH13, GH18, GH2, GH20, GH28, GH31, GH5, GH45, GH47 and GH48. Of these proteins, 11 candidate proteins were identified from the transcriptome as GH1  were highly expressed in the treatment group and the top two GH transcripts (evgtrinloc27093t1 and evgtrinloc16407t0) belonged to GH5  were highly expressed in the treatment group and the top two transcripts (evgtrinloc13823t0 and evgtrinloc44713t0) belonged to AA3  were highly expressed in the treatment group and the top transcript (evgtrinloc23745t0) belonged to CE10  remained for analysis. A cluster analysis of the CAZyme gene transcripts revealed that the expression patterns of GH, GT, CE, AA and CBM were divided mainly into two categories: one with no obvious difference between the control and treatment groups and the other in which the expression in the treatment group was higher than that in the control group , two β-glucosidase genes (evgtrinloc709t0 and evgtrinloc1536t1), two exoglucanase genes (evgtrinloc145t1 and evgtrinloc766t0), one CE10 gene (evgtrinloc23745t0) and two laccase genes (evgtrinloc15173t0 and evgtrinloc11252t0), using the primers described in Additional file EF1-ɑ acting as the reference gene. The expression of each of these ten genes in the gut at 0, 0.5, 1 and 2 h after feeding was determined. Evgtrinloc3368t0, evgtrinloc27093t1, evgtrinloc16407t0, evgtrinloc145t1 and evgtrinloc766t0 were upregulated after feeding, while evgtrinloc23745t0, evgtrinloc1536t1, evgtrinloc709t0, evgtrinloc15173t0 and evgtrinloc11252t0 were upregulated initially and then decreased after feeding  in the insects’ faeces. We found that cellulose, hemicellulose and lignin had decreased Fig. e. By exaC. buqueti, the crude enzymes were extracted and used to degrade BSPs in vitro for 6 days, after which BSPs were collected for SEM examination. The surface of the untreated BSPs was smooth  is an important step in lignocellulose conversion. The degradation of bamboo lignocellulose, including its pre-treatment and enzyme hydrolysis, has focussed mainly on using physical, chemical and commercial enzymes –5, and frsicolor  and GalaCCZU11-1 . In addiC. buqueti, degraded lignocellulose from bamboo shoots both in vivo and in vitro. In addition, the expression levels of many CAZyme genes were upregulated, including those for cellulase, xylanase and ligninase genes in the transcriptome. Therefore, we proposed a scheme for applying the lignocellulolytic enzymes from C. buqueti to degrade bamboo lignocellulose using genetic, enzyme and fermentation engineering to overexpress the lignocellulolytic enzymes genes in vitro and obtain massive amounts of enzymes that could efficiently degrade bamboo lignocellulose and be used for lignocellulose bioconversion.The digestive system of termites has been described by many scientists as the world’s smallest but most efficient bioreactor . Xie et Cyrtotrachelus buqueti is an extremely harmful bamboo borer and acts as a ‘natural biomass utilisation system’. However, little work has been done for C. buqueti and bamboo degradation. In this study, we determined the degradation efficiency of bamboo lignocellulose, and showed that the degradation efficiencies of cellulose, hemicellulose and lignin in BSPs were 61.82%, 87.65% and 69.05%, respectively, in vivo, and were 24.98%, 37.52% and 26.67%, respectively, in vitro. It indicated the degrading ability in bamboo lignocellulose of the insect. Moreover, the transcriptome showed that, after feeding, many CAZyme gene expressions changed and that lignocellulosic enzyme genes, such as endoglucanase genes (evgtrinloc27093t1 and evgtrinloc16407t0) and laccase genes (evgtrinloc15173t0 and evgtrinloc11252t0), were upregulated, which was in accordance with the enzyme activity. This indicates that lignocellulosic enzymes and those related to bamboo fibre degradation within the insect itself co-degraded bamboo lignocellulosee, which may provide new enzyme resources and ideas for the bioconversion of bamboo lignocellulose.The bamboo snout beetle C. buqueti were collected from Dannan, Sichuan Province  in early August 2017 and were used 3 days after emergence [Adult mergence . The insTotal RNA was extracted from the samples using the DP431 RNAprep Pure Tissue Kit . The yield and purity of the RNA were assessed using 1% agarose gel. An Agilent 2100 Bioanalyzer  was used to detect RNA integrity. RNA was stored at − 80 °C. Five micrograms of total RNA was used to construct the cDNA library and Illumina sequence using Illumina HiSeq 4000, which was managed by Chengdu Basebiotech Co., Ltd.The following eight databases were used for unigene annotation: Conserved Domain Database (CDD), NCBI protein (Nr), Protein family (PFAM), euKaryotic Orthologous Groups (KOG), Translation from EMBL (TrEMBL), Swiss-Prot, GO, and KEGG Orthology. Blast2GO and KEGG Automatic Annotation Server tools were used for annotation. Trinity was used to assemble the transcriptome , and RSEq value ≤ 0.05 were considered to be significantly enriched.GOseq was used for GO analysis based on Wallenius’ non-central hypergeometric distributions . KOBAS 2To identify the genes involved in lignocellulose degradation, coding sequences (CDS) were analysed using the dbCAN CAZyme annotation algorithm, which gives the hidden Markov model index files of various carbohydrate enzyme domains using hmmscan .C. buqueti adults that had been denied food for 24 h were placed on bamboo to feed and then collected 0, 0.5, 1 and 2 h after feeding. Their intestines were dissected in an aseptic environment and made into a crude enzyme solution for each of the different feeding periods and then philtre sterilised with a 0.22-μm philtre. The solution was then used to determine lignocellulolytic enzyme activity; assays were conducted five times each. The tissues were then ground, added to 1 mL pH 5.6 phosphate-buffered saline (PBS) extraction buffer, and centrifuged at 13,000×g for 10 min at 4 °C. The supernatant was collected, which represented the crude enzyme solution. Each replicate sample contained tissues from at least five insects. The process was conducted five times on each sample.To verify changes in the lignocellulolytic enzymes cellulase, xylanase and ligninase in the intestinal tract after eating bamboo shoots, 30 The assays for endoglucanase (EC 3.2.1.4) and exoglucanase (EC 3.2.1.91) were conducted according to the method of Ghose et al.  and the LiP-like activity was measured according to the method of Yan et al. . BrieflyC. buqueti adults that were denied food for 24 h were collected 0, 0.5, 1 and 2 h after feeding, and their intestines were rapidly extracted. A DP431RNAprep Pure Tissue Kit  was used to extract total RNA from the intestines of the insects. All treatments were conducted three times. The primers used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis are listed in Additional file −ΔΔCT method [Thirty T method .Bambusa emeiensis were collected from Muchuan  and cut into 5 × 5 cm pieces. The pieces were divided into two parts: one was directly used as food for C. buqueti; the other was strained through a 40-mesh sieve, dried at 65 °C for 72 h and stored at 4 °C.Bamboo shoots 50–100 cm high from The insects were fed bamboo shoot pieces that measured 30 cm in diameter, placed in a high 10-cm-diameter plastic box and reared in the laboratory at 25 °C ± 1 °C and 70% ± 10% relative humidity with a 12L:12D photoperiod . Faeces BSPs and faeces were examined by SEM. The surface morphology of the samples was observed by SEM  and the samples were sprayed with gold to a thickness of approximately 10 nm using an E-1010 sputtering film coating machine (Japan) before imaging. The operating current and voltage of the SEM were 81 mA and 10 kV, respectively.To prepare BSPs for assay, the shoots were dried to a uniform weight at 65 °C, then crushed into particles using a pulverising machine and strained through a 40-mm-mesh sieve.C. buqueti adults were used for the process of extracting the crude enzyme proteins. The tissues were ground into 10 mL pH 5.6 PBS extraction buffer, the crude extract was then centrifuged at 13,000×g for 10 min at 4 °C and the supernatant collected and then philtre sterilised with a 0.22-μm philtre. Each replicate sample contained tissues from 10 insects, and the process was conducted five times on each sample.The tissues of 50 For the assay, 10-mL 80 g/L tetracycline solution was prepared. As shown in Table Reducing sugar was identified using 3,5-dinitrosalicylic acid . FirstlyThe cellulose, hemicellulose, and lignin contents of the dried BSPs were determined using the Van Soest method  and the The following equations were used for calculating degradation efficiency:From the co-cultures, 2-mL samples were withdrawn at 0.5, 1, 2, 6, 24, 48, 72, 96, 120 and 144 h for assays of lignocellulolytic enzyme activity. The determination method was same as that used above.2SO4, filtered through philtre paper and concentrated to ~ 1 mL using a rotary vacuum evaporator. Then, 100-µL dioxane and 10-µL pyridine were added to the sample followed by silylation with 50-µL trimethyl silyl . Gas chromatography–mass spectrometry (GC–MS) was used to analyse the silylated compounds according to the procedure of Chen et al. [Cultures of BSP treated with crude enzymes were collected on day 6 and centrifuged at 8000 rpm for 15 min to remove biomass. The supernatants were collected and treated using the method of Raj et al. . In brien et al. . The TMSt test was used to compare the means from two groups. Intergroup comparisons of more than two groups were conducted using analysis of variance. A p value of less than 0.05 indicated a statistically significant difference.Statistical analyses were performed using SPSS 19.0 . Descriptive data were expressed as the mean ± standard error of mean (SEM). A Student’s Additional file 1: Table S1. Number and length of transcripts and unigenes and unigenes annotated in different databases.Additional file 2: Table S2. Percentage numbers of the abundant annotated species.Additional file 3: Fig. S1. The number of CAZymes in the transcriptome. GHs: glycoside hydrolases, GTs: glycosyltransferases, CEs: carbohydrate esterases, CBMs: carbohydrate-binding domains, PLs: polysaccharide lyases, AAs: auxiliary activities, CAZyme: carbohydrate-active enzymes.Additional file 4: Table S3. Unigenes annotated in CAZyme.Additional file 5: Table S4. The CAZyme classification at the family level.Additional file 6: Table S5. The annotation of genes in CAZyme in the transcriptome.Additional file 7: Fig. S2. The lignocellulolytic enzyme activities of C. buqueti displayed by enzymes of the adults 0.5, 1, 2, 6, 24, 48, 72, 96, 120 and 144 h after being co-cultured with BSP in vitro.Additional file 8: Fig. S3. Total ion chromatograms of dichloromethane extract analysed as trimethylchlorosilane derivatives from control (a) and treatment (b) in vitro.Additional file 9: Table S6. The primer sequence of qRT-PCR.


For decades, genetic aberrations including chromosome and molecular abnormalities are important diagnostic and prognostic factors in acute myeloid leukemia (AML). ATRA and imatinib have been successfully used in AML and chronic myelogenous leukemia, which proved that targeted therapy by identifying molecular lesions could improve leukemia outcomes. Recent advances in next generation sequencing have revealed molecular landscape of AML, presenting us with many molecular abnormalities. The individual prognostic information derived from a specific mutation could be modified by other molecular lesions. Therefore, the genomic complexity in AML poses a huge challenge to successful translation into more accurate risk stratification and targeted therapy. Herein, a summary of these mutations and targeted therapies are described. We focus on the prognostic information of recent identified molecular lesions and emerging targeted therapy. Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by multiple somatically acquired driver mutations, coexisting competing clones, and disease evolution over time , 2. Spec2) derived an increase in median overall survival (OS) compared with Standard dose daunorubicin (45 mg/m2) therapy (16.9 m vs 75.9 m) [NPM1 mutations are reported in approximately one-third of AML adults, and more than half of them are with normal cytogenetics (CN-AML). It often co-occurs with mutations in epigenetic modifiers such as DNA methyltransferase 3A (DNMT3A), Ten-eleven translocation gene-2(TET2) and Isocitrate dehydrogenase1/2 (IDH1/2) mutations . Numerou 75.9 m) . Besides 75.9 m) , 22.In addition to mutations in NPM1, mutations leading to aberrant activation and proliferation of cellular signaling pathways, including FLT3, KRAS, NRAS, PTPN11, NF1, and KIT, are present in approximately two-thirds of AML cases.n = 267) [n = 717) [n = 23) [Mutations in FLT3 mostly involves internal tandem duplications within the juxta membrane region (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD). Previous studies have confirmed that, FLT3-ITD mutations are associated with higher relapse rate and poorer overall survival, particularly with a high ratio of mutant allelic burden , 24. In n = 267) . A phasen = 717) , 14. Secn = 717) –29. The n = 717) . It is t[n = 23) . Adding [n = 23) . FLT3 in[n = 23) . CurrentThe receptor tyrosine kinase KIT is frequently mutated in Core-binding factor AML (CBF-AML), which is defined by the occurrence of t  or inv.(16)/t  rearrangements. It has been demonstrated that the frequency of inv.(16)/t  AML in CBF leukemia is higher in Caucasian than in Chinese and Japanese . The maiMutations in transcription factors occur in 20- 25% of patients with AML, including myeloid transcription factors, Runt-related transcription factor 1 (RUNX1) and CCAAT/enhancer binding protein α (CEBPA).P = 0.002; 14 m vs 22 m, P = 00.048). Concomitant ASXL1 mutation and ≥ 2 additional mutations correlated with shorter OS  [RUNX1 mutations were reported in 5% to 10% of AML, and more in patients with secondary AML evolving from myelodysplastic syndrome , 41. The= 0.017) .Mutated CEBPA gene occur in 5-14% of AML patients, mainly in CN-AML, and was related to better prognosis. Recent studies showed that, the favorable impact of mutant CEBPA on prognosis is associated with biallelic mutations , 45. TheIn recent years, the discovery of epigenetic regulators has provided great insight into the pathogenesis of AML. Mutations in these genes such as IDH1/2, DNMT3A, TET2, Additional sex comb-like 1(ASXL1), and Enhancer of zeste homolog 2(EZH2), which appear to impact on DNA methylation or histone posttranslational modifications, may serve as prognostic markers for risk stratification and therapeutic decision –49.n = 179) [Mutations in genes encoding IDH1 and IDH2 gene mainly involves IDH1-R132, IDH2-R140, and IDH2-R172, and IDH1 and IDH2 mutations rarely co-occur. IDH1-R132 or IDH2-R140 frequently occur in AML patients with normal cytogenetics and advanced age, with concurrent mutations of NPM1 . While In = 179) . Based on = 179) .P = 0.29) [Mutations in epigenetic regulators also include DNMT3A, TET2 and ASXL1, namely DTA mutations. These mutations are most common in people harboring age-related clonal hematopoiesis. Nowadays, no consensus has been reached on the prognosis of DNMT3a mutation. Some studies reported its poor prognosis , while o = 0.29) , 66. But = 0.29) .Mutations in splicing factors accounts for 10% of AML patients, which are defined by mutations in genes regulating RNA splicing . They are likely to cause aberrant splicing, affecting the transcriptome and proteome of cells. Accumulating evidence shows that spliceosome mutations are associated with older age, less proliferative disease, low response rate to standard treatment, and poorer survival . Recent Although a lot of mutations are predicted to be activating or neomorphic, many of them have been demonstrated to be loss-of-function mutations, such as mutation in tumor protein 53(TP53), rendering them less tractable targets. TP53 is a key tumor suppressor gene. TP53 mutations accounts for 8% of patients with AML, and are associated with complex cytogenetics, therapy-related AML, chemoresistance, high relapse rates and poor survival , 58, 70.The discovery of the molecular landscape of AML not only provides us a chance to better understand the pathogenesis of the disease, but also refines the risk stratification and management of patients. However, evolving evidence demonstrated that the individual prognostic information derived from a specific mutation could be modified by other molecular characteristic and clinical parameters. Therefore, development of risk group classification schemes based on comprehensive genomic assessment should be considered a work in future. Targeted therapy to these mutations achieved huge progress in recent years. For instance, both IDH1/2 and FLT3 inhibitors showed promising results. And the combination of venetoclax with azacitidine produced a median OS longer than 12 months in older AML patients with a favorable safety profile , 77. ConIn addition to genetic aberrations, epigenetics or posttranscriptional regulations may also play a pivotal role in determining the biological behavior of AML. Current researches demonstrated that DNA methylation patterns and long noncoding RNAs contribute to many critical signaling pathways in AML development, even exert effects on diagnosis classification and outcome of AML , 79. AndIn addition, encouraging efficacy of immunotherapeutic agents, especially the chimeric antigen receptor T (CAR-T) cell therapy, has brought huge advance to ALL treatment in the past decade –83. Howe


Laparoscopic cholecystectomy - associated bile duct injury is a clinical problem with bad outcome. The study aimed to analyze the outcome of surgical management of these injuries.We retrospectively analyzed 69 patients underwent surgical management of laparoscopic cholecystectomy related major bile duct injuries in the period from the beginning of 2013 to the beginning of 2018.Regarding injury type; the Leaking, Obstructing, leaking + obstructing, leaking + vascular, and obstructing + vascular injuries were 43.5%, 27.5%, 18.8%, 2.9%, and 7.2% respectively. However, the Strasberg classification of injury was as follow E1 = 25, E2 = 32, E3 = 8, and E4 = 4. The definitive procedures were as follow: end to end biliary anastomosis with stenting, hepaticojejunostomy (HJ) with or without stenting, and RT hepatectomy plus biliary reconstruction with stenting in 4.3%, 87%, and 8.7% of patients respectively. According to the time of definitive procedure from injury; the immediate (before 72 h), intermediate (between 72 h and 1.5months), and late (after1.5 months) management were 13%, 14.5%, and 72.5% respectively. The hospital and/or 1month (early) morbidity after definitive treatment was 21.7%, while, the late biliary morbidity was 17.4% and the overall mortality was 2.9%, on the other hand, the late biliary morbidity-free survival was 79.7%. On univariate analysis, the following factors were significant predictors of early morbidity; Sepsis at referral, higher Strasberg grade, associated vascular injury, right hepatectomy with biliary reconstruction as a definitive procedure, intra-operative bleeding with blood transfusion, liver cirrhosis, and longer operative times and hospital stays. However, the following factors were significantly associated with late biliary morbidity: Sepsis at referral, end to end anastomosis with stenting, reconstruction without stenting, liver cirrhosis, operative bleeding, and early morbidity.Sepsis at referral, liver cirrhosis, and operative bleeding were significantly associated with both early and late morbidities after definitive management of laparoscopic cholecystectomy related major bile duct injuries, so it is crucial to avoid these catastrophes when doing those major procedures. •Sepsis at referral was associated with poor outcome after management of LC related MBDIs.•Liver cirrhosis and operative bleeding were associated with poor outcome after management of these injuries.•It is crucial to avoid these catastrophes when doing those major procedures. ACCAcute calcular cholecystitisASAAmerican society of anesthesiaBDIBile duct injuryBIBiliary injuryCBDCommon bile ductCDSClavien Dindo scoreCTcomputerized tomographyCUSACavitron ultrasonic surgical aspiratorERCPEndoscopic retrograde cholangio-pancreatographyHAHepatic arteryHJHepaticojejunostomyHPBHepatopancreatobiliaryIOCIntra-operative cholangiogramIRBInstitutional review boardKMKaplan–MeierLCLaparoscopic cholecystectomyLFTLiver function testMBDIsMajor bile duct injuriesMRCPMagnetic resonance cholangiopancreatographyMRIMagnetic resonance imagingNLINational Liver InstituteOCOpen cholecystectomyPDSpolidioxanonePODPost-operative dayPTCPercutaneous transhepatic cholangiographyPTDPercutaneous transhepatic drainagePVPortal veinRUQRight upper quadrantUSUltrasonography1Despite increased surgical skills and experience regarding laparoscopic choelcystectomy (LC), the rate of LC related bile duct injury (BDI) is still higher in comparison to open cholecystectomy (0.2%–1.5% vs. 0.1–0.2% respectively) . LC relaThe mechanisms of these injuries involve thermal injuries, scissors, ligatures or clips . These iSurgical biliary-enteric reconstruction including the Hepp-Couinaud approach at a specialist hepatobiliary center by an experienced surgeon, is the most effective treatment of these injuries with perfect long-term results . HoweverThere is little literature on the long-term outcomes after surgical reconstruction of MBDIs, moreover, the factors predicting those outcomes have not been studied extensively . Our stu1.1Ninety patients underwent definitive surgical management of LC related MBDIs (>E1), in the period from the beginning of 2013 to the beginning of 2018 either in the department of hepato-pancreato-biliary (HPB) surgery , National Liver Institute (NLI), University of Menoufia, Menoufia, Egypt (88 patients), or in the calling hospitals in Menoufia (2 patients). After exclusion of cases with data loss and cases that refused research; our study included 69 patients , and one patient who had the injury in our hospital). After approval of institutional review board (IRB), we did this cohort study which is a single-institution retrospective analysis of a prospectively collected database that analyzed the outcome of surgical management of LC related MBDI in the period from mid 2017 to mid 2018, where patients were observed from POD1 until the end of June 2018 or until death of patients with median follow up period of 43 ms, range (0.7–66 ms), with researchregistry2211.The data were collected from our records in our HPB surgery department including the data of the 3 patients operated in the calling hospitals. Written informed consents regarding surgery and researches were obtained from our patients. Our work has been reported in line with the STROCSS criteria .The recorded data included patient demographics, indications of cholecystectomy, IOC performance during cholecystectomy, direct cause of biliary injury (BI) and timing of its discovery, referral time after injury, presentation of patients and presence of sepsis at referral, tools of biliary anatomy determination and BI classification, associated vascular injury, intervention before definitive operation and preoperative American society of anesthesia (ASA) score, time of definitive procedure from injury and type of operations, intra-operative liver biopsy results, operative bleeding and time, patients outcome, and lastly follow-up data.Patient presentation  was known by good history, clinical examination, and laboratory investigations especially liver function tests (LFTs))), furthermore, sepsis at referral was known by leukocytosis, abdominal pain, fever, and/or peritonitis . HoweverFor determination of biliary anatomy, abdominal US was done for all referred patients, abdominal CT, Magnetic resonance cholangio-pancreatography(MRCP) and fistulogram were done as indicated, however CT angiography was done when co-vascular injury was suspected, lastly ERCP and percutaneous trans-hepatic cholangiography/drainage (PTC)/PTD were done for diagnostic and therapeutic purposes. The anatomic extent of BI was classified according to the Strasberg- Bismuth classification system ,41 Fig. After referral to our center, multidisciplinary staff meeting occurred including surgeons, endoscopists, radiologists, and anaesthetists for controlling initial patient condition where different intervention procedures were performed before the definitive operation ; the laparotomy included drainage of biliary collection ± external biliary diversion, where the endoscopy included ERCP ± sphinectrotomy ± dilatation ± stenting and lastly, the radiology included percutaneous pigtail drainge ± PTD.).The time of performance of operation from injury was classified into immediate (during the 1st 72 h), intermediate (between 72 h and 1.5 months) and late (after1.5 months).Operation type: Our institutional guidelines is performing HJ with or without trans-anastomotic stent (Hepp-Couinaud approach) , Fig. 4,The surgical techniques: After opening the abdomen, adhesiolysis and mobilization of the liver were done. Intraoperative doppler-ultrasound was used to detect vascular involvement (if suspected), Pringle maneuver was systematically done, hilar structures were exposed and IOC was performed to assess the biliary tree .In HJ cases, the anastomosis was performed with a Roux-en-Y HJ, with extension to the left hepatic duct (Hepp– Couinaud approach)  to achieStents were internal or external; the internal stents were 4–7 French straight biliary stent (9–11 cm) or it was 4–7 French ureteric catheters that were placed into the common bile duct (CBD) and blindly directed across the ampulla of Vater. However, the external stent tip was positioned into the bile duct mostly the left one crossing the HJ anastomosis and pulled out of the jejunum . They weThe outcome of patients: It was classified into 1- Early  morbidities that occurred during the initial hospital stay or after discharge until the 1st month after surgery and classified according to Clavien-Dindo grading system . 2-EarlyLong-term follow-up: For detection of late biliary morbidity, patients were followed-up every 3 months in the 1st year then yearly until the end of follow-up period by clinical assessment, LFT, ultrasonography, and others if needed (i.e. MRCP).Qui square or Fisher exact tests. Numerical data were expressed as the mean and standard deviation and were compared with the T or Mann whitteny tests. Univariate analysis and then multivariate analysis (by Binary logistic regression method) were done to detect the relationship between the different pre- and intra-operative variables and early morbidity as well as the relation between these variables and late biliary morbidity. The Kaplan–Meier method was used for survival analysis to assess the overall and late biliary morbidity-free survivals, a P value of <0.05 was significant.Statistical Techniques: Th data were processed with SPSS software . Nonnumerical data were expressed in frequency and % and analyzed with the 22.1They were classified as 31(44.9%) males, and 38 (55.1%) females; their mean age was 38.08 ± 9.6. The previous cholecystectomy was done due to acute calcular cholecystitis (ACC) and biliary colic in 66.7% and 33.3% of them respectively; furthermore, IOC was done in 15.9% of patients during LC. Clipping, diathermy, ligature, and scissors were the direct cause of injury in 17.4%, 62.3%, 8.7% and 11.6% of them respectively. BI was discovered during cholecystectomy in 20.3% of patients, on the other hand, it was diagnosed in the early (during 7 days from cholecystectomy), intermediate (7 days–3 months) and late (after 3 months) periods after cholecystectomy in 44.9%, 26.1%, and 8.7% of them respectively. Furthermore, those patients with BI discovery after cholecystectomy were mainly presented with jaundice, cholangitis, bilious drain, and peritonitis that affected 23.2%, 17.4%, 30.4%, and 8.7% of them respectively. The mean time of referral to our center after injury diagnosis was7.1 ± 8.9 days, however, 10 (14.5%) of patients had sepsis at referral; this sepsis was due to biliary peritonitis or severe cholangitis with or without cholangectitic abscesses. The abdominal US, CT abdomen, abdominal CT angiography, MRCP, PTC, and ERCP were done in 65(94.2%), 14 (20.3%), 11 (15.9%), 40(58%), 10(14.5%), and 35(50.6%) of them respectively.According to the nature of biliary injury, it was classified into Leaking, obstructing, leaking + obstructing, leaking + vascular, and obstructing + vascular that affected 43.5%, 27.5%, 18.8%, 2.9%, and 7.2% of patients respectively. On the other hand and regarding Strasberg classification of injury, the E1, E2, E3, and E4 types affected 36.2%, 46.4%, 11.6% and 5.8% of them respectively. In our biliary injured patients, the associated vascular injury was as follow: RT hepatic artery (HA) injury, RT HA + RT portal vein(PV) injuries, and RT PV injury that affected 5(7.2%), 1(1.4%), and 1(1.4%) of our patients respectively. 2.2Fifty-six (81.2%) of patients underwent intervention procedures before the definitive operation in the form of laparotomy, endoscopy and/or intervention radiology; laparotomy was done in 21 patients where 16 of them underwent exploration for leak and drainage, and the other 5 patients underwent external biliary diversion, however, endoscopic intervention was done in 35 ones where 25 of them underwent ERCP and stenting, 7 patients underwent ERCP but failed dilatation or stenting, and the other 3 ones had cannulation failure, on the other hand, 26 patients underwent intervention radiology where 16 of them underwent pigtail drainage(single or multiple), 9 patients underwent PTD, and the last one underwent both pigtail drainage and PTD. Patients were graded as regard ASA score into 55(79.7%), 10(14.5%), and 4(5.8%), ASA 1, 2, and 3 respectively. The mean pre definitive procedure total and direct bilirubin were 6.5 ± 6.3 and 5.1 ± 4.9 (mg/dl) respectively.The mean time of definitive procedure from injury was 74.5 ± 61.8 days  where 9 (13%), 10 (14.5%), and 50(72.5%) of patients underwent the definitive operation immediately (during the 1st 72 h), in the intermediate period (between 72 h and 1.5 months) and in the late period (after1.5 months) from injury respectively. The definitive procedures were as follow: End to end direct biliary anastomosis with stenting in 3 patients, HJ without stent in 20 patients, HJ with stent in 40 patients, and hepatectomy with biliary reconstruction with stenting in 6 patients. Regarding the 3 patients who underwent end to end anastomosis with stent; the 1st one was due to our center injury that was discovered during LC, so conversion to open surgery was decided and after performing IOC; anastomosis was done using PDS5/0 interrupted sutures with internal 4 French straight biliary stent (9 cm), however, the other 2 patients had the injury in other centers where we were called and did the repair immediately in these centers using PDS 5/0 interrupted sutures with internal 7 French ureteric catheters. On the other hand, regarding the 6 patients who underwent hepatectomy; the 1st one had E3 injury and associated RT PV injury and liver atrophy, the 2nd one had E4 injury and associated RT HA injury, RT PV injury and liver abscesses, the 3rd one had E4 injury and associated RT HA injury and liver abscesses, while the last 3 ones had associated RT HA injury and liver atrophy (2 of them had E4 and one had E3 injuries). Thirteen (18.8%) and 10(14.5%) of patients had cirrhotic liver and operative bleeding during the definitive operation respectively. Lastly, the mean blood transfusion and operative time after the definitive procedure were 0.2 ± 0.7 units, and 227.8 ± 85.5 min respectively. 2.3The early  morbidity affected 15(21.7%) of patients (N.B some of the patients had more than one complication) where, early infection (pulmonary and/or wound infection), abdominal collection, bile leak, and cholangitis affected 10(14.5%), 4(5.8%), 4(5.8%), and 4(5.8%) of them respectively. However, these complications were graded regarding Clavien grading (CDS) as 6(8.7%), 6(8.7%), 1(1.4%) and 2(2.9%) grades 2, 3, 4, and 5 respectively . Furthermore, the early mortality was 2.9% of patients (2 patients); the 1st one had associated vascular injury (HA + PV injuries) and liver atrophy and underwent RT hepatectomy + biliary reconstruction with stenting but unfortunately died 24 days after operation from liver failure and sepsis, however the other one had associated HA injury with multiple liver abscesses and underwent RT hepatectomy + biliary reconstruction with stenting and died 20 days from surgery due to liver failure and sepsis too. The mean hospital stay was 8.6 ± 5.2 days.On the other hand, the late biliary morbidity was 12(17.4%), in the form of recurrent cholangitis 5(7.25%); where the initial attacks developed at 9 months, 10 months, 20 months, 33 months and 39 months from definitive surgery, stricture 5(7.25%), that occurred at 20 months, 21 months, 22 months, 25 months, and 35 months from surgery, and both stricture and recurrent cholangitis 2(2.9%), that happened at 40 months and 45 months from surgery; as regard McDonald's grading, grades A, B, C, and D were 45/67(67.2%),10/67(14.9%),5/67(7.5%), and 7/67(10.4%) respectively; these complications were managed as follow: Medical treatment for the 5 cases with recurrent cholangitis, PTD for 3 cases with stricture, and redo HJ for the 2 cases with both stricture and recurrent cholangitis and the other 2 cases with stricture only, moreover; all these patients improved after management. There was no other late mortality except the 2 cases with early mortality, however, the overall survival and the late biliary morbidity-free survival were 67(97.1%), and 55(79.7%) respectively. 2.4On univariate analysis, the following factors were significant predictors of early morbidity: Ligature followed by clipping as mechanisms of injury, injury discovery in the intermediate period after cholecystectomy (between 1 week and 3 months), sepsis at referral, obstructing and vascular form of injury, higher Strasberg grade, associated vascular injury, intervention before definitive repair, performing the definitive surgery later after injury (mean 140 ± 60.4 days), RT hepatectomy with biliary reconstruction as the definitive procedure, operative bleeding and blood transfusion (mean 0.9 ± 1.4 units), cirrhotic liver during definitive procedure and longer mean operative time and hospital stay(296 ± 79.2 min and 16.6 ± 5.5 days respectively). On the other hand, there was a trend towards the occurrence of significant early morbidity with later referral after injury discovery (mean 10.8 ± 10.05 days). In contrast, there was no independent predictor of early morbidity on multivariate analysis. 2.5On univariate analysis, the following factors were significant predictor's of late biliary morbidity: Sepsis at referral, performing the definitive surgery later after injury (mean 114.8 ± 84.9 days), end to end anastomosis with stenting as the definitive procedure, the absence of stenting, operative bleeding, cirrhotic liver, and early morbidity. Conversely, on multivariate analysis, there was no independent risk factor of our late biliary morbidity. 3BDI is considered the most significant complication of LC ,20,47. TA detailed preoperative evaluation by, and imaging, improving of patient general condition before the definitive procedure by co-operation of surgeons, endoscopists and intervention radiologists, correction of nutritional, fluid-electrolyte disorders and controlling sepsis; and then finally performing meticulous wide anastomosis by experienced surgeons in specialized hepato-biliary units are required for achieving long-term success after repair of MBDIs . SimilarIn the current cohort, we analyzed the early and late outcomes after the definitive procedures.The surgical principles associated with a successful repair of MBDI are exposure of well-vascularized healthy proximal bile ducts that drain the entire liver, and preparation of a Roux-en-Y limb of jejunum>60 cm for a mucosa to-mucosa, tension-free anastomosis between them ,15,53. THepatectomy as a management of MBDIs is associated with a high postoperative morbidity rate ,53. In sPrimary end-to-end repair of MBDI is a method of repair used when there is no loss of tissue . When usThe use of trans-anastomotic stents is controversial ,7,15,67.The reported both early and late morbidity rates after repair of BIs ranged from 38% to 65% ,24,50,71Despite the presence of scarce literature on the factors affecting post repair stricture, some factors were mentioned,  ,50,53,60In our study, referral time to our center after injury diagnosis had no significant impact on early complications or late biliary morbidity; despite the trend towards occurrence of significant early complications with longer time, similarly, In a multivariate analysis done by Lillemoe et al., 2000 , the intIntra-abdominal sepsis and abscesses even if drained effectively may remain active in the period after surgery, predisposing patients to fibrosis, resulting in late anastomotic stricture. Furthermore, inflammatory changes in the surgical bed produce tissue friability, resulting in increased technical difficulty at repair time ,73. In sRepair in patients with higher strictures , and the surgeon experience . PerformIn another line, Arora et al., 2015  found exChronic extra-hepatic biliary obstruction results in dilatation of the extra and intrahepatic biliary tree and proliferation of biliary ductules within the portal triads leading to fibrosis of portal tracts that results in secondary biliary cirrhosis and portal hypertension ,51,54. TTo the best of our knowledge, previous literature did not mention any correlation between operative bleeding and the occurrence of early or late morbidities after surgical repair of MBDIs, however, we found a significant correlation between intra-operative bleeding and both early and late morbidities. Our explanation is that bleeding was related to cases that underwent hepatectomy (P = 0.00) that had a negative impact on early morbidity, and also, was associated with cases with liver cirrhosis (P = 0.00) that had a negative impact on both early and late morbidities.The occurrence of major postoperative complications (CDS>3) were associated with an increased risk of biliary stricture after surgery in Sulpice et al., 2014  and BooiThe main limitation of the study is being retrospective with relatively small NO of patients. So, it is advisable to do further studies with larger no and longer follow-up period with stress on the effect of blood transfusion on morbidities.The approval by National liver institute, Menoufiya university.No source of funding for this research.Emad Hamdy Gad: Study design, data collection, writing, statistical analysis and publication.Eslam Ayoup: Reference update.Yasmin Kamel: data collection, writing, statistical analysis.Talat Zakareya: data collection, writing.Mohamed Abbasy: data collection, writing.Ali Nada: data collection, writing.Mohamed Housseni: data collection, writing.Mohammed Al-sayed Abd-elsamee: Reference update.No conflict of interest to declare.researchregistry2211.All the authors of this paper accept full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish.Not commissioned externally peer reviewed.