Carex L., a grass genus commonly known as sedges, is distributed worldwide and contributes constructively to turf management, forage production, and ecological conservation. The development of next-generation sequencing (NGS) technologies has considerably improved our understanding of transcriptome complexity of Carex L. and provided a valuable genetic reference. However, the current transcriptome is not satisfactory mainly because of the enormous difficulty in obtaining full-length transcripts.Carex breviculmis. We generated 60,353 high-confidence non-redundant transcripts with an average length of 2302-bp. A total of 3588 alternative splicing events, and 1273 long non-coding RNAs were identified. Furthermore, 40,347 complete coding sequences were predicted, providing an informative reference transcriptome. In addition, the transcriptional regulation mechanism of C. breviculmis in response to shade stress was further explored by mapping the NGS data to the reference transcriptome constructed by SMRT sequencing.In this study, we employed PacBio single-molecule long-read sequencing (SMRT) technology for whole-transcriptome profiling in C. breviculmis using the SMRT sequencing method for the first time. The transcriptome atlas obtained will not only facilitate future functional genomics studies but also pave the way for further selective and genic engineering breeding projects for C. breviculmis.This study provided a full-length reference transcriptome of  Carex L. consists of more than 2000 grassy species of the family Cyperaceae, commonly known as sedges, has a worldwide distribution in temperate and cold regions, and contribute constructively to turf management, forage production, and ecological preservation [Carex L., with previous studies being limited to physiological investigation and stress-resistance evaluation [Carex L. breeding urgently needs a theoretical basis at the molecular level and further exploration of genetic resources. Although Li et al. (2018) firstly reported the salt-responsive mechanism of regulation in Carex rigescens utilizing next generation sequencing (NGS), the current description of that transcriptome remains unsatisfactory due to the inborn limitations of NGS technology in reads length.Genus ervation . The widaluation , 4. ConsTriticum aestivum [Oropetium thomaeum [Trifolium pretense [Medicago sativa [Fragaria vesca [Arabidopsis thaliana [Phyllostachys edulis [The PacBio single-molecule long-read sequencing technology (SMRT sequencing) can obtain full-length splice isoforms directly, without assembly, thus providing a better opportunity to investigate genome-wide full-length cDNA molecules . To dateaestivum , Oropetithomaeum , Trifolipretense , Medicago sativa , Fragariia vesca , Arabidothaliana  and Phyls edulis . These ss edulis , 14.Carex breviculmis is a perennial grass with wide distribution in China that lives mainly under tree crowns, as it is highly shade tolerant. As afforestation in China accelerates, C. breviculmis is expected to be planted more widely. However, to date, the genetic resources of C. breviculmis have not been properly exploited, hampering the progress of C. breviculmis breeding efforts.C. breviculmis, we generated high quality full-length non-chimeric reads (FLNC) in the present study by taking advantage of SMRT sequencing technology combined with NGS sequencing methods. In addition, AS events and long non-coding RNAs (lncRNAs) were predicted. Our results provided new insights into the possible mechanism underlying the transcriptional regulation of shade tolerance in C. breviculmis.Aiming to provide a full-length reference transcriptome atlas for C. breviculmis grown under two different conditions  in equal amounts to obtain a full-length reference transcriptome using PacBio sequencing. Three cDNA libraries of different sizes  were constructed and then sequenced using the PacBio RSII sequencing platform, thereby generating 11.52 Gb of SMRT sequencing raw data consisting of 751,460 raw polymerase reads. These reads resulted in 5,086,638 post-filter subreads (length > 50 bp and accuracy > 0.75) with an average of 1,017,327 subreads per cell  from each of the three libraries, respectively , while transcripts in the 300–400 bp range accounted for the least percentage (0.02%). Particularly, the shortest transcript was 305-bp (F01.PB2138) while the longest was 24,616-bp (F01.PB60208).The 73,508 consensus FLNC reads were first clustered using Iterative isoform-clustering program (ICE) program and then polished using the quiver program and non-full-length (NFL) reads. We obtained 56,080 high-quality isoforms (HQ) from 73,508 consensus isoforms Fig. . With thC. breviculmis based on high-quality full-length isoforms. The results showed that 5052 AS events were identified among the transcripts which had two or more alternative isoforms , Coding-Non-Coding Index (CNCI), Protein family (pfam) and Coding Potential Assessment Tool (CPAT), 13,965 transcripts were primarily found to be putative non-coding RNAs Fig. a. FinallC. breviculmis, the CDS-containing transcripts generated by SMRT were used as queries against those of rice. The results showed that 68.57%  of the transcripts identified in C. breviculmis were homologous to those of rice, while the other 31.43%  were specific to C. breviculmis. The homologous transcripts and C. breviculmis specific transcripts are listed in Additional file The TransDecoder program was used to predict coding sequence (CDS) and untranslated regions (UTRs). These unique full-length transcripts involved 57,816 CDS with a mean length of 1189.23 nucleotides, including 40,347 transcripts with complete open reading frames (ORFs) (data not shown). Full-length transcripts consisting of 600–900 nucleotides were most abundant and corresponded to19.89% of the identified CDS  and Musa acuminate (11.13%)  on several databases, 60,353 non-redundant transcripts were annotated for the reference transcriptome. In general, 42,604, 27,264, 39,038, 49,017, 43,321, 27,160, 57,429, and 58,130 transcripts were annotated in the GO, KEGG (Kyoto Encyclopedia of Genes and Genomes), KOG (euKaryotic Orthologous Groups), Pfam (a database of conserved protein families or domains), Swissprot , COG (Clusters of Orthologous Genes), eggNOG  and NR (NCBI non-redundant protein databases), respectively. Finally, based on the annotation results, 58,328 integrate annotated transcripts were generated, providing a comprehensive reference transcriptome for 2 concentration (Ci), transpiration rate (Tr) and stomatal conductance (Cd) were examined to investigate the photosynthetic changes induced by shade treatment. Overall, shade stress reduced Pn and Ci, but increased Tr  function as key players in plant responses to biotic or abiotic stresses conditions. Rationally, we focused on the expression patterns of TFs in responses to shade stress of We assembled the ~ 20% Illumina short reads which were not aligned with the full-length transcriptome build in this study using reference free method. The results contained 56,920 unigenes, among which 32,031 unigenes could be annotated. BLAST analysis was carried out using the unigenes against the full-length transcriptome constructed in this study. Finally, 34,663 unigenes were proved to have not been detected by SMRT sequencing. The assemble results of the Illumina short reads was provided in Additional file Carex, one of the largest genus in Cyperaceae, contains more than 2000 species and plays important roles in turfgrass and forage production as well as in ecological conservation [Carex species transcriptomes is mainly based on gene expression analysis of Carex rigescens using NGS technologies [Carex. transcriptome had not been fully explored due to lack of full-length cDNA. In this study, we adopted the SMRT sequencing strategy with assistance of NGS sequencing to provide a comprehensive transcriptomic landscape of C. breviculmis by generating 11.52 Gb of raw data from 5 SMRT cells and 43.67 Gb of NGS raw data. After clustering, correction, and redundancy elimination, we finally obtained 60,353 high-quality non-redundant transcripts were obtained from C. breviculmis. This information provides the first reported SMRT sequencing atlas of the full-length transcriptome of C. breviculmis.ervation . Howevernologies . MoreoveC. breviculmis, although with lower frequency than that found in strawberry [C. breviculmis.In diverse biological responses, AS serves as an effective mechanism to increase the complexity and flexibility of the entire transcriptome and proteome . Becauserawberry , bamboo rawberry , and Ararawberry . This lorawberry . FurtherlncRNAs, a recently identified class of non-coding RNAs function as essential regulators in a wide range of biological processes , 17. By C. breviculmis. However, we did not validate the authenticity of the prediction results, so further experimental confirmation of the targets is still needed in the future.The LncTar has advantages in accuracy and running speed in predicting lncRNA targets, providing a valuable and efficient tool for large-scale prediction of the targets of candidate lncRNAs . In thisC. breviculmis generated by SMRT were considered homologous to that of rice. This revealed the reliability of the full-length sequences obtained by SMRT and encouraged the functional annotation of the full-length transcripts which provided a reference transcriptome for C. breviculmis including 58,328 integrate annotated transcripts. NR alignment results showed that 25.41% of sequences could be aligned to Elaeis guineensis, indicating that C. breviculmis was most closely related to E. guineensis in terms of protein alignment based on the current NR database. E. guineensis belongs to Palmae while C. breviculmis belongs to Cyperaceae. Considering plant taxonomy, E. guineensis and C. breviculmis seem distantly related. The un-expected protein alignment results may, at least in part, be due to the lack of Cyperaceae related species data in the current NR database, reflecting the urgent need to improve the genetic database for this genus.Using the rice transcriptome as reference, 68.57% of the CDS-containing transcripts of C. breviculmis. Chlorophyll a-b binding proteins are probably the most abundant membrane protein complexes and play important roles in light-harvesting activities in higher plants. Photosystem I/II reaction subunit mediates light-driven electron transfer, protecting plants from phototoxic damage [Light irradiance levels are known to affect the photosynthetic capacity and chlorophyll content in plants . Stay-grc damage . The supC. breviculmis is widely used as an important cover plant in China because of its outstanding level of shade tolerance [FTSH1 and FTSH12) and two malate synthase isoforms ranked the top 10 up-regulated genes. The FTSHs are correlated with in vivo photosystem II function [olerance . Howeverolerance . Two ATPfunction , and malfunction . Wall-asfunction . Two walC. breviculmis. Glutathione peroxidase (GPX) was reported to help photosynthetic organs reduce oxidative damage in transgenic tobacco seedlings under stressful conditions [C. breviculmis, at least partly, at the transcriptional level. Transcription factors such as WRKY, AP2-ERF, NAC and GRAS have been extensively reported as key players in plant responses to biotic and abiotic stresses [C. breviculmis leaves, indicating their various roles in the response of this specie to shade stress.In this study, global expression analysis revealed that biological processes related to ‘proline transport’, ‘chlorophyll biosynthesis’, and ‘photosystem II assembly’ were shade responsive activities in nditions . Phototrnditions . Thus, tstresses –35. CompC. breviculmis shade tolerance. Plants have evolved a complex sucrose transport reaction to avoid shade stress [C. breviculmis shade tolerance. To the best of our knowledge, this is the first transcriptome-wide investigation of C. breviculmis in response to shade stress using reference transcriptome data generated by SMRT sequencing.In addition, pathways related to ‘Nitrogen metabolism’, ‘Starch and sucrose metabolism’, and ‘Photosynthesis-antenna proteins’ seem to actively participate in e stress . Sucrosee stress . The oveAlthough SMRT sequencing has clear advantages over Illumina sequencing in investigating full length transcripts, it has distinct limitations in throughput . In addiC. breviculmis. The analysis of NGS data elucidated on the regulatory transcription mechanism underlying this plant response to shade. This study provides, for the first time, a full-length transcriptome of C. breviculmis using the SMRT sequencing method. The transcriptome atlas generated here will facilitate future studies on functional genomics and provided basis for further selective and genetic engineering breeding of C. breviculmis.Collectively, we have generated 60,353 high-quality non-redundant full-length transcripts, 3588 AS events, 40,347 complete CDS and 1273 lncRNAs of C. breviculmis cultivar ‘Four seasons’ (China accession No. S-SV-CL-006-2010) was identified and cultivated at the Beijing Research and Development Center for Grass and Environment, and it has undergone inbreeding for at least five generations. It was collected from the National Experiment Station for Precision Agriculture  for sequencing due to its wide application area in Beijing. The plants were planted in 20 cm diameter, 20 cm deep plastic pots filled with nutrition medium containing peat, vermiculite and pearlite (1:1:1 in volume). Plants were cultivated in a growth chamber  at 28/24 °C(day/night), 60% relative humidity, 14-h photoperiod, and average photosynthetic active radiation (PAR) of 400 μmol m− 2 s− 1. Plants were weekly fertilized with half-strength Hoagland’s solution [− 2 s− 1 m− 2 s− 1 for two weeks. Whole plants were respectively collected for RNA extraction on each sampling day.solution . For shaTm Size Selection System . These three SMRT libraries were generated at Biomarker Technology Co.  using the Pacific Biosciences DNA Template Prep Kit 2.0.  according to the standard protocol. SMRT sequencing was then performed on the Pacific Bioscience RS II  platform at Biomarker Technology Co.  using the protocol provided by the manufacturer.Total RNA was extracted using the Plant RNA Kit . Equal amounts of RNA collected from ‘Four season’ plants at each sampling day  were pooled together. The quantity and integrity of RNA samples were assessed in the NanoDrop ND-1000 spectrophotometer  and in the 2100 Bioanalyzer , respectively. Qualified RNA samples were then used for constructing cDNA libraries. The SMARTer PCR cDNA Synthesis Kit  was utilized to synthesize full-length cDNA and cDNA fraction and length selection  were performed using the BluePippinTotal RNA was extracted from three independent biological replicates, each comprising one independent plant subject to either. Next, the six prepared RNA samples prepared (three from control and three from shade treatment) were evaluated as described above. The strand-specific cDNA libraries were constructed using a NEBNext® Ultra™ RNA Library Prep Kit  following the manufacturer’s protocol. Qualified libraries were sent to Biomarker Technology Co.  for second-generation sequencing on the Illumina HiSeq4000 platform . To verify the digital expression level of RNA-seq data, a qRT-PCR was carried out to assess the expression levels of ten representative genes. Relative expression level was figured out using the comparative ΔΔ Ct method [http://www.pacificbiosciences.com). FLNC and NFL reads were classified using RS-IsoSeq (v2.3) by identifying poly (A) signal and 5′ and 3′ adaptors. To identify all possible reads, a relaxed standard with a minimum full pass of 0 and an accuracy of 75% was adopted in the filtering panel. The FLNC reads generated from the same isoform were clustered into one consensus isoform using ICE. Subsequently, the consensus isoforms were further polished by the Quiver program with the assistance of NFL reads, resulting in HQ (accuracy> 99%) and LQ. The raw Illumina reads were filtered to remove adaptor sequences, ambiguous reads with ‘N’ bases, and low-quality reads. Filtered Illumina data were then used to polish the LQ reads using the proovread 213.841 software [C. breviculmis was constructed.ROIs were generated from SMRT subreads using the standard protocols in the SMRT analysis software suit  to generate a UniTransModel file. Subsequently, error-corrected non-redundant transcripts were mapped to UniTransModels using GMAP  , CNCI , CPAT  and Pfam  databases were combined to sort non-protein coding RNA candidates from putative protein coding RNAs in the transcripts. Putative protein-coding RNAs were filtered out using a minimum length and exon number thresholds. As so, transcripts longer than 200 bp with more than two exons were selected as lncRNAs candidates and further screened using CPC/CNCI/CPAT/Pfam, as these tools can distinguish protein-coding from non-protein coding genes. Only the transcripts identified in the four databases were regarded as lncRNAs. To investigate the target genes of lncRNAs, the LncTar tool  was utilized following two different strategies: the first was based on the localization of lncRNA and mRNA, whereas the second was based on the interactivity results of lncRNA and mRNA caused by base pairing.https://transdecoder.github.io/) was utilized to define the putative CDS of the 60,353 non-redundant transcripts. The predicted CDS were then annotated and confirmed by BLAST . Those transcripts containing complete ORFs as well as 5′ and 3′ UTR were designated as full-length genes. The CDS-containing transcripts were used as queries against those of rice (reference genome MSU_v7.0) in Orthomcl software [TransDecoder v2.0.1 . Proline content was examined using the 5-sulfosalicylic acid method, as described in a previous report . SolubleC. breviculmis reference transcriptome using Bowtie2 program [< 0.01. The identified DEGs were mapped to each term of the GO database (www.geneontology.org) and calculated using GOseq-R package [p-value for enrichment was corrected via Kolmogorov-Smirnov test and the corrected p-value of ≤0.05 was believed to be significantly enriched. KOBAS [C. breviculmis TFs were predicted and distinguished using HMMER 3.0 [The Illumina clean reads were mapped to the constructed  program . The unm program . After e program . The par package . The p-vd. KOBAS  was usedd. KOBAS  as the rMMER 3.0 .Additional file 1: Figure S1. NGS sequencing data evaluation and biological repetitions dependency examination. A. Statistics of FPKM results for each sample. B. biological repetitions dependency examination.Additional file 2: Figure S2. Analysis of differential expressed genes (DEGs) for NGS. A. Volcano plot of DEGs. B. GO classification. C. KEGG annotation. D. KEGG enrichment.Additional file 3: Figure S3. Heatmap of representative transcription factors.Additional file 4: Table S1. Summary of the candidate alternative splice (AS) events.Additional file 5: Table S2. Summary of the lncRNAs.Additional file 6: Table S3. Prediction results of the target mRNAs for lncRNAs.Additional file 7: Table S4. The identified homologous transcripts between C. breviculmis and rice.Additional file 8: Table S5. Sequences of the primers used for qRT-PCR verification.Additional file 9: Table S6. GO classification of the biological processes.Additional file 10: Table S7. Summary of the transcription factors predicted in this study.Additional file 11: Table S8. Illumina short read assemble results.


Rhodobacter is taxonomically well studied, and some members are model organisms. However, this genus is comprised of a heterogeneous group of members. 16S rRNA gene-based phylogeny of the genus Rhodobacter indicates a motley assemblage of anoxygenic phototrophic bacteria (genus Rhodobacter) with interspersing members of other genera (chemotrophs) making the genus polyphyletic. Taxogenomics was performed to resolve the taxonomic conflicts of the genus Rhodobacter using twelve type strains. The phylogenomic analysis showed that Rhodobacter spp. can be grouped into four monophyletic clusters with interspersing chemotrophs. Genomic indices (ANI and dDDH) confirmed that all the current species are well defined, except Rhodobacter megalophilus. The average amino acid identity values between the monophyletic clusters of Rhodobacter members, as well as with the chemotrophic genera, are less than 80% whereas the percentage of conserved proteins values were below 70%, which has been observed among several genera related to Rhodobacter. The pan-genome analysis has shown that there are only 1239 core genes shared between the 12 species of the genus Rhodobacter. The polyphasic taxonomic analysis supports the phylogenomic and genomic studies in distinguishing the four Rhodobacter clusters. Each cluster is comprised of one to seven species according to the current Rhodobacter taxonomy. Therefore, to address this taxonomic discrepancy we propose to reclassify the members of the genus Rhodobacter into three new genera, Luteovulum gen. nov., Phaeovulum gen. nov. and Fuscovulum gen. nov., and provide an emended description of the genus Rhodobacter sensu stricto. Also, we propose reclassification of Rhodobacter megalophilus as a sub-species of Rhodobacter sphaeroides.The genus  Rhodobacter (Rba.) was proposed by Rhodopseudomonas  with vesicular intracytoplasmic membrane (ICM) architecture as Rba. capsulatus (type species of the genus), Rba. sphaeroides, Rba. adriaticus and Rba. sulfidophilus. Subsequently, based on 16S rRNA gene sequence comparisons, requirement of NaCl for optimal growth, final oxidation product of sulfide, polar lipid composition and sulfide tolerance, Rba. sulfidophilus, Rba. euryhalinus, and Rba. adriaticus were reclassified as Rhodovulum sulfidophilum, Rhodovulum euryhulinum, and Rhodovulum adriaticum , ICM architecture (vesicular/lamellar), sulfur metabolism and cell division (budding/binary fission). The phenotypic diversity among the taxa of the genus Rhodobacter was critically commented upon 21/2 decades back  tool  tool . A concaCG) tool  in a paiCG) tool  for the 3. In brief, the Patric server begins with amino acid sequence files for each genome. Homologous proteins were identified in two rounds. BLAST was used in first round in which the genome of each distinct species is searched against other genomes and an MCL (Markov Cluster) algorithm was used to cluster the top scoring hits, which are initial seed sets for the homology groups. Alignment of seed sets was carried out by MUSCLE, and hmmbuild was used to build Hidden Markov Models (HMMs). The phylogenomic tree was generated with FastTree and RAxML from the concatenated alignment. Instead of bootstraps, trees are built from random samples of 50% of the homology groups used for the main tree, in a process referred to as gene-wise jackknifing. Hundred of these 50% gene-wise jackknife trees are made using FastTree/RAxML, and the support values shown indicate the number of times a particular branch was observed in the support trees.The phylogenomic tree was also deduced from the genome translated amino acids using the automated pipeline of the Patric online serverRhodobacter was carried out using the BPGA pipeline  by two-dimensional chromatography using chloroform/methanol/water (65:25:4 by vol.) in the first dimension and chloroform/methanol/acetic acid/water (80:12:15:4 by vol.) in the second dimension  as described by imension , b. The y vol. asimension .Rhodobacter form five distinct clades  have 94.0% sequence similarity  also showed . The results of dDDH also matched with the data obtained using the Type (Strain) Genome Server (TYGS)  . Ortholos 97.96% .Rhodobacter ranged from 54.2 to 88.2%. The lowest POCP value was between Rba. megalophilus JA194T and Rba. aestuarii JA296T and highest POCP value was between Rba. megalophilus JA194T and Rba. sphaeroides 2.4.1T. The POCP values among the clade I species ranged from 66.5 to 88.2%, whereas for clade II values were between 72.1 to 79.9%, and was 71.1% between Rba. veldkampii and Rba. vinaykumarii. The POCP value between Rba. capsulatus ATCC 11166T and Rba. sphaeroides 2.4.1T was 59%. POCP values among Rhodobacter clades and related genera are above 50%  and C. changlensis (formerly Rba. changlensis) form RC-LH1 (photosynthesis reaction center-light-harvesting 1) complex dimers instead of monomers, as in Rba. capsulatus, Rba. veldkampii and Rba. vinaykumarii. The dimer formation is facilitated by the PufX protein, by interacting with two monomers of RC–LH1 complex, without disturbing their structure . Phylogenetic analysis of one or two photosynthetic genes  is not ster spp.  revealedus clade . In the tic tree , which wc clades .Rhodobacter spp. typically includes phosphatidylethanolamine, phosphatidylglycerol, uncharacterized aminolipid, phosphatidylcholine (except Rba. veldkampii) and other unidentified lipids. In addition, clade I members have DPG and an unidentified glycolipid (GL). Polar lipid profiles of the representative strains are shown in The polar lipid composition of Rhodobacter spp., genome based analysis was carried out in this study. The genome size of clade I members is relatively larger than that of the remaining clades, with the exception of Rba. ovatus. The clade I members have higher G+C content (68.2–69.1 mol%) when compared with clade II (61.0–66.6 mol%), clade III (66.4–66.5 mol%), clade IV (65.0 mol%) and clade V (68.2 mol%). According to Rhodobacter sensu stricto (Clade II). This is also in agreement with the separation of Rba. capsulatus strains from Rba. sphaeroides in the phylogenomic analysis of The use of genomics in taxonomic studies improves the credibility, reliability and reproducibility of the data, serves as an informative tool in establishing phylogenetic relationships among prokaryotes and helps in eliminating unreliable methods and subjective difficult-to-replicate data . To resoRhodobacter revealed that only 1239 core genes are present  can be used to determine the phylogenetic relationships of species when the 16S rRNA gene sequence based phylogenetic tree is unable to resolve the phylogenetic position of taxa, based on the fact that the house keeping genes are less conversed and evolve faster than the 16S rRNA gene  are not essential to resolve the taxonomic positions of members of the genus Rhodobacter.The conflict observed in the current classification of the genus RNA gene  or rpoB sed tree , which iRNA gene . Based oRba. sphaeroides clade (clade I) were isolated from freshwater environments only (Rba. capsulatus clade (clade II) members have been isolated from freshwater , were isolated from freshwater habitats  has a requirement of NaCl for optimum growth and cannot assimilate sulfate (Rhodobacter azotoformans) of clade I has denitrification ability  have glycolipid and DPG  we conclude that they are not two different species. Based on phenotypic differences . This clade accommodates the type species of the genus Rhodobacter, Rba. capsulatus and members of this clade are proposed to be included in the genus Rhodobacter sensu stricto. Rba. veldkampii and Rba. vinaykumarii formed two different clusters with interspersing chemotrophs. However, in the genome-based trees  they are clustered together. Genome based phylogenomic trees have better resolution than single gene-based trees, so for the time being we are not separating these two into two different genera, which can be done in future based on multiple species/strains. For the Rba. veldkampii clade accommodating Rba. vinaykumarii and Rba. veldkampii, we propose the new genus Phaeovulum gen. nov. To accommodate Rba. blasticus, we propose the new genus Fuscovulum gen. nov. The classification of Rba. blasticus itself controversial when it was transferred from the genus Rhodopseudomonas to Rba. blasticus, despite having lamellar ICM structures in contrast to all other members of genus Rhodobacter, which have vesicular ICM structures. It was proposed that ICM systems may be of questionable value as genus specific characters, because they are not coincident with the 16S rRNA gene phylogenetic relationships  a small egg; N.L. neut. n. Luteovulum small yellow egg].a and carotenoids of the spheroidene series. Facultative aerobes and mesophilic. Phototrophic growth occurs on a number of organic substrates. The growth factors biotin, niacin and thiamine, alone or in combination, are required for growth. NaCl requirement is not obligatory, can tolerate up to 2–3%. C18:1ω7c/C18:1ω6C, C18:0, C16:0, C10:0 3OH, C16:1ω7C/C16:1ω6c, C18;1ω7c11 methyl are the major fatty acids. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid are the major polar lipids. Hopanoids are not produced. Q10 is the major quinone. Delineation of the genus is based on 16S rRNA and rpoB gene-based phylogeny, phylogenomics, genome comparison and chemotaxonomic differences.Members can be isolated from freshwater ponds, paddy soils, wastewater treatment plants, alkaline ponds and lake sediments. Gram-stain negative, oval-to-rod shaped cells and have vesicular ICM architecture. Cells are mostly motile with a single polar flagellum and multiply by binary fission. Catalase and oxidase positive. Primarily phototrophic and contain BChl-Luteovulum sphaeroides subsp. sphaeroides.The type species is Luteovulum sphaeroides is identical to that of Rba. sphaeroides  and LMG (LMG 2827T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain is X53853 and CP030271, CP030272, CP030273, CP030274, CP030275, and CP030276 are the genome sequence accession numbers of the type strain.The description of aeroides  except fLuteovulum megalophilum  friend, loving; N.L. neut. adj. megalophilum, wide (temperature)-loving). The description of Luteovulum megalophilum is identical to that of Rba. megalophilus  and KCTC (KCTC 5602T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain AM421024 and that of the genome sequence is FZOV00000000.lophilus  except fLuteovulum johrii .Basonym:Rhodobacter johriiLuteovulum johrii is identical to that of Rba. johrii (T) and DSMZ (DSM 18678T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain is and that of the genome sequence is MABH00000000.The description of . johrii . The typLuteovulum ovatum Basonym:Rhodobacter ovatusLuteovulum ovatum is identical to that of Rba. ovatus (T) and CCUG (CCUG 55049T). AM690348 is the 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number and OAOQ00000000 is the genome sequence accession number.The description of . ovatus . Type stLuteovulum azotoformans ], nitrogen; N.L. pref. azo-, pertaining to nitrogen; L. part. adj. formans, forming; N.L. part. adj. azotoformans, nitrogen forming).Basonym:Rhodobacter azotoformansLuteovulum azotoformans is identical to that of Rba. azotofarmans (T) and NBRC (NBRC 16436T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain is D70846 and that of the genome sequence is QAOT00000000.The description of ofarmans . The typLuteovulum alkalitolerans .Basonym:Rhodobacter alkalitoleransLuteovulum alkalitolerans is identical to that of Rba. alkalitolerans (T) and LMG (LMG 28749T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain is LN810645.The description of tolerans . The typFuscovulum .a and carotenoids of the spheroidene series. Growth occurs under aerobic, anaerobic and mesophilic conditions. Phototrophic growth occurs on a number of organic substrates. Nicotinic acid and thiamine are required for growth. NaCl is not required for growth. Q-10 is the major quinone. The G+C mol% of the type strain of the type species is 66.5%. Delineation of the genus is based on 16S rRNA and rpoB gene-based phylogeny, phylogenomics, genome comparison and chemotaxonomic differences.Members can be isolated from freshwater habitats. Gram-stain negative, rod-to-oval shaped cells have lamellar ICM architecture. Cells are mostly non-motile and multiply by sessile budding. Catalase and oxidase positive. Primarily phototrophic and contain BChl-Fuscovulum blasticum.The type species is Fuscovulum blasticum .Basonym:Rhodobacter blasticus  and NBRC (NBRC 16437T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain is DQ342322 and that of the genome sequence is PZKE00000000.The description of blastica  and Rhodlasticus . The typPhaeovulum .a and carotenoids of the spheroidene series. Growth occurs under anaerobic and mesophilic conditions. Phototrophic growth occurs on a number of organic substrates. Biotin, para-aminobenzoate and thiamine are required for growth either singly or in combination. Some strains do not contain phosphatidylcholine. Some require NaCl for growth and can tolerate up to 4%. C18:1ω7c/C18:1ω6C, C18:0, C16:0, C10:0 3OH, C16:1ω7C/C16:1ω6c, C18;1ω7c11methyl are the major fatty acids. Q-10 is the major quinone. G+C mol% is 65–68.25. Delineation of the genus is based on 16S rRNA and rpoB gene based phylogeny, phylogenomics, genome comparison and chemotaxonomic differences.Members can be isolated from marine habitats including tidal waters, fresh water. Gram-stain negative, rod to oval shaped cells have vesicular ICM architecture. Cells are mostly non-motile and multiply by binary fission. Catalase and oxidase positive. Primarily phototrophic and contain BChl-Phaeovulum veldkampii.The type species is Phaeovulum veldkampii .Basonym:Rhodobacter veldkampiiPhaeovulum veldkampii is identical to that of Rhodobacter veldkampii (T) and DSMZ (DSM 11550T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number is D16421 and that of the genome sequence is PZKF00000000.The description of ldkampii . The typPhaeovulum vinaykumarii .Basonym:Rhodobacter vinaykumariiPhaeovulum vinaykumarii is identical to that of Rhodobacter vinaykumarii (T) and DSMZ (DSM 18714T). The 16S rRNA gene sequence GenBank/EMBL/DDBJ accession number of the type strain is AM408117 and that of the genome sequence is OBMN00000000.The description of ykumarii . The typThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.CVR and CS designed the work. GS maintained the bacterial cultures. TL and GS genomic data retrieval from databases, pangenomic, phylogenomic, and phylogenetic data analysis. GS, TL, and BI calculated the genomic relatedness indices. All authors made the manuscript and accepted the work for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We characterized and identified five karyotypes from 21 individuals of Nomascus. Using karyotypes and mitochondrial and nuclear genes, we identified three purebred species and three hybrids, including one F2 hybrid between N. gabriellae and N. siki. Our results also supported that N. leucogenys and N. siki shared the same inversion on chromosome 7, which resolves arguments from previous studies. Our results demonstrated that both karyotyping and DNA-based approaches were suitable for identifying purebred species, though neither was ideal for hybrid identification. The advantages and disadvantages of both approaches are discussed. Our results further highlight the importance of animal ethics and welfare, which are critical for endangered species in captivity.Gibbons and siamangs (Hylobatidae) are well-known for their rapid chromosomal evolution, which has resulted in high speciation rate within the family. On the other hand, distinct karyotypes do not prevent speciation, allowing interbreeding between individuals in captivity, and the unwanted hybrids are ethically problematic as all gibbon species are endangered or critically endangered. Thus, accurate species identification is crucial for captive breeding, particularly in China where studbooks are unavailable. Identification based on external morphology is difficult, especially for hybrids, because species are usually similar in appearance. In this study, we employed G-banding karyotyping and fluorescence  Hoolock, Hylobates, Nomascus, and Symphalangus; Symphalangus) possess a unique and large gular sac (throat pouch), gibbons share very similar external morphology, and were long acknowledged as a single genus (Hylobates). However, Hoolock and Nomascus were subsequently recognized as full genera based on karyotypic studies, which revealed a unique diploid number (2n) of chromosomes among each genus: Hoolock (2n=38), Hylobates (2n=44), Nomascus (2n=52), and Symphalangus (2n=50)  . This su (2n=50) . Today,  (2n=50) .N. leucogenys) and human genomes revealed 100 synteny breakpoints, which can facilitate chromosome breakage and rearrangement , N. gabriellae (yellow-cheeked gibbon), N. hainanus (Hainan gibbon), N. leucogenys (northern white-cheeked gibbon), N. nasutus (eastern black-crested gibbon), N. siki (southern white-cheeked gibbon) (N. annamensis (northern buffed-cheeked gibbon) (The crested gibbon genus  gibbon) , and the gibbon) . These s gibbon) .Nomascus species have a unique diploid number of 52 . We also sequenced mitochondrial and nuclear fragments across a known inversion breakpoint on chromosome 7 identified in a previous study , and BP primers should result in positive amplification only for N. leucogenys. The mitochondrial and nuclear amplicons were sequenced using a ABI 3730 sequencer .After karyotyping, we extracted total DNA from the cultured cells using a commercial DNA extraction kit . The DNA extracted from 10 samples was considered acceptable for subsequent analyses as other samples were not well preserved after karyotyping. We amplified and sequenced cyt b gene against the GenBank nucleotide database; (4) we estimated a gene tree to verify the BLAST results using our cyt b genes accompanied by a set of sequences representing the recognized Nomascus species downloaded from GenBank; and (5) we repeated the karyotyping and amplification/sequencing in triplicate in cases where the mitochondrial, nuclear sequencing, and karyotyping results conflicted . A final purebred or hybrid and species identification was determined.We identified species based on the karyotyping and sequencing results using the following strategy: (1) we identified the species based on its karyotype; (2) we verified the karyotyping results using PCR with primer sets BP and HSA; (3) we BLAST each cyt b sequences, 17 cyt b sequences representing seven recognized species were downloaded from GenBank (Supplementary Table S1) and aligned with our sequences using MAFFT v7.3 (We constructed the maximum likelihood gene tree using RAxML v8.2.10 and the CIPRES Science Gateway . In addiFFT v7.3 . We partn) of all analyzed gibbons was 52. G-banding revealed different lengths of chromosomes 1 and 22, and FISH revealed two, three, or four fluorescence signals on chromosome 7  were similar to the G-banded  and R-baucogenys . Chromosucogenys A.N. siki reported by N. leucogenys, a balanced translocation between chromosome pairs 1 and 22 was detected, resulting in one pair of shortened chromosome 1 and one pair of derived chromosome 22 ). Similar to that observed in N. leucogenys, FISH demonstrated a pericentric inversion event on chromosome 7  were identical to those of mosome 7 B. The saN. gabriellae . Only one pair of FISH signals were observed on chromosome 7, thus differing from N. siki and N. leucogenys  were the same as that of briellae . This kaucogenys C.N. leucogenys, and the other chromosome 1 and 22 were similar to that of N. siki, indicating one translocation t . Two pairs of FISH signals were observed on chromosome 7, indicating a pericentric inversion event . FISH detected three fluorescence signals on chromosome 7, indicating heterozygous pericentric inversion  and two hybrids (n=3) identified in the karyotypic analyses. All new sequences are available in GenBank under accession Nos. MH188408–MH188428 (Supplementary Table S1). Five out of the seven purebred animals were supported by BLAST and phylogenetic analyses using the cyt b gene and the other two were not =98). Sample 14 was identified as N. leucogenys based on its karyotype but could not be identified unambiguously as it was equally similar to N. leucogenys and N. siki. It was closely related to (BS=84) and formed a sister lineage of these two species on the phylogenetic tree (BS=52). The cyt b genes of the two hybrids identified in the karyotypic analyses (Xiao-Xiao and Mei-Mei) corresponded to one of their parental species (BS≥88). On our cyt b genetic tree (N. siki+N. leucogenys (BS=52).We determined the complete cyt were not . One samtic tree , sample N. leucogenys, N. siki, and the N. leucogenys×N. siki hybrid (Mei-Mei) and were negative when using the HSA primers for these samples (N. gabriellae but positive when using the HSA primers. For the hybrid identified as N. gabriellae×N. siki (Xiao-Xiao), PCR and sequencing were successful using both BP and HSA primers. For Da-Shan, the PCR results were positive using BP primers and negative using HSA primers, which were congruent with its karyotype (siki type) but disagreed with its mitochondrial gene results.The PCR results using the HSA and BP primer sets were concordant with the karyotypic results. The PCR results using the BP primers (confirming the inversion) were positive for purebred  samples . The PCRN. cf. leucogenys as it did not cluster with the other N. leucogenys sequences downloaded from GenBank. We considered Da-Shan as an F2 hybrid of N. gabriellae and N. siki due to the inconsistent patterns revealed by karyotyping and PCR using the HSA and BP primers (siki), and the mitochondrial phylogeny (gabriellae)  was identified as riellae) .Nomascus species characterized by high karyotypic diversity. We determined the specific karyotypes of each species as well as the existence of an inversion on chromosome 7 in N. siki and N. leucogenys. We also revealed the definitive existence of hybrids in zoos in China, which calls attention to the animal ethics and welfare issues related to endangered species breeding in captivity. Using the Nomascus species as our focal taxa, we evaluated the accuracy and efficiency of both approaches in identifying purebred and hybrid species.We used karyotypic- and PCR-based approaches to examine the affinities of N. gabriellae, N. leucogenys, and N. siki are distinguishable based on translocation t  and inversion on chromosome 7 , and therefore has a distinct karyotype. It would be interesting to examine the G-banded karyotypes and FISH results of N. concolor as well as N. annamensis, which is sister to N. gabriellae and recognized only recently (Hylobates species such as H. lar×H. pileatus, H. lar×H. agilis, and H. muelleri×H. agilis/H. albibarbis has been observed (Nomascus species in the wild has not been reported, which is likely due to their allopatric distribution. However, we observed heterozygous karyotypes in captive Nomascus animals, congruent with previous study (Our results supported that mosome 7 , even threcently . Due to observed . Hybridius study . Our resus study , which iN. leucogenys and N. siki (N. siki, which is known for low DNA quantity as well as the existence of PCR inhibitors.Our FISH and DNA analyses supported the existence of an inversion on chromosome 7 in both  N. siki , consist N. siki , though  N. siki . The negN. leucogenys samples (N. leucogenys and N. siki, resulting in non-monophyletic relationships. This situation has never been observed because the effective population sizes of gibbon species are usually small. Alternatively, this sample might represent a distinct and unknown taxon, which is a close relative but not identical to N. leucogenys or N. siki. The sample was obtained from the Kunming Institute of Zoology, Chinese Academy of Sciences, in 1993 and the information on the animal was not recorded in detail. Unfortunately, neither hypothesis could be supported or rejected in the current study.The phylogenetic position of sample 14 was an interesting finding. Its karyotype was identical to the other  samples , which w samples . We repeN. gabriellae and N. siki, despite there being no available studbook. This is the first record of a cross-back F2 hybrid in Nomascus as most other countries have well-established systems in place to prevent interbreeding of gibbons in captivity, with many animals also previously karyotyped. Continuous interbreeding and production of fertile offspring may spread across zoos in China because there are only limited numbers of gibbons in zoos, and correct species identification of their offspring will become far more difficult after several generations and recombination. Our findings call for the introduction of a system to prevent gibbon interbreeding in captivity and for better welfare and awareness of these animals.Similar to previous studies, our study supported that hybridization has occurred in captivity. However, fertility may be less impacted as identification of Da-Shan showed it to be a likely F2 hybrid of Nomascus species (N. leucogenys/N. siki from N. gabriellae, but could not easily distinguish N. leucogenys/N. siki from the hybrid of N. leucogenys×N. siki.The R-banding technique can distinguish different karyotypes of  species , but is  species . As per  species , we agre


Amomum villosum has been historically used in China as food and in the auxiliary treatment of digestive system disorders. Numerous studies have shown that gastrointestinal function is closely related to the development of nonalcoholic fatty liver disease via the “gut-liver” axis. The dried mature fruit of A. villosum. The present study aimed to explore whether the mechanism underlying the regulation of lipid accumulation in nonalcoholic fatty liver disease (NAFLD) may affect related disorders using the active ingredients in A. villosum (WEAV), volatile oil of A. villosum (VOAV), or bornyl acetate. After treatment, serum and liver total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured. The regulatory role of A. villosum in the microecology of the intestines was assessed using the V4 region of the 16S rDNA sequencing. The expression of the intestinal tight junction proteins occludin and ZO-1 was also measured. The influence of A. villosum on TLR4-mediated chronic low-grade inflammation was evaluated based on the concentrations of key proteins of the TLR4/NF-кB signaling pathway. Results. A. villosum effectively inhibited endogenous lipid synthesis, reduced TG, TC, and FFA accumulation, regulated the expression of LDL-C, and decreased lipid accumulation in liver tissues. VOAV effectively regulated the intestinal microflora, improved chronic low-grade inflammation by promoting ZO-1 and occludin protein expressions, and inhibited the TLR4/NF-кB signaling pathway. Male Sprague-Dawley rats on a high-fat diet (HFD) to induce NAFLD were administered water extract of A. villosum in NAFLD prevention and treatment. Additional chemical constituents other than bornyl acetate also contributed to the preventive effects of A. villosum on NAFLD. The present study provides scientific basis for the potential application of Nonalcoholic fatty liver disease (NAFLD) is a common disorder that is characterized by accumulation of excess fats in the liver of individuals who drink little or no alcohol. Epidemiological studies have shown that NAFLD may affect individuals of any age and race. The prevalence of NAFLD in Western countries is about 18%–35%, and up to 80% of obese individuals develop NAFLD . Recent  Amomum villosum Lour., A. longiligulare T.L. WU, and A. villosum Lour. var. xanthioides T.L. Wu et Senjen of Zingiberaceae , Parabacteroides, CF231, and Bacteroides to normal levels. LPS is a major constituent of the outer membranes of gram-negative bacteria. LPS recognition and signal transmission are key events in the host defense reaction towards gram-negative bacteria and are associated with various disorders [ A. villosum might be responsible for this.Third,isorders , 35. VOAisorders . Tight jisorders . The proisorders . The regκB (NF-κB), which belongs to a family of transcription factors, is the principal player in the regulation of inflammatory gene expression. Inhibitors of NF-κB (I-κB) kinase (IKKs) are a central component of the signaling cascade that controls NF-κB-dependent inflammatory gene transcription. IKKs activate the NF-κB pathway by phosphorylating I-κB and NF-κB P65, making it an attractive target for therapeutic intervention [κB protein expression was inhibited by VOAV and WEAV. A. villosum was found to downregulate IKK protein expression in the TLR4/NF-кB signaling pathway. The production of IкB, whose interactions with NF-кB prevented NF-кB translocation and activation, was enhanced by A. villosum treatment. The release of downstream inflammatory cytokines in the LPS/TLR4/NF-κB signaling pathway was found to be mitigated by A. villosum. Studies showed that a high-fat diet could increase NF-кB activation in mice, which led to a sustained elevation in the level of IкB kinase epsilon (IKK epsilon) in the liver, adipocytes, and adipose tissue macrophages. IKK epsilon knockout mice were protected from high-fat diet-induced obesity, chronic inflammation of the liver and fat, hepatic steatosis, and whole-body insulin resistance [Endotoxemia and TLR4 signaling control the production of proinflammatory cytokines in target tissues, which lead to chronic inflammation and insulin resistance in HFD-fed rats . TLR4 isrvention .TLR4 andsistance –42.P < 0.001), followed by the middle-dose group of volatile oil from Amomum villosum. However, VOAV showed better effect on the increasing of liver AST and ALT. In the meantime, A. villosum could effectively inhibit lipids accumulation in circulation system.Both ezetimibe and VOAV could inhibit the increasing of TC, TG, and FFA in liver homogenates. Among them, ezetimibe displayed better inhibitory effect on the content of TC, TG, and FFA in liver homogenate , while A. villosum was found to effectively inhibit lipid accumulation in liver tissue, regulate the intestinal microflora, strengthen intestinal mucous membrane barrier, inhibit the TLR4/NF-кB signaling pathway, and relieve chronic low-grade inflammation. VOAV showed great potential in NAFLD prevention and treatment. This work could provide a scientific basis for further application and development of A. villosum. In sum, A. villosum in the treatment of NAFLD. Results showed that A. villosum could improve the permeability of intestinal epithelial cells and regulate the balance of intestinal flora. In China, A. villosum is used in both medicine and cuisine food [ A. villosum water extract, which could be considered scientific evidence that is in agreement with the traditional findings that A. villosum should be extracted promptly to prevent the oil from undergoing volatilization.The present study has investigated the role ofine food . This ma


Zea mays systematically, and 8 of 12 circRNAs were validated by experiments. By analysing circRNA sequence, the events of alternative circularization phenomenon were found prevailed in maize. By comparing circRNAs in different species, it showed that part circRNAs are conserved across species, for example, there are 273 circRNAs conserved between maize and rice. Although most of the circRNAs have low expression levels, we found 149 differential expressed circRNAs responding to heat, cold, or drought, and 1782 tissue-specific expressed circRNAs. The results showed that those circRNAs may have potential biological functions in specific situations. Finally, two different methods were used to search circRNA functions, which were based on circRNAs originated from protein-coding genes and circRNAs as miRNA decoys. 346 circRNAs could act as miRNA decoys, which might modulate the effects of multiple molecular functions, including binding, catalytic activity, oxidoreductase activity, and transmembrane transporter activity. In summary, maize circRNAs were identified, classified and characterized systematically. We also explored circRNA functions, suggesting that circRNAs are involved in multiple molecular processes and play important roles in regulating of gene expression. Our results provide a rich resource for further study of maize circRNAs.Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs, which increasingly drawn researchers' attention in recent years as their importance in regulating gene expression at the transcriptional and post-transcriptional levels. With the development of high-throughput sequencing and bioinformatics, circRNAs have been widely analysed in animals, but the understanding of characteristics and function of circRNAs is limited in plants, especially in maize. Here, 3715 unique circRNAs were predicted in  Drosophila, mice, and humans many years [Circular RNAs (circRNAs) are a class of endogenous noncoding RNA molecules formed by backsplicing –5. Althony years –10, theyArabidopsis thaliana), rice (Oryza sativa), tomato (Solanum lycopersicum), barley (Hordeum vulgare), maize (Zea mays L.) and trifoliate orange (Poncirus trifoliata L. Raf.) [Unlike mRNA, circRNA transcripts usually lack of the 5′ cap and 3′ poly(A) tail, and the majority of circRNAs are expressed at low levels compared with linear RNAs . HoweverL. Raf.) , 24–30.Zea mays L.) is one of the most important crops worldwide and serves as model organism in biological research. With the development of high-throughput sequencing technology, more and more data have been produced, now it is possible for us to study circRNAs in maize systematically. In this article, maize circRNAs were firstly identified from multiple resources. Then circRNA characteristics, such as genomic distribution, alternative circularization, conservation and expression patterns, were analyzed. Whether CircRNAs act as miRNA decoys to mediate the regulation of gene expression in maize or not were analysed. Finally, maize circRNA functions were inferred in our study. The discovery of maize circRNAs enriches the repositories of plant circRNAs.Maize   . The twoum test) , suggestP < 2.2e-16, Wilcoxon rank sum test)  , as was Z. mays [Z. mays.Because different forms of linear mRNAs are produced during the alternative splicing (AS) process in Z. mays , we hypoZ. mays with circRNAs in O. Sativa and A. thaliana [Z. mays and O. sativa (Z. mays higher than the proportion in O. sativa (12.2%) in a previous publication [Z. mays and A. thaliana, 109 orthologous gene pairs were found, and a total of 122 circRNAs were conserved between Z. mays and A. Thaliana. In addition, the ratio of orthologous genes to the total parent genes (10.9%) in Z. mays was slightly lower than that in A. thaliana (14.5%) [To evaluate circRNA conservation among different species, we firstly compared circRNAs in thaliana . 291 ort. sativa , which clication . For par (14.5%)   , 39, 115To confirm our identification of maize circRNAs, twelve randomly selected circRNAs from the highly expressed maize circRNAs were used in experimental validation. Divergent primers  were firTwenty-one samples from five resources  were used to measure the expression abundance of the circular RNAs. The result of two different measurements both showed that expression levels of maize circRNAs were primarily concentrated in 0–0.5 ratio or RPM bin . ApparenTo investigate whether circRNAs have tissue/developmental stage-specific expression patterns , 13, traTo examine the differential expression levels of the circRNAs, we compared the expression profiles of circRNAs and genes under control and stress conditions in the different transcriptome data groups. We found that 149 circRNAs were differentially expressed, of which 88 and 91 were up-regulated and down-regulated under heat or cold in seedling, and under drought in stem, leaf, or root, respectively . CircRNAetc), carbon fixation in photosynthetic organisms, photosynthesis, biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, etc , which may be due to our strict standard [O. sativa and A. thaliana  [O. sativa, A. thaliana, Solanum lycopersicum, and Glycine max L. Merr [Maize circRNAs were identified by a widely used tool find_circ in our study, and the total number of circRNAs in maize was relatively lower compared to the circRNAs identified in ctively) , 39. Sim L. Merr , 39, somTo date, the functions of circRNAs are largely unknown. By using our developed methods, the function of circRNAs can be inferred. To investigate whether circRNAs acted as miRNA decoys in maize, the method used in our previous reports was used to identify circRNAs as miRNA decoys . OverallIn addition, the function of circRNAs was further predicted based on the ceRNA hypothesis and GO analysis. By comparing the potential function of the circRNAs from the parent gene and acting as miRNA decoys, the GO items (53) of the circRNAs as miRNA decoys more than the items (14) of circRNAs from the parent gene. But the functional prediction results of circRNAs from this two parts were consistent in several aspects, such as components of cytoplasmic, catalytic and binding activity, response to stress and intracellular metabolic processes. Several pathway refer to energy transformation  suggests circRNAs may contribute to crop products. Therefore, these results further prove that circRNAs could participate in a variety of biological processes, constitute different cell components and exert a variety of molecular functions.Although the results of circRNAs' functional prediction and expression profiling in this paper show that maize circRNAs may be involved in a variety of metabolic processes and stresses in plants, the function of plant circRNAs need to be further validated.This study found 3715 unique circRNAs in maize across different experiments by employing a computational pipeline for the genome-wide identification of circRNAs. Furthermore, the property of circRNAs, patterns of differential expression and tissue-specific expression are investigated. Finally, possible functions of circRNAs are inferred by two different methods. Our method and results provide an in-depth analysis of maize circRNAs and can be expanded to more plant species. In addition, future experimental studies are required to elucidate the mechanisms and functions of circRNAs in plants.Zea mays genomes and gene annotation files were downloaded from Phytozome v9.0 . Transcriptome data were collected from 5 resources, including 21 samples. They were downloaded from NCBI under accession number SRP006965 , SRP011480 , SRP041183 , SRP052520  and SRP061631  (http://plantrepeats.plantbiology. msu.edu/downloads.html). Bioinformatics tool find_circ used to identify maize circRNAs was downloaded from https://github.com/marvin-jens/find_circ.The drought) , respectThe algorithm find_circ  was mainZ. mays, A. thaliana and O. sativa [Z. mays protein sequences were blasted against with A. thaliana and and O. sativa protein sequences , and the best paired genes were selected as orthologs by using our in-house Perl scripts. Then circRNAs from orthologous parental genes in both species were reanalyzed and regarded as conserved circRNAs by using BLAST . Another two species, S. lycopersicum and G. max were analyzed with the same method [P < 0.05).To explore circRNA conservation, circRNA candidates predicted as exonic circRNAs were selected in . sativa . To detee method , 27. TheZea mays L.) seedlings were first grown for 2-week-old in the greenhouse . Total RNA was isolated from leaves of the maize seedlings using TRIzol reagent (Ambion) according to the manufacturer’s instructions. Partial total RNA samples were treated for 15 min at 37°C with 3 units μg−1 of RNase R (Epicentre). RNase R could digest essentially linear RNAs. First-strand cDNA was synthesized from untreated and RNase R-treated total RNA using the iScript cDNA synthesis kit (Bio-Rad) respectively. Polymerase chain reaction (PCR) primers (divergent and convergent) were designed for circRNA validation  , 36. Then is the number of tissues, expi is the expression value of each circRNA in tissue i, and expmax is the maximum expression value of each circRNA or linear gene among 10 selected tissues. The circRNAs with an index greater than 0.9 were deemed as exhibit specific expression in one tissue. EdgeR from R packages was used to identify differential expressed circRNAs with FDR  less than 0.05 and fold change > 2 or < 0.5 [The degree of tissue specificity for circRNAs was evaluated with a tissue-specific index that ranged from 0 to 1 . The tisor < 0.5 .To explore whether circRNAs were generated from parent genes with special functions, the protein sequences of the parent genes corresponding to the circRNAs were firstly blasted against the nr database, then GO enrichment analyses were performed for the parent genes generating circRNAs by using Blast2GO with Fisher's exact test (FDR < 0.05). KEGG enhttp://mirbase.org/) were aligned to the circRNA candidate sequences using GSTAr.pl (https://github.com/MikeAxtell/GSTAr). The method used to predict miRNA decoys from circRNAs was built from previous reports [To test whether circRNAs could act as miRNA decoys, 203 sequences of plant miRNAs downloaded from miRBase Click here for additional data file.S2 FigA. thaliana and Z. mays. The label is the same as (A) Example of single and multiple circularized exon and type of alternative circularization. Backspliced reads are marked. Orange rectangles: exons in transcripts; yellow rectangles: exons in another transcript derived from the same gene in the third example. Black line at the exon boundaries: backsplice site overlapped with known exon boundaries; red line: novel splice site. Absolute green arrows: backsplice using both annotated splice sites; green arrows with a blue rim: backsplice using two annotated splice sites from different transcripts; blue arrows: backsplice using no known exon boundaries. (B) Another example of sequence conservation analysis of circRNAs between (PDF)Click here for additional data file.S3 FigRelated to (PDF)Click here for additional data file.S4 Fig(A) Enrichment analysis for genes overlapped with circRNAs that were significant . The x- and y-axes are the same as (PDF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 TableArabidopsis Thaliana. (C) Result of Conservation analysis between maize and Soybean. (D) Result of Conservation analysis between maize and tomato.(A) Result of Conservation analysis between maize and rice. (B) Result of Conservation analysis between maize and (XLS)Click here for additional data file.S4 Table(A) Divergent primers for validation of candidate circRNAs. (B) Convergent primers for validation of candidate circRNAs.(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(A) CircRNAs and the function of their parent genes. (B) Enrichment of pathway analysis.(XLS)Click here for additional data file.S7 TablemiRNA were listed in the first column. CircRNA in the second column acting as the miRNA decoys. The following columns mean the starting and terminating sites between circRNA and miRNA, MFE of a perfectly matched site, MFE of the alignments, MFEsite/MFEperfect, respectively. MFE: minimum free energy.(XLS)Click here for additional data file.S8 Table(A) Enrichment of Gene Ontology analysis. (B) Enrichment of pathway analysis.(XLS)Click here for additional data file.


Biomphalaria glabrata thioester-containing proteins (BgTEP) to other well-annotated homologues. We performed functional characterization studies of BgTEP to homologues in Anopheles gambiae and in disparate invertebrates.There is paucity of information on functional relationship and characterization of B. glabrata genome. In addition, TEP sequences deposited in NCBI protein database were also retrieved and utilized for sequence analysis. BgTEP relatedness to its other homologues as well as functional domain and protein-protein interaction analysis was performed.Genomic sequences of TEPs were retrieved after annotation of the B. glabrata, A. gambiae, and Chlamys farreri. In addition, we identified 19 TEP sequences spread across 10 animal species. The B. glabrata genome contains 14190 unannotated proteins after filtration with about 85% of its proteome annotated. The phylogenetics, functional domain and protein-protein interaction analyses suggest an immunological role for BgTEP in B. glabrata.Our analysis resulted in the identification of TEPs in a number of organisms including, B. glabrata may have a strong effect on resistance to infection.The predicted role of thioester-containing proteins to be involved in immunological role in  Biomphalaria glabrata is the most important host in the transmission of human schistosomiasis in the Caribbean and South America. Hence, recent molecular and immunological work has focused on Biomphalaria glabrata   to that of A. gambiae, since such findings will give a better understanding of the role the protein may play in Schistosoma infection and development.Some identified important immune factors are the nuclear factor kappa B (NF-κB) homologues –29 and bnfection –30. The labrata) , is faci species , 18. Difglabrata –34. TEP immunity . HoweverB. glabrata genome sequence for the present of thioester-containing proteins and carried out comparative analysis with homologues in A. gambiae and a disparate number of invertebrate; predict the possible role TEP might play in defense and protection of B. glabrata against infection.In the present study, we explored the recently completed B. glabrata” separately in Oct 2016 of The National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/guide/proteins/) website for invertebrates TEP proteins and the resulting protein sequences were confirmed on UniProt (http://www.uniprot.org). Protein sequence homologues of a variety of insects such as Anopheles mosquitoes, Drosophila were retrieved from NCBI. These were used, as they are recognized invertebrate organisms for which numerous studies have described key aspects of their innate immune system response to pathogens. Other Mollusca such as Azumapecten farreri and Euphaedusa tau with TEP protein been sequenced were also retrieved and included in the analyses. The different proteoforms of B. glabrata TEP protein, deposited by  with the accession No: ACL00841.1 and AHH81765.1 were retrieved. Partial sequences were avoided during retrieval and collection. All protein sequences were retrieved in FASTA format.We performed a thorough manual literature search using “Thioester-containing Protein plus Mollusca” and “Thioester-containing Protein plus B. glabrata (BB02) , containing more than 14,000 none annotated sequences were downloaded from VectorBase, http://www.vectorbase.org,  protein database, 1.0xE3 for blast expectation value and sequences with a maximum hit of 20 sequences on the average. The Blast2Go cut-off parameters used to filter out poor quality BLAST hits for the annotation were as follows: Annotation rule cut-off = 55; E-value = 1e–6; Hit-HSP overlap = 0; and the GO weight = 5.The publicly available genomic data of ase.org,  and convase.org, . Exportease.org, –43. SequB. glabrata and other species. Prediction of transmembrane helices was done by TMHMM Server v. 2.0 and validated using CCTOP webtool . The tree file in Newick format was exported and visualized in FigTree software version 1.4.2 for proper annotation. We went further to estimate the pairwise distance using the same version of MEGA. Analyses were conducted using the Poisson correction model. The analysis involved 19 amino acid sequences. Comparative functional domain analysis arranged along the branch of the constructed tree using MEGA was employed in the prediction of the possible functional role of BgTEP.Phylogenetic trees were constructed using MEGA version 7 software and neighbor-joining method was applied in the analysis. The trees were drawn to scale, with branch lengths measured for the number of substitutions per site. We removed 19 amino acids and all ambiguous positions were removed for each sequence pair. There were 127 positions in the final dataset. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test was 1000 replicates. The constructed phylogenetic trees were validated using Robust Phylogenetic Analysis tools . Nineteen invertebrate TEP sequences were used in this study that spread across 10 species including B. glabrata. The B. glabrata proteome dataset downloaded from vectorbase consist of 14190 unannotated proteins after filtration. After annotation using Blast2Go about 85% of these protein sequences were annotated while the other 15% sequences remained unannotated. About 15 proteins out of the 19 were predicted to have signal peptides computed to be localized in the secretory pathway with a strong reliability class (RC), while the remaining 4, lack signal peptides. Ten of the selected protein sequences were secreted using the non-classical pathway. The other proteins were predicted to secrete through the classical pathways. Few of the proteins were computed to have just one TMH; A. gambiae (AAG00600.1), A. sinensis (KFB36250.1), Drosophila mojavensis (EDW13040.2), D. virilis (EDW64840.1), D. mojavensis (EDW12892.1) and D. mojavensis (EDW11960.2).A comprehensive search for Thioester-containing Proteins from Most of the protein-protein interactions prediction show that TEP binds with different proteoforms of leucine-rich immune protein (LRIM1), which in turn binds to APL1C and REL2 and other uncharacterized proteins . The binAnopheles and Drosophila show similarities as seen between A. darling (gb|ETN60497.1) and D. virilis (gb|EDW64840.1) as well as D. mojavensis (gb|EDW12892.1). The two proteoforms of B. glabrata TEP shows similarity to the two other Mollusca species; Azumapecten farreri and Euphaedusa tau (B. glabrata TEP identified from vectorbase dataset do not conform to this possessing just a few of the motifs. A. darling (gb|ETN60497.1), D. virilis (gb|EDW64840.1) and D. mojavensis (gb|EDW12892.1) lack Alpha-macro-globulin thiol-ester bond-forming regions  is important for snail defenses against schistosome infections in Biomphalaria . TEP had a highly reactive thioester motif, which can undergo spontaneous hydrolysis. This research identified these functional motifs as Alpha-macro-globulin thiol-ester bond-forming region. However, this vital motif was absent from A. darling (gb|ETN60497.1), D. virilis (gb|EDW64840.1) and D. mojavensis (gb|EDW12892.1) TEP homologues as presented in D. virilis and D. mojavensis. One strain of D. virilis and D. mojavensis with the accession no gb|EDW65046.2, and gb|EDW11960.2 respectively have the TE motif while the same species with accession number gb|EDW64840.1 and gb|EDW12892.1 respectively lacks the TE motif.Although, we employed A. gambiae; therefore, this could open a new era of schistosomiasis control research by disrupting schistosome–snail compatibility. Moreover, our exploitation of the recently released B. glabrata genome for TEP was successful and an ongoing work on shotgun proteomic and bioinformatics analyses of the hemolymph of B. glabrata in our laboratory reveals some proteoforms of this protein. However, further experimental studies could involve sensitive genomic, transcriptomic and proteomics tools.The presence of the TE motif in TEP-like protein and this motif is similar to that found in


Escherichia coli is one of the most common strains of extended-spectrum β-lactam (ESBL)-producing bacteria, and the prevention and treatment of ESBL-producing E. coli infections is an ongoing challenge. The clinical characteristics and outcomes of ESBL-producing E. coli bacteremia in non-transplant patients remain to be elucidated.E. coli bloodstream infections (BSIs) from January 2013 to December 2016 and was conducted to investigate the risk factors, clinical features, and outcomes of these infections.This retrospective study included 491 non-transplant patients with E. coli BSI patients, 57.6% suffered from infections with ESBL-producing strains. A multivariate analysis showed that urinary tract infection, prior use of cephalosporin, and treatment with β-lactam-β-lactamase inhibitor (BLBLI) combination antibiotics were independent risk factors for the development of ESBL-producing E. coli BSIs. The overall mortality rate in E. coli BSI patients was 14.46%, and there was no significant difference in the 28 day mortality rate between ESBL-producing E. coli and non-ESBL-producing E. coli BSI patients . Similarly, there was no difference between the community-acquired infection group and the nosocomial infection group. Hepatobiliary disease, carbapenem exposure, high APACHE II score, and hypoproteinemia were independent risk factors for death in E. coli BSI patients. Multivariate analysis showed that hypoproteinemia and severe disease were independent risk factors for death from ESBL-producing E. coli BSIs. Furthermore, there was no significant difference in the 28 day mortality between patients with ESBL-producing E. coli BSIs treated with carbapenem monotherapy versus those treated with BLBLI combination antibiotics .Of the 491 ESBL-producing E. coli infection. Hypoproteinemia and severe disease are independent risk factors for death in patients with E. coli BSIs. There was no significant difference in the 28 day prognosis of patients with ESBL-producing E. coli and those with non-ESBL-producing E. coli BSIs. These data do not support the conclusion that carbapenems might be more effective than BLBLI antibiotics for treatment of patients with BSIs caused by ESBL-producing E. coli.Prior use of cephalosporin or BLBLI combination antibiotics increased the risk ratio for The online version of this article (10.1186/s13756-019-0599-y) contains supplementary material, which is available to authorized users. Escherichia coli is one of the most common strains of extended-spectrum β-lactamase (ESBL)-producing bacteria , ESE. coli infection were evaluated by comparing the ESBL-producing and non-ESBL-producing patient groups. Second, to investigate the risk factor of mortality, the 491 E. coli BSI patients were divided into survival and death groups according to their survival status after 28 days of infection. Finally, 283 patients with ESBL-producing E. coli BSIs were analyzed to assess the risk factors associated with 28 day mortality and various antibiotic treatments.Demographic and clinical data were collected from the electronic case system, including age, gender, underlying disease, comorbidities, length of hospital stay, admission to the ICU, invasive procedures before and after infection, and antibiotic treatments. The severity of the disease was assessed via the Acute Physiology and Chronic Health Evaluation (APACHE) II scores and Pitt scores . The ChaE. coli BSI was defined as an infection manifested by the presence in at least one blood culture that grew a E. coli strain. BSI refers to the systemic inflammatory response syndrome caused by toxins and metabolites produced by pathogens. Possible sources of E. coli BSIs are based on the Centers for Disease Control (CDC) and Prevention/National Healthcare Safety Network (NHSN) surveillance definitions ). For categorical variables, the percentages for each group are reported. Variables with P values ≤0.05 in the univariate analysis were included in the multivariate analysis, and binary logistic regression (backward: condition) was used to identify independent predictors. The survival distribution functions in different groups were compared via the Kaplan-Meier product limit method. All data were statistically analyzed using SPSS version 23.0, and P values ≤0.05 were considered statistically significant.In the univariate analysis of ESBL-producing E. coli BSIs were included in this study, except for 67 patients who received solid organ or hematopoietic stem cell transplants and 22 patients with CRE BSIs. Among the included patients, 283 (57.6%) had ESBL-producing E. coli BSIs and 208 (42.4%) had non-ESBL-producing E. coli BSIs. There were no significant differences in age and gender between the two groups . Of the primary infection sites of the 491 patients with E. coli BSIs, abdominal infections were the highest (n = 249), followed by respiratory infections (n = 108) and urinary tract infections (n = 84). The annual incidence rates of ESBL-producing E. coli BSIs from 2013 to 2016 were 66.2% (86/130), 59.1% (88/149), 49.1% (53/108), and 53.8% (48/104), respectively, and while the incidence rate fluctuated, it did not increase significantly (P = 0.638).From January 2013 to December 2016, 491 patients with E. coli BSIs included urinary system infection, prior surgery, prior invasive procedures , and antibiotic use within 30 days before infection . In a multivariate analysis, logistic regression analysis showed that urinary tract infections were the primary site of E. coli BSIs  and that cephalosporin exposure  and treatment with BLBLI combination regimens in the 30 days prior to the BSI  were independent risk factors for ESBL-producing E. coli BSIs.A univariate analysis Table  showed tE. coli BSIs were classified into survivor and death groups based on the outcome at 28 days. The overall all-cause 28 day mortality rate of the 491 patients was 14.46% (71/491). A survival curve analysis , and the difference between the mortalities was not statistically significant. Furthermore, the patients were divided into community infection and nosocomial infection groups . There was also no significant difference in the mortalities between these two groups in the patients with nosocomial infections .The non-transplanted patients with E. coli BSI survivor and non-survivor subgroups are shown in Table E. coli BSIs: hepatobiliary disease , prior use of carbapenem antibiotics , and higher APACHE II scores . Normal serum total protein  and appropriate definite antibiotic treatment were independent protective factors  for the outcomes of patients with E. coli BSIs.The main characteristics of the E. coli BSI patients were included in this analysis, and the 28 day mortality was 14.8%. In the univariate analysis  and higher APACHE II scores  were independent risk factors for poor outcomes.A total of 283 ESBL-producing E. coli, 87.99% (249/283) received appropriate empirical antibiotic therapy and 94.35% (267/283) received appropriate definite antibiotic therapy. Appropriate empirical antibiotic treatment was a protective factor in the univariate analysis, and the rate of patients receiving appropriate empirical antibiotic treatment in the death group was significantly lower than that in the survival group . Based on our multivariate analysis, appropriate empirical antibiotic treatment also appeared to be a protective factor that can reduce the mortality rate, although this effect was marginally significant .As shown in Additional file E. coli BSIs were treated with carbapenem monotherapy, and 95 patients were treated with BLBLI combination monotherapy regimens. Figure n = 117) or a BLBLI regimen (n = 95) resulted in no differences in 28 day mortality between the two groups , and their APACHE II scores were 10 and 9, respectively. Considering the potential confounding effect of severity, the patients were divided into two groups according to their APACHE II scores (< 9 and ≥ 9), and the difference in the prognoses of the two groups was compared ; similarly, no significant difference was observed between the two groups in patients with APACHE II scores < 9 .In terms of empirical treatment, 117 patients with ESBL-producing Escherichia coli is one of the most common pathogens in clinical infections. The primary mechanism of drug resistance in these bacteria is the production of ESBLs, and this mechanism underlies a high proportion of the antibiotic resistance cases in China [E. coli BSIs in non-transplanted patients.in China . This stE. coli infections and prior antibiotic therapy; for example, Denis et al. reported that there was no significant difference in the prevalence of antibiotic exposure between the ESBL-producing E. coli and non-ESBL-producing E. coli groups, which included 82 patients (P = 0.32) [E. coli BSIs, which is consistent with the findings of many previous studies. Regarding the likely infectious source of E. coli BSIs, intra-abdominal infections were most common, followed by respiratory tract infections and urinary tract infections. However, most studies showed that ESBL-producing E. coli strains are primarily derived from urinary tract infections [E. coli urinary tract infections (n = 29) and 66% of the patients with intra-abdominal infections (n = 93); therefore, this significant difference between the frequencies of patients with infections in these two sites is likely due to a higher likelihood of acquiring an abdominal infection in the hospital. Based on this observation, we suspect that the reason for the high proportion of intra-abdominal infections in this study may be related to the high number of patients undergoing hepatology-related procedures and hepatobiliary surgery in our hospital. It is well known that E. coli is an opportunistic bacterial pathogen that can invade the body when invasive procedures disrupt the mucosa. The multivariate analysis of the risk factors associated with ESBL-producing E. coli in this study showed that urinary tract infection was an independent risk factor (P = 0.014), consistent with previous studies [E. coli BSIs originating from urinary tract infections are more commonly studied, while articles focusing on abdominal infections are rare, suggesting a further research direction.Antimicrobial use prior to BSI is believed to be an important factor in drug-resistant infections , 20, alt = 0.32) . Our resfections , 9, 22,  studies –25. CurrE. coli BSIs, we evaluated the patient characteristics and the treatments they received. In this study, there was no significant difference in the 28 day mortality between patients infected with ESBL-producing E. coli and those infected with non-ESBL-producing E. coli (P = 0.953), similar to the findings of some previous studies [E. coli than in those infected with non-ESBL-producing E. coli [E. coli. For patients with end-stage liver disease, bacterial infection is one of the most common causes of death [E. coli infection by Kang et al. yielded similar results [E. coli BSIs, which is also similar to the results of previous studies [E. coli and ESBL-producing E. coli BSIs. Serum albumin level is a common indicator for assessing a patient’s nutritional status, organ function, and comorbidity. The inflammatory state resulting from bacterial infection, which leads to the production of IL-1, TNF, and other cell mediators, can interfere with liver albumin synthesis, resulting in hypoalbuminemia [E. coli infections. Akirov et al. [E. coli infections and found that prior use of BLBLI combination antibiotics increased the mortality rate [To explore the high mortality rate associated with  studies , 26. Conof death , which e studies . We alsouminemia . There iv et al.  studied ity rate . In the ity rate  have shoE. coli BSIs, we believe that appropriate, defined antibiotic treatments are a protective factor for reducing death due to E. coli BSIs, as reported in this study , and this observation is consistent with those of some other studies [E. coli have been inconclusive, although carbapenems have long been considered the best antibiotic for treating infections with ESBL-producing E. coli [E. coli infections is becoming more and more accepted by researchers [E. coli BSIs, including 95 patients with BLBLI combination monotherapy regimens and 117 with carbapenem monotherapy (median APACHE II 9 and 10). Their 28 day mortality rates were 12.8 and 17.9%, respectively, and the difference was not statistically significant . Because disease severity may affect prognoses, we divided the ESBL-producing E. coli BSI patients into two groups according to their APACHE II scores  and assessed their 28 day mortality rates. We found no statistically significant difference in 28 day mortality between the two groups , which confirms that carbapenem antibiotics are no longer more effective than BLBLI combination antibiotics against ESBL-producing E. coli BSIs. These findings are consistent with previous reports that proposed the use of BL/BLIs to treat ESBL-producing E. coli BSIs [In terms of the antibiotic options for treating  studies . The fin E. coli . Howeverearchers –38. We aoli BSIs , 39. ForE. coli BSI patients to better understand the drug resistance profiles involved as well as the relevant treatment options.Regarding the limitations of this study, our findings are based mainly on retrospective data and studies, which makes it impossible to eliminate certain types of bias. Furthermore, while this study excluded patients with solid organ and hematopoietic stem cell transplantation, some patients with underlying diseases may have been treated with immunosuppressive agents. Finally, we were unable to perform further stratification of the outcomes based on patient genotyping due to a lack of phenotypic testing. In this context, we should perform more detailed drug resistance gene detection and drug susceptibility testing in ESBL-producing E. coli infections. Prior carbapenem use and a poor systemic condition were risk factors for increased mortality in patients with ESBL-producing E. coli and non-ESBL-producing E. coli BSIs; however, no significant difference was found in the prognoses of patients with ESBL-producing E. coli and non-ESBL-producing E. coli BSIs. Hypoproteinemia and severe disease lead to worse outcomes for patients with ESBL-producing E. coli BSIs. Carbapenem offered no significant advantage over BLBLI combination antibiotics for improving the 28 day mortality of patients with ESBL-producing E. coli BSIs.In summary, prior exposure to cephalosporin and BLBLI combination antibiotics increases the risk of acquiring ESBL-producing Additional file 1:Figure S1. Kaplan-Meier survival estimates among non-transplant patients with BSIs caused by non-ESBL-producing E. coli and ESBL-producing E. coli. (A) Community-acquired infection. (B) Nosocomial-acquired infection. Figure S2. Kaplan-Meier 28 day survival estimates. (A) E. coli BSI patients (APACHE II score < 9) treated with carbapenem and β-lactam-β-lactamase inhibitor (BLBLI) combination antibiotics. (B) E. coli BSI patients (APACHE II score ≥ 9) treated with carbapenem and BLBLIs. (DOCX 122 kb)Additional file 2:Table S1. Analysis of risk factors for 28 day mortality in 283 patients with ESBL- producing E. coli BSI. (DOCX 31 kb)


Background: More than 700 plants are reported to be used for medicinal purposes in Nepal; however, many of them are not studied for their scientific evidences. The aims of the present study were the estimation of the total phenolic and flavonoid contents, and the evaluation of the free radical scavenging and α-amylase inhibitory activities of five selected medicinal plants from western Nepal: Aeschynanthus parviflorus Wall. (Gesneriaceae), Buddleja asiatica Lour. (Loganiaceae), Carica papaya L. (Caricaceae), Drepanostachyum falcatum (Nees) Keng f. (Gramineae) and Spondias pinnata (L. f.) Kurz (Anacardiaceae). Methods: The total phenolic content (TPC) and total flavonoid content (TFC) were measured using Folin-Ciocalteu’s phenol reagent and aluminium chloride methods, respectively. A 1,1–diphenyl–2–picrylhydrazyl (DPPH) free radical scavenging assay was used to evaluate the free radical scavenging activity and an α-amylase inhibitory assay was carried out to determine the in vitro antidiabetic activity. Results: The phytochemical screening of five hydroalcoholic plant extracts revealed the presence of various secondary metabolites, including alkaloids, flavonoids, reducing sugars, saponins, terpenoids and tannins. The amounts of total phenolics and flavonoids were found to be the highest in B. asiatica leaf extract, which also showed the most potent free radical scavenging activity. Extract of C. papaya fruits showed the highest α-amylase inhibitory activity, whereas the extracts of B. asiatica leaves and S. pinnata leaves exhibited moderate activity. Conclusions: Some of the medicinal plants selected in this study showed high TPC and TFC values and potent bioactivities. These results may provide the scientific evidences of the traditional uses of these plants. However, further detailed studies on bioactive compounds isolation and identification and evaluation of in vivo pharmacological activities should be performed in future. Aeschynanthus parviflorus Wall. (Gesneriaceae), Buddleja asiatica Lour. (Loganiaceae), Carica papaya L. (Caricaceae), Drepanostachyum falcatum (Nees) Keng f. (Gramineae) and Spondias pinnata (L. f.) Kurz (Anacardiaceae), from Western Nepal and screened for their phytochemical constituents and biological activities.In most developing countries, medicinal plants and their products are utilized as important therapeutic agents for treating various diseases ,2. CountA. parviflorus is an epiphytic sub-shrub distributed in India, Nepal, Sikkim, Bhutan, Southern China, Burma, Thailand and Vietnam –β–D–glucopyranoside was isolated from the leaves [B. asiatica has been reported to exhibit different pharmacological activities, including antibacterial, antifungal, antispasmodic, cytotoxic, anti-inflammatory and antihepatotoxic activities [C. papaya is well known for its nutritive and medicinal values. The unripe pulp of C. papaya contains phytochemicals, such as saponins and cardinolides, as well as minerals, including potassium, sodium, calcium, iron, phosphorus, zinc, copper and magnesium [C. papaya leaves showed flavonols–manghaslin, clitorin, rutin, nicotiflorin, and piperidine alkaloids–carpamic acid, methyl carpamate and carpaine [C. papaya leaves is reported to exhibit an anti-tumor activity against human lymphocytes and showed immunomodulatory effects [D. falcatum and A. parviflorus have not been reported yet. In this study, a preliminary phytochemical screening of A. parviflorus and D. falcatum hydroethanolic extracts only showed the presence of terpenoids among the different phytochemicals that were tested. Both plant extracts exhibited a poor free radical scavenging activity and α-amylase inhibitory activity, and they also possess a low quantity of total phenols and flavonoids. S. pinnata is known to exhibit various pharmacological activities, such as antimicrobial, antidiabetic, ulcer-protective, anticancer, antidiarrhoeal, anthelmintic, cytotoxic and hepatoprotective activities [S. pinnata bark showed a promising hypoglycemic activity in normal and alloxan induced diabetic rats [S. pinnata bark exhibited an anticancer activity by the induction of a sustained extracellular signal-regulated kinase 1/2 activation and apoptosis in human glioblastoma [S. pinnata has also revealed that its fruits are rich in amino acids, calcium, phosphorus, ascorbic acid and malic acid [There have been some previous studies on the chemical constituents and bioactivities of these selected plants. ompounds . 6–O–(3″asiatica . Similare leaves . B. asiativities . C. papaagnesium . Similarcarpaine . The aqu effects . Extensitivities . The mettic rats . Methyl blastoma . A studylic acid . Furtherα-amylase inhibitory activities. The results concluded that S. pinnata and B. asiatica leaf extracts were found to possess a strong antioxidant activity and higher contents of total phenol and flavonoid. The α-Amylase inhibitory activity of C. papaya fruits was found to be higher among the tested plant samples, whereas B. asiatica and S. pinnata leaves exhibited a moderate activity. Further studies should focus on the bioassay-guided chemical analysis and in-vivo bioactivity evaluations.In this study, five medicinal plants from western Nepal were screened for their phytochemical constituents, and their free radical scavenging and