Misgurnus anguillicaudatus, and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach,  Before embryos begin to utilize newly synthesized products derived from the zygotic genome, maternally inherited components such as RNA transcripts and proteins direct virtually all aspects of early development in animal embryos ,2,3,4. MFish embryos and early larvae are often exposed to various potential pathogens in the environment. Accordingly, a need to protect themselves from pathogenic invaders is crucial during this early life interval. The immune system in vertebrates including teleost fishes utilizes complex and highly organized gene sets of molecular signaling pathways that protect against environmental invasions and regulate dynamic changes in an organism ,14. ThisMisgurnus anguillicaudatus , also known as oriental weather loach, is widely distributed in freshwater areas. M. anguillicaudatus prefers a habitat with muddy bottoms as a spawning ground, which is known to contain abundant and diverse microbial populations. The loach has been considered a valuable source of food owing to its high nutritional values in East Asia including Korea and, has become an important freshwater aquaculture species (https://www.fishbase.de/summary/Misgurnus-anguillicaudatus.html). Moreover, this species has many advantageous merits as an experimental model organism, particularly with respect to developmental and reproductive researches. They include short generation time, year-round multiple spawning under controlled laboratory conditions, high fecundity, and fast and transparent embryonic development [The cyprinid loach, elopment ,27,28,29M. anguillicaudatus transcriptome newly constructed from RNA-Seq libraries derived from unfertilized eggs, ovary, testis, and muscle tissues. We employed a paired-end Illumina HiSeq 2500 sequencing technology and the subsequent de novo assembly, to generate a comprehensive set of reference transcripts. The transcriptome assembly was used to analyze expression profiles and identify maternal gene transcripts related to immune functions. Further, we analyzed transcriptional levels of identified maternal immune-related candidate genes during the development of M. anguillicaudatus. This study is the first transcriptome analysis of maternal gene transcripts associated with immune function in the loach, M. anguillicaudatus.Here, we report the The RNA-Seq libraries from muscle, testis, ovary, and unfertilized eggs were constructed to carry out paired-end sequencing using the Illumina HiSeq 2500 platform. The statistics for the sequencing and de novo assembly are shown in M. anguillicaudatus [M. anguillicaudatus achieved a reasonable degree of completion and an acceptable coverage and quality of protein-coding transcripts for subsequent analysis.Thus, the N50 value (1533 bp) of our transcriptome assembly was comparable to those obtained for published de novo transcriptomes of caudatus ,35,36,37caudatus ,39, whicSinocyclocheilus, which include S. rhinocerous , S. anshuiensis , and S. grahami . Then, these species were followed by Danio rerio , and Cyprinus carpio . In contrast, a previous study reported that transcripts of M. anguillicaudatus skin transcriptome showed relatively high homology to D. rerio [D. rerio transcriptome database) [To find homologous proteins and the collection of biological information from various sources, all of 267,111 transcripts were used to search homology against several public databases based on the BLAST algorithm. Overall, 161,091 (60.3%) transcripts were successfully annotated to a least one database and 1394 (0.5%) transcripts shared annotation in all databases used a. Of theD. rerio . This maatabase) .M. anguillicaudatus are somewhat limited. Nevertheless, the representation of GO distribution was consistent between our transcriptome and previous transcriptome assemblies for all three main categories [The GO annotation is a standardized system for functional classification and inference of the biological significance of genomic/transcriptomic datasets . Therefotegories ,36,37,43M. anguillicaudatus [M. anguillicaudatus transcriptome.The KEGG pathway provides an important insight into different biological processes and interactions across different tissues or conditions within an animal, enabling further understanding of the biological function and interaction of the transcripts in caudatus ,45. ConsM. anguillicaudatus unfertilized eggs, we evaluated the expression levels and classified the transcripts from each RNA-seq library. Overall, more than 89% of transcript sequences for each library were mapped to the reference transcriptome assembly, of which 95% of transcript sequences in unfertilized eggs were mapped  transcripts were coexpressed in more than two tissues, of which 20,868 (86.5%) transcripts were coexpressed between unfertilized eggs and other tissues  and the KEGG pathway of immune system (5.1), which was closely associated with immune functions, processes, and interactions and often used to identify genes/transcripts associated with the immune functions. A total of 61 maternal transcripts were classified into 16 different GO terms (level 3) in the GO immune system process and 218 maternal transcripts with 171 KOs were assigned into 19 signaling pathways in the KEGG immune system  were further studied with qPCR analysis. They included genes, analysis , indicatM. anguillicaudatus  [RAC1 mRNA clearly showed the involvement in the host immune response by microbial infection in fish [CTNB1 is well-known as a maternal gene, which mediates the canonical Wnt signaling pathway and plays a crucial role in the generation of the body plan in zebrafish embryo [RAC1, CTNB1, and ITB1 seem to be “typical” for maternal transcripts that could be degraded during midblastula transition (MBT). These transcripts are responsible for defense against pathogen invasion and/or embryonic development of M. anguillicaudatus. MAPKs generally are known as a protein kinase group and are involved in directing a diverse array of cellular functions including proliferation, gene expression, differentiation, mitosis, and apoptosis. A knock-down experiment in zebrafish showed MAPK3 and MAPK14 mRNA expression was required at an early embryonic stage  in the gilthead sea bream (Sparus aurata) [MAPK3 and MAPK14 were upregulated by the immune challenge in several fish species [MAPK14 in M. anguillicaudatus declined around the gastrulation stage. Meanwhile, the expression pattern of MAPK3 was steady, so we were unable to determine maternal to zygotic transition, suggesting the expression of MAPK3, whether maternal or zygotic expression, is required above a certain transcriptional level for immune function and embryonic development (IL1B was evident in rohu (Labeo rohita) and brown trout  [IL1B in M. anguillicaudatus gradually declined until the end of gastrulation. Furthermore, there was no change during the myotome stages  during apoptosis [FADD knockout mouse model demonstrated that the absence of FADD is lethal [FADD during our study   and the  in fish ,61,62. Ch embryo ,64. Thus aurata) ,66. Zygo species ,67. Exprelopment c,d. Stud trutta) ,73. Expre stages e. This ipoptosis . The funs lethal . We did ur study g. Many mhttp://www.ncbi.nlm.nih.gov/sra) under the accession numbers SRX4452604, SRX4452605, SRX4452606, and SRX4452607 for ovary, testis, muscle, and unfertilized eggs, respectively. The Transcriptome Shotgun Assembly (TSA) project has been deposited in the DDBJ/EMBL/GenBank under the accession GGUH00000000. The version described in this paper is the first version, GGUH01000000.The cleaned short read sequences from the RNA-Seq data were deposited in the NCBI Sequence Read Archive . Fishes were fed with a commercial diet (50% crude protein) on an ad libitum basis. For tissue sampling, 2-year-old mature adult fishes were euthanized with tricaine methanesulfonate . Gonads (yolk-laden mature ovary or milky white-colored testis) were surgically dissected from 3 females (average body weight (BW) = 24.3 ± 3.5 g) and 3 males (average BW = 12.3 ± 1.4 g). In addition, muscles  were obtained from the same individuals above in order to increase transcriptome coverage of the gene expressed in fertilized eggs during de novo transcriptome assembly. Collected samples were immediately frozen on dry ice and stored at −80 °C until RNA extraction. Unfertilized eggs were obtained by hormone-induced spawning, as described previously [n = 3; same-aged and similar-weighed as above) were given an intraperitoneal injection of carp pituitary extract  at a dose level of 10 μg/g body weight. Thirteen hours after injection at 25 °C, eggs were hand stripped and directly subjected to RNA extraction. This study was approved by the Animal Ethics Committee of Pukyong National University  and performed according to the guidelines for the care and use of laboratory animals.The cyprinid loach, eviously . Mature Total RNAs were extracted from individual tissue and egg batch samples using the Trizol reagent  and purified using the RNeasy Plus Mini Kit  according to the manufacturer’s instruction, including a genomic DNA elimination step. RNA purity, quality, and quantity were determined by NanoDrop-2000 spectrophotometer , ethidium bromide staining of 28S and 18S ribosomal bands on a 1% MOPS-formaldehyde agarose gel, and Bioanalyzer 2100 system . Only high-quality RNAs  were pooled within a given sample type  to construct cDNA libraries. Briefly, 1 μg of total RNA was purified, fragmented, and primed to synthesize the first- and second-strand cDNA according to the TruSeq Stranded Total RNA Sample Pre protocol . Synthesized second-strand cDNA was purified, end-repaired, and ligated to index adapters. The ligated products were separated to select suitable sequencing templates that were amplified by PCR to generate cDNA libraries. Finally, the libraries were sequenced using the Illumina HiSeq 2500  platform as paired-end reads to 100 bp in length.M. anguillicaudatus in this study, the clean reads were pooled together to maximize the chance of obtaining as many transcripts as was possible, and de novo assembly used to create a reference transcriptome using Trinity Assembler v2.2.0 with default parameters [http://busco.ezlab.org/) against the eukaryote (eukaryote_odb9) lineages with default parameters in the CyVerse Discovery Environment (https://de.cyverse.org/) [After sequencing was completed, the Trimmomatic v0.32 software was used to process Illumina paired-end raw reads to obtain clean reads through trimming the adaptor sequences, plus trimming of sequences with an ambiguous base (N) ratio >5% and low-quality . Subsequrameters . The assse.org/) ,39,78.https://trinotate.github.io/) pipeline was used to search for any additional homologies of the transcripts: the open reading frames (ORF) for each transcript were predicted by TransDecoder v3.0.1 (https://github.com/TransDecoder) [p-value < 0.05 were defined as significantly enriched GO terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assignments were performed on the KEGG Automatic Annotation Server (KAAS) [https://www.kegg.jp/kegg/tool/map_pathway.html). Enrichment analysis was conducted by comparison of a transcript set against the reference transcriptome background using the Pearson Chi-Square test in R package. GO terms and KEGG pathways with a p-value less than 0.05 were considered to be enriched significantly.Initially, transcripts were annotated by homology search using BLASTx algorithm against NCBI NR protein database. In addition, Trinotate v3.0.1 (Decoder) . NucleotDecoder) . OrtholoDecoder) . FurtherDecoder)  and tmHMDecoder) . The NR Decoder) . After rDecoder) . GO termr (KAAS) . KEGG anExpression levels of transcripts from four tissue libraries  were calculated by mapping to the reference transcriptome that was created by the de novo assembly mentioned above. To obtain the expression levels of each transcript in each tissue, the transcript abundances were quantified for each library using Kallisto v0.43.0 . Low abu18S rRNA, accession no. EU120032) as an endogenous control [M. anguillicaudatus that has been previously described [-ΔΔCT method [p < 0.05 was considered statistically significant.Seven genes from the RNA-Seq analysis and 18s ribosomal RNA ( control  were anaescribed . Three iT method . The resM. anguillicaudatus, and for the first time, maternal gene transcripts and their immune system involvement were identified from unfertilized eggs of M. anguillicaudatus. These findings are needed to understand maternal gene transcripts in early embryonic development and the immunological understanding of maternal immune-related gene transcripts before zygotic gene expression. This information can be utilized to assess developmental competence concerning cellular mechanism and/or immune system in embryonic development, which can be associated with the egg quality of teleost fish. The data generated could be a valuable transcriptomic/genomic resource for further analysis of maternal genes involved in the cellular mechanisms and/or immune system in early embryonic development of M. anguillicaudatus and other teleost fishes.Our study presented the transcriptome expression profiles of the loach,


Background: Machine perfusion (MP) and static cold storage (CS) are two prevalent methods for liver allograft preservation. However, the preferred method remains controversial.Aim: To conduct a meta-analysis on the impact of MP preservation on liver transplant outcome.Methods: PubMed, EMBASE, and Cochrane Library databases were systematically searched to identify relevant trials comparing the efficacy of MP vs. CS. Odds ratios (OR) and fixed-effects models were calculated to compare the pooled data.Results: Ten prospective cohort studies and two randomized controlled trials (RCTs) were included (MP livers vs. CS livers = 315:489). Machine perfusion demonstrated superior outcomes in posttransplantation aspartate aminotransferase levels compared to CS (P < 0.05). The overall incidence of early allograft dysfunction (EAD) was significantly reduced with MP preservation than CS . The incidence of total biliary complications  and that of ischemic cholangiopathy  were significantly lower in recipients with MP preservation compared with CS preservation. Hypothermic machine perfusion (HMP) but not normothermic machine perfusion (NMP) was found to significantly protect grafts from total biliary complications and ischemic cholangiopathy (P < 0.05). However, no significant differences could be detected utilizing either HMP or NMP in primary nonfunction, hepatic artery thrombosis, postreperfusion syndrome, 1-year patient survival, or 1-year graft survival (P > 0.05).Conclusions: Machine perfusion is superior to CS on improving short-term outcomes for human liver transplantation, with a less clear effect in the longer term. Hypothermic machine perfusion but not NMP conducted significantly protective effects on EAD and biliary complications. Further RCTs are warranted to confirm MP's superiority and applications. Liver transplantation (LT) has been the optimal treatment for patients with end-stage liver disease. Rapidly increasing demands for LT have also caused severe shortage of donor liver organs in clinical application. To overcome this discrepancy, donation after circulatory death (DCD), extended criteria donors (ECDs), living liver donation, or marginal liver grafts are increasingly accepted to expand the limited donor pool. However, studies have suggested that these additional liver donation sources often suffered from longer warm ischemic periods and worse ischemia–reperfusion injury (IRI) during LT, resulting in increased risk of early allograft dysfunction (EAD), primary nonfunction (PNF), biliary complications, and poorer long-term graft and patient survival .During transplantation, the quality of donor liver preservation is an important factor on maintaining and improving transplant outcomes of patients. Standard static cold storage (CS) remains the gold standard method for liver graft preservation. Nevertheless, the effectiveness of CS preservation is currently unable to provide sufficient protection of liver grafts against IRI, especially for prolonged CS of DCD and marginal livers, often resulting in increased risk of EAD, PNF, and biliary complications . As an aMachine perfusion has been widely used for preservation in kidney transplantation. Plenty of evidence suggested MP's priority in improving early graft function, reducing the risk of delayed graft function (DGF), and enhancing ECD graft survival after renal transplantation . Unlike Large animal and human experimental studies have suggested that MP may provide better protection of liver grafts against IRI than CS , 6. ExprIn this systematic review and meta-analysis, we systematically analyzed the available published data to compare MP preservation with CS preservation on outcomes of LT, including EAD, PNF, biliary complications, and graft and patient survival after transplantation, with the aim of identifying the optimum preservation method for donor livers and achieving preferable outcomes in clinical application.A comprehensive literature search was carried out on PubMed/MEDLINE, EMBASE, and Cochrane library databases using the following terms: ((machine perfusion OR machine preservation) AND (liver OR liver transplant OR liver transplantation)). The literature search was performed in November 2019. Publications were limited to articles written in English. The titles and abstracts of all relevant publications were reviewed, and full texts of those potentially matching the inclusion criteria were retrieved. Reference lists of the retrieved full-text articles were also searched manually to help identify potentially eligible studies. This systematic review and meta-analysis was performed in line with the PRISMA  recommendations .Clinical studies comparing outcomes of human LT using MP preservation vs. static CS were eligible for this analysis.Exclusion criteria were as follows:  studies Newcastle–Ottawa Quality Assessment Scale (NOS) was used to evaluate the methodological quality of the included prospective cohort studies . The quaThe primary outcomes extracted included the incidence of EAD, PNF, hepatic artery thrombosis (HAT), total biliary complications, and ischemic cholangiopathy (IC) rates after transplantation. Secondary outcomes included liver graft function, postreperfusion syndrome (PRS), and 1-month, 6-month, and 1-year graft and patient survival rates.All of the studies defined EAD according to Olthoff et al. . To be s2) and the I2 statistical test were adopted to evaluate the heterogeneity among the included studies. Heterogeneity was considered significant when P < 0.10 or I2 > 50%. In the absence of heterogeneity among the included studies, a fixed-effects model was adopted to combine studies and pool the total effect size; otherwise, a random-effects model was adopted. Funnel plots were created for assessment of publication bias (publication bias was examined in funnel plots by performing Begg and Egger tests). P < 0.05 was considered statistically significant. All statistical analyses were performed using Cochrane Review Manager 5.3. (http://ims.cochrane.org/revman).Pooled odds ratios (ORs) were used to evaluate the summary event rates (dichotomous data) with 95% confidence interval (CI). Standardized mean difference (SMD) was used to compare continuous data. Cochran Q  (P = 0.04) (P = 0.06) (Reperfusion injury and liver graft function were determined by AST and/or ALT serum levels after transplantation. As shown in  < 0.05) , 26, 28, < 0.05) , 29, 30. < 0.05) , 28, 29. = 0.04) . There w = 0.06) .P < 0.05)  , 26, 30.P < 0.0001) .Eleven of the 12 included studies reported the incidence of EAD within the first week posttransplantation. The overall rate of EAD was 15.2%  for the MP group and 30.6% (0–46.4%) for the CS group. Pooled meta-analysis of the HMP and NMP studies revealed that the overall rate of EAD was significantly reduced in the MP preservation compared with the CS preservation  . There wI2 = 0%; P = 0.78). In the subgroup analysis, EAD rates were significantly lower in the HMP subgroup  compared to those of CS. There is statistically significant heterogeneity in the NMP subgroup . Thus, a random-effects model was adopted. However, subgroup analysis showed no significant difference between NMP preservation and CS preservation . In the NMP subgroup, a sensitivity analysis was conducted by excluding Bral and colleagues' study (P = 0.0001). The overall risk of EAD in the MP group remained statistically significant  (figure not shown).Subgroup analysis showed no significant difference between two subgroups ; thus, a fixed-effects model was used. A meta-analysis indicated that there was no statistical significance in PNF rates between MP preservation and CS preservation   .2 = 8.08, P = 0.33, I2 = 13%); thus, a fixed model was adopted. Meta-analysis of these studies indicated that total biliary complications at 1 year after transplantation were significantly lower in MP preservation group compared with CS preservation group  (P = 0.007). However, no significant difference could be detected between the NMP subgroup and CS subgroup   . Subgrou = 0.34) .P = 0.02). No significant heterogeneity was found between the MP and CS groups . The subgroup analysis showed that the incidence of IC was significantly lower in HMP preservation patients compared with CS preservation patients  (P = 0.64)  , but the = 0.64) .P = 0.26) and NMP group  compared to those of the CS group. However, there was no statistical significance (P > 0.05). A pooled meta-analysis of HMP and NMP studies indicated that there was no statistical significance in HAT rates between MP preservation and CS preservation   in the MP group and 5.2%  in the CS group, respectively. Hepatic artery thrombosis rates were lower in both the HMP group  .P = 0.61). However, it had no statistical significance , 6 months , and 1 year , 6 months , and 1 year .The results of this meta-analysis provide a comprehensive and up-to-date insight into the current evidence regarding the priority of MP preservation to CS in the clinical setting. Our analysis concludes that compared with CS preservation MP can reduce the risk of post-LT EAD and total biliary complications. However, MP preservation could not improve PNF, 1-year graft, and patient survival.ex vivo after donor liver procurement by effectively providing continuous circulation of oxygen, essential nutrients, and adequate adenosine triphosphate substrates to restore the normal physiological and also supplying vasodilators to maintain the peribiliary vascular microcirculation while flushing out the metabolites to avoid accumulation of toxic substances. Evidence from animal models and human studies suggests that MP can avoid cold ischemia injury during preservation and reduce posttransplant IRI and sterile inflammation, thus causing less damage to hepatocytes and the biliary epithelia in both deceased brain donor (DBD) and DCD LT compared with CS preservation. As a result, MP has been found to bring about significantly reduced posttransplant peak enzyme release, lower EAD rates, less biliary complications, and shorter hospital stay in MP livers.Theoretically, MP preservation can simulate physiological condition P < 0.05), and there was a trend favoring MP in ALT levels (P = 0.06) (P < 0.0001). In line with animal studies, our meta-analysis suggested that MP can achieve definite protecting effects for reducing IRI by decreasing transaminases releasing.Liver graft reperfusion injury was usually determined by AST and/or ALT serum levels after transplantation. Previous evidence suggested that episodes of EAD and elevation of hepatic transaminases may adversely affect survival rates after LT . Althoug = 0.06) . NotablyP < 0.0001)  , indicat 0.0001) , suggestP = 0.006) (P = 0.02)  . Further = 0.02) . Van RijP = 0.49). This may be true, or more likely, there may be insufficient data to draw the conclusions about the benefits of MP on reducing PNF rates as clinical investigations on MP in LT are still limited. Primary nonfunction rates are relatively low in clinical practice, occurring in 5% to 8% of liver transplants, but it may be a life-threatening circumstance requiring immediate retransplantation . Although there was a trend favoring MP preservation in Patrono and colleagues' study (P = 0.18), a longer follow-up result was lacking. In Dutkowski and colleagues' recent serial study   , suggest= 0.024) . Therefoex vivo graft repair through delivery of targeted additives, MP seems to provide more obviously protective effects in relatively low-quality donors such as ECDs or DCDs. Thus, MP has the potential to resuscitate the donor graft from IRI to a better state, reduce the discarded rates, and increase the overall organ utilization. The results of this study on MP's benefits are consistent with those findings in kidney transplantation. In kidney transplantation, large RCTs and recent meta-analyses have drawn the conclusions of MP's superiority to CS on improvement in short-term outcomes, such as enhanced early renal function and decreased risk of DGF in DCD and ECD grafts. Similar to our analysis, however, no significant differences could be found in PNF rates and patient survival between MP and CS for DCD LT (ex vivo and in situ NMP could significantly reduced peak AST and ALT levels compared with CS for DCD livers (P < 0.00001). It is worth noting that total bile production was also significantly higher  in the NMP group, indicating the excellent effect of NMP in preserving liver functions and viability assessment. In Zhang and colleagues' recent meta-analysis on HMP, they found that HMP could significantly reduce the incidences of EAD  and biliary complications , respectively. And there was no difference in the incidence of PNF  and vascular complications  between HMP and CS preservation. These results were consistent with our present analysis. However, Zhang et al. found that 1-year graft survival was significantly increased in HMP preservation compared to CS preservation , in which there was no difference in our analysis . To be noted, Zhang and colleagues' meta-analysis included overlapping studies from the same institution [Guarrera et al. , and perioperative parameter among different publications may still result in analytical bias. Notably, a recent meta-analysis did not observe any differences in post-LT outcomes such as PNF, biliary complication, and 1-year survival while using different perfusion fluids, volumes, or perfusion routes , indicatIn conclusion, this meta-analysis demonstrates that MP preservation, especially HMP, could significantly improve short-term outcomes by reducing the rates of EAD, total biliary complications, and IC after transplantation compared to CS preservation. However, it was not correlated with the incidence of PNF, HAT, and 1-year graft and patient survivals. Considering the benefits, MP preservation is still recommended for LT to expand the donor pool and improve posttransplant outcomes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcherJJ and YN designed the research. JJ, YN, JL, and HX retrieved literature, extraction data, analyzed the data, and wrote the manuscript. JL, HX, LZ, JY, and S-SZ revised the manuscript.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.


Gerres species play significant roles in marine fishery, but its evolution has received little attention. To date, only two Gerres mitochondrial genomes were reported. In the present study, three mitogenomes of Gerres  were systemically investigated. The lengths of the mitogenome sequences were 16,673, 16,728, and 16,871 bp for G. filamentosus, G. erythrourus, and G. decacanthus, respectively. Most protein-coding genes (PCGs) were initiated with the typical ATG codon and terminated with the TAA codon, and the incomplete termination codon T/TA could be detected in the three species. The majority of AT-skew and GC-skew values of the 13 PCGs among the three species were negative, and the amplitude of the GC-skew was larger than the AT-skew. The genetic distance and Ka/Ks ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, were which most likely due to the difference in their living environment. The phylogenetic tree was constructed by molecular method (Bayesian Inference (BI) and maximum Likelihood (ML)), providing further supplement to the scientific classification of fish. Three Gerres species were differentiated in late Cretaceous and early Paleogene, and their evolution might link with the geological events that could change their survival environment.Mitochondrial genome is a powerful molecule marker to explore phylogenetic relationships and reveal molecular evolution in ichthyological studies.  Mitochondrial genomes (mitogenome) have become a powerful molecule marker to explore phylogenetic analysis and taxonomic diagnosis in ichthyological studies because of its simple genetic structure, maternal inheritance, fast evolutionary rate, high specificity and easy detection . The mitMitogenome is typically circular in vertebrates, generally composed of protein-coding genes (PCGs), transfer RNA (tRNA) genes, ribosomal RNA (rRNA) genes, and one non-coding control region (D-loop) with a whole length of approximately 15–20 kb . As the Gerres (with 28 species), Eucinostomus (10 species), Gerres (six species), Diapterus (four species), Parequula (two species), Pentapion (monotypic), Deckertichthys (monotypic), and Ulaema (monotypic) [Gerres is the domination of the fishery capture with higher exploitation [Gerreidae fishes belong to Perciformes, commonly known as mojarras or silver biddies, and comprises eight genera: notypic) . As marinotypic) ,15. Gerrnotypic) . The datoitation . Gerres mainly concentrate on morphology and description of new species [Gerres have received little attention. To date, 178 complete mitochondrial genomes of Perciformes have deposited in the MitoFish GenBank, but only two mitogenomes for Gerres fishes (G. oyena and G. filamentosus) were sequenced [Recently, related studies on genus  species ,19,20,21 species ,23. Withequenced . Gerres species  which were widely distributed in the South China sea [Gerres species. The relative synonymous codon usage (RSCU) and AT skew value of PCGs were analyzed to better understanding the functional inference of related genes. Moreover, phylogenetic analysis among Gerres species and related species in Perciformes were conducted to investigate the kinship between them. The evolution rate and divergence time of the three species were also calculated to evaluate the adaptive capacity to environment, the situation of selection pressures, and how the environment influences their differentiation. In the present study, the complete mitogenomes of three hina sea , were seG. filamentosus, G. erythrourus, and G. decacanthus were 16,673, 16,728, and 16,871 bp in length, respectively. The mitogenome contained 13 PCGs , two rRNA genes (12SrRNA and 16SrRNA), 22 tRNA genes and one D-loop region  in the length of 35 bp to 37 bp. The OL was always found in the intergenic region between two conserved genes [L was usually observed between tRNA-Asn and tRNA-Cys in bony fishes, and the secondary structure that is folded into a stable stem-loop was the main feature of OL [Spacers in animals’ mitochondrial genes were short, and can be used for evolutionary studies due to quickly changing ratio than gene regions . There wentified . The lenariation . The sized genes , and there of OL .G. filamentosus, G. erythrourus, and G. decacanthus, respectively /synonymous substitution (Ks) is a common indicator to assess selective pressure and evolutionary relationships of species in molecular studies . Ka/Ks <l waters ,16,54. Pl waters ,57. The Gerres mitogenomes comparing to other genes, while COXI and Cytb genes were low  values across three were low . Low mutThe AliGROOVE analysis of 13 PCGs showed that there were no strongly divergent patterns among 26 species and all sites displayed positive scores. Besides, the Gerreidae exhibited higher heterogeneity than other families . The sitG. decacanthus had the closest relationship with G. oyena and the farthest with G. erythrourus in family Gerridae.The phylogenetic analysis contained 27 species from nine families  . The phyGerres species was separated around 104.5 million years ago (Mya) (G. erythrourus and other Gerres species was the earliest (70.01 Mya). However, the G. decacanthus and G. oyena had latest differentiation time (50.18 Mya). There were 7.78 million gaps between G. erythrourus and G. filamentosus, and 12.05 between G. filamentosus and G. decacanthus. The accumulation of changes in genetic composition resulted species reproductive isolation and evolution, indicating evolution was a long-term process as shown in the earlier study [Gerres species could trace back to the late Cretaceous (66–145 Mya). At that time, many mountains were formed, angiosperms began to appear and shale were extensively deposit to the ocean and the Oceanic Anoxic Event 2 occurred [G. filamentosus differentiation from other species was around 62.23 Mya, and G. decacanthus and G. oyena were differentiated around 50.18 Mya. Cenozoic-Paleogene, approximately occurred at 2.4–65 Mya, a period with significantly shrank of transgressive range in the continent and appeared of marine sediments in the marginal areas of China [The present divergence time calculations showed that the initial of the go (Mya) . In famier study . And theer study ,63. The occurred . The geoG. filamentosus , G. erythrourus , and G. decacanthus  were collected in Pearl River Estuary, and were deposited in the South China Agriculture University, Guangzhou, China. One individual of each species was used for DNA extraction. The dorsal muscle was collected and the genomic DNA was extracted by the TIANamp Marine Animals DNA Kit  following the manufacturer’s protocol, except that the final step was eluted with sterilized water instead of TE. The integrity of DNA samples was firstly characterized by 0.8% gel electrophoresis and UV spectroscopy. DNA concentration and purity were measured by NanoDrop 2000 spectrophotometer  and Qubit 2.0 Flurometer . Qualified DNA samples were stored at −20 °C for the next experiment. All animal experiments were conducted in accordance with the guidelines and approval of the Animal Research and Ethics Committees of South China Agricultural University.Wild specimens of High-quality DNA samples were randomly broken into fragments with the length of 350 bp for paired-end sequencing, and the DNA libraries were constructed according to the standard procedure of Illumina DNA library construction. The quality of the library was control by qPCR method and Agilent 2100 Bioanalyzer . The quality-qualified DNA library was run on an Illumina HiSeq 4000 instrument  with paired-end reads of 150 bp, and the sequencing data of per sample was not less than 2 GB.http://mitofish.aori.u-tokyo.ac.jp/) and ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). The protein-coding regions and ribosomal genes were determined by align with the reported mitochondrial genomes of close related species base on the methods of Blastl and Blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The complete mitogenomes of G. filamentosus, G. erythrourus and G. decacanthus were uploaded to GenBank with accession number MG587039, MN075144, and MT023107, respectively.The original sequences obtained by Illumina HiSeq 4000 sequencing were filtered to get high-quality sequences, with the following principle: when the content of N in any sequencing reads exceeded 10% of the number of read bases or any sequencing reads containing low-quality (Q ≤ 5) bases exceeded 50%, the paired reads were removed. The obtained high-quality fragments were aligned with Gerridae mitochondrial genomes on NCBI to remove sequence repeats and inaccurate sequencing, and then assembled by SPAdes v.3.5.0  softwarehttp://stothard.afns.ualberta.ca/cgview_server/). The secondary structure of tRNAs were obtained by ARWEN (Version1.2) [http://lowelab.ucsc.edu/tRNAscan-SE/) if any tRNA structure was abnormal. Sequence length, nucleotide composition and codon usage were calculated via DNAStar. The following formulas were used to calculate the values of AT/GC-skew to assess the nucleotide bias: AT-skew = (A – T)/(A + T) and GC-skew = (G – C)/(G + C). The data of RSCU were received by MEGA 7 software [Mitochondrial gene structure maps were drawn using CGView Server  (http://sion1.2) , and version1.2)   was used as the out-group to determine the root of phylogenetic tree [http://www.timetree.org/) [To establish evolutionary relationships among software  was utiltic tree . The Baytic tree . Mtmam +tic tree  with 100tic tree . The divtic tree . The calee.org/) .G. filamentosus, G. erythrourus, and G. decacanthus were obtained by high-throughput sequencing. Their mitogenomes were with a total length of 16,673 bp in G. filamentosus, 16,728 bp in G. erythrourus, and 16,871 bp in G. decacanthus, respectively. Each of the mitogenome composed of 13 PCGs, 2 rRNAs, 22 tRNAs and one D-loop. Most PCGs were initiated with the typical ATG codon and terminated with TAA codon. The ratio of Ka and Ks indicated that three species were suffering a purifying selection, while the COI and Cytb showed the highest Ka/Ks values. The three Gerres species were differentiated in late Cretaceous and early Paleogene, and their evolution might link with the geological events that could change their survive environment. The phylogenetic tree provided further supplement to the scientific classification of Gerres fishes. This study could provide basis information for genetic characters, phylogenetic position and evolution profile for these fishes, which could benefit for resource management or selective breeding in fishery and aquaculture.In the present study, mitogenome sequnences of


The spectra had five vibrations occurring at approximately 473, 477, 482, 607 and 616 cm−1 for all samples, which can be attributed to the vibrations of CuO, validating the formation of highly pure CuONPs.Green synthesis of nanoparticles using citrus peel extracts is known to be environmentally friendly and non-toxic when compared to chemical methods. In this study, different citrus peel extracts obtained with the solvents acetone and distilled water were used to synthesize copper oxide nanoparticles (CuONPs). The nanoparticles were characterized using cyclic voltammetry, ultraviolet-visible spectroscopy, energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). The absorption spectrum of CuONPs prepared with acetone exhibited characteristic peaks at the wavelengths between 280–293 nm, while those with distilled water had peaks at 290 nm. The acetone-synthesized CuONPs were spherical while those produced using distilled water were rod-shaped. Based on EDS, the analysis revealed a trace spectrum of CuO nanoparticles with different weight compositions that varied with the type of citrus peel and solvent used. FTIR measurements were carried out in the range of 500–4000 cm Escherichia coli, Salmonella typhi and Klebsiella pneumonia [According to the National Institute of Communicable Diseases (NICD), there are constant outbreaks and re-emergence of infections caused by pathogenic bacteria such as neumonia . Infectineumonia ,3. This neumonia . Furtherneumonia . To achineumonia . Much atneumonia ,8,9,10. neumonia ,12.Green synthesis is a set of principles that eliminates the use of hazardous substances in manufacturing and applications of chemical products . There iNanoparticles are tiny solid materials with a structural length of a few nanometers . They arAfter the process of synthesis, it is necessary that nanoparticles are characterized primarily, to assess the safety properties especially if they are intended to be used at full potential to enhance human welfare  in the area of nanomedicine . Before Copper oxide is one of the metallic multifunctional nanoparticles with useful physical properties applied in biomedicine, electron chemical effects, antimicrobial studies, solar power generation and catalytic causes ,24. CoppCitrus aurantifolia leaves could be used as an antibacterial agent and photocatalyst for dye removal and purification of contaminated water in industries. Moreover, they reported that CuONPs from the same plant extract showed sensitivity against K. pneumonia and S. aureus, revealing that they have antibacterial properties capable of inhibiting drug-resistant bacteria. The tangerine plant extract has been used to remove inorganic and organic contaminants from wastewater [El-Moslamy et al.  reportedstewater . Furtherstewater .E. coli O177, E. coli O157, E. coli O26, Salmonella typhi and S. pneumonia that are most often resistant to antibiotics [One health challenge is resistance to treatments administered against various infections . Accordiibiotics . Antimicibiotics .Comparative studies on citrus peel extracts used to synthesize metal oxide nanoparticles are limited in the literature. Therefore, the present study aimed to report a comparative study on spectroscopy, morphological, cyclic voltammetry and antimicrobial properties of CuO nanoparticles amalgamated from citrus peel extracts of orange, lemon and tangerine.3)2·5H2O) as a precursor. Acetone and distilled water were solvents. The color of the extracts was orange. The resultant precipitates were brownish-black for the acetone solvent and brown for the distilled water solvent. The extract and precursor solution changed from light blue to a green color solution, indicating a formation of copper oxide nanoparticles. A similar color change was observed in the literature [For this study, copper oxide nanoparticles were synthesized using citrus peels  with copper (II) nitrate (VI) pentahydrate  extracts was round, which agrees with what was observed in this study. In contrast to the study of [SEM was used to confirm the morphology of nanoparticles synthesized. SEM images of CuONPs clearly show that the particles are rod and spherical, not forming any clusters shown in study of , the SEMEDS (energy-dispersive X-ray spectroscopy) was used to confirm the presence of both copper and oxygen in the nanoparticles. CuONPs had different weight compositions varying with the type of citrus peel and solvent used. The weight composition spectra of copper and oxygen for the distilled water solvent shown in The CuO nanoparticle size and morphology were investigated with TEM images presented in C. coli ATCC 33559, E. coli ATCC 25922, M. catarrhalis ATCC 25240, S. diarizonae ATCC 12325, P. aeruginosa ATCC 27853) and Gram-positive  bacteria using the agar well diffusion method. The results of the agar well diffusion assays indicating the antibacterial activity of CuO-NPs-O on pathogenic bacterial strains are summarized in C. perfringens (ATCC 13124), E. coli (ATCC 25922), S. aureus (ATCC 25923), L. monocytogenes (ATCC 19115), S. pneumonia (ATCC 13883), P. aeroginosa (ATCC 27853) and M. catarrhalis (ATCC 25240). C. perfringens ATCC 13124 (12 mm and 19 mm), E. coli ATCC 25922 (18 mm and 24 mm), M. catarrhalis ATCC 25240 (7 mm and 16 mm), C. coli ATCC 33559 (20 mm and 26 mm) and S. aureus ATCC 25923 (13 mm and 25 mm) while CuO-NPs-ODI showed no activity. From data in S. pneumonia with a ZBGID of 8 mm and 14 mm, respectively, while those from acetone showed no activity against the pathogens. Aqueous lemon peel mediated CuO-NPs (CuO-NPs-LDI) showed activity against C. coli ATCC 33559, with a ZBGID of 16 mm at 25 µg/mL and 25 mm when 50 µg/mL of the nanoparticle was used (S. aureus) with a ZBGID of 17 mm and 23 mm for concentrations 25 µg/mL and 50 µg/mL, respectively. CuO-NPs-LDI produced ZBGID of 9 mm and 13 mm against L. monocytogenes while those for the Gram negative P. aeruginosa were 6 mm and 20. Acetone lemon peel (CuO-NPs-LA) mediated CuONPs on the other hand showed no antibacterial activity. Generally, the antibacterial properties of the CuONPs increased with increase in concentration, as revealed by an increase in the respective ZBGIDs. In a previous study, copper oxide nanoparticles showed mild inhibitory effects against E. faecalis, Salmonella, E. coli and S. aureus [viz E. aerogenes (Gram negative) and S. aureus (Gram positive) [E. coli, P. aeruginosa, K. pneumonia, E. faecalis, S. flexneri, S. typhimurium, P. vulgaris and S. aureus. Similar to these findings, a previous report [The antibacterial efficiency of the citrus peel mediated CuONPs was evaluated against both Gram-negative (was used . Antibacositive) . A studyositive)  also rev7 mm and  mm, C. c3)2·5H2O; GlassWorld, Johannesburg, South Africa), sodium hydroxide , acetone  and distilled water. All reagents were of analytical grade. The pathogenic bacterial strains were obtained from the American type culture collection strains purchased from Bio Mérieux, South Africa.The citrus peels were obtained from citrus fruit bought at Food Lover’s Market in Mafikeng, North-West South Africa. Reagents used included copper (II) nitrate (VI) pentahydrate 2·5H2O was treated with an extract of 20 mL and was magnetically stirred at room temperature for 30 min until the light blue color changed to a green color, which indicated the formation of CuONPs. The solution was stirred for 1 h, then heated at 80 °C for 10 min. Then, 5 mL of 1 M sodium hydroxide (reducing agent) was added twice at 5 min intervals. The solution resulted in a brown precipitate that was repeatedly washed with deionized water followed by ethanol to remove impurities found in the precipitate. Lastly, it was dried at 60 °C and put in storage for analysis.CuONPs were synthesized with slight modifications following the protocol described by . A 200 mThe resulting synthesized metal oxide nanoparticles were characterized by energy-dispersive X-ray spectroscopy (EDS), UV-Visible spectroscopy, scanning electron microscope (SEM), transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). UV-Vis spectroscopy is a simple, fast, sensitive and selective characterization technique that enables the determination of nanoparticle formation and stability . The metMorphology, microstructure and the elemental composition of the synthesized metal oxide nanoparticles were examined using SEM with EDS. Scanning electron microscopy (SEM) images were recorded using a Hitachi 3600 SEM instrument  and energy dispersive X-ray analysis (EDS) was done using the Thermo Fisher Scientific Ultradry  instrument. TEM analysis aims to provide information on the shape and size distribution of synthesized nanoparticles while confirming metal oxide nanoparticle existence in the synthesized samples. TEM analysis was done using the transmission electron microscope, JEOLJEM 2100 electron microscope  operated at 200 kv accelerating voltage and connected to an energy-dispersive spectrophotometer (EDS). In performing the analysis, 2 mg of each sample was weighed out into a sample bottle and about 4 mL ethanol solution was added. The mixture was sonicated for 10 min to disperse the particles, using a digital ultrasonic cleaner. A drop of the sonicated mixture was placed on a carbon-coated copper grid and allowed to dry for about 5 min.3)2·5H2O [E. faecalis ATCC 29212, S. aureus ATCC 25923, L. monocytogenes ATCC 19115, S. pneumonia ATCC 13883 and C. perfringens ATCC 13124) and five strains of Gram-negative  bacteria were used in the analysis. This study’s pathogenic bacterial strains were American type culture collection strains purchased from Bio-Mérieux, South Africa. Wells were made using sterile cork borer (10 mm) into each MHA Petri plate and were used to evaluate the antimicrobial activity of the synthesized CuO nanoparticles. The microbial culture was exposed to CuONPs of different concentrations (25 and 50 mg/mL). The zones of inhibition were observed and measured. DMSO (dimethyl sulfoxide) was used as negative control and tetracycline used as a positive control. The bacteria were cultured overnight on MHA at 37 °C in an incubator.The antibacterial potency of the synthesized metal oxide nanoparticles was evaluated using ten pathogenic bacterial strains associated with both food poisoning diseases and nosocomial infections in humans. Each bacterial strain was sub-cultured in the nutrient broth and incubated aerobically at 37 °C for 24 h. (CuNO3)2·5H2O , as a prC. perfringens ATCC 13124, C. coli ATCC 33559, E. coli ATCC 25922, L. monocytogenes ATCC 19115, S. pneumonia ATCC 13883, P. aeroginosa ATCC 27853 and M. catarrhalis ATCC 25240. S. aureus ATCC 25923 and E. coli ATCC 25922 showed a significant zone of inhibition to CuONPs compared to the positive control (tetracycline). C. perfringens ATCC 13124, L. monocytogenes ATCC 19115, S. pneumonia ATCC 13883, P. aeroginosa ATCC 27853 and M. catarrhalis ATCC 25240 strains showed a moderate zone of inhibition compared to the positive control (tetracycline). This study successfully validates the use of copper oxide nanoparticles with citrus extracts to exert antimicrobial properties on various microorganisms that are resistant to antibiotics.This study reports on the green synthesis of CuONPs prepared by the biosynthesis technique using citrus extracts. The UV-Vis absorption spectra show that copper oxide nanoparticles synthesized from selected peel extracts exhibit characteristic peaks at the wavelengths 280–320 nm. SEM images reveal that the particles appear to be almost rod-like and sphere-shaped. TEM images showed spherical shapes and the size estimation, and the subsequent presentation in different histograms revealed an average size ranging between 48–76 nm. The CuO nanoparticles exhibited significant antibacterial activity against


In vitro study revealed that LINC01419 promotes growth and migration of HCC cells. Genes that affected cell proliferation and cell migration were identified using RNA-sequence. Subsequently, it was confirmed that LINC01419 binds to EZH2, leading to histone methylation of the RECK promoter. Interaction between LINC01419 and FUS stabilized EZH2 mRNA thereby enhancing EZH2 expression. Conclusively, the results of this study confirm that LINC01419 may serve as a potential target for HCC diagnosis and treatment.Long non-coding RNAs (lncRNAs) have been reported to play significant roles in human tumorigenesis, for example, in hepatocellular carcinoma (HCC). This study explored the role of LINC01419, a new lncRNA, in HCC.  Hepatocellular carcinoma (HCC) is one of the most prevalent and fatal human cancers globally. Current HCC treatments include surgical resection, molecular-targeted therapy, and immunotherapy . AdvanceLong non-coding RNAs (lncRNAs) are implicated in diverse cellular processes and disease pathogenesis . LncRNAsThis study aims to investigate the biological role of LINC01419 and the underlying mechanism in HCC.http://app.cgu.edu.tw/circlnc/) and TANRIC (http://ibl.mdanderson.org/tanric/_design/basic/index.html). Several abnormal lncRNAs were detected and showed significant statistical differences. This study focused on LINC01419 because it showed the most significant difference , , . To confex.php), . In vitr ability . Analysi ability .The relationship between LINC01419 expression and the clinicopathological characteristics of patients has been highlighted in In evaluating the biological function of LINC01419 in HCC, siRNA was used to knockout the endogenous expression of LINC01419 . MTT assin vitro proliferation and invasion of HCC cells.In summary, these results implicated that LINC01419 promoted RNA transcriptome sequencing was used to identify the potential target genes correlated with LINC01419.Series of genes were either up-regulated or down-regulated (fold change≥4-fold) after the LINC01419 knockout. Genetic ontology analysis was performed to determine the most significant biological behavioral pathways for protein binding, RNA binding, and DNA binding . The KEGhttp://genome.ucsc.edu/), , . It was,c.edu/), . RNA pulc.edu/), . To detec.edu/), . Elevatec.edu/), , 3G. Subc.edu/), . Contrarc.edu/), . The abohttp://starbase.sysu.edu.cn/starbase2/index.php). This study determined whether FUS enhanced EZH2 expression inLINC01419. Through RIP analysis, it was reported that LINC01419 and EZH2 were significantly captured by FUS in HepG2 and Huh7 cells  were used to analyze the 1000bp region, upstream of the LINC01419 transcription initiation site. Two c-jun-binding motifs at-992 to -986 and −107 to −101 were identified in the promoter region, upstream of the LINC01419 transcription initiation site. The two transcription factor-binding sites (TFBSs) were named A and B, respectively .Furthermore, we determined whether c-jun modulates LINC01419 expression. c-jun down-regulation by si-RNAs inhibited LINC01419 in HCC cells . To confMany reports have recommended lncRNAs as vital regulators of gene expression and cancer development over the past few decades. From our previous studies, for example, we reported that LINC01287 and XIST could function as oncogenic lncRNA in human HCC , 7. LINCin vivo experiments, LINC01419 downregulation was shown to suppress HCC proliferation. In addition, an in vivo study revealed that inhibiting LINC01419 impaired proliferation ability of HCC cells. Despite having reported that LINC01419 functions as an onco-lncRNA in HCC, its relevant regulatory mechanism in HCC remains unclear. Therefore, we performed an RNA sequence to explore the underlying mechanism. Reports indicate that several lncRNAs work in conjunction with chromatin-modifying enzymes to either increase or decrease epigenetic target gene expression. Results from this study indicated that LINC01419 could bind to EZH2, a key component of methyltransferase PRC2.This suggested that LINC01419 may promote the development of HCC through transcriptional regulation of target genes. The genes are associated with HCC cell progression as they interact with EZH2. The oncogene, EZH2, plays a significant role in HCC by promoting cell proliferation and metastasis [In this study, we determined that the LINC01419 expression level was elevated in HCC. This was verified using lncRNA raw data from TCGA. Our results indicated that LINC01419 inhibition could decrease HCC cell proliferation and migration ability. Moreover, through tastasis . Besidestastasis . Again, Among the target genes regulated by LINC01419, we focused on RECK because it is known to suppress tumors in various cancers, for example, HCC . The RECLINC01419 upregulation in HCC may be caused by several mechanisms. Transcriptional regulation, for example, majorly leads to abnormal expression of lncRNA . AnalysiIn conclusion, this study reports that elevated LINC01419 expression level results in poor outcomes in HCC. LINC01419 potentially suppresses RECK expression epigenetically via EZH2.This, therefore, promotes HCC progression. LINC01419 upregulation is induced by the transcription activation of c-jun. These findings proved that LINC01419 could provide a theoretical basis for clinical diagnosis and treatment of hepatocellular carcinoma.HCC samples and paired non-cancerous specimens were obtained from the People's Hospital of Shanxi Province and Affiliated Cancer Hospital and Institute of Guangzhou Medical University. Tissues were immediately frozen in liquid nitrogen after hepatectomies and stored in a refrigerator at −80°C. The study was reviewed and approved by the Institutional Review Board of People's Hospital of Shanxi Province and Affiliated Cancer Hospital and Institute of Guangzhou Medical University.2 at 37 °C. The cells were maintained in RPMI 1640 supplemented with 10% FBS.HCC cell lines  were maintained in our laboratory. Cells were cultured in 5% COpcDNA3.1-RECK, pcDNA3.1-EZH2, and pcDNA3.1-LINC01419 were obtained from RiboBio .siRNAs that targetLINC01419 were obtained from Genechem . Cells were transfected with oligonucleotides using Lipofectamine 2000 .Total RNA was extracted from tissues or cells using TRIzol reagent  following the manufacturer’s protocol. The PrimeScript RT reagent Kit (TaKara) was used during RNA reverse transcription. RT-PCR analyses were performed using the Gotaq® Green Master Mix (TaKara). The results were normalized with GAPDH. All the primer sequences used for RT-qPCR are listed in For the colony formation assays, HCC cells were inoculated into 6-well plates. Two weeks later, the forming colonies were fixed with 4 % paraformaldehyde and stained with Giemsa. The colonies were counted.Cell migration assay was performed as previously described .2. The mixture was added into cell lysates. Subsequently, these mixtures were incubated at 4 °C for 4 hours and then washed in lysis buffer. The precipitated proteins were separated by SDS-PAGE and subjected to western blot assay.The sense and antisense of full-length lncRNA- 01419 were labeled with biotin. The labeled RNAs were added into MgClThe Magna RIP RNA binding protein immunoprecipitation kit  was used for the RIP assay. The assay was performed following the manufacturer’s protocol.The ChIP assay kit  was used for CHIP assay. The experiment was performed following the manufacturer’s instructions. The PCR products were visualized on an agarose gel.Total protein was extracted from cells using RIPA. The proteins were separated by SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were blocked in 5 % fat-free milk and incubated with specific primary antibodies at 4 °C overnight. Subsequently, the membranes were incubated with the second antibody for one hour at room temperature. Finally, the protein was detected using an enhanced chemiluminescence system.Graph Pad Prism 5.0software and SPSS 13.0 were used for statistical analyses. The values were presented as the mean ± S.E.M. Analyses of different groups were performed using one-way ANOVA or two-tailed Student’s t-test. P < 0.05 was considered statistically significant.Supplementary FiguresSupplementary Table 1


Schistosoma haematobium, was thought to be the only schistosome species transmitted through Bulinus snails on Unguja and Pemba Island . For insights into the environmental risk of S. haematobium transmission on Pemba Island, malacological surveys collecting Bulinus globosus and B. nasutus, two closely related potential intermediate hosts of S. haematobium were conducted across the island in November 2016. Of 1317 B. globosus/B. nasutus collected, seven B. globosus, identified through sequencing a DNA region of the mitochondrial cytochrome oxidase subunit 1 (cox1), were observed with patent infections assumed to be S. haematobium. However, when the collected cercariae were identified through sequencing a region of the cox1 and the nuclear internal transcribed spacer (ITS1 + 2), schistosomes from five of these B. globosus collected from a single locality were in fact S. bovis. The identified presence of S. bovis raises concerns for animal health on Pemba, and complicates future transmission monitoring of S. haematobium. These results show the pertinence for not only sensitive, but also species-specific markers to be used when identifying cercariae during transmission monitoring, and also provide the first molecular confirmation for B. globosus transmitting S. bovis in East Africa.The causative agent of urogenital schistosomiasis,  The snail-borne neglected tropical disease (NTD), schistosomiasis, is the most important freshwater parasitic disease of humans associated with poverty, poor sanitation and lack of safe water supplies . AmbitioSchistosoma species groups  have been historically identified as ‘model islands’ for implementing multiple effective infectious disease control and elimination programmes in sub-Saharan Africa , examined and individually induced to shed cercariae following previous methods  for long-term deoxyribonucleic acid (DNA) storage. After shedding, all infected snails were preserved in 100% ethanol for future morphological and molecular characterization.In November 2016, as part of a larger ongoing molecular xenomonitoring study on Pemba, cox1) and partial nuclear internal transcribed spacer (ITS1 + 2) DNA regions  before being compared with reference sequence databases for Bulinus , with minor changes to the standard protocol in that quantities of the digest reagents were doubled and digests were incubated for at least 12 h. From each snail, a 623 base pair region of the mitochondrial istosoma  to confiB. globosus and B. nasutus were collected, seven of these snails (B. globosus with two cox1 haplotypes recognized (GenBank accession numbers: MH014040 and MH014041) which matched those snails previously reported from Pemba , were identified as S. bovis (S. bovis cox1 haplotypes [Genbank accessions: S.b (i) MH014042 and S.b (ii) MH014043]  and a different stream site in Kinyasini (Kinya2). The S. haematobium cercariae from Kinyasini and Chambani, respectively, were of two different S. haematobium cox1 haplotypes [Genbank accessions: S.h (i) MH014046 and S.h (ii) MH014045] with only single haplotypes produced from each snail. These haplotypes matched those identified as group 2 S. haematobium cox1 haplotypes found only in the Indian Ocean Islands (The other two infected snails shed S.h MH014047 and S.b MH014044) and were identified as either S. bovis or S. haematobium by the three inter-specific single nucleotide polymorphisms  endemic to Zanzibar, should be determined to assess the impact on livestock and wildlife health. However, diagnosing S. bovis from the definitive host remains challenging, with the detection of S. bovis eggs in the stool being difficult and the more sensitive method of observing adult worms in the host being only possible post-mortem via dissection. An antigen-based test with promising diagnostic performance has been developed (S. bovis in Pemba.d vigour . Investia Island  facilitaBulinus africanus species complex (B. globosus naturally transmitting S. bovis in East Africa gives credibility to a previous observation (B. globosus being naturally refractory (B. nasutus with S. bovis in East Africa is also tainted with contradicting evidence, some showing natural infections (B. nasutus in other endemic areas (S. bovis has a broad intermediate host range in East Africa utilising several Bulinus species, as it has also been identified from B. ugandae (B. africanus (B. forskalii (S. bovis to B. globosus or indeed other endemic Bulinus species on Pemba, including B. nasutus and B. forskalii, are required to determine the transmission potential and possible spread of this emerging schistosome in Zanzibar.Due to the difficulty in classifying species within the ervation , and disfections b going  ugandae , B. afriorskalii . Therefo


Leptospira isolates at the species level fromclinical samples. Unfortunately, this method cannot differentiate betweensome intermediates and saprophytic species. Phylogenetic analysis of the 16S ribosomal gene initial region is used toidentify Leptospiraspecies to find new molecular targets for Leptospiraspecies identification. We used comparative genomic analysis between 35 rpoC gene, encoding theDNA-directed RNA polymerase β-subunit, for identifying 35Leptospira species.  We proposed the use of the rpoC gene can be a molecular target to identify the mainspecies of the Leptospira genus directly from clinicalsamples.The  Leptospira genusL. biflexa/L. wolbachii, L. meyeri/L. macculloughii/L.levettii/L. yanagawae, L. licerasiae/L. saintgironsiae/L.neocaledonica, L. brenneri/L. harrisiae, and L. venezuelensis/L.haakeii/L. hartskeerlii/L. wolffii speciesL. meyeri and L.yanagawaeLeptospira species, with the aim of finding newmolecular targets for the identification of Leptospira species.Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria ofthe Leptospira species were downloadedfrom the NCBI database. These species represent pathogenic, intermediate, andsaprophytic subgroups. The evolutionary history was inferred using theNeighbor-Joining method. The tree topologies were evaluated by using a bootstraptest, and the values were obtained after 1000 replicates of the dataset. Theevolutionary distances were computed using the Kimura 2-parameter method. A total of331 positions were obtained in the final dataset. Evolutionary analyses wereconducted with the Molecular Evolutionary Genetics Analysis (MEGA)-V7 softwareThe reference sequences of 35 ,Leptospiraspecies was determined using the Rapid Annotation Using Subsystem Technology (RAS)serverLeptospira species because the RAST webserver only allowed for analyses by groups of 10 species. Subsequently, the presenceof orthologous proteins was verified in the remaining 16 species. The proteomes were used for the identification of orthologous proteins. The OrthoMCL(for the identification of orthologous groups in eukaryotic genomes) and OrthoVenn(a web server for genome-wide comparison and annotation of orthologous clustersacross multiple species) bioinformatics tools were used to cluster proteins intoorthologous groups based on the identity of their sequencesLeptospira specieswere selected for manual extraction of their coding sequences (CDS) from theirgenomes. These CDS were aligned using the MEGA-V7 software to identify conservedregions among CDS for the design of genus-specific primers and polymorphic regionsthat allow for the differentiation of species.The proteins that were conserved among the 35 The primers were designed using the Primers3 bioinformatics program. Dimers,heterodimers, and hairpins were evaluated with the Oligo Analyzer software, and thecross-amplification test was performed with Primer-BLAST NCBI bioinformaticstool.The evolutionary history was inferred using the Neighbor-Joining method. Thepercentage of replicate trees in which the associated taxa clustered together in thebootstrap test (1000 replicates) was determined. The evolutionary distances werecomputed using the Kimura 2-parameter method. There were a total of 353 positions inthe final dataset. Evolutionary analyses were conducted in MEGA-V7.rpoC gene was amplified by polymerasechain reaction (PCR). The reagent concentrations used for PCR standardization wereas follows: primers , 0.4 μM; dNTPs, 0.2 mM; buffer, 1×; MgCl2, 1.5 mM;Taq polymerase, 1 unit/reaction; and DNA, 200 ng/μL. The final volume for eachreaction was 25 μL. The PCR was performed in a Perkin Elmer 9700 thermocycler. Thethermal cycling profile was as follows: one initial denaturation cycle at 94ºC for 5minutes, followed by 35 cycles at 94ºC for 35 seconds, 58ºC for 45 seconds, 72ºC for1 minute, and a final cycle at the extension temperature of 72ºC for 5 minutes.A 353 bp fragment from the L. interrogans, L. noguchii,L. kirschnerii, L. santarosai, and L.borgpetersenii), two intermediates , and one saprophytic species (L.biflexa) were used for the PCR-rpoC standardization.Six samples from humans, rodents, and monkeys naturally infected withLeptospira were used to verify the usefulness of the PCR indetecting Leptospira from clinical samples (the samples weredonated by the leptospirosis research group of the CES University). Additionally,the detection limit of PCR assay for the rpoC gene was evaluatedusing serial dilutions (1/10), starting with 100 ng of DNA from the L.interrogans reference strain.Five pathogenic (rpoC gene from each sample was amplified andpurified using a gel extraction kit (Qiagen). The concentration and purity weredetermined using a Nanodrop, whereas integrity was assessed by electrophoresis witha 1% agarose gel. All amplification products were sent to the Macrogen® company for sequencing. For each sample, both forward and reverse sequenceswere used to generate a consensus sequence using Mega7. The phylogenetic analysis ofthe rpoC gene was performed as previously described.To confirm the species identification from the phylogenetic analysis, a 353 bpfragment of the Leptospira genus from the genetically closestgenus (Leptonema illini), an adequate separation of the pathogenic,intermediate, and saprophytic subgroups, and the ability to differentiate 20/35species currently describedL. biflexa/L.wolbachii, L. meyeri/L. macculloughii/L. levettii/L.yanagawae, L. licerasiae/L. saintgironsiae/L.neocaledonica, L. brenneri/L. harrisiae, andL. venezuelensis/L. haakeii/L. hartskeerlii/L. wolffii species . Additionally, the reference sequences of therpoC and 16S ribosomal genes were extracted manually from thesegenomes for the subsequent phylogenetic analyses.Successful extraction of 35 reference genomes from the NCBI database corresponding todifferent Leptospira speciesrevealed 1650 genus-specific proteins [Supplementary data ]. An amino acididentity analysis among the different species show a high genetic diversity betweendifferent Leptospira species. The saprophytic species were the mostconserved, while the pathogenic and intermediate species showed a high genomicplasticity [Supplementary data ]. Additionally,the most polymorphic proteins, but with conserved regions, were selected for primerdesign and subsequent phylogenetic analysis from its coding regions.A query of orthologous proteins between the 35 Leptospira species. Interestingly, high variability innucleotide sequences and low variability in amino acid sequences were found; thesefindings could be related to genetic differences between the three species-subgroupsand may have an important role in virulence. From these conserved proteins, weselected the DNA-directed RNA polymerase subunit beta protein (rpoCgene) because this protein was conserved in all species of theLeptospira genus, and its polymorphism permits genus,subgroups, and species identification. Additionally, this protein contains conservedregions that allow for the design of gender-specific primers. A manual evaluation of orthologous proteins conserved in the genus was made with thefollowing inclusion parameters for the proteins: (1) the protein must be present inall species, (2) the gene must have conserved regions for genus-specific primerdesign, and (3) the genetic polymorphisms should allow for the differentiation ofthe 35 rpoC-F and rpoC-R primers were designed in twoconserved regions of the rpoC gene using the Primers3bioinformatics tool. The in silico amplification product was 353bp. Dimers , heterodimers , and hairpins  were evaluated with the Oligo Analyzer software. A cross-amplificationtest was performed with the Primer-BLAST NCBI bioinformatics tool. Nocross-amplification of sizes close to 353 bp were obtained. Only one possibleamplification of 476 bp was detected in Runella sp,Bacteroidetes and Spirosoma radiotolerans, butthe primers did not have a 100% alignment with these species; therefore, anamplification would not be obtained. The rpoC gene sequences of 35 Leptospira specieswere obtained from the NCBI database. To delimit the phylogenetic analysis, therpoC-F and rpoC-R primers were used. A common353 bp fragment for each species was used for the phylogenetic analysis. As a resultof the sequence alignment, we found that 58.07% (205 nucleotides) and 41.92% (148nucleotides) were conserved and variable, respectively. A phylogenetic analysis ofthe rpoC gene resulted in the correct separation of theLeptospira genus, an adequate separation of the pathogenic,intermediate, and saprophytic subgroups, and the ability to differentiate 35currently described Leptospira species with branch supports between11% and 100% (data not shown).The rpoC, it was possible to amplify therpoC gene from eight reference strains belonging to pathogenic,intermediate, and saprophytic Leptospira species[Supplementary data ], and the other species were theoreticallyinferred. Additionally, the usefulness of the PCR assay for detectingLeptospira from human, rodent, and monkey samples naturallyinfected with Leptospira was verified[Supplementary data ] with an analytical sensitivity of 10 DNAfemtograms per sample [Supplementary data ]. Furthermore,we were able to correctly identify the species in human, rodent, and monkey samplesby a phylogenetic analysis of the rpoC gene[Supplementary data ]. The phylogenetic analysis for therpoC gene correctly identified the unknown samples at thespecies level, and these results were 100% concordant with those obtained by thephylogenetic analysis of the 16S ribosomal gene (data not shown). Human, rodent, andmonkey sequences were deposited in the NCBI database under the following codes:MK510855, MK510852, MK510856, MK510851, MK510854, MK510853, MK529909, MK529910,MK521701, MK521702, MK521699, and MK521700.With the PCR assay for Leptospira species,finding that the Leptospira species shared approximately 1650orthologous proteins, and there was a high variability in the amino acid compositionof these proteins. These proteins are ideal molecular targets for the implementationof new molecular characterization tools for Leptospira species. Inour study, we found approximately 1650 orthologous proteins that were related tovital processes of the bacteria (data not shown). A notable finding was that most ofthese proteins reflect the molecular speciation process of theLeptospira genus, which may be related to the independentevolutionary process of each subgroup. By taking advantage of this evolutionaryprocess, we propose using the rpoC gene (which encodes theDNA-directed RNA polymerase subunit beta) to implement a phylogenetic identificationsystem that will allow for the identification of unknown isolates at the specieslevel, and to classify them according to their pathogenicity status directly fromclinical samples. Additionally, the gene polymorphisms allow for the identificationand differentiation of the 35 Leptospira species currentlydescribed via the amplification of a 353 bp fragment . The rpoCgene fragment offers better advantages than the initial region of the 16S ribosomalgene for species identification, since it can be used to differentiate betweenL. biflexa/L. wolbachii, L. meyeri/L. macculloughii/L.levettii/L. yanagawae, L. licerasiae/L. saintgironsiae/L.neocaledonica, L. brenneri/L. harrisiae, andL. venezuelensis/L. haakeii/L. hartskeerlii/L. wolffii. We present a comparative genomic analysis of 35 rpoC gene can be used toidentify all 35 species. Another advantage of the rpoC gene is thatit can be used to differentiate between intermediate species, which may be presentin environmental water sources and soils and can be sources of infection for humansand animalsrpoC wassuccessfully used to detect species belonging to pathogenic, intermediate, andsaprophytic subgroups. Additionally, the PCR assay for rpoC withunknown samples from humans, rodents, and monkeys was successful in detectingLeptospira with an analytical sensitivity of 10 femtograms ofDNA. Furthermore, the phylogenetic analysis for the rpoC genecorrectly identified the unknown samples at the species level, and these resultswere 100% concordant with those obtained by the phylogenetic analysis of the 16Sribosomal gene (data not shown). Therefore, the PCR assay for rpoCcould be a valuable tool for the detection and identification ofLeptospira species directly from unknown samples with a highanalytical sensitivity in addition to the absence of cross-reactions with otherbacterial species and uncultivable microorganisms. Currently, therpoB gene, wzy gene,S10-spc-α locus, and 23S rRNA gene are used to identifyLeptospira species, but the 35 species of theLeptospira genus currently described have not been evaluated inthese studies-Leptospira genusdirectly from clinical samples, environmental water sources, and soils.The initial fragment of the 16S ribosomal gene can only discriminate 20 out of the 35species currently described, while the


Site-selective antibody fragment conjugation is achieved by using a disulfide bond to ‘hook’ and deliver thioester acylating agents to specific lysines. The modification of lysine residues with acylating agents has represented a ubiquitous approach to the construction of antibody conjugates, with the resulting amide bonds being robustly stable and clinically validated. However, the conjugates are highly heterogeneous, due to the presence of numerous lysines on the surface of the protein, and greater control of the sites of conjugation are keenly sought. Here we present a novel approach to achieve the targeted modification of lysines distal to an antibody fragment's binding site, using a disulfide bond as a temporary ‘hook’ to deliver the acylating agent. This cysteine-to-lysine transfer (CLT) methodology offers greatly improved homogeneity of lysine conjugates, whilst retaining the advantages offered by the formation of amide linkages. NHS esters) to form robustly stable, clinically validated amide bonds.Ka's of lysines or local environments.32Labeling We chose to develop the CLT strategy for antibody conjugation on the Fab fragment of Her2-targeting breast cancer drug trastuzumab. Specifically, trastuzumab fragment conjugates are of widespread interest for drug delivery and imaging applications,S,N-acyl transfer onto proximal lysines. They are >100 times more reactive than the corresponding oxo-esters to nucleophiles such as amines, whilst being less reactive to hydrolysis.e.g. in acetoacetyl CoA in the Krebs cycle,via an S,N-acyl transfer involving a 5-membered ring intermediate.Thioesters present an ideal reactivity profile to achieve the desired initial cysteine acylation and subsequent 2 along with a common acylating agent NHS ester 1. The number of acylations were identified by LC-MS  along with unmodified LC confirmed that a small amount of background lysine conjugation was occurring  was determined to be 1.5 by UV absorbance  selectively afforded bridged conjugate 8  methodology allows the construction of highly homogenous antibody fragment conjugates, whilst incorporating robustly stable, clinically validated amide linkages. The readily available thioester reagents are shown to react selectively with the cysteines obtained from the reduced interchain disulfide bond in a Fab, and then transfer at raised pH to specific proximal lysine residues, which are ideally placed distal from the binding site. By employing either mono- or bisthioesters we have shown it is possible to control the stoichiometry, to afford major products containing 2 or 1 acylations per disulfide. Whilst hydrolysis of the thioesters represents an expected competing background reaction, the efficiencies of the macrocyclic N. F., V. C. and J. R. B. conceived and designed the project; N. F., V. C. and J. R. B. conceived and designed the chemistry and chemical biology experiments; N. F. and I. B. performed the chemistry and chemical biology experiments; K. K. performed the mass spectrometry experiments; N. F., V. C., K. K. and J. R. B. analysed the data; N. F., V. C. and J. R. B. co-wrote the paper.The authors have filed a patent application on this technology with UCL Business.Supplementary informationClick here for additional data file.


Helicobacter pylori (H. pylori) infection remains high in China though the incidence of inflammatory bowel disease (IBD) has increased. Our aim was to investigate the relationship between the prevalence of H. pylori and inflammatory bowel disease.H. pylori antibody were enrolled. Controls were chose from age- and sex- matched healthy physical examination people who had H. pylori antibody test in a 1:2 fashion (IBD patients:controls). IBD medical history was recorded. All patients were typed by the Montreal classification. Mayo Clinic score and the Harvey-Bradshaw Severity Index were used to evaluate their disease activity. Patients and controls that had H. pylori eradication therapy before were excluded.Hospitalized IBD patients including Crohn’s disease (CD) and ulcerative colitis (UC) who had tested H. pylori infection in IBD patients  and IBD newly diagnosed patients , as well as CD patients  including CD newly diagnosed patients  and UC patients  was significantly lower than controls  , while there was no statistically difference between UC newly diagnosed patients and the controls, and IBD patients with different disease type, disease activity and treatment history.Two hundred and sixty IBD patients including 213 CD patients and 47 UC patients, and 520 controls were involved in this study. The prevalence of H. pylori infection had a negative association with IBD, especially CD. Helicobacter pylori (H. pylori), a common Gram-negative, gastric mucosa colonized microaerobic bacteria, classified as a Class I carcinogen by the World Health Organization, can cause infectious disease and is associated with peptic ulcers, gastric cancer and MALT lymphoma. It has also been reported to be negatively connected to asthma and esophageal adenocarcinoma [H. pylori in China has gradually declined, but still at a high level [H. pylori infection was a protective factor for IBD [H. pylori in colitis [et al. didn’t identify any Helicobactor species in the colonic biopsies of IBD patients by multiple polymerase chain reactions (PCR) and indicated that it was not related to the occurrence of IBD [H. pylori status and IBD patients in China.arcinoma . In recegh level , 3. Meangh level  includin for IBD  and some colitis . Other re of IBD . HoweverH. pylori serology test during their hospitalization in the Department of Gastroenterology of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, between June 2019 and July 2020. Hospitalized IBD patients included newly diagnosed patients (naïve patients), patients with IBD flare, day hospitalization patients for IV infliximab or adalimumab, and regular reexamination patients. The diagnosis of CD and UC was based on the 2018 Chinese Consensus on diagnosis and treatment of inflammatory bowel disease [H. pylori eradication therapy records before were excluded. Previous medical history including age of onset and IBD treatment  was obtained from medical records. CD and UC patients were classified according to The Montreal classification [H. pylori serology test results were collected from medical records and were fully anonymized before, during and after our access. The Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine approved this retrospective study (KY2020-139) and waived the requirement for informed consent.Study population consisted of 260 hospitalized IBD patients, who had fication  and the fication . All of H. pylori infection analysis. Peripheral venous serum samples of patients and controls were tested by H. pylori antibody kit (Latex Immunoturbidimetric method) . Reference range: negative: <22 AU/ml; positive: ≥22 AU/ml.Serology test was used as a reference basis for H. pylori prevalence in China is 55.8% [H. pylori infection is 0.43 [According to previous study, the is 55.8% , and the is 0.43 . The minH. pylori infection and IBD onset. All computations were performed with the SPSS 26.00 statistical software.Categorical variables were analyzed by Chi Square/Fisher’s exact test and continuous variables by T-test/Mann Whitney test. A statistical p value of 0.05 (two sided) was considered significant for all comparisons. Odds ratios (OR) with corresponding 95% confidence intervals (CIs) were calculated for analyzing the association between Two hundred and sixty seven patients (213 CD and 47 UC) and 520 sex- and age-matched controls were enrolled in this study. The detailed demographic and clinical characteristics are shown in H. pylori serology, which were significantly lower than controls  (p = 2.796*10−10). The H. pylori prevalence of CD  and UC  patients was also separately lower than their controls  .Overall, only 25 IBD patients  had positive H. pylori prevalence  was slightly higher than all IBD patients  (p = 0.547), but still lower than the control group  (p = 0.007). And CD naive patients  were also significantly lower than their controls  while the UC ones  has no statistically difference with their controls . . .H. pylori infection rate in CD and UC patients with different ages, lesion parts, disease behaviors, perianal lesions and activity levels. , the infection rate relatively decreased. The reason might be more chance to use antibiotics in the progression and exacerbation of the disease.The low reatment . Our stuH. pylori positive patients and controls, and found H. pylori related to increased fecal microbes diversity [H. pylori may regulate lower digestive tract microbiota through the change of gastric acid secretion [H. pylori chromosomal DNA can prevent colitis by inhibiting the production of type I interferon and IL-12 in the mouse model [H. pylori infection may contribute the pathogeneses of IBD, especially CD.In addition, Frost et al. analyzed fecal microbiota in 212 iversity . Other siversity , 20 incliversity . Therefose model , while tse model . The decH. pylori infection in IBD patients, especially CD, is lower than general population. H. pylori infection might be a protective factor for CD, or it might be two outcomes influenced by some common factors, which still need further study.Our study shows that the prevalence of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced ‘omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests.The parasitic mite  S. scabiei. This NTD has a major adverse impact in disadvantaged communities around the world, particularly when associated with secondary bacterial infections and clinical complications. Here we report the first high-quality genome and transcriptomic data for S. scabiei and explore molecular aspects of S. scabiei/scabies. This genome  provides a solid foundation for fundamental investigations of the molecular biology of the mite, host-parasite interactions and disease processes as well as for translational research to develop new treatments, vaccines and diagnostic tests.Scabies is a highly significant parasitic disease caused by the mite  Sarcoptes scabiei is a parasitic mite of the skin that causes scabies, one of the commonest dermatological diseases worldwide that results in major morbidity, disability, stigma and poverty  that are known to function in dsRNA/siRNA uptake and secondary siRNA dissemination in nematodes [S. scabiei, the presence of the RNA-dependent RNA polymerase coding gene (rdrp) suggested an endogenous synthetic machinery for secondary siRNAs, which might link to a novel spreading mechanism. In addition, although homologous piRNA-binding proteins aubergine (AUB) and PIWI were not detected , small-interfering RNA (siRNA) and/or piwi-interacting RNA (piRNA) pathways , 48. Thenogaster , very li scabiei . The res scabiei . Althougematodes  were notdetected , the genst mites .S. scabiei provides improved insights into the molecular landscape of one of the most important mite pathogens of animals worldwide. This study has inferred molecules involved in host-parasite interactions and immune responses/allergy. The improved genome assembly and associated data sets for S. scabiei should accelerate post-genomic explorations of molecules involved in mite reproduction and development, metabolism, parasite-host interactions, disease pathogenesis, and the genetics and mechanisms of drug resistance.The present genomic and molecular exploration of S. scabiei could assist functional genomic work on selected stages  of the parasite. Given that gene-specific knockdown by double-stranded RNA interference (RNAi) has been demonstrated [S. scabiei but not harm the host animal. Combined with the bioinformatic prediction and prioritisation of essential genes from functional information  available for other metazoan organisms, particularly D. melanogaster, using machine learning approaches [S. scabiei stages provides a powerful functional genomics tool to validate prioritised targets. Focusing on groups of molecules, such as the complex array of peptidases, GPCRs, kinases and ion channels, and understanding their involvement in the host-mite interplay would likely assist in the design of new drugs or a vaccine against scabies. Moreover, future studies should focus on defining a spectrum of key molecules involved in pathways associated with the development of the nervous system in different life-stages of the mite, and on evaluating their potential as drug targets. The availability of a gene knockdown system [in vivo pig-scabies model [in vitro and in vivo. Although the present study focused on S. scabiei, the results and methods employed here should be readily applicable to other ectoparasites of major animal and human health importance. We believe that the substantially improved genome of S. scabiei should accelerate both fundamental and applied investigations of scabies, enabling the development of new interventions for this important neglected tropical disease.Inferring the RNAi machinery in nstrated , we belinstrated . The aimproaches , RNAi-ban system , a drug n system , 57 and es model  provide Animal ethics approval was granted by the QIMR Berghofer Medical Research Institute (permit nos. P630 and P2159) and the Ethics Committee of the Queensland Animal Science Precinct (permit SA 2015/03/504).Sarcoptes scabiei was produced on pigs (3 months of age), isolated and stored using a well-established protocol [S. scabiei-infected pigs, washed extensively, and directly snap frozen and stored at -70°C. In addition, faecal samples (n = 5) were collected from five different batches of mites (same number and stages) isolated from skin crusts taken from pigs on different days; from these faecal samples, crude protein extracts were prepared, freeze-dried and resuspended in 200 μl 8M urea in 100 mM triethylammonium bicarbonate (pH 8.5) with protease inhibitor cocktail set I  [protocol . Mites (Denmark) .Qubit fluorometer dsDNA HS Kit (Invitrogen), according to the manufacturer’s instructions. Genomic DNA integrity was verified by agarose gel electrophoresis and using a Bioanalyzer 2100 (Agilent). Long-read sequencing of libraries constructed using the 20 kb Template Preparation employing BluePippin Size-Selection System was conducted using an established Pacific Biosciences (PacBio) protocol [High molecular weight genomic DNA was isolated from six samples each containing 1,000 motile adults, nymphs, larvae and eggs, collected on different days, using the Gentra Puregene Tissue Kit (Qiagen) according to manufacturer’s instructions. Total DNA amount was determined using a protocol . Short-rprotocol . LibraryS. scabiei var. suis employing the ToTally RNA Kit (Ambion). RNA yields were estimated spectrophotometrically (NanoDrop 1000), and the integrity of RNA was verified using a BioAnalyzer 2100 (Agilent). Following mRNA isolation using the MicroPolyAPurist kit (Ambion), RNA-seq was carried out as described previously [Total RNA was isolated separately from eggs  and mixed larvae, nymphs and adults  of eviously . SequencS. scabiei eggs, nymphs and adults was investigated using an established in-solution digestion protocol [The proteome of faecal matter (“excretome”) from protocol . In brieprotocol , trypticS. scabiei. First, raw LC-MS/MS data were processed with the program MaxQuant using the Andromeda search engine [−8) against the NCBI protein nr database, the allergens identified for S. scabiei var. canis [Dermatophagoides farinae and D. pteronyssinus [Excretory/secretory proteins were inferred from LC-MS/MS  data against the proteome inferred from the genome of h engine . Fixed mh engine  and Multh engine . To clasr. canis , and knonyssinus ; gene moAn established pipeline  was usedS. scabiei protein-coding gene set was inferred utilizing available evidence data, including the transcriptomic data for egg and mixed-sex, motile stages, and protein sequence data were deposited in the UniProtKB/SwissProt database  [de novo and genome-guided transcript assemblies acquired using the Trinity pipeline. Transcripts with unknown nucleotide positions (“Ns”) were removed, and cd-hit-est [de novo-gene prediction program AUGUSTUS [T. urticae were also given to MAKER3 [S. scabiei. Genes inferred to encode peptides of ≥ 30 amino acids in length were preserved. Next, the PASA pipeline [The 4, 2019) . First, 4, 2019)  and simp4, 2019) . Transcr4, 2019) , 78 and 4, 2019)  and RNAs4, 2019) . CAP3-as-hit-est  was used-hit-est , and cd--hit-est . The valAUGUSTUS  that proo MAKER3  to provipipeline  employedpipeline  and tbl2pipeline .S. scabiei, each inferred amino acid sequence was assessed for conserved protein domains using InterPro (release 75.0) [−8) against the following protein databases: Swiss-Prot within UniProtKB [First, following the prediction of the protein-coding gene set for se 75.0)  employinniProtKB ; Kyoto EniProtKB , 90; andniProtKB . Genes e−8) [Gene models were curated employing protein domain architecture information from the InterPro database (release 75.0) and from transcriptomic data. Kinase gene models were curated using an established approach –i.e. kin−8)  and manu−8)  and InteS. scabiei were inferred using the program RepeatModeler [S. scabiei genome assembly using the program RepeatMasker [Genomic repeats specific to tModeler  that mertModeler  and RepetModeler . Custom tModeler  were theatMasker .S. scabiei, T. urticae and D. pteronyssinus were inferred by comparison among all proteins using the program OrthoMCL v2.0.4 . The counts for shared homologous genes among these species were displayed in a Venn diagram.Homologs among S. scabiei var. suis, S. scabiei var. canis [D. pteronyssinus [Metaseiulus occidentalis [Psoroptes ovis [Tetranychus urticae [http://tree.bio.ed.ac.uk/software/figtree).Single-copy orthologous (SCO) genes were inferred from homologous genes shared by r. canis , D. pternyssinus , Metaseidentalis , Psoropttes ovis  and Tetr urticae , and con urticae , employi urticae  and MAFF urticae  for the  urticae  for the  urticae  were mer urticae  to assig urticae  from fou urticae  and the D. pteronyssinus [S. scabiei var. canis [S. scabiei var. suis and T. urticae [Density diagrams were created using standard commands in the R language . Gene-, nyssinus , S. scabr. canis , S. scab urticae .S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.