Ephemerella sp. Yunnan-2018, Serratella zapekinae, Serratella sp. Yunnan-2018, Serratella sp. Liaoning-2019, Torleya grandipennis and T. tumiforceps. These mitogenomes were employed to reveal controversial phylogenetic relationships among the Ephemeroptera, with emphasis on the phylogenetic relationships among Ephemerellidae. The lengths of the six mayfly mitogenomes ranged from 15,134 bp to 15,703 bp. Four mitogenomes of Ephemerella sp. Yunnan-2018, Serratella zapekinae, Serratella sp. Yunnan-2018 and Serratella sp. Liaoning-2019 had 22 tRNAs including an inversion and translocation of trnI. By contrast, the mitogenomes of T. tumiforceps and T. grandipennis had 24 tRNAs due to an extra two copies of inversion and translocation of trnI. Within the family Ephemerellidae, disparate gene rearrangement occurred in the mitogenomes of different genera: one copy of inversion and translocation trnI in the genera Ephemerella and Serratella, and three repeat copies of inversion and translocation of trnI in the genus Torleya. A large non-coding region (≥200 bp) between trnS1 (AGN) and trnE was detected in T. grandipennis and T. tumiforceps. Among the phylogenetic relationship of the Ephemeroptera, the monophyly of almost all families except Siphlonuridae was supported by BI and ML analyses. The phylogenetic results indicated that Ephemerellidae was the sister clade to Vietnamellidae whereas Teloganellidae was not a sister clade of Ephemerellidae and Vietnamellidae.As a small order of Pterygota (Insecta), Ephemeroptera has almost 3,500 species around the world. Ephemerellidae is a widely distributed common group of Ephemeroptera. However, the relationship among Ephemerellidae, Vietnamellidae and Teloganellidae is still in dispute. In this study, we sequenced six complete mitogenomes of three genera from Ephemerellidae (Insecta: Ephemeroptera):  BecauseSiphluriscus chinensis (Siphluriscidae) had one extra trnK-like gene (trnK 2 (AAA)  had an inversion of trnI (Alainites yixiani (GU479735) (Baetidae) showed a gene arrangement of trnI-trnW-trnQ-trnY-trnM. Therefore, these different sets of data suggested that specific gene rearrangements may occur in the mitogenomes of different families. The present study is important and original in that it explores the relationships between mitogenome rearrangements and taxonomic categories of Ephemeroptera.Gene rearrangements can be frequently observed in insect mitogenomes , whereas 2 (AAA) . Within  of trnI . AdditiotrnI inversion in the Family Ephemerellidae and explore the phylogenetic relationships of Ephemerellidae, we sequenced six complete mitogenomes belonging to three genera of Ephemerellidae. The compositional and structural features of these six mitogenomes are described and gene rearrangements were analyzed to explain the mechanism of mitochondrial gene rearrangements.In order to discuss the characteristics of Ephemerella sp. Yunnan-2018, S. zapekinae, Serratella sp. Yunnan-2018, Serratella sp. Liaoning-2019, T. grandipennis and T. tumiforceps were separately captured from Ning’er Yunnan province, Xiuyan Liaoning province, Ning’er Yunnan province, Kuandian Liaoning province, Longquan Zhejiang province and Tonglu Zhejiang province, China, respectively. After morphological identification, the specimens were deposited at −40 °C in the Animal Specimen Museum, College of Life Sciences and Chemistry, Zhejiang Normal University, China. Total genomic DNA was extracted from tissues of each complete specimen by Ezup Column Animal Genomic DNA Purification Kit . The Animal Research Ethics Committees of Zhejiang Normal University approved the experimental design.The voucher specimens of Takara Taq and Takara LA Taq DNA polymerase in a reaction volume of 50 μL. The amplifications for both normal PCR and Long-PCR were performed under the conditions as described in Several partial fragments were amplified using common primers , as deschttp://mitos.bioinf.uni-leipzig.de/index.py) (http://rna.tbi.univie.ac.at/forna) (12S and 16S rRNA). The trnI sequences of T. grandipennis and T. tumiforceps were aligned using the Clustal W program implemented in Mega 7.0 (MT274127–MT274132. The mitogenome maps of the six mayfly species were drawn using GenomeVx (http://wolfe.ucd.ie/GenomeVx) (http://tandem.bu.edu/trf/trf.submit.options.html)   using tht/forna) . After ut/forna)  to compaMega 7.0 . Then, wMega 7.0 . The sixenomeVx) . Codon uenomeVx) . Using tenomeVx) , we calcns.html) . Additions.html) .Siphluriscus chinensis acted as the outgroup  to −0.017 (Serratella sp. Yunnan-2018) whereas the GC-skew for the majority strand ranged from −0.234 (Ephemerella sp. Yunnan-2018) to −0.159 (S. zapekinae). In addition, we analyzed the sizes and nucleotide compositions of the previously published mayfly mitogenomes. Differences in mitochondrial sequences of Ephemeroptera were mainly determined by different sizes of the CR. Among all sequenced Ephemeroptera mitogenomes  was the longest because of the longest CR  (Baetis sp. PC-2010 (GU936204)  was the shortest CR (340 bp). The AT-skews of mitogenomes on the majority strand were negative ranging from Baetis sp. PC-2010 (GU936204) (−0.093) to Caenis sp. YJ-2009 (GQ502451) (−0.001) except for positive skews in Ephemera orientalis (EU591678) (0.03), S. chinensis (0.019) (Ameletus sp. MT-2014 (0.01) (Habrophlebiodes zijinensis (GU936203) (−0.296) to S. chinensis (−0.139) (Baetis sp. PC-2010 (GU936204) (0.006), A. yixiani (GU479735) (0.14) and Ameletus sp. MT-2014 (0.202)  , whereas (0.019)  and Amel4 (0.01) . The GC-(−0.139)  except f (0.202) .ND3 (Ephemerella sp. Yunnan-2018) and ND6  along with GTG assigned to ND1 (Serratella sp. Yunnan-2018), ND3 (Serratella sp. Yunnan-2018), ND4 (Serratella sp. Liaoning-2019), and ND5 . Typical stop codons (TAA/TAG) were found in most PCGs, except for incomplete terminal codons, such as T used for COX2 , COX3 , Cyt b , ND4 (Serratella sp. Liaoning-2019), ND5  and ND6 (Serratella sp. Liaoning-2019), as well as TA used for ND4 . Incomplete terminal codons perform a significant function in polycistronic transcription cleavage and polyadenylation processes  of the six mayfly mitogenomes . The anaEphemerella sp. Yunnan-2018, S. zapekinae, Serratella sp. Yunnan-2018 and Serratella sp. Liaoning-2019 had 22 tRNA genes  in S. zapekinae (trnQ and U-G in the T ψC (T) arm of trnN and trnS1 (AGN). In general, these mismatches are fundamental units of tRNA secondary structure and are nearly isomorphic to normal base pairs, as commonly observed in mayflies or other insects  and Epeorus sp. MT-2014 (A. yixiani ( GU479735)]. It has also been observed in other insect orders such as Mantodea , such as Anthonomus rectirostris, A. rubi, A. eugenii and A. pomorum  and trnV. Unexpectedly, the 12S rRNA was located between trnV and trnI due to the translocation of trnI. The sizes of the 16S rRNA genes in these six mayfly species varied from 1,212 bp (T. grandipennis) to 1,230 bp (Serratella sp. Yunnan-2018), and the sizes of 12S rRNA ranged from 769 bp (T. grandipennis and T. tumiforceps) to 778 bp (Serratella sp. Yunnan-2018). Their length fit within the lengths detected in other Ephemeroptera mitogenomes (Serratella sp. Yunnan-2018 had the highest A+T content of the rRNA genes (68.8%) whereas the mitogenome of T. grandipennis had the lowest (63.1%). In the six mayfly mitogenomes, the AT bias of both 16S rRNA and 12S rRNA was slightly positive (≤0.075), whereas the GC bias was strongly positive (≥0.22), which revealed that the contents of A and G outnumbered T and C nucleotides, respectively  to 68.5% (Serratella sp. Liaoning-2019)  . The CRsng-2019) . We obseapekinae . It has apekinae . The secapekinae , are shoslippage . These sslippage .Ephemerella sp. Yunnan-2018, S. zapekinae and Serratella sp. Yunnan-2018 were obviously lower than the A+T content of corresponding whole mitogenomes, which was also observed in other mayflies  (Mecopoda niponensis) and Hemiptera  , Caenis sp. JYZ-2018  and Epeorus sp. MT-2014 (Siphlonurus immanis (FJ606783)] and Isonychiidae [Isonychia ignota (HM143892)].Interestingly, the A+T content of CRs in us youi) . This phrostris) . The abnrostris) . In the of 43.6% . Also, tsequence . Moreove insects . HoweverJYZ-2018  and CaenJYZ-2020 ], Heptag MT-2014 ], SiphloS. zapekinae, Serratella sp. Yunnan-2018 and Serratella sp. Liaoning-2019 contained 4, 6, and 6 non-coding intergenic spacer sequences, respectively, with total lengths of 22 bp, 32 bp and 29 bp, whereas Ephemerella sp. Yunnan-2018 had 7 non-coding intergenic spacer sequences of 92 bp in total length due to an intergenic spacer of 68 bp between ND4L and trnT. An intergenic spacer between trnS2 (UCN) and ND1 ranging from 17 bp to 19 bp was also detected in these four mitogenomes and is commonly found in Ephemeroptera. In terms of the intergenic spacer between ND4L and trnT in Ephemerella sp. Yunnan-2018, we also detected a similar sequence of 62 bp with a similarity of 72.58% in Ephemerella sp. MT-2014 , we observed a large intergenic spacer between trnS1 (AGN) and trnE within the mitogenomes of T. tumiforceps and T. grandipennis, with lengths of 531 bp and 200 bp, respectively. The large intergenic region in T. tumiforceps and T. grandipennis had 52.17% and 52% A+T content, respectively, with a strongly positive GC-skew (0.31 and 0.13). The large intergenic region in T. tumiforceps and T. grandipennis had a similar starting sequence of 20 bp (GGGSCKAAAGGSCCTACCTA) and a conserved sequence of 19 bp (TTTTTAGCGAACTTAGGGG), respectively, with 4 tandem repeats of 87 bp and 3 tandem repeats of 42 bp along with 4 partial sequences, respectively. The repeat unit of T. tumiforceps could be folded into three stem-loop secondary structures, whereas T. grandipennis folded into two stem-loop secondary structures  and trnE could be synapomorphic for the genus Torleya owing to the fact that sequences located between trnS1 (AGN) and trnE within mitogenomes of other Ephemeroptera were short (<13 bp) and the large intergenic spacer has only been found in Torleya. This is the first finding of a large intergenic region in the mitogenomes of mayflies. As a consequence of the non-coding particularity of the intergenic spacer sequence, these species may well have gone through further sequence divergence swiftly ], whereas an intergenic spacer between trnQ and trnM ranging from 26 bp to 53 bp was found in Caenidae  and Ephemeridae [E. orientalis (EU591678)]. In terms of the mitogenomes of Heptageniidae, we detected an intergenic spacer between trnA and trnR, ranging from 33 bp to 47 bp ))), as reported by P. youi + (P. cupulatus + (Epeorus sp. JZ-2014 + (Epeorus sp. MT-2014 + E. herklotsi)))), as reported by P. youi, P. cupulatus and (Epeorus sp. JZ-2014 + (Epeorus sp. MT-2014 + E. herklotsi)), with low support. On the whole, the monophyly of all Ephemeroptera families was supported except the family Siphluriscidae.Derived from both BI and ML analyses, the phylogenetic trees showed an identical topology, excluding the internal relationship among Heptageniidae  and 5. T12S, 16S, 18S, 28S and H3 genes, Ephemerellidae was supported as a monophyletic group, and their relationship with Teloganellidae and Vietnamellidae remains problematic  + Serratella sp. Yunnan-2018) + (T. tumiforceps + T. grandipennis)) + (Ephemerella sp. Yunnan-2018 + Ephemerella sp. MT-2014)). The inversion and translocation of one copy of trnI was found in Ephemerella and Serratella, whereas the inversion and translocation of three copies of trnI was found in Torleya (Serratella + Torleya) + Ephemerella), we can deduce that the inversion and translocation of one copy of trnI is characteristic of the ancestral Ephemerellidae. Unfortunately, the presence of only one mitogenome of Teloganellidae restricted a discussion of its monophyly, which requires more species to be added.Our findings indicated that Ephemerellidae was the sister clade to Vietnamellidae, but Teloganellidae was the sister clade to Baetidae , which mblematic . In addi Torleya . AccordiEphemerella sp. Yunnan-2018, S. zapekinae, Serratella sp. Yunnan-2018, Serratella sp. Liaoning-2019, T. grandipennis and T. tumiforceps. The mitogenomes of the genera Ephemerella and Serratella showed similar gene features to Ephemerella sp. MT-2014 (KM244691) and had the same inversion and translocation of trnI, whereas two extra copies of trnI were found in the genus Torleya. The translocation and extra copies of trnI could be explained by the tandem duplication/random loss model, whereas the inversion of trnI could be interpreted by the mitogenome recombination model. The evidence from this study suggests that these specific mitogenome rearrangements may be molecular markers of the family Ephemerellidae, especially the three copies of trnI as a synapomorphy for the genus Torleya. The occurrences and mechanisms of the large intergenic region between trnS1 (AGN) and trnE, detected in T. tumiforceps and T. grandipennis, require more mitogenomes of Torleya species to be investigated. Phylogenetic analyses based on BI and ML trees both supported the monophyly of Ephemerellidae and Vietnamellidae. Moreover, Ephemerellidae acted as the sister clade to Vietnamellidae whereas Teloganellidae existed in the other clade. Considerable work may be needed to increase the number of mitogenomes to resolve the phylogenetic relationships of Ephemeroptera.We successfully determined the complete mitogenomes of 10.7717/peerj.9740/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9740/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9740/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.9740/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9740/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.9740/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.9740/supp-7Supplemental Information 7 Ephemerella sp. Yunnan-2018, Serratella zapekinae, Serratella sp. Yunnan-2018. and Serratella sp. Liaoning-2019; (B) Torleya grandipennis and Torleya tumiforceps. The figure is written permission from Yu-Rou Cao to publish. She helped drawing under our CC BY 4.0 license.External genes on the circle are encoded by the positive strand (5’ →3’) and internal genes are encoded by the negative strand (3’ →5’). (A)Click here for additional data file.10.7717/peerj.9740/supp-8Supplemental Information 8trnI; (B) trnQ; (C) trnM; (D) trnW; (E) trnC; (F) trnY; (G) trnL (CUN); (H) trnK; (I) trnD; (J) trnG; (K) trnA; (L) trnR; (M) trnN; (N) trnS (AGN); (O) trnE; (P) trnF; (Q) trnH; (R) trnT; (S) trnP; (T) trnS (UCN); (U) trnL (UUR); (V) trnV.A) Click here for additional data file.10.7717/peerj.9740/supp-9Supplemental Information 9trnI; (B) trnQ; (C) trnM; (D) trnW; (E) trnC; (F) trnY; (G) trnL (CUN); (H) trnK; (I) trnD; (J) trnG; (K) trnA; (L) trnR; (M) trnN; (N) trnS (AGN); (O) trnE; (P) trnF; (Q) trnH; (R) trnT; (S) trnP; (T) trnS (UCN); (U) trnL (UUR); (V) trnV.(A) Click here for additional data file.10.7717/peerj.9740/supp-10Supplemental Information 10trnI; (B) trnQ; (C) trnM; (D) trnW; (E) trnC; (F) trnY; (G) trnL (CUN); (H) trnK; (I) trnD; (J) trnG; (K) trnA; (L) trnR; (M) trnN; (N) trnS (AGN); (O) trnE; (P) trnF; (Q) trnH; (R) trnT; (S) trnP; (T) trnS (UCN); (U) trnL (UUR); (V) trnV.(A) Click here for additional data file.10.7717/peerj.9740/supp-11Supplemental Information 11trnI; (B) trnQ; (C) trnM; (D) trnW; (E) trnC; (F) trnY; (G) trnL (CUN); (H) trnK; (I) trnD; (J) trnG; (K) trnA; (L) trnR; (M) trnN; (N) trnS (AGN); (O) trnE; (P) trnF; (Q) trnH; (R) trnT; (S) trnP; (T) trnS (UCN); (U) trnL (UUR); (V) trnV.(A) Click here for additional data file.10.7717/peerj.9740/supp-12Supplemental Information 12trnI; (B) trnI; (C) trnI; (D)trnQ; (E) trnM; (F) trnW; (G) trnC; (H) trnY; (I) trnL (CUN); (K) trnK; (K) trnD; (L) trnG; (M) trnA; (N)trnR; (O) trnN; (P) trnS (AGN); (Q) trnE; (R) trnF; (S) trnH; (T) trnT; (U) trnP; (V) trnS (UCN); (W) trnL (UUR); (X) trnV.(A) Click here for additional data file.10.7717/peerj.9740/supp-13Supplemental Information 13trnI; (B) trnI; (C) trnI; (D)trnQ; (E) trnM; (F) trnW; (G) trnC; (H) trnY; (I) trnL (CUN); (K) trnK; (K) trnD; (L) trnG; (M) trnA; (N)trnR; (O) trnN; (P) trnS (AGN); (Q) trnE; (R) trnF; (S) trnH; (T) trnT; (U) trnP; (V) trnS (UCN); (W) trnL (UUR); (X) trnV.(A) Click here for additional data file.10.7717/peerj.9740/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.9740/supp-15Supplemental Information 15Serratella zapekinae;(B) the repeat unit (100 bp) in Serratella zapekinae;(C) the repeat unit (109 bp) in Serratella sp. Liaoning-2019; (D) the repeat unit (72 bp) in Serratella sp. Liaoning-2019.(A) the repeat unit (90 bp) in Click here for additional data file.10.7717/peerj.9740/supp-16Supplemental Information 16T. tumiforceps; (B) the repeat unit (42 bp) in T. grandipennis.(A) the repeat unit (87 bp) in Click here for additional data file.10.7717/peerj.9740/supp-17Supplemental Information 17trnS1 (AGN) and trnE. The CS indicates the 19 bp conserved sequence TTTTTAGCGAACTTAGGGG. Gene sizes are not drawn to scale. Genes located on the majority strand are shown along the top of the boxes whereas genes located on the minority strand are shown on the bottom. White boxes represent genes with the same relative position as in the ancestral insect arrangement pattern. Grey boxes represent non-coding regions. The remaining genes and gene orders that were identical to the ancestral insect are left out.The duplication/random loss model can explain the NCR between Click here for additional data file.10.7717/peerj.9740/supp-18Supplemental Information 18Click here for additional data file.10.7717/peerj.9740/supp-19Supplemental Information 19Click here for additional data file.10.7717/peerj.9740/supp-20Supplemental Information 20Ephemerella sp. Yunnan-2018. Blue shading represents Serratella zapekinae. Yellow shading represents Serratella sp. Yunnan-2018. Green shading represents Serratella sp. Liaoning-2019.Orange shading represents Click here for additional data file.10.7717/peerj.9740/supp-21Supplemental Information 21Click here for additional data file.10.7717/peerj.9740/supp-22Supplemental Information 22Click here for additional data file.10.7717/peerj.9740/supp-23Supplemental Information 23Click here for additional data file.


Macadamia integrifolia is a representative of the large basal eudicot family Proteaceae and the main progenitor species of the Australian native nut crop macadamia. Since its commercialisation in Hawaii fewer than 100 years ago, global production has expanded rapidly. However, genomic resources are limited in comparison to other horticultural crops. The first draft assembly of M. integrifolia had good coverage of the functional gene space but its high fragmentation has restricted its use in comparative genomics and association studies. Here we have generated an improved assembly of cultivar HAES 741  using a combination of Illumina paired and PacBio long read sequences. Scaffolds were anchored to 14 pseudo-chromosomes using seven genetic linkage maps. This assembly has improved contiguity and coverage, with >120 Gb of additional sequence. Following annotation, 34,274 protein-coding genes were predicted, representing 90% of the expected gene content. Our results indicate that the macadamia genome is repetitive and heterozygous. The total repeat content was 55% and genome-wide heterozygosity, estimated by read mapping, was 0.98% or an average of one SNP per 102 bp. This is the first chromosome-scale genome assembly for macadamia and the Proteaceae. It is expected to be a valuable resource for breeding, gene discovery, conservation and evolutionary genomics. As a member of the Gondwanan family Proteaceae  (Macadamia (F.Muell.) is a basal eudicot and is phylogenetically divergent from other tree crops  , Macadamtivation , is a mitivation . MacadamMacadamia  were then anchored and oriented to 14 pseudo-chromosomes using methods to maximize colinearity across seven genetic linkage maps . An herbarium specimen was submitted to the Southern Cross Plant Science Herbarium [accession PHARM-13-0813]. On collection, samples were snap frozen in liquid nitrogen or placed on dry ice and stored at -80° prior to extraction. Previously described methods for and DNA/RNA extraction for Illumina , new libraries with 200, 350 and 550 bp insert sizes were prepared with TruSeq DNA PCR-free kits and sequenced with Illumina HiSeq 2500 producing 57.0, 29.8 and 29.6 Gb paired-end sequence data. A PacBio library (20-Kb) was prepared and sequenced across 10 SMRT cells on a PacBio RSII system (P6-C4 chemistry) generating 6.44 Gb data. Transcriptome sequencing of leaf, shoot and flower tissue followed previously described methods .de novo assembly, scaffolding and gap closing was performed using MaSuRCA , Eucalpytus grandis and Coffea canephora (coffee) genomes. Pairwise sequence similarities were determined applying a BLASTP E-value cut-off of 1E-05 and an inflation value of 1.5 for OrthoMCL Markov clustering. Macadamia specific gene clusters were tested for GO enrichment using OrthoVenn2.Orthologous gene clusters were identified and compared using OrthoVenn2   with 180 × coverage of the genome and a scaffold N50 of 413 kb  and 93.5% of corrected PacBio reads tagged as primary alignments by Minimap2 aligned reliably to the assembly. No Pacbio reads aligned to two different scaffolds at a cutoff of 1/2 read length. At 1/3 read length, 130 reads (mean length 7833 bp representing < 0.14% of the assembly) aligned to two scaffolds and no reads aligned to more than two scaffolds.M. integrifolia of 652 Mb (600-700 Mb) that was based on 51.6 Gb Illumina short read data  of the assembly. As reported for many other plant genomes, long terminal repeat (LTR) retrotransposons were the most abundant repeat type (30.5%). Small RNAs including pseudogenes and active small RNA genes including tRNA and snRNA accounted for 0.54% of the assembly .N. nucifera, A. thaliana, P. persica, E. grandis and C. canephora identified 8,961 orthologous clusters of which 2,051 contained a single gene from each of the six eudicot species. M. integrifolia and N. nucifera both belong to the basal eudicot order Proteales. These species and contained similar numbers of clusters with 13,491 and 13,321 respectively and shared the largest number of clusters (503) of any species pair (P = 4.8E-05) and fruit ripening .Annotation of the final assembly, identified 34,274 high-confidence gene models. Of these, 25,779 (75.2%) were anchored to pseudo-chromosomes and 8,495 were located on unanchored scaffolds . Comparaies pair . Tests fHere we present the first chromosome-scale genome assembly for the nut crop macadamia and the large Gondwanan plant family Proteaceae. This provides a platform for unraveling the genetics of macadamia and is expected to underpin future breeding, and comparative and horticultural genomics research.Versions and parameters of the tools implemented in data analysis are provided below:BBMap version 36.62: ktrim = r k = 23 mink = 11 hdist = 1 tpe tbo maxns = 1 minlen = 50 maq = 8 qtrim = rl trimq = 20LoRDEC version 0.4: lordec-correct -2 input_for_read_correction.fastq -k 19 -S out.stat.txt -s 3 -T 12 -i PacBio_filtered_reads.fasta -o out.pacbio.corrected.fastaNUM_THREADS = 16, JF_SIZE = 20000000000 (jellyfish hash size)MaSuRCA version 3.2.1. Default parameters except: vs.macaAssembly.psl -noHead -out = pslL_RNA_scaffolder: blat -t = dna -q = dna scaffolds_gapClosed_min1000.fa Trinity.fasta transcript.ALLMAPS version 0.7.7: Default parametersJellyfish version 2.0: jellyfish count -t 14 -C -m 27 -s 8G -o 27mer_maca_illumOnly_out <all Illumina-only WGS fastqs > jellyfish histo -t14 27mer_maca_illumOnly_out> 27mer_maca_illumOnly_out.histofindGSE: Default parametersGenomeScope version 2.2.6:kmer length 27, read length 125bp, Max kmer coverage 1000Trinity, version 2.0.3: Trinity–seqType fq–max_memory 100G –left reads_S_R1_clean.fq,reads_F2_R1_clean.fq,reads_YL_R1_clean.fq–right reads_S_R2_clean.fq,reads_F2_R2_clean.fq,reads_YL_R2_clean.fq–CPU 8RepeatModeler version 2.0.1. Default parametersRepeatMasker version 4.0.9. Default parametersMAKER, version 2.31.10. Default parameters except: Gene prediction methods Augustus and SNAP (trained with previously generated macadamia gene models); AED score = 0.40; Minimum protein length: = 50 amino acidsBUSCO version 3.0.2: busco -i proteins.fasta -l viridiplantae_odb10 -m proteins -o output_nameMinimap2 version 2.17. Default parameters (paftool.js bundled with Minimap2): minimap2 -ax sr ref_assemblyfasta fastq1stReadPair fastq2ndReadPair > ref_readPairs_aln.paf; k8 paftools.js stat ref_readPairs_aln.paf > alignment_mapstatscf.gz -ERC GVCF -heterozygosity 0.01GATK HaplotypeCaller version 4.1.4.1: gatk–java-options “-Xmx8g” HaplotypeCaller -R refGenome.fasta -I input.bam -O output.g.vcf.gz -O output.vcf.gz–heterozygosity 0.01GATK GenotypeGVCFs version 4.1.4.1: gatk–java-options “-Xmx4g” GenotypeGVCFs -R refGenome.fasta -V input.g.vcf.gz -F CHROM -F POS -F TYPE -F REF -F ALT -F HET -GF GT -GF AD -O output_genoGVCF.table.txt.GATK VariantsToTable version 4.1.4.1: gatk-4.1.4.1/gatk–java-options “-Xmx8g” VariantsToTable -V output.v


RAS wild-type metastatic colorectal cancer (mCRC) treated with anti-EGFR  based first-line treatment is still debated. Methods: This multicenter, real-world, retrospective study is aimed at evaluating the effectiveness of second-line Bevacizumab- and Aflibercept-based treatments after an anti-EGFR based first-line regimen. Clinical outcomes measured were: objective response rate (ORR), progression free survival (PFS), overall survival (OS) and adverse events (AEs) profiles. Results: From February 2011 to October 2019, 277 consecutive mCRC patients received Bevacizumab-based  or Aflibercept-based  regimen. No significant difference was found regarding ORR. The median follow-up was 27.7 months (95%CI: 24.7–34.4). Aflibercept-treated group had a significantly shorter PFS compared to Bevacizumab-treated group  (HR = 1.34 (95%CI: 0.95–1.89); p = 0.0932). The median OS of the Bevacizumab-treated group and Aflibercept-treated group was 16.2 (95%CI: 15.3–18.1) and 12.7 (95%CI: 8.8–17.5) months, respectively (HR= 1.31 (95%CI: 0.89–1.93) p = 0.16). After adjusting for the key covariates  Bevacizumab-based regimens revealed to be significantly related with a prolonged PFS (HR = 1.44 (95%CI: 1.02–2.03); p = 0.0399) compared to Aflibercept-based regimens, but not with a prolonged OS (HR = 1.47 (95%CI: 0.99–2.17); p = 0.0503). The incidence of G3/G4 VEGF inhibitors class-specific AEs was 7.5% and 26.5% in the Bevacizumab-treated group and the Aflibercept-treated group, respectively (p = 0.0001). Conclusion: Our analysis seems to reveal that Bevacizumab-based regimens have a slightly better PFS and class-specific AEs profile compared to Aflibercept-based regimen as second-line treatment of RAS wild-type mCRC patients previously treated with anti-EGFR based treatments. These results have to be taken with caution and no conclusive considerations are allowed.Background: The optimal anti-angiogenic strategy as second-line treatment in  RAS genotype  1–2 and 3–4). Toxicities were summarized and compared among subgroups according to three key subgroups: VEGF inhibitors class-specific AEs , hematologic AEs , and non-hematologic AEs . Only AEs which occurred in more than 5% of patients were included in the safety analysis.KRAS, NRAS and BRAF mutational status was assessed with Sanger sequencing, real-time PCR techniques and next-generation sequencing (NGS) ; Pyromark Q96 ID System, Qiagen ; EasyPGX and Myriapod Colon Status, Diatech Pharmacogenetics ). MSI (microsatellite instability) status and/or MMR (mismatch repair) proteins expression were assessed with molecular sequencing  and Immunohistochemistry (IHC) ; Ultraview Universal Detection Kit and Ventana platform, Roche Tissue Diagnostics and Ventana Medical Systems ).All the molecular analyses were performed according to the local clinical practice of the participating centers. p < 0.05. Hazard Ratios (HRs) with 95% confidence intervals (CIs) were calculated using the logistic regression model. All statistical analyses were performed using MedCalc Statistical Software version 18.11.3 .Baseline patients’ characteristics were reported with descriptive statistics and compared among subgroups with the Chi-square test. Chi-square test was also used to compare ORR and the incidence of AEs across subgroups. Logistic regression was used for the multivariate analysis of ORR. Median PFS and median OS were evaluated using the Kaplan–Meier method. Median period of follow-up was calculated according to the reverse Kaplan-Meier method. Cox proportional hazards regression was used for the univariate and multivariate analysis of PFS and OS. The alpha level for all analyses was set to RAS wild-type mCRC patients were treated with Bevacizumab-based  or Aflibercept-based  second-line regimens. The median age was 64.5 years (range: 29–84). Patients features  are summarized in p < 0.0001). According to the clinical indication of Aflibercept, also the previously received first-line regimens (p < 0.0001) and second-line chemotherapy backbone (p = 0.0148) were significantly different. A total of 277 consecutive The activity profile for the overall population and according to subgroups is summarized in p = 0.0932) (p = 0.1600) (p = 0.0399) compared to Aflibercept-based regimens, but not with a prolonged OS (HR = 1.47 (95%CI: 0.99–2.17); p = 0.0503).The second-line median follow-up for the study population was 27.7 months (95%CI: 24.7–34.4); median PFS and median OS were 7.1 months  and 15.7 months . Median PFS of the Bevacizumab-treated group was 7.1 months , while median PFS of the Aflibercept-treated group was 5.6 months , without statistically significant difference at the univariate analysis (HR = 1.34 (95%CI: 0.95–1.89);  0.0932) A. Median 0.1600) B. Table p = 0.1908). The incidence of G3/G4 VEGF inhibitors class-specific AEs was 7.5% and 26.5% in the Bevacizumab-treated group and in the Aflibercept-treated group, respectively (p = 0.0001) (p = 0.0033), while the incidence of G3/G4 non hematologic AEs was 4.4% and 10.2%, respectively (p = 0.1032). The incidence of G1/G2 hematologic AEs was 24.6% and 22.4% in the Bevacizumab-treated group and in the Aflibercept-treated group, respectively (p = 0.7545), while the incidence of G3/G4 hematologic AEs was 3.1% and 18.4%, respectively (p < 0.0001).The toxicity profile for the overall study population and according to subgroups is summarized in  0.0001) . The incp = 0.2236). A total of 136 patients (69.4%) and 24 patients (60%) were treated with a third-line systemic therapy, among those who discontinued second-line treatment in the Bevacizumab-treated group and Aflibercept-treated group, respectively (p = 0.5930). A total of 67 patients (29.4%) and nine patients (18.4%) underwent a maintenance therapy after an induction phase, in the Bevacizumab-treated group and in the Aflibercept-treated group, respectively , and this might have affected the clinical outcomes [BRAF mutant patients [BRAF mutational status , identifying a study population with good prognosis overall [Despite the unbalanced grouping of the study population according to the received regimens (82.3% Bevacizumab-based vs. 17.7% Aflibercept-based), most of the patients characteristics were balanced between the subgroups, such as elderly patients, number of metastatic sites and primary tumor location see . On the outcomes . The clipatients , are ali overall .RAS wild-type patients were eligible) might also partially explain these discrepancies. Interestingly, genotype based post-hoc analyses reported an OS of 15.4 months for KRAS wild-type patients of the experimental arm of the ML18147 [RAS wild-type patients of the experimental arm of the VELOUR trial [Even though studies results comparisons are not methodologically correct, some speculations are allowed. The median PFS of the Bevacizumab-treated group (7.1 months) was comparable to the PFS reported in the E3200 and ML18147 trials  ,13, wher ML18147 , and an UR trial . Moreovep = 0.0399), whereas a not significant trend towards a shorter OS was reported (HR = 1.47 (95%CI: 0.99–2.17); p = 0.0503). Concerning safety data, we found a significant higher incidence of G3/G4 VEGF inhibitors class-specific AEs among Aflibercept-treated patients, compared to the Bevacizumab-treated patients . This aspect might be also related to the different pharmacodynamic mechanisms of action of Bevacizumab  and Aflibercept ) [p = 0.0033) and G3/G4 hematologic AEs (3.1% vs. 18.4%) to the detriment of the Aflibercept-treated patients, was found. The latter aspect could be related to the different chemotherapy backbone .Intriguingly, the multivariate analysis revealed that the Aflibercept-treated group had a statistically significant shorter PFS compared to the Bevacizumab-treated group (HR = 1.44 (95%CI: 1.02–2.03);  (PIGF)) . FurtherOur results suggest a slightly better clinical performance for second-line Bevacizumab-based regimens compared to Aflibercept-based regimens. In our opinion, the different safety profile might had affected the effectiveness of Aflibercept-based regimens compared to Bevacizumab-based regimens, leading to a higher discontinuation rate  and a worse PFS.According to the RAISE trial results , it woulRAS wild-type patients, are awaited. The STRATEGIC-S1 trial (NCT01910610) [RAS wild-type mCRC patients: an oxaliplatin-based second-line regimen with Bevacizumab after fist line FOLFIRI-Cetuximab vs. an irinotecan-based second-line regimen with Bevacizumab after a first-line OPTIMOX Bevacizumab, followed by an anti-EGFR based third-line treatment. The DISTINCTIVE trial (NCT04252456) [RAS wild-type mCRC patients after an oxaliplatin/fluoropyrimidines-based first-line regimen combined with either Panitumumab or Cetuximab. Results from important prospective phase II-III studies, comparing different sequencing strategies of available biological agents for 1910610)  is an in4252456)  is a proRAS wild-type mCRC patients.There are some obvious limitations in this study, including its retrospective design, which expose to selection bias, therefore the results must be taken with caution. Further analysis with a larger sample size and a prospective translational design are certainly needed to better define and personalize the anti-angiogenic strategy as a second-line treatment in RAS wild-type mCRC patients who received first-line anti-EGFR based treatments. These results have to be taken with caution and no conclusive consideration are allowed.Our analysis seems to reveal that Bevacizumab-based regimens have a slightly better efficacy and safety profile compared to Aflibercept-based regimens as second-line treatment of


Kernel weight and morphology are important traits affecting cereal yields and quality. Dissecting the genetic basis of thousand kernel weight (TKW) and its related traits is an effective method to improve wheat yield.QTkw.cas-1A.2 and QTkw.cas-4A explained the largest portion of the phenotypic variance for TKW and kernel length (KL), and the other two stable QTLs QTkw.cas-6A.1 and QTkw.cas-7D.2 contributed more effects on kernel width (KW). Conditional QTL analysis revealed that the stable QTLs for TKW were mainly affected by KW. The QTLs QTkw.cas-7D.2 and QKw.cas-7D.1 associated with TKW and KW were delimited to the physical interval of approximately 3.82 Mb harboring 47 candidate genes. Among them, the candidate gene TaFT-D1 had a 1 bp insertions/deletion (InDel) within the third exon, which might be the reason for diversity in TKW and KW between the two parents. A Kompetitive Allele-Specific PCR (KASP) marker of TaFT-D1 allele was developed and verified by PG-RIL and a natural population consisted of 141 cultivar/lines. It was found that the favorable TaFT-D1 (G)-allele has been positively selected during Chinese wheat breeding. Thus, these results can be used for further positional cloning and marker-assisted selection in wheat breeding programs.In this study, we performed quantitative trait loci (QTL) analysis using recombinant inbred lines derived from the cross ‘PuBing3228 × Gao8901’ (PG-RIL) to dissect the genetic basis of kernel traits. A total of 17 stable QTLs related to kernel traits were identified, notably, two stable QTLs TaFT-D1 could be the candidate gene for QTLs QTkw.cas-7D.2 and QKw.cas-7D.1.Seventeen stable QTLs related to kernel traits were identified. The stable QTLs for thousand kernel weight were mainly affected by kernel width.  Triticum aestivum L.) is one of the most important cereal crops for feeds 40% of population in the world (http://www.fao.org/). Wheat yield is determined by thousand kernel weight (TKW), kernel number per spike, and effective tiller number [Common wheat  in tetraploid wheat [Recently, high-density single nucleotide polymorphism (SNP) arrays technology provides a superior approach to identify QTLs for wheat kernel-related traits –31. To did wheat , 42. HowUsing a RIL population derived from ‘PuBing 3228 (P3228) × Gao8901 (G8901)’, the objectives of this study were to (i) identify stable and major QTLs for TKW, KL, KW and KL/W under different field conditions; (ii) reveal the contribution of the other kernel traits to TKW using conditional QTL analysis; (iii) predict candidate gene(s) for targeted QTLs interval based on reference genome annotation information; (iv) develop KASP markers of the candidate gene(s) and verified by PG-RIL and a natural population consisted of 141 cultivar/lines for marker-assisted selection in high-TKW wheat breeding.H) of four traits were higher than 0.60  are listed in Table A total of 47 putative QTLs were detected for TKW, KL, KW and KW/L Figs. .Fig. 3GQTkw.cas-1A.2, QTkw.cas-4A, QTkw.cas-5D, QTkw.cs-6A.1 and QTkw.cas-7D.2  ranged from 8.31 to 11.84%  were significant in at least two environments . The favorable alleles of the five QTLs  were derived from the parent G8901 , 1B, 4B, 6A, 7A (two) and 7D, respectively  were found in at least two environments .A total of 10 QTLs for KL/W were identified on chromosomes 1A, 1B, 2A, 5A (two), 5D, 7A (two) and 7D (two), with PVE of individual QTL ranging from 1.79 to 22.41%  = 21.93%), indicating that the major QTLs QTkw.cas-4A and QKl.cas-4A for TKW and KL, respectively, were significantly affected by the environment  interactions were detected at 43 loci for TKW, KL, KW and KW/L  were identified to be associated with KW, but independent of KL and KL/W , a homolog of Arabidopsis FLOWERING LOCUS T, was considered as the candidate gene for QTkw.cas-7D.2 and QKw.cas-7D.1  closely linked to the two stable QTLs (QTkw.cas-7D.2 and QKw.cas-7D.1) and 1 bp InDel of TaFT-D1 were further converted to KASP markers -allele) and P3228-allele (TaFT-D1(−)-allele) plants -allele was significantly higher than those of the TaFT-D1(−)-allele .Two SNPs markers -allele in modern cultivars was higher in the seven agro-ecological wheat production regions , suggesting that TaFT-D1(G)-allele have undergone positive selection during wheat breeding process -allele can be used in different wheat production regions.To determine whether the two g season , 52. ComQTkw.cas-1A.2, QTkw.cas-5D, QTkw.cas-6A.1, QTkw.cas-7D.2) account for TKW, while two (QTkw.cas-4A and QTkw.cas-5D) on KL -allele associated with TKW and KW has been positively selected during Chinese wheat breeding. In addition, the current study provided new options for dissecting the genetic basis of yield and molecular-assisted breeding.In this study, we performed QTL analysis using the PG-RIL population in four environments for kernel-related traits , which were mainly distributed on chromosomes 1A, 1B, 4A, 5D, 6A, 7A and 7D Fig. . Notably6–9 RILs derived from ‘PuBing3228 × Gao 8901 ‘was developed by single seed descent method. The wheat germplasm P3228 was developed by Dr. Lihui Li . G8901 is a commercial cultivar released by Gaocheng institute of agricultural science, Hebei, China. The P3228 has higher kernel number per spike and the G8901 has higher thousand kernel weight . The H was calculated using the QGAStation 2.0 (http://ibi.zju.edu.cn/software/qga/v2.0/index c.htm) and the following formula H = VG/VP; where VG and VP are the genetic variance and phenotypic variance, respectively.For the four environments, 10 representative plants were sampled from each plot to investigate kernel-related traits. At seed maturity, the TKW and kernel morphometric traits  of at least 500 kernels were measured three times using the rapid SC-G grain appearance quality image analysis system . Analysis of variance (ANOVA), mean values of traits, standard deviations and variation coefficients (CV) were performed with SPSS Statistics v20.0 software . Effects among genotypes, environments, and GE interaction were estimated by ANOVA. BLUP for all four traits across four environments was calculated using R software ) of TKW in wheat were obtained by the mixed-model approach. The conditional phenotypic value can be partitioned as.Conditional QTL analysis was performed to analyze the genetic contributions of kernel-related traits to TKW, by the procedure of inclusive composite interval mapping . The cony(TKW|KL)=μ(TKW|KL) + G(TKW|KL) + E(TKW|KL) + e(TKW|KL).y(TKW|KL) is the conditional phenotypic value of TKW on KL; μ(TKW|KL) is the conditional population mean, G(TKW|KL) is the conditional general genotypic effect; E(TKW|KL) is the conditional effect for the environment and e(TKW|KL) is the conditional residual error.where (TKW|KL) denote TKW conditional on KL; y(TKW|KL), y(TKW|KW) and y(TKW|KL/W)) are the conditional phenotypic value of TKW on KL, KW or KL/KW in the corresponding environment, which were estimated using QGAStation2.0 (http://ibi.zju.edu.cn/software/qga/). MapChart 2.2 (http://www.biometris.nl/uk/Software/MapChart/) was used to draw the genetic map.The conditional phenotypic values  and physical (right) positions for SNPs mapped on the chromosome 7DS in PG-RIL genetic map. Fig. S2 A 1 bp InDel in TaFT-D1 caused a frameshift mutation of the protein. (a) Sequence alignment of TaFT-D1 showing 1 bp InDel between P3228 and G8901. (b) Protein alignment of TaFT-D1 showing frameshift mutation in P3228. Fig. S3 Heatmap showing the expression profile of DEGs at 15 development stages. Fig. S4 Allelic segregation of KASP markers AX-111061288 (a) and AX-111184541 (b) for QTkw.cas-7D.2 and QKw.cas-7D.1.


Chronic isoleucine supplementation prevents diet-induced weight gain in rodents. Acute-isoleucine administration improves glucose tolerance in rodents and reduces postprandial glucose levels in humans. However, the effect of chronic-isoleucine supplementation on body weight and glucose tolerance in obesity is unknown. This study aimed to investigate the impact of chronic isoleucine on body weight gain and glucose tolerance in lean and high-fat-diet (HFD) induced-obese mice. Male C57BL/6-mice, fed a standard-laboratory-diet (SLD) or HFD for 12 weeks, were randomly allocated to: (1) Control: Drinking water; (2) Acute: Drinking water with a gavage of isoleucine (300 mg/kg) prior to the oral-glucose-tolerance-test (OGTT) or gastric-emptying-breath-test (GEBT); (3) Chronic: Drinking water with 1.5% isoleucine, for a further six weeks. At 16 weeks, an OGTT and GEBT was performed and at 17 weeks metabolic monitoring. In SLD- and HFD-mice, there was no difference in body weight, fat mass, and plasma lipid profiles between isoleucine treatment groups. Acute-isoleucine did not improve glucose tolerance in SLD- or HFD-mice. Chronic-isoleucine impaired glucose tolerance in SLD-mice. There was no difference in gastric emptying between any groups. Chronic-isoleucine did not alter energy intake, energy expenditure, or respiratory quotient in SLD- or HFD-mice. In conclusion, chronic isoleucine supplementation may not be an effective treatment for obesity or glucose intolerance. The branched-chain amino acids (BCAAs), isoleucine, leucine and valine, are essential amino acids accounting for ~35% of the essential amino acids comprising muscle proteins in humans and ~40% of the pre-formed amino acids required by all mammals . In popudb/db) mice, a model of morbid obesity and hyperglycaemia [Acute administration of isoleucine and leucine in rats improved glucose tolerance, with isoleucine showing greater effectiveness than leucine . This walycaemia . This evIt is known that postprandial blood glucose levels are influenced by the rate of gastric emptying . In partThe current study aimed to determine whether chronic dietary supplementation with the BCAA isoleucine, alters body weight gain, adiposity, glucose tolerance, and energy metabolism in mice with HFD-induced obesity.This study was approved  by the South Australian Health and Medical Research Institute Animal Ethics Committee. All experimental protocols were performed in alignment with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.n = 54) were group-housed in a 12:12 h light-dark cycle within a temperature (24 ± 1 °C) controlled facility. Mice were provided ad libitum access to either a standard laboratory diet  or HFD . Consistent with the previous literature, the HFD-induced obese mouse model was chosen as both a model of obesity [n = 10; SLD-Chronic (Ch)) and HFD-mice  received ad libitum isoleucine  supplemented in the drinking water. The remaining SLD (n = 20) and HFD-mice (n = 16) continued with ad libitum access to normal drinking water. At 16 weeks, all mice were singly housed and underwent an OGTT (at 1400 h) and gastric emptying breath test (at 0900 h) in random order with a three-day recovery between tests. In each diet group, the mice provided normal drinking water were subdivided into two groups, receiving either an oral gavage of water ; n = 8, HFD-C) or isoleucine ; n = 8, HFD-A) 30 min before the OGTT or GEBT. The SLD and HFD-Ch mice received an oral gavage of drinking water similar to the control groups. The doses for acute and chronic isoleucine treatments were chosen based on previous studies [Eight-week-old male C57BL/6 mice  to consume voluntarily within 1 min. Breath samples were collected before isoleucine administration  and again at regular intervals  after egg consumption. Breath samples were analysed for the 13CO2 content using an isotope ratio mass spectrometer . The 13CO2 excretion data were analysed by non-linear regression analysis for curve fitting and for calculation of gastric half emptying time (t ½) [Gastric emptying of a solid meal was determined using a non-invasive breath test as previously described ,22. Miceme (t ½) . Gastric2/VO2), and total activity  were measured and analysed using the ExpeData data analysis software .Mice were individually housed in Promethium metabolic cages  for 72 h of continuous metabolic monitoring. Energy intake (kJ), energy expenditure (kJ/lean mass), respiratory quotient  then anaesthetised with isoflurane . The nose-to-tail length and abdominal circumference of mice were measured. Blood was collected from the abdominal aorta and transferred to ethylenediaminetetraacetic acid (EDTA) tubes . Plasma was extracted by centrifugation at 1000 g and 4 °C for 15 min, and snap-frozen in liquid nitrogen prior to storage at −80 °C until further analysis. Liver, gonadal fat pads, and inter-scapula brown fat pads were collected and weighed. Lean mass was determined by the final body weight minus the weight of collected fat pads. A section of the liver was fixed in 4% paraformaldehyde for 4 h, cryoprotected overnight in 30% sucrose in a phosphate buffer, frozen in Tissue-Tek O.C.T. compound  and stored at −80 °C before processing for histology. Plasma total triglycerides, total cholesterol, and high-density lipoprotein (HDL)-cholesterol concentrations were measured using commercial enzymatic kits ) on a Beckman AU480 clinical analyser . Plasma low-density lipoprotein (LDL)-cholesterol concentrations were estimated using the Friedewald equation : LDL-cho2 area using the ImageJ-win64 software.The histological lysochrome lipid stain Oil Red O was performed on liver sections using a standard protocol . Slides 0–120 min blood glucose area under the curve (AUC) was generated using the IBM SPSS Statistics 26 software . A correlation between gastric half emptying time (t ½) and OGTT0–120 min blood glucose AUC for SLD groups was performed in the GraphPad Prism v8 software. The coefficient of determination value (r2) was considered significant at p < 0.05.Results are expressed as the mean ± SEM. A two-way ANOVA was performed to assess diet and isoleucine treatment effects with a Tukey’s post hoc test for multiple comparisons, using the GraphPad Prism v8 software . The OGTTp < 0.001, unpaired t-test; p < 0.0001, F  = 28.17, diet effect; p = 0.1262, F  = 2.418, isoleucine effect; SLD-C/A; 34.8 ± 0.7 g (n = 20), SLD-Ch; 37.2 ± 1.3 g (n = 10), HFD-C/A; 42.3 ± 1.6 g (n = 16), and HFD-Ch; 44.1 ± 1.7 g (n = 8)).At the beginning of week 12, prior to isoleucine supplementation, HFD-mice gained significantly more weight than SLD-mice  = 19.21, diet effect; In weeks 12–15, HFD-mice continued to gain more weight compared to SLD-mice  = 24.78, diet effect; SLD-C, 9.3 ± 0.2 cm, SLD-Ch, 9.9 ± 0.5 cm, HFD-C, 11.4 ± 0.3 cm, and HFD-Ch, 11.2 ± 0.3 cm). In addition, HFD-mice had heavier gonadal fat pads and brown fat pads than SLD-mice  = 11.13 and F  = 11.44, respectively, diet effect; At week 18, abdominal circumference was greater in HFD-mice than SLD-mice, but was not affected by chronic isoleucine treatment  = 18.34, diet effect; p < 0.0001, F  = 37.31, diet effect; HFD-mice had heavier livers than SLD-mice  = 4.157, diet effect; p < 0.05, F  = 5.715, diet effect; HFD-mice consumed more energy across 24 h  = 9.151, p < 0.05, F  = 6.499, p < 0.01, F  = 11.25, respectively, diet effect; p < 0.05, F  = 5.416, interaction; p < 0.05, F  = 6.468, interaction; p < 0.05, Sidak’s post hoc test), but not in HFD-mice.HFD-mice had a significantly lower energy expenditure  compared to SLD-mice across 24 h, during the light phase or dark phase  = 19.66, diet effect; p < 0.05, F  = 4.082, diet effect; p < 0.001, F  = 38.4, diet effect; HFD-mice had lower RQ values compared to SLD-mice across 24 h  = 31.28, diet effect; p < 0.001, F  = 40.02, diet effect; p < 0.001, F  = 40.16, diet effect; p < 0.0001, F  = 25.36, diet effect; HFD-mice had elevated plasma total triglycerides  = 19.34, diet effect; HFD-mice had higher fasting blood glucose levels than SLD-mice  = 34.44, diet effect; p < 0.05, one-way ANOVA; HFD-mice had a greater glucose AUC than SLD-mice (n = 8); SLD-A, 144.4 ± 15.3 min (n = 8) and SLD-Ch, 126.7 ± 5.1 min (n = 7)).There was no significant difference in the gastric half emptying time (t ½) between different isoleucine groups in SLD-mice ; SLD-C, r2 = 0.0197, SLD-A, r2 = 0.2602, and SLD-Ch, r2 = 0.004292).There was no correlation between gastric half emptying time and blood glucose AUC in SLD-mice  mice, significantly reduced blood glucose levels in response to an OGTT, albeit at a higher dose of 0.5 g/kg [In the current study, acute isoleucine orally administered at a dose of 0.3 g/kg, had no effect on postprandial blood glucose levels in SLD- or HFD-mice. Previously, acute administration of 0.3 g/kg isoleucine in lean  and diet0.5 g/kg . ConsideConsistent with a previous report , the chr13CO2 content in the breath following a gastric emptying breath test, but did not significantly affect the 13CO2 AUC, Cmax, or Tmax values (peak concentration and the time at which it occurred) compared to controls [Slowing gastric emptying allows for efficient digestion and absorption of nutrients, including glucose ,36,37. Icontrols .The acute and chronic isoleucine supplementation had no beneficial effect to limit HFD-induced body weight gain, adiposity, fasting blood glucose levels, and glucose tolerance. In contrast, the chronic isoleucine treatment impaired glucose tolerance in SLD-mice. Therefore, the chronic isoleucine supplementation is unlikely to be an effective dietary intervention for the treatment of obesity and type 2 diabetes.


Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element.Campylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic  Campylobacter is the most commonly reported foodborne bacterial pathogen causing human gastroenteritis in the European Union (EU) and Ireland, most often associated with the broiler reservoir [Campylobacter spp. of food animal origin continues to limit the spectrum of clinically useful antimicrobials and is internationally recognised as a major societal challenge. Veterinary antimicrobials used therapeutically and prophylactically are often the same as, or belong to the same class as those used clinically [eservoir . Irelandeservoir . Frequeninically .Campylobacter spp. isolates, while resistance to erythromycin is typically low to moderate across Europe [Macrolides, fluoroquinolones and aminoglycosides are classified as critically important antimicrobials, while tetracycline is considered a highly important antimicrobial . Resistas Europe ,5,6. Macs Europe ,8,9. Syss Europe  and resigyrA and gyrB, respectively), catalysing ATP-dependent negative supercoiling of DNA to regulate replication, repair and gene expression [Campylobacter spp. is largely mediated by chromosomal mutations in the quinolone resistance-determining region (QRDR) of gyrA, typically conferred by the C257T nucleotide mutation (Thr-86-Ile) [In Gram negative bacteria, DNA gyrase is the primary target of fluoroquinolones . DNA gyrpression ,13,14. R-86-Ile) . The QRD-86-Ile) . rplD and rplV, respectively) or the presence of the emerging ermB gene contribute to macrolide resistance [ermB was reported recently for the first time in thermophilic Campylobacter spp., located on a chromosomal multidrug resistance genomic island (MDRGI) (likely originating from a Gram positive species) in a high-level erythromycin-resistant Campylobacter coli (C. coli) isolate (ZTC113) of swine origin in China [Macrolides act by binding to the 50S bacterial ribosomal subunit and inhibit translational elongation, and interfere with protein synthesis and subsequent ribosomal subunit assembly ,17. Polysistance ,18. Βetasistance ,20. Mutasistance . The ribin China . ErmB diin China . Campylobacter spp. contributes to baseline resistance against structurally diverse antimicrobials [cmeABC operon encodes a tripartite multidrug efflux pump that consists of an outer membrane channel protein (cmeC), an inner membrane efflux transporter (cmeB) and a periplasmic fusion protein (cmeA) [cmeR) binding to an inverted repeat (IR) (TGTAATAAATATTACA) in the intergenic region between cmeR and cmeA transcriptionally represses the cmeABC operon [The intrinsic, chromosomally encoded resistance-nodulation-division (RND) CmeABC (Campylobacter multidrug efflux) efflux pump in crobials ,24,25,26n (cmeA) . RepressC operon ,29. ConsC operon ,30.Campylobacter spp. is largely conferred by a ribosomal protection protein (RPP), TetO, capable of displacing tetracycline from its primary binding site on the 30S ribosomal subunit [Campylobacter tetO can be located chromosomally but is often plasmid-mediated [tetO gene exists in at least eleven bacterial genera, including four Gram negative and seven Gram positive genera [tetO between members of different bacterial genera indicates that conjugative plasmids, transposons, or recombination events contribute to the dissemination and maintenance of the tetO gene [Tetracycline resistance in  subunit ,31. Bact subunit ,33,34. Cmediated ,37,38,39tetO acquisition is the most prevalent genetic event conferring tetracycline resistance among Campylobacter spp., mosaic tet genes  have also been reported within the genus [tetO, W, 32 [tetM, S [tet genes are widespread among Gram positive and Gram negative genera in human and animal isolates [Although he genus . Mosaic O, W, 32 ,40,41,42[tetM, S ) to form[tetM, S . Mosaic Campylobacter spp. resistance to aminoglycosides is mediated by reduced antimicrobial binding affinity for target sites due to enzymatic modification, via acetylation, phosphorylation or adenylation of amino or hydroxyl groups of the aminocyclitol nucleus or sugar moieties [ant(6)-I-sat4-aphA3), first isolated from Staphylococci [Campylobacter spp. isolates [Aminoglycosides are broad spectrum antimicrobials and inhibit protein synthesis by binding to 16S rRNA of the 30S ribosome ,45. Campmoieties ,45,46. Amoieties ,48,49, wmoieties ,51. The ylococci ,53. ANT and C. coli isolates recovered from Irish broiler neck skin and caecal samples over a one-year period (2017–2018).Despite the high prevalence of  broiler ,57,58,59 broiler ,59,60,61 broiler  or rumin broiler ,63 isolagyrA gene and 100% of isolates (n = 99) harboured a C257T point mutation, which is the dominant mutation conferring resistance among campylobacters. Resistant isolates were grouped into C. jejuni gyrA and C. coli gyrA  sequence types based on the presence of synonymous and nonsynonymous mutations present in the portion of the gyrA gene sequenced (C. jejuni isolates (n = 85) were grouped into three GTJs . A large proportion (47.1%) carried the Thr-86-Ile substitution exclusively (GTJ1). Synonymous mutations T72C, C243T, T357C, C360T, C471T, T483C, and C622T were exclusively associated with C. jejuni and were present in both GTJ-II and GTJ-III. Nonsynonymous Ser-22-Gly (A64G) and Ala-206-Thr (G616A) mutations were present in 35.3% and 17.7% of isolates of ciprofloxacin-resistant C. jejuni isolates, respectively and were the basis of defining GTJ-II and GTJ-III, respectively. Both GTJ-II and GTJ-III were associated with the Asn-203-Ser (A608G) substitution. All CIP-resistant C. coli isolates tested (n = 14) harboured one nonsynonymous mutation only (Thr-86-Ile), but were grouped into seven GTCs based on the presence of various synonymous mutations quinolone-resistant isolates tested and minimum inhibitory concentrations (MICs) ranged from 0.5–8 mg/L.rplD gene was detected in the five isolates, and the partial sequences shared 100% homology with the rplD gene of erythromycin-sensitive C. coli isolates (GenBank accession numbers: MH084640.1 and MH084639.1) [rplV sequence in all erythromycin-resistant isolates: double point mutation at positions 308 and 309 , A325G (Thr-109-Ala), a double point mutation at positions 332 and 333 (Ala-111-Glu), G340A (Ala-114-Thr) and C358A (Pro-120-Thr). Nonsynonymous mutations  in rplV were also identified in all erythromycin-resistant isolates. Partial rplV sequences in this study were homologous to a high-level erythromycin-resistant clinical C. coli isolate rplV gene (GenBank accession number: GU384982.1) [cmeR-cmeA intergenic region, upstream of the IR (positions 66-80), homologous to sequences derived from erythromycin-resistant (GenBank accession number: FJ797673.1) and erythromycin-sensitive (GenBank accession number: FJ797671.1) strains [ermB gene was not detected among the erythromycin-resistant isolates tested.Five erythromycin-resistant isolates were screened for mutations contributing to erythromycin resistance. All five erythromycin-resistant isolates harboured the A2075G mutation in the 23S rRNA gene. A T82C mutation (Ser-28-Pro substitution) in the  strains . The ermtetO gene was detected in 100% of tetracycline-resistant isolates (n = 119). Three isolates , accounting for 2.5% of the tetracycline-resistant isolates, harboured the mosaic tetO/32/O type II gene, confirmed by PCR/partial sequencing and genomic sequencing (Streptococcus suis (S. suis) (GenBank accession number: KY994102.1) tetO/32/O gene (Campylobacter spp. (GenBank accession numbers: WP_052855148.1) and Clostridiales (WP_117823345.1) TetO/32/O sequences. A portion of the quencing B. Isolat2/O gene . EquallyC. jejuni isolates CITCj625-18 and CITCj727-18 were found to harbour multiple resistance genes, including a truncated tetO , mosaic tetO/32/O type II (1920 bp), aminoglycoside-6-nucleotidyltransferase (ant(6)-Ib)), and streptothricin acetyltransferase (satA). These antimicrobial resistance genes were located circumjacent to proteins involved in replication and recombination (C. jejuni isolates CITCj625-18 (first thin)  and CITCj727-18   were nearly identical with an average nucleotide identity (ANI) of 99.99% and 30.5% GC content, indicating that the isolates had been circulating within and had been maintained by the flock. The pair belonged to sequence type ST-45 clonal complex (ST-137) and harboured LOS locus class C.bination A. The MDant(6)-Ie gene (900 bp), with almost identical sequences (99.89% identity). ANT(6)-Ie in this study shared 99.66% amino acid identity with each other, where CITCc3448-18 harboured a nonsynonymous G820A mutation  and was identical to a C. coli ANT(6)-Ie protein (GenBank: WP_052786298.1). CITCc1631-18 and CITCc3448-18 ANT(6)-Ie shared between 29.7-36.1% identity to ANT(6)-Ia, ANT(6)-Ib, ANT(6)-Ic, and ANT(6)-Id amino acid sequences  [CITCc1631-18 and CITCc3448-18 belonged to ST-828 clonal complex  and harboured the ctively) ,51,65. N-acetyltransferase (AAC(3)) (261 amino acids) was also detected in both CITCj625-18 and CITCj727-18, 44.3% identical to a aminoglycoside 3-N-acetyltransferase (AAC(3)) variant (239 amino acids) in both CITCc1631-18 and CITCc3448-18. However, these isolates were gentamicin-susceptible, although this protein may confer resistance to other aminoglycoside antibiotics [C. coli variant was identical to AAC(3) genes reported in Campylobacter spp., 37.5% identical to AAC(3)-Ia described in Serratia marcescens (GenBank accession number: Q7B9H0), and 19–32.25% similar to AAC(3) orthologous, including types AAC(3)-Ib/Ic/Id/Ie/IIa/IIb/IIc/IId/IIe/IIf/IIIa/IIIb/IIIc/IVa/VIa/VIIIa/Ixa/Xa/XIa. The AAC(3) variant in CITCj625-18 and CITCj727-18 was identical to Campylobacter AAC(3) variants and was 19.3–25.84% similar to orthologues reported in other bacterial genera.Aminoglycoside 3-ibiotics . The C. Campylobacter spp. isolates  have been detailed previously [The antimicrobial resistance rates of this pool of broiler-associated thermophilic eviously , and areCampylobacter isolates, collected throughout the Republic of Ireland, from the three largest poultry processors, over a one-year period (2017–2018). This study reports the antimicrobial resistance determinants circulating among Irish broiler-associated Campylobacter spp. [n = 99) tested in the current study, similar to reports published worldwide [Campylobacter spp. isolates [The Thr-86-Ile mutation is the predominant genetic alteration conferring (fluoro)quinolone resistance among ter spp. , and wasorldwide ,70,71. Sisolates ,62,72, iCampylobacter spp. are ecologically competitive and persistent even in the absence of antimicrobial selective pressure [Campylobacter isolates (3.1% to 28.9%) in Ireland between 1998 and 2000 [Fluoroquinolone-resistant pressure ,74,75. Dand 2000 ,76, in 2and 2000 . Clonal and 2000 ,78,79.C. jejuni isolates harbouring multiple amino acid substitutions in the gyrA QRDR (GTJ-II and GTJ-III)  were necessary to produce high level moxifloxacin resistance [gyrA [gyrA QRDR have a negligible effect on (fluoro)quinolone resistance.GTJ-III)  had ciprsistance ,85. MoxigyrA alleles within a population of Campylobacter isolates have been identified as epidemiological markers and may serve as a supplementary approach to classical epidemiological typing methods [C. coli gyrA type in 0.23% (n = 1) of 430 C. jejuni isolates tested, and 1.4% (n = 4) C. coli isolates harboured a typical C. jejuni gyrA type. The amino acid substitutions present in each of the three GTJ , while MICs ranged from 128 mg/L to ≥ 128 mg/L. Three of these isolates were collected from the same flock in north-central Ireland while one isolate was collected from a farm approximately 10 km away, the following week. The fifth erythromycin-resistant isolate was recovered from a farm in the mid-south-west of Ireland, two months previously, but all birds from these farms were processed in the same processing plant. Identical mutations in the 23S rRNA, rplV gene, encoding the 50S ribosomal protein L22 were detected in the C-terminal region (amino acids 109–142) [cmeR regulatory IR. The effects of mutations detected in this study in rplD, rplV, and the intergenic region of cmeR-cmeA are unknown, but may contribute to erythromycin resistance.The A2075G point mutation in the 23S rRNA gene remains the most prevalent genetic event conferring macrolide resistance ,92 and w109–142) , includi109–142) . Mutatio109–142) ,93) are ermB gene was not detected among the erythromycin-resistant isolates tested. Resistance mediated by ermB in Campylobacter spp. is largely confined to China, which may reflect the extensive use of antimicrobials in food producing animals in China [ermB-positive C. coli isolates recovered from poultry exist in Europe [ermB-positive isolate was detected for the first time in the United States in a C. jejuni isolate of clinical origin [ermB gene was recently detected in 18.3% of 240 thermophilic Campylobacter spp. retail meat associated-isolates in South Africa [C. jejuni 23S rRNA has been associated with a fitness cost and reduced doubling times [The in China . Three rn Europe ,95,96 anl origin , while th Africa . Mutationg times ,100,101,tetO gene, while three isolates harboured the mosaic tetO/32/O type II gene were detected. Mosaic tetracycline resistance genes in Campylobacter spp. are typically derived from tetO and tet32 sequences in the type II conformation, with a shorter central tet32 segment [Campylobacter spp. The first mosaic tetracycline RPP gene was detected in Megasphera elsdenii, composed of a central tetW region flanked by two tetO regions [tetO primers [tetO gene and enable the detection of mosaic tet genes with a central portion flanked by an initial tetO region until position 228. The tetO/32/O type II gene reported here was associated exclusively with an MIC of 64 µg/L. However, 27 of 119 (22.7%) tetracycline-resistant Campylobacter spp. isolates tested had MICs of 64 µg/L or ≥ 64 µg/L, indicating that other factors contribute to enhanced tetracycline resistance. It should be noted that in this study, all three isolates harbouring mosaic tetracycline genes were also co-resistant to streptomycin, enabling co-selection and persistence of these antimicrobials. The burden of mosaic tetracycline resistance genes within the genus should be considered as part of the approach to elucidate developing and newly acquired antimicrobial resistance determinants within the genus. All tetracycline-resistant isolates harboured a portion of the  segment , althoug regions . However regions ,43. The  primers  used in n = 4) isolates had MICs of ≥ 16 mg/L. Streptomycin-resistant C. jejuni isolates CITCj625-18 and CITCj727-18 (ST-137) harboured sialylated LOS locus class C, which has been identified as a risk factor for post-infectious Guillain-Barré syndrome and increased severity of enteric disease [C. jejuni ST-45 clonal complex [All streptomycin resistant  revealed identical genes in a multidrug resistance genomic island  and Campylobacter spp. (GenBank accession number: WP_002823161.1). The presence of multiple tet genes (coding for similar or different mechanisms) in Gram negative isolates has also been documented [tetO genes have also been reported in C. coli MDGRI containing aadE (ant(6)-Ib) and ermB [ant(6)-Ib (867 bp)) and streptothricin resistance (satA) genes were also located within the multidrug resistance island (satA) is frequently reported in Gram positive bacilli [sat4) reported in Campylobacter [Genomic sequencing of tetracycline-/streptomycin-resistant c island A. Both icumented . Howeverand ermB . Aminogle island A. Strept bacilli  and sharlobacter ,111,113.C. coli of swine origin in Spain, America, and Grenada [C. coli ST-6543 on the PubMLST Campylobacter database at the time of writing [CITCc3448-18 belonged to ST-828 clonal complex (ST-1096). ST-1096 has been isolated from a case of gastroenteritis in the United Kingdom (UK) in 2016  and has  Grenada ,115,116. Grenada . There a writing , includiC. coli isolates (CITCc1631-18 and CITCc3448-18) harboured the emerging ant(6)-Ie gene (900 bp), found widely disseminated among clinical and animal C. coli isolates [Campylobacter ant(6) genes, ant(6)-Ie appears to be inherent to C. coli and does not have a Gram positive ancestor [ant(6)-Ie gene was originally detected in a hypervariable genomic region, unaccompanied by other resistance genes [ant(6)-Ie genes were located chromosomally, and were not located near other resistance determinants. Both streptomycin-resistant C. coli isolates were co-resistant to ciprofloxacin/nalidixic acid (GTC-V) and tetracycline. C. coli isolate 1631-18 harboured tetO (1920 bp), while C. coli isolate CITCc3448-18 also harboured the mosaic tetracycline resistance gene, tetO/32/O (1290 bp), highly homologous with that detected in CITCj625-18 and CITCj727-18. Both streptomycin-resistant isolates ,65. UnliCampylobacter isolates (296 C. jejuni and 54 C. coli) were recovered from free range and intensively-reared broiler carcasses  using ISO 10272-2:2017 [C. jejuni and 18 C. coli) isolates tested were resistant to at least one antimicrobial and were subsequently tested for resistance determinants.A total of 350 thermophilic 2-2:2017  for thei2-2:2017 ,119. Ove2, 10% CO2, 85% N2) and subcultured. DNA was extracted using the PureLink Genomic DNA Mini Kit , according to manufacturer’s instructions and DNA was standardised to 50–100 ng/μL.Briefly, isolates were recovered from −80 °C on Columbia blood agar  and incubated for 24 h at 42 °C, under microaerobic conditions (5% OtetO/32/O and tetO/W/O) were designed on SnapGene2.3.2 software  and regions of primer complementarity were assessed on Primer-BLAST [Primer sets, target genes and annealing temperatures are listed in er-BLAST .PCRs were performed with 2.5 U of Amplitaq polymerase , 1 × PCR buffer I and 2.5 mM magnesium chloride (Applied Biosystems), 0.2 mM of each deoxyribonucleotide , 0.2 pmol/μL of each primer, and 1 µL (1–2 ng/μL) of DNA. Reaction conditions were denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing  for 30 sn = 99) were screened for mutations in the QRDR of the gyrA gene [gyrA of the reference C. jejuni (GenBank accession number: L04566.1 and AL111168.1) and C. coli sequences (GenBank accession number: AF092101.1 and NZ_UIGM01000003.1) on SnapGene 2.3.2. Ciprofloxacin-resistant isolates  and the regulatory site of the cmeABC operon in five erythromycin-resistant C. coli isolates are listed in C. coli type strain NCTC 11366 (ATCC 33559) 23S rRNA (GenBank accession number: GQ167698.1), rplD (GenBank accession number: DQ639752.1 and UIGM01000003.1), rplV (GenBank accession number: UIGM01000003.1) and cmeABC operon (GenBank accession number: FJ797670.1) sequences was performed on SnapGene 2.3.2. Isolates were also screened for the presence of ermB, according to Wang et al. (2014). The primers used for the amplification and sequencing of domain V of 23S rRNA, n = 119) were screened for the presence of tetO, according to Aminov et al. (2001) and products were visualised on 2% agarose gel electrophoresis. The tetO amplicon of a tetracycline-resistant C. jejuni isolate (CITCj382-18) from this study was purified and sequenced (as described above) as a positive control. Consensus sequences were aligned to the C. jejuni tetO gene (GenBank accession number: M18896.2).Tetracycline-resistant isolates (tetO/W/O (tetO (GenBank accession number: M18896.2) and mosaic tetO/WO genes (GenBank accession numbers: EF065524.1 and AY196921.1). Two tetO/32/O-targeting primers (tetO (GenBank accession number: M18896.2) and tetO/32/O type I genes with a longer central tet32 region (GenBank accession numbers: AJ295238.3 and JQ740052.1) and tetO/32/O type II genes with a shorter central tet32 segment  [Primers were designed to target tetO/W/O  based on primers  were des00033.1) .n = 4) were screened for sialylated LOS locus class A/B and C (C. jejuni 81-176 (ATCC BAA2151) and C. jejuni NCTC 11168 (DSM 27585), respectively, as positive controls. Streptomycin-resistant isolates  were tested for moxifloxacin susceptibility. Briefly, isolates were recovered from −80 °C on CBA (Fannin Ltd) for 24 h at 42 °C under microaerobic conditions, and subcultured to CBA for 20 ± 2 h at 42 °C under microaerobic conditions. In microtiter plates, 100 µL serial dilutions of moxifloxacin (Sigma Aldrich) were prepared in of Mueller Hinton broth with lysed horse blood  ranging from 0.125–16 mg/L. A 0.5 McFarland inoculum was prepared in 5 mL demineralised water (Thermo Fisher Scientific) and 100 µL was transferred to 11 mL of Mueller Hinton broth with lysed horse blood (Thermo Fisher Scientific). Moxifloxacin serial dilutions were inoculated with 100 µL of cell suspension and incubated for 20 ± 2 hat 42 °C under microaerobic conditions.All (fluoro)quinolone-resistant isolates (C. jejuni isolates CITCj625-18 and CITCj727-18 and C. coli isolates CITCc1631-18 and CITCc3448-18) were sequenced. DNA was quantified in triplicate with the Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific). Genomic DNA libraries were prepared using the Nextera-XT protocol , with changes including 2 ng of input DNA and a minute PCR elongation time. DNA quantification and library preparation were performed on a Hamilton Microlab STAR system. Pooled libraries were quantified using the Kapa Biosystems library quantification kit on a Roche light cycler 96 qPCR machine. Libraries were sequenced on the Illumina HiSeq using a 250 bp paired-end protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cut-off of Q15 [The genomes of four streptomycin-resistant isolates (f of Q15 . De novof of Q15  and assef of Q15 .Genomes were annotated using Prokka 1.14.3 . SimilargyrA GT was submitted for each type. This whole-genome project has been deposited at DDBJ/ENA/GenBank under the accession number PRJNA612628. CITCj625-18, CITCj727-18, CITCc1631-18, and CITCc3448-18 genomes can be accessed using SAMN14379027, SAMN14379028, SAMN14379029, and SAMN14379030. The partial and complete gene sequences deposited in GenBank are listed in Campylobacter spp. hosts. The broiler industry serves as a reservoir for the dissemination of resistant campylobacters. The enrichment and stability of Campylobacter spp. resistance determinants is noteworthy but the natural competence and potential of recombination or acquisition of mobile genetic elements contributes to the Campylobacter. Taken together, the data collected in this study point to the diversity of resistance determinants circulating among Irish broilers, contributing to the development of resistance to clinically relevant antimicrobials. Although non-poultry sources contribute to campylobacteriosis incidence, poultry are natural thermophilic


Splenocyte proliferation was tested using thymidine incorporation. Th1 and Th17 cells were analyzed by flow cytometry. Results: Oral KLHTT treatment (50 and 100 mg/kg) ameliorated mouse CIA by decreasing the levels of interleukin (IL)-1β, IL-6, IL-17A, and tumour necrosis factor-α in the paw homogenates and serum. KLHTT also suppressed anti-CII antibody formation, splenocyte proliferation, and splenic Th1 and Th17 cell numbers. Additionally, KLHTT showed antioxidant activity by reducing the concentrations of MDA and H2O2 in paw tissues. Conclusions: The therapeutic effects of KLHTT in CIA mice were through regulating oxidative stress and inflammatory responses. Our results suggest that KLHTT has potential to treat RA.Background: Kan-Lu-Hsiao-Tu-Tan (KLHTT) exhibits anti-psoriatic effects through anti-inflammatory activity in mice. However, the therapeutic effects of KLHTT on rheumatoid arthritis (RA), another significant autoimmune inflammatory disorder, have not been elucidated. Herein, we explored the anti-arthritic effects of KLHTT on collagen-induced arthritis (CIA) in mice. Methods: KLHTT was extracted by boiling water and subjected to spectroscopic analysis. Chicken collagen type II (CII) with complete Freund’s adjuvant was intradermally injected to induce CIA in DBA/1J mice. Anti-CII antibody, cytokines, malondialdehyde (MDA), and hydrogen peroxide (H Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1% of the global population, which is characterised by synovitis, cartilage damage, and bone resorption in the joint , cervicaRA is characterised by synovitis accompanied by the infiltration of immune cells , includiKan-Lu-Hsiao-Tu-Tan (KLHTT), a Chinese medicine (CM), has been used to treat inflammatory conditions such as RA, systemic lupus erythematosus, dermatomyositis , sinusit3H labelled thymidine was purchased from Amersham Pharmacia Biotech, Arlington Heights, IL, USA. Brefeldin A and Freund’s adjuvant were bought from Sigma-Aldrich, St. Louis, MO, USA. Phycoerythrin (PE)-conjugated anti-mouse CD4 (clone GK1.5), FITC-conjugated anti-mouse IL-17A (clone TC11-18H10.1), and FITC-conjugated anti-mouse interferon (IFN)-γ (clone XMG1.2) antibodies were ordered from Biolegend, San Diego, CA, USA. Formalin was bought from AVANTOR, Center Valley, PA. Bovine serum albumin (BSA) was purchased from EMD Millipore, Billerica, MA, USA. Tween 20 was obtained from EMD Millipore, Alsace, France.KLHTT (batch number: 0503-2-403-01) was supplied by Sun Ten Pharmaceutical Corporation, New Taipei City, Taiwan. Chicken collagen type II (CII) was purchased from Chondrex, Inc., Woodinville, WA, USA. Mycobacterium tuberculosis H37RA was bought from Difco Laboratories Inc., Detroit, MI, USA. Methotrexate (MTX) and EDTA were ordered from Bio Basic Inc., Toronto, ON, Canada. Enzyme-linked immunosorbent assay (ELISA) kits  were obtained from eBioscience, San Diego, CA, USA. An IL-17A ELISA kit was purchased from R&D Systems Inc., Minneapolis, MN, USA. A hydrogen peroxide assay kit was purchased from Cell Biolabs, Inc., San Diego, CA, USA. Goat anti-mouse IgG1 and goat anti-mouse IgG2a secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, PA, USA. 2,2’-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) substrate solution was ordered form Roche Diagnostic Systems, South San Francisco, CA, USA. A Artemisia capillaris Thunb. (seedling), 4.17 g Scutellaria baicalensis Georgi (root), 2.50 g Acorus gramineus Soland. (rhizome), 2.08 g Clematis armandii Franch. (rattan and stem), 2.08 g Fritillaria cirrhosa D. Don (bulb), 1.67 g Pogostemon cablin (Blanco) Benth. (plant shoot), 1.67 g Forsythia suspensa (Thunb.) Vahl (fruits), 1.67 g Amomum kravanh Pierre ex Gagnep. (fruits), 1.67 g Mentha haplocalyx Briq. (stem and leaf plot), and 1.67 g Belamcanda chinensis (L.) DC (rhizome)) was extracted by boiling water (12 times the weight of the herbs) for 1 h, and then concentrated to a voucher specimen (CGU_KLHTT-01) by the freeze dryer  [The herbs of KLHTT were purchased and identified by Sun Ten Pharmaceutical Corporation, New Taipei City, Taiwan. A total of 27.93 g of herbs  comprising an LC-30AD pump, SIL-30AC auto-sampler, CTO-20AC column, and SPD-M20A photodiode array detector . Prior to being loaded onto the UPLC column, 1 mg of KLHTT extract was first dissolved in 1 mL of methanol and filtered through a 0.45 μm membrane. Sample injections of 1 μL were then performed automatically. Liquid chromatography was performed using a CORTECS UPLC C18 column  . The mobile phase was a mixture of MeCN (A) and water . A gradient sequence was executed as follows: 0–10 min, 10–20% A; 10–14 min, 20–25% A; 14–24 min, 25–30% A; 24–28 min, 30–40% A; 28–33 min, 40–50% A; 33–38 min, 50–75% A; 38–40 min, 75–100% A; and 40–43 min, 100% A. The column temperature was set at 35 °C. The flow rate was at 0.4 mL/min. The range of detection wavelengths was fixed in the 190–500 nm.Multiple reaction monitoring (MRM) experiments (in negative) were carried out using Shimazu LCMS-8045 triple quadrupole mass spectrometry to identify the constituents of KLHTT extract. The precursor ion settings of the corresponding profiling peaks were determined using the full scan experiment (50–1000 amu). The product ions were settled according to previously reported data. The dwell time was fixed at 100 ms and the collision energy was set at 25–45 eV. All MS data were acquired and processed using LCMS LabSolutions software Version 5.93 .DBA/1J mice  were purchased from Jackson Laboratories  and maintained at 20–25 °C with half day light/dark cycle under a specific pathogen-free condition. All mice were treated according to the guidelines of the Institutional Animal Care and Use Committee of National Chung Hsing University (NCHU). The study protocol was approved by NCHU ethics committee .2O, KLHTT, or MTX was administered orally once a day from day 21 to 42. Mice were divided into four groups (n = 6/group) randomly as follows: Group I, Normal; Group II, Vehicle (ddH2O) + CII; Group III, KLHTT (50 mg/kg) + CII; Group IV, KLHTT (100 mg/kg) + CII. MTX (0.5 mg/kg) was used as a positive control. Mice were euthanized with CO2 exposure (100% CO2 for 5 min) by experienced experimenters humanely on day 42. The arthritis severity score was recorded every 3 days after the treatment of drugs. The biochemical and histological assays were performed on day 42.CIA was induced by active immunisation with chicken CII . BrieflyThe body weight and arthritis severity score were obtained . The artAfter the mice were humanely sacrificed, the hind limbs were fixed in 10% buffered formalin, decalcified in 15% EDTA, and embedded in paraffin. Serial paraffin sections (5 μm) were stained with haematoxylin and eosin. The severity of histopathological lesions was scored  as folloHind paw was dissected and homogenised in ice-cold saline using a tissue homogeniser. After being centrifuged at 3000 rpm , the hind paw homogenates were harvested. Blood was collected from the heart. The levels of cytokines in hind paw homogenates and serum were measured by ELISA .2O2) concentration was measured using a colorimetric OxiSelect™ hydrogen peroxide assay kit at 560 nm [Malondialdehyde (MDA) concentration was determined by thiobarbituric acid reactive substances assay at 532 nm. The standard curve was obtained using 1,1,3,3-tetramethoxypropane. Hydrogen peroxide  at 4 °C overnight. The plates were washed and incubated with goat anti-mouse secondary antibodies IgG1 (1:500 dilution) or IgG2a (1:500 dilution) at 25–27 °C for 1 h. After being washed, ABTS substrate was added and the reactions were stopped by adding H2SO4. The level of IgG1 and IgG2a was measured at 450 nm by an ELISA reader  [Serum samples were diluted 1:250 for IgG1 or 1:125 for IgG2a in Tris-buffered saline , and then transferred to CII (10 μg/mL) pre-coated 96-well plates (Microtiterzerland) .5 cells/well) were cultured with chicken CII (50 μg/mL) at 37 °C for 40 h, and then incubated with 3H for 8 h. Cell proliferation was evaluated by radioactive thymidine incorporation [Splenocytes (4 × 10poration .6 cells/well) were cultured with chicken CII (50 μg/mL) at 37 °C for 48 h, and then brefeldin A (5 μg/mL) was added for 6 h. Cells were harvested and extracellularly stained with PE-conjugated anti-mouse CD4 antibodies. After being fixed and permeabilised with Cytofix/Cytoperm solution (BD Pharmingen), cells were then intracellularly labelled with FITC-conjugated anti-mouse IL-17A and anti-mouse IFN-γ antibodies. Splenocytes were detected by an Accuri C5 flow cytometer  and analysed by BD Accuri™ C6 Plus software [Splenocytes , chrysin 6-C-glucoside-8-C-arabinoside (2), baicalin (3), norwogonin-7-O-β-d-glucuronide (4), chrysin 7-O-β-d-glucuronide (5), oroxylin A 7-O-β-d-glucuronide (6), wogonoside (7), and baicalein (8)  B (Table lein (8) .The CIA mouse model is a well-established and commonly used model mimicking the clinical symptoms and immunopathogenesis of human RA . ImmunisHistopathological analysis of the CIA mice revealed inflammatory cell infiltration into articular tissues, exudates within the synovial space, synovial hyperplasia, and cartilage erosion. KLHTT-treated mice demonstrated well-preserved joint spaces with minimal inflammatory exudates, normal cartilage structure, and clear synovial spaces, along with improved histological arthritis severity scores . MTX 0..The pathogenesis of RA involves activated T cells promoting macrophages to release pro-inflammatory cytokines . Therefo2O2 were noted in the hind paw homogenates of CIA mice. KLHTT (50 and 100 mg/kg) significantly reduced the levels of MDA  and MDA (a lipid peroxidation marker) in joint homogenates from CIA mice. MDA-related reactions are highly immunogenic. MDA levels correlate with RA severity and can be used to predict RA severity [Oxidative stress is involved in the pathogenesis of RA ,43. In tseverity . The newseverity . RA patiseverity ,47. Inflseverity . Adalimuseverity . In thisC-arabinoside-8-C-glucoside, chrysin 6-C-glucoside-8-C-arabinoside, baicalin, norwogonin-7-O-β-d-glucuronide, chrysin 7-O-β-d-glucuronide, oroxylin A 7-O-β-d-glucuronide, wogonoside, and baicalein. Previous studies have reported that baicalin ameliorated CIA in mice by down-regulating Janus kinase 1/signal transducer and activator of transcription 3 signalling and inhibiting IL-17-mediated joint inflammation [We identified eight flavonoids from KLHTT, including chrysin 6-ammation ,51. Baicammation . Intrapeammation . Chrysinammation ,55. The ammation . ObservaIn summary, our results indicate that KLHTT, a CM formulation, shows significant anti-inflammatory effects, antioxidant activities, and immunomodulatory functions in CIA mice. The present study also demonstrates that KLHTT has potential to treat RA.