Atherosclerosis is a complex disease process. It is increasingly recognised that both lipoprotein retention and inflammatory cellular components are intricately related in the initiation and development of atherosclerotic plaque. LDL-c (cholesterol) has been long established as a cause for atherosclerosis; additionally, inflammatory cells such as monocytes and subsequently foam cells have also been directly linked to the progression of atherosclerotic disease. Emerging data suggest that structures outside vascular intima and media are also closely related to atherosclerosis. Perivascular adipose tissue (PVAT) may be a determinant of the inflammatory status of the atherosclerotic plaque. All these features are becoming extremely relevant as therapies against atherosclerosis are targeting both lipid retention and inflammation. Recently, there has been some success in these novel therapies, such as the proprotein convertase subtilisin-kexin type 9 (PCSK-9) inhibitor evolocumab and the interleukin-1ß neutralising antibody, canakinumab, in reducing cardiovascular events when added to standard therapy such as statin. This review will discuss the pathogenesis of atherosclerosis, including some novel features, and its management using new anti-atherosclerotic drugs. Atherosclerosis is a complex disease with multiple processes implicated in early atherogenesis. Lipoprotein retention within the intima of the vascular wall coupled with inflammatory flux of monocytes and lymphocytes in addition to changes in endothelial function and migration of vascular smooth muscle cells leads to the formation of atherosclerotic plaque . Early eProgression of atherosclerotic plaque is accelerated by the increased number of intimal monocytes and the ingestion of LDL particles by macrophages generating lipid-rich foam cells . The endP < 0.001) [P = 0.018) [Lipoprotein retention is the cornerstone of the development of atherosclerosis , 11. The< 0.001) . Moreove< 0.001) , 16. Lik< 0.001) . The Fra< 0.001) . In addi< 0.001) . Subsequ< 0.001) –21. Howe< 0.001) . While p< 0.001) , 24. Non< 0.001) . The res< 0.001) . The ear< 0.001) . Followi< 0.001) . The inc< 0.001) . However< 0.001) . In some< 0.001) . Secondl< 0.001) . Reverse< 0.001) . Yet, re< 0.001) . Moreove< 0.001) . To the = 0.018) . Subsequ= 0.018) . This qu= 0.018) .+CD16− and CD14lowCD16+. The former one is presumably more inflammatory and classically considered to be the source of M1 macrophages responsible for marinating chronic inflammatory state [LDL-c retention within the intima of vascular wall triggers a cascade of inflammatory cellular influx mainly monocytes. The susceptible LDL particles to oxidation are chemo-attractant to monocytes, lymphocytes and vascular smooth muscle cells . In addiry state . This crry state . The funry state . Vasculary state . Extracery state .h1 response also enhances protease and chemokine secretion, with less formation of collagen fibre, upregulation of adhesion molecules and activation of macrophages and endothelial cells [h2 response has been less clear with some studies suggesting protective role while others showing neutral effects [Despite the central role of monocytes/ macrophages, other cell types take part in the development of atherosclerosis, including dendritic, eosinophils, mast cells and lymphocytes. The adaptive immune system (including T and B lymphocytes) secretes a number of cytokines, depending on their local environment, which has an impact on atherosclerotic plaque. These cytokines are either pro-inflammatory like IL1ß, IL6 and TNF or promote resolution of inflammation such as IL4 and IL10 . The proal cells . On the  effects .Circulating microparticles have also been shown to play a significant role in the pathogenesis of atherosclerosis . These vAtherosclerosis is a disease affecting the vascular media and intima. Nonetheless, structures like the adventitia, through vasa vasorum, or more recently PVAT have been recognised as active participants in the progression of atherosclerotic plaque . The priDespite better understanding into the pathogenesis of atherosclerosis, it continues to be the most common cause of death worldwide . TargetiThis ongoing debate has probably put into a halt following data from ezetimibe in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT), where incremental cardiovascular benefits were demonstrated when adding a non-statin drug . CombiniP < 0.001). These benefits were derived from reduction in non-fatal myocardial infarction, ischaemic stroke and unstable angina but not on cardiovascular mortality. Interestingly, there was a statistically significant reduction in all-cause mortality.More recently, PCSK9 inhibitors have led to unprecedented decrease in LDL-c which was translated into reduction in cardiovascular events . PCSK9 iP = 0.021) [P = 0.02) [The recent advances in pharmacotherapies tackling lipoproteins were also matched with similar progress in the inflammatory pathway of atherosclerosis. Canakinumab is a human monoclonal antibody against IL1ßwhich was studied in the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) trial . There w= 0.021) . However = 0.02) . This la = 0.02) , 57. Thi = 0.02) , 57.The recent relative success of novel therapies in reducing cardiovascular events has promoted an important debate as to whether these drugs are applicable for the real world. These drugs have typically produced an absolute risk reduction of 1–2% of combined clinical outcomes, including death, myocardial infarction, stroke, unstable angina and coronary revascularisation. This reflects a small margin of benefits challenging their unselective use in atherosclerotic patients. Numerous factors may explain the modest effects of these drugs. Firstly, all these therapies have been tested against placebo in statin-treated patients. The significant effects when using statin in reducing cardiovascular events would make detection of any potential benefits more challenging. Moreover, the current guidelines mandate the use of anti-platelet therapy in addition to angiotensin converting enzyme inhibitors and beta blocker aggregating further reduction in vascular events . This woOne promising strategy is to characterise atherosclerotic disease and to identify the prominent disease substrate, e.g. lipid accumulation and inflammation, and to match a drug with a mechanism of action that targets the particular substrate , 22. SucThere is currently better understanding regarding the pathogenesis of atherosclerosis. Although significant progress has been made since Anitschkow’s work, the main pathways of atherosclerotic plaque continue to be lipid retention and inflammation. Novel successful therapies targeting atherosclerosis are promising, but further refinements are required prior to their application in the real world.


Scutellaria indica var. coccinea (Lamiaceae) is an endemic taxon in Jeju Island, Korea. We present the complete chloroplast genome sequence of S. indica var. coccinea. Its genome size is 151,956 bp in length comprising a large single copy of 83,951 bp, a small single copy of 17,539 bp, and a pair of inverted repeats measuring 25,233 bp. The complete genome contains 115 genes including 80 protein-coding genes, four rRNAs, and 31 tRNAs. Phylogenetic analysis of this species with 10 published chloroplast genomes in Lamiaceae showed that S. insignis is a sister to S. indica var. coccinea. Scutellaria L. is the second largest genus in the Lamiaceae with approximately 470 recognized species that are mainly distributed in temperate regions. Plants are annual or perennial herbs and rarely subshurubs or aquatic species. Eighteen taxa of Scutellaria are distributed in Korea  of Jeju Island, which is located to the south of the Korean peninsula . In this species-group category, S. indica L. is included under ‘S. violacea species-group’ of the section Scutellaria, which is characterized by nutlets with acuminate papillae terminating in hooks. In S. indica, S. indica var. coccinea is distinguished from other varieties having widely cordate leaves, conspicuously sunken veins in adaxial leaf surface and dark pink corolla color.The recent classification system of ns Paton . In thisS. indica var. coccinea (GenBank accession: MN047312). Genomic DNA was extracted from leaves of a plant cultivated in Sungshin University, which is propagated from seeds of a wild plant collected in Jeju Island . Voucher specimen is deposited in Sungshin University herbarium (S. Kim 2015-0298). The whole genome sequencing was conducted with 100 bp paired-end reads on the BGISEQ-500 sequencer . To obtain the chloroplast genome (cp) sequence, paired-end reads were mapped against a reference cp genome (GenBank accession: NC_028533) which is previously reported from Scutellaria insignis using Geneious  of 83,951 bp, a small single copy (SSC) of 17,539 bp, and a pair of inverted repeats (IR) measuring 25,233 bp. It includes 115 genes comprising 80 protein coding genes, four rRNA genes and 31 tRNA genes. Among these genes, 16 genes carry a single intron, and two genes (clpP and ycf3) contain two introns, respectively.The complete cp genome of Scutellaria and seven in other Lamiaceae; S. indica var. coccinea is a sister to S. insignis. In this study, we report the cp genome of S. indica var. coccinea as an endemic taxon in Korea. The findings contribute to the conservation of this species and the phylogenetic study of Scutellaria.The phylogenetic tree was reconstructed with this cp genome and previously published cp genomes (three in


Helicobacter pylori (H. pylori) infection and the risk of developing irritable bowel syndrome (IBS) has yet to be investigated; thus, we conducted this nationwide cohort study to examine the association in patients from Taiwan. Methods: A total of approximately 2669 individuals with newly diagnosed H. pylori infection and 10,676 age- and sex-matched patients without a diagnosis of H. pylori infection from 2000 to 2013 were identified from Taiwan’s National Health Insurance Research Database. The Kaplan–Meier method was used to determine the cumulative incidence of H. pylori infection in each cohort. Whether the patient underwent H. pylori eradication therapy was also determined. Results: The cumulative incidence of IBS was higher in the H. pylori-infected cohort than in the comparison cohort . After adjustment for potential confounders, H. pylori infection was associated with a significantly increased risk of IBS (adjusted hazard ratio (aHR) 3.108, p < 0.001). In addition, the H. pylori-infected cohort who did not receive eradication therapy had a higher risk of IBS than the non-H. pylori-infected cohort . The H. pylori-infected cohort who received eradication therapy had a lower risk of IBS than the comparison cohort . Conclusions: Based on a retrospective follow-up, nationwide study in Taiwan, H. pylori infection was associated with an increased risk of IBS; however, aggressive H. pylori infection eradication therapy can also reduce the risk of IBS. Further underlying biological mechanistic research is needed.Background: The association between  Irritable bowel syndrome (IBS), a functional disorder of the gastrointestinal tract, is formalized in the Rome criteria, which include chronic abdominal pain and altered bowel habits . IBS is Helicobacter pylori (H. pylori) infection has a potential pathogenic role in IBS given the significant overlaps between IBS and dyspepsia [H. pylori, which is a widespread gram-negative bacterium that colonizes the human gut [H. pylori infection irritates immune neutrophils and lymphocytes in gut epithelial cells and liberates cytokines, including IL-1, IL-6, IL-8, IL-12, TNF-α and IFN-γ [H. pylori infection rates in patients with IBS compared to healthy groups [H. pylori infection and IBS [H. pylori infection and IBS [H. pylori-infected population in Taiwan.The pathophysiology of IBS is multifactorial and remains uncertain . Recentlyspepsia . Approxiuman gut . H. pylond IFN-γ . Some sty groups ,15. Howe and IBS ,17. The  and IBS . TherefoIn 1995, the government of Taiwan implemented a single-payer universal health insurance system, the Taiwan National Health Insurance (NHI) program, which covers more than 99% of the 23 million residents in Taiwan and has been widely used in academic studies . The NHIH. pylori infection (ICD-9-CM codes 041.86) in the period from 2000 to 2013. The ICD-9-CM diagnosis of H. pylori infection was based on endoscopic biopsy urease testing or non-invasive tests, such as urea breath testing. The diagnosis of H. pylori infection was confirmed at least twice. H. pylori infection is usually treated with triple eradication therapy  for at least two weeks to help prevent the bacteria from developing resistance to one particular antibiotic. Those with a documented H. pylori infection before 1 January 2000 or with incomplete medical information were excluded to ensure the data were from individuals with a first diagnosis of H. pylori infection. In Taiwan, the diagnoses of IBS (ICD-9-CM code: 564. 1) were made by board-certified gastroenterologists, and were diagnosed according to the serial versions of the Rome criteria [H. pylori infection. For each identified patient with H. pylori infection, four comparison patients were randomly identified and frequency-matched according to the age, sex, and year of index date for the non-H. pylori-infected cohort.As shown in criteria . The NHIcriteria . We alsoThe sociodemographic variables used in this study comprised age, sex and urbanization level. The level of urbanization was divided into four levels (Level 1 is the most urbanized and level 4 is the least urbanized) based on the NHRID report. We defined baseline comorbidities, coronary artery disease (ICD-9-CM codes 410-414), cerebrovascular accident (ICD-9-CM codes 430-438), hypertension (ICD-9-CM codes 401-405), hyperlipidemia (ICD-9-CM codes 272), diabetes (ICD-9-CM codes 250), end-stage renal disease (ICD-9-CM codes 585.6), asthma (ICD-9-CM codes 493) and medication use of tetracycline , minocycline  and doxycycline .Helicobacter pylori infection (HPI) cohort and the comparison cohort using the chi-squared test to examine categorical variables and the t-test to examine continuous variables. Kaplan–Meier analysis was performed to estimate IBS in these two cohorts, and the log-rank test examined the difference between the curves. We computed the incidence density rate of IBS  at follow-up for each cohort. Univariable and multivariable Cox proportion hazard regression models were used to examine the effect of HPI on the risk of IBS, shown as a hazard ratio (HR) with a 95% confidence interval (CI). The multivariable models were adjusted for age, sex, urbanization level and all comorbidities. Further analysis was performed to assess the effect of the dose response of H. pylori infection to the antibiotic medicine that was prescribed on the risk of IBS. All statistical analyses were performed using SPSS software . The comparisons used the significance level of 0.05 for two-sided testing.The distribution of sociodemographic data and comorbidities were compared between the http://www.mohw.gov.tw/cht/DOS/DM1.aspx?f_list_no=812).Data are available from the National Health Insurance Research Database (NHIRD) published by the Taiwan National Health Insurance (NHI) Administration. Due to legal restrictions imposed by the government of Taiwan in relation to the “Personal Information Protection Act”, data cannot be made publicly available. Requests for data can be sent as a formal proposal to the NHIRD .p = 0.593). In addition, there was no significant difference in sex or age for both groups. Regarding insurance premiums (in New Taiwan dollars (NT$)) in both cohorts, almost all of the enrolled patients were in the <18,000 group (98.25%), followed by the 18,000 to 34,999 group (1.44%) and the >35,000 group (0.31%). There was no significant difference in the insurance premiums (NT$) in either group (p = 0.136). In terms of the comorbidity, patients with H. pylori infection had lower rates of coronary artery disease (CAD), cerebrovascular accident (CVA), hypertension (HTN), hyperlipidemia, diabetes mellitus (DM) and asthma than the control patients. The CCI_R value was 1.27 ± 0.77 in the study cohort and 0.27 ± 0.69 in the control cohort (p < 0.001). In addition, more individuals in the study cohort than in the control cohort lived in southern and eastern Taiwan and lower urbanized areas, and received therapy in local hospitals (p < 0.001).H. pylori infection, sex, age group, insurance premium and pre-existing comorbidities, including sleep apnea, HTN, and only H. pylori infection  = 1.934–4.995, p < 0.001), patients in the study cohort showed an increased risk of IBS diagnosis compared with the control cohort. In addition, patients with CVA had a decreased risk of developing IBS than those without CVA . Patients with other comorbidities, age groups and insurance premiums, as well as other chronic diseases, were not significantly associated with IBS diagnosis according to the hazard ratios .The IBS rate was calculated by the Kaplan–Meier method . The resH. pylori infection . In the age group analysis, patients with H. pylori infection in the 44 to 64-year age group were independently associated with an increased risk following IBS diagnosis compared with patients without H. pylori infection . In the insured premium group analysis, H. pylori-infected patients in the <18,000 group showed an increased risk of developing IBS . In the pre-existing condition group analysis, H. pylori-infected patients with CAD, CVA, HTN or DM displayed an increased risk of developing IBS . In the season group analysis, patients infected with H. pylori in the summer and autumn groups showed an increased risk of developing IBS . In the level of care group analysis, H. pylori-infected patients in the hospital center and regional hospital groups displayed an increased risk of developing IBS compared with those in clinics . Patients with H. pylori infection were associated with a higher risk of IBS than patients without H. pylori infection, and patients with H. pylori infection who received aggressive eradication therapy had a decreased risk of IBS compared with patients with H. pylori infection who did not receive eradication therapy   . H. pylori infection and the risk of IBS. After adjusting for covariates, the overall adjusted HR was 3.108 . In other words, adult patients with H. pylori infection had a nearly 3.1-fold increased risk of developing IBS than those without H. pylori infection. The Kaplan–Meier analysis revealed that the study subjects had a significantly higher 14-year IBS rate than the controls after adjusting for potential confounders; this is further strong evidence for a potential association between IBS and H. pylori infection.This is the first large-scale, population-based study with long-term follow-up on the association between H. pylori can produce bacterial urease, which hydrolyses in gastric luminal urea to form ammonia, which helps neutralize gastric acid and form a protective cloud around the organism, enabling it to penetrate the gastric mucus layer [H. pylori infection and the incidence of gastric cancer and colorectal cancer [H. pylori includes endoscopic biopsy urease testing, urea breath testing, stool antigen assay, and serological ELISA testing [H. pylori infection is usually treated with at least two different antibiotics at once to help prevent the bacteria from developing resistance to one particular antibiotic [us layer . Severall cancer ,27. Diag testing . H. pylotibiotic . Triple H. pylori infection. It seems that chronic inflammation caused by H. pylori infection is related to the pathogenesis of atrophic gastritis, intestinal metaplasia and peptic ulcers [H. pylori infection may increase inflammatory marker levels [The pathogenesis of IBS is still uncertain. In our study, we tried to find a relationship between IBS and c ulcers . H. pylor levels , increasr levels ,33,34, cr levels ,36,37,38H. pylori infection who did not receive eradication therapy had a higher risk of IBS than the non-H. pylori-infected cohort. . Patients with H. pylori infection who received eradication therapy had a lower risk of IBS than the untreated group . In the long term observation, the risk of IBS is reduced in the H. pylori infection-treated population. There seems to be some connections between H. pylori infection and IBS. Clinically, it is also possible to add a supplemental H. pylori test for drug treatment to reduce the symptoms of IBS.According to our study, patients with H. pylori has been challenged. For example, H. pylori is thought to commonly influence the upper GI tract rather than the lower GI tract. Additionally, findings by Xiong indicate that there is no relationship between H. pylori infection and IBS [H. pylori infection globally [H. pylori infection drugs fail or resistance develops in approximately 10% of patients [The association between IBS and  and IBS . Howeverglobally . Clinicapatients , and theThis study has a number of strengths. First, the sample size was very large, which enhanced the statistical power of the data. We used stratified analyses based on the confounding factors of age, comorbidities, and a wide range of demographic characteristics. Second, because we used a nationwide database with a very high coverage rate, almost all patients’ follow-up data were available. Third, the population-based data were considered representative of the general population in Taiwan.Our study also had some limitations. First, this was a retrospective cohort study; therefore, it has lower statistical quality. Bias from unknown confounders and errors of primary records may have affected our results, and a well-designed prospective, randomized, controlled study is needed to help establish causal relationships. Second, the NHIRD does not report information such as clinical symptoms, signs, or laboratory data, which may help distinguish different types of IBS for further analysis. H. pylori infection have a higher proportion of IBS, and aggressive H. pylori infection eradication therapy can reduce the risk of IBS. Further underlying biological mechanistic research is needed. Clinically, IBS patients seem to be able to undergo routine H. pylori diagnostic tests to determine the direction of treatment.Patients with


Rheum officinale Baill. was determined based on the Illumina Sequencing data. The cp genome is 161,563 bp in length. The overall G + C content of the cp genome was 37.31%. The R. officinale cp genome contains 129 genes, including 84 protein-coding genes, eight rRNA genes (four rRNA species), and 37 tRNA genes (20 tRNA species). A neighbour-joining phylogenetic tree clarified that the cp genome of R. officinale was closely related to that of R. palmatum in Polygonaceae.The complete chloroplast (cp) genome of  Rheum officinale Baill., a valuable medicinal herb, is widespread in Shaanxi, Sichuan, Hubei, Guizhou, Yunnan, and Henan provinces of China. The root and rhizome of called Rhei Radix et Rhizoma is a very important traditional Chinese medicine, possessing many pharmacological actions, such as purgative and cathartic, anti-oxidative, and anti-inflammatory, etc. . A voucher specimen (Ro170820LY) was deposited at Shaanxi University of Chinese Medicine Herbarium. Total genomic DNA of fresh leaves from a single individual plant was extracted with EASYspin plus Total DNA Isolation Kit  following the manufacturer’s instructions and 2 μg genome DNA was sequenced using Illumina Hiseq 2500 Sequencing Platform  at Majorbio Biotechnologies Inc. .R. officinale was reconstructed with MITObim v1.8  was set as the reference  with standard settings. The cp genome of R. officinale cp genome is a double-stranded circular DNA of 161 563 bp in length and deposited in GenBank under the accession of MK674495. Its structure is consistent with most cp genomes in various plant species , eight ribosomal RNA (rRNA) genes (four rRNA species), and 37 transfer RNA (tRNA) genes (20 tRNA species). Most of the gene species occur as a single copy while 15 gene species occur in double copies, including all rRNA species, five tRNA species, and six PCG species.The R. officinale and R. palmatum are phylogenetically related to each other (R. officinale and its phylogenetic tree analysis clarified here should be useful for the population genetics, molecular ecology, molecular authentication, and evolution studies of genus Rheum.A neighbour-joining phylogenetic tree constructed with 55 proteins-coding genes extracted from the cp genomes of 14 taxa suggested that ch other . The cp


Horseshoe crabs are ancient marine arthropods with a long evolutionary history extending back approximately 450 million years, which may benefit from their innate immune systems. However, the genetic mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear.Tachypleus tridentatus with 24,222 predicted protein-coding genes. Comparative genomics shows that T. tridentatus and the Atlantic horseshoe crab Limulus polyphemus have the most orthologues shared among two species, including genes involved in the immune-related JAK-STAT signalling pathway. Divergence time dating results show that the last common ancestor of Asian horseshoe crabs (including T. tridentatus and C. rotundicauda) and L. polyphemus appeared approximately 130 Mya (121–141), and the split of the two Asian horseshoe crabs was dated to approximately 63 Mya (57–69). Hox gene analysis suggests two clusters in both horseshoe crab assemblies. Surprisingly, selective analysis of immune-related gene families revealed the high expansion of conserved pattern recognition receptors. Genes involved in the IMD and JAK-STAT signal transduction pathways also exhibited a certain degree of expansion in both genomes. Intact coagulation cascade-related genes were present in the T. tridentatus genome with a higher number of coagulation factor genes. Moreover, most reported antibacterial peptides have been identified in T. tridentatus with their potentially effective antimicrobial sites.Here, we describe the 2.06 Gbp genome assembly of T. tridentatus would provide important evidence for further clarifying the taxonomy and evolutionary relationship of Chelicerata. The expansion of conserved immune signalling pathway genes, coagulation factors and intact antimicrobial peptides in T. tridentatus constitutes its robust and effective innate immunity for self-defence in marine environments with an enormous number of invading pathogens and may affect the quality of the adaptive properties with regard to complicated marine environments.The draft genome of  Horseshoe crabs are marine arthropods, representing an ancient family with an evolutionary history record extending back approximately 450 million years . Based oTachypleus tridentatus , an extant horseshoe crab species, is mainly distributed from coastal Southeast China to western Japan and in a few islands in Southeast Asia [T. tridentatus relies entirely on its innate immune system, including haemolymph coagulation, phenoloxidase activation, cell agglutination, release of antibacterial substances, active oxygen formation and phagocytosis [ast Asia . Similarocytosis –8, whichocytosis . Previouocytosis , 12. CurLimulus polyphemus (Linnaeus 1758), is the most extensively investigated species of horseshoe crabs, occupying a large latitudinal range of coastal and estuarine habitats along the west Atlantic coast from Maine to Florida in eastern North America and along the eastern Gulf and around the Yucatán peninsula of Mexico [L. polyphemus with a high assembly quality has published, focusing on the full repertoire of Limulus opsins, which could provide insight into the visual system of horseshoe crabs [T. tridentatus but also of the xiphosuran lineage and try to reduce errors of only using a single draft-quality genome, the comparative genomic study of immune systems within T. tridentatus and L. polyphemus were included.The Atlantic horseshoe crab, f Mexico , 13, 14.oe crabs . In ordeT. tridentatus genome sequence together with comparative genomic and divergence time analyses on other available Chelicerata genomes to date, including the previously released L. polyphemus assembly [Here, we present an analysis of the assembly . ParticuT. tridentatus was sequenced to 124× coverage and assembled into a 2.06-Gb genome. The k-mer analysis yielded an estimated genome size of 2.22 Gb with a depth peak of 78×. The final draft assembly consists of 143,932 scaffolds with an N50 scaffold size of 165 kb, among which the longest scaffold size is 5.28 Mb and the shortest is 1 kb. The GC content of the genome is 32.03% . To estimate of repeat sequences which are more difficult to detect in the draft assembly, RepeatModeler [T. tridentatus, including a 13.26% proportion of transposable elements. Meanwhile, long interspersed elements (LINEs) composed the largest portion at 6.21%. LTR elements (1.72%) and DNA elements (5.33%) were also detected in the T. tridentatus genome. To determine the reliability of the repeat contents screening by RepeatMasker and RepeatModeler, we also performed repeat analysis of the L. polyphemus genome for reference. Similar results were obtained with the identification of repeat sequences representing 1.11 and 34.24% in L. polyphemus, respectively. Given that RepeatMasker use similarity of known repeat sequences in the Repbase database to identify repeats in the input sequence, this suggests that the repeat sequences from horseshoe crabs have a great difference compared with existing homologous repeats.The screening of repeat contents from the RepeatMasker  analysistModeler  was usedT. tridentatus genome assembly was assessed using the transcriptome data of the embryonic sample at Stage 21 (the hatch-out stage) of T. tridentatus [− 30. To further confirm the completeness of the predicted genes, the commonly used genome assembly validation pipeline BUSCO [T. tridentatus reveals 98.7% conserved proteins of homologous species with 1052 BUSCOs . Only 1.3% of the benchmarked universal single-copy orthologous groups of arthropod genes were missing in the assembly. This demonstrated that most of the evolutionarily conserved core genes were found in T. tridentatus genome, suggesting a remarkable completeness of genome assembly and predicted gene repertoire of T. tridentatus.The completeness of the dentatus . It was L. polyphemus assemblies have been previously documented [T. tridentatus and L. polyphemus. Of these, 10,968 orthologues contained genes found in both horseshoe crab genomes, with 15,905 T. tridentatus and 20,390 L. polyphemus genes included; moreover, approximately 6880 of the shared genes were single-copy. Functional enrichment analysis showed that these shared genes were involved in several important pathways , such as metabolic pathways , ribosome biogenesis and DNA replication. The analysis also identified 1418 protein-coding genes that were only present in T. tridentatus. In total, 1956 genes were specific to L. polyphemus. To place T. tridentatus with the most current understanding of the evolution of Chelicerata species, phylogenetic and comparative genomic analyses of T. tridentatus and 11 other Chelicerata as well as one Myriapoda outgroup were conducted. The phylogenetic tree was rooted using the centipede S. maritima as the outgroup  and 2648 (21.5%)). Pathway enrichment of these genes showed significant enrichment  for neuroactive ligand-receptor interaction, FoxO signalling pathway and AGE-RAGE signalling pathway in diabetic complications. The latter two KEGG pathways include the important JAK-STAT signalling pathway genes related to innate immunity in arthropods. With respect to species-specific genes, 1124 genes were unique to T. tridentatus. C. sculpturatus had the most (7328) expanded species-unique genes, followed by 6247 N. clavipes-specific gene families. In contrast, only 161 genes were unique to T. mercedesae. The numbers of species-specific genes in T. tridentatus and L. polyphemus were in between, with 1124 and 857, respectively. Nevertheless, considering the fragmentation of the draft genome, there may be unidentified coding genes in the analysed genomes. The species-specific genes described here only refer to the results based on the draft genomes.Two cumented , 22, onecumented  calculatT. tridentatus) and L. polyphemus was dated to 130 Mya (121–141) and that the split of the Asian horseshoe crabs T. tridentatus and C. rotundicauda was dated to 63 Mya .The divergence time estimate results for the 7 Chelicerata species showed that the last common ancestor of Asian horseshoe crabs , thioester-containing proteins (TEPs), fibrinogen-related proteins (FREPs), down syndrome cell adhesion molecules (Dscams), galectins and C-type lectins (CTLs). The results revealed 42 FREPs and 117 Dscams in T. tridentatus that were extensively present in both horseshoe crab genomes with functional domains.Immune-related genes can be broadly classified into pattern recognition receptors (PRRs), signaling transduction pathways and effectors. We manually searched the ion Fig. . We examT. tridentatus and L. polyphemus. We found that IMD and JAK-STAT pathway genes in T. tridentatus and L. polyphemus exhibited a certain degree of expansion. The orthologue analysis for shared genes in horseshoe crabs with their close evolutionary related species showed that horseshoe crabs have the most unique (more than twenty percent) uniquely shared gene orthologues, including the abovementioned expanded gene families.Recognition of PAMPs by PRRs triggers signal transduction pathways through transcriptional activation. All known gene family components that play important roles in innate immune signal transduction in arthropods  –41 are pT. tridentatus and L. polyphemus. For Dredds, the phylogeny tree shows one branch including 7 corresponding genes identified in the two horseshoe crabs and 1 gene in C. sculpturatus. Another branch encompasses 2 genes in P. tepidariorum , MAPKKK transforming growth factor - β (TGFβ) - activated kinase 1 (Tak1) and Relish proteins within rum Fig. a. The Drion Fig. b. Moreov shrimps . Plausibrum Fig. c.Fig. 4T. tridentatus host defence system is the production of antimicrobial peptides, which act as innate immune effectors [T. tridentatus genome for antimicrobial peptide genes and identified most of the antibacterial peptides that have been reported, including one anti-LPS, two tachyplesin and two big defensin peptides  region between G23 to R83 with two conserved cysteine residues as well as a hydrophobic NH2-terminal and cationic residues clustered in its disulphide loop, which are supposed to act as an affinity site in combination with LPS [Bonamia ostreae, Perkinsus marinus and Vibrio P1, and can also have strong inhibitory effects on the growth of fungi [T. tridentatus, each of which consists of 77 amino acids encompassing a putative signal peptide sequence, a mature tachyplesin peptide sequence, a C-terminal arginine followed by the amidation signal residues Gly-Lys-Arg and a 22-aa peptide in the C-terminal portion [T. tridentatus genome, one of which is 118 amino acids in length and contains a hydrophobic N-terminal half and a cationic C-terminal half, which may be closely related to its biological activity for broad antimicrobial properties [The anti-LPS gene found in the with LPS , 45. Theof fungi , 47. In operties .T. tridentatus and L. polyphemus have all the coagulation-related genes while other related species lack a part of the coagulation pathway  but higher than that from L. polyphemus  [Limulidae and T. tridentatus lineages was congruent approximately 121–141 Mya, and the lineages of the two Asian horseshoe crabs T. tridentatus and C. rotundicauda was also congruent approximately 57–69 Mya. According to the continental drift theory, before the Triassic Period, virtually all continents were joined to form the supercontinent Pangea, with the breakup of Pangea commencing in the Triassic Period [L. polyphemus. The second group migrated to the east through the Tethys, is found along Asia from Japan to India, and evolved into T. tridentatus, T. gigas, and C. rotundicauda. There is evidence showing that the India–Asia collision was underway in northern Pakistan ca. 56–55 Mya [T. tridentatus and C. rotundicauda may be related to the India–Asia collision.The draft genome of oe crabs , 53. Usi –20. Consc Period . Approxic Period . The subc Period , amphibic Period , mammalsc Period  and evenc Period  matches c Period , but untc Period . The ancc Period , 64. Aft6–55 Mya . The divT. tridentatus and L. polyphemus genomes in this study revealed two Hox gene clusters, suggesting that one round of whole-genome duplication occurred in T. tridentatus and L. polyphemus. Considering that draft genome data was utilized, the identification and scaffold positions in the Hox genes analysis in both horseshoe crab genomes probably provide a minimum estimate. More intact Hox clusters may be recovered with more complete assemblies; thus, we can assume that at least one round of whole-genome duplication occurred in both T. tridentatus and L. polyphemus.Hox genes have been well studied to play essential roles in determining the anterior-posterior axis as well as organogenesis during embryonic development , 25. ResL. polyphemus genomes and our presented T. tridentatus genome. Among them, the immune system undoubtedly provides an important guarantee. We searched the two horseshoe crab genomes to investigate the molecular basis of immune signalling pathways, which affect the specificity of foreign pathogen recognition and performance for releasing immune response substances. Genes that are not found in the T. tridentatus genome were supplemented by transcriptome data to restore as accurate of numbers as possible. Although the draft genome is incomplete, the gene count errors should be small enough for the predicted genes according to the BUSCO evaluation results of 98.7% conserved homologous proteins. From the results we have obtained, the two horseshoe crabs showed high sequence homology for most immune signaling-related gene families, which have been studied previously in other arthropods. Homologs of the FREP and Dscam families play an important role, and pattern recognition receptors with their corresponding functional domains were extensively present in the two horseshoe crab genomes. The Dscam family in arthropods has evolved to recognize a variety of pathogens, and this is supported by the abundant receptor diversity due to alternative splicing of hypervariable regions [T. tridentatus transcriptome suggest an effective ability to recognize a broad range of pathogens, which may be inducted to cope with a great diversity of invading microorganisms in the marine environment. Pathogen recognition would further lead to the activation of signal transduction and the amplification of immune responses, thus producing immune factors that are resistant to microbial activity. Several gene family members in the IMD and JAK-STAT signalling transduction pathway exhibit a certain degree of expansion in T. tridentatus and L. polyphemus. Research in Drosophila has shown that the IMD pathway preferentially recognizes the peptidoglycan molecules present on the surface of Gram-negative bacteria through peptidoglycan recognition receptors (PGRPs), leading to the generation of specific AMPs. The JAK-STAT pathway in arthropods, which is analogous to a cytokine-signalling pathway in mammals, has been implicated as having defence abilities against viral, bacterial or protozoan pathogens [T. tridentatus has strong signal reception and lineage-specific signal transduction abilities. The interactions among the components in T. tridentatus should be paid more attention.Horseshoe crabs are an independent ancient group with distributions in Southeast Asia and North America. There must be inherent reasons for their survival against complicated marine environmental changes. Some clues could be found from the comparison of the previously determined  regions –30. The athogens –33. The T. tridentatus assembly, including anti-LPS, tachyplesin, and big defensin peptides. However, in the L. polyphemus genome, we failed to find any of the tachyplesin family genes, probably because these antimicrobial peptides are usually shorter and have a high degree of species specificity. There is increasing evidence that AMPs in horseshoe crabs not only possess broad-spectrum antimicrobial capabilities but also have a strong resistibility to enveloped viruses, parasites and tumour cells [T. tridentatus.The generation of AMPs is an important aspect of immune responses in horseshoe crabs. The transcription of specific AMPs, which act as important innate immune effectors, are activated through the Toll, IMD, JAK-STAT or JNK pathway based on the recognition of bacteria, fungi, viruses or parasites. We identified most of the antimicrobial peptides isolated in previous studies in ur cells –69. Moreur cells –72. ThusT. tridentatus assembly, indicating a complete coagulation cascade in horseshoe crabs. Other Chelicerata species can lack factor G and coagulogen. This might be because of a limited annotation of the draft genome assemblies or there might be other special biosensors involved in their antifungal recognition process. Although the more direct internal cause for the long-term evolutionary conservation and success of horseshoe crabs with abilities to maintain morphological stability in a fluctuating marine environment may be the combination of a wide feeding spectrum, substantial saline tolerance and insensitivity to temperature by horseshoe crabs [Serine protease-dependent haemolymph coagulation is a major component of the innate immune system in horseshoe crabs. Activation of the horseshoe crab coagulation cascade consists of four coagulation factors including factor C, factor B, factor G and proclotting enzyme , 74. Facoe crabs , 76, theoe crabs , 78.T. tridentatus was sequenced into a 2.06 Gb of genome consisting of 143,932 scaffolds with an N50 scaffold size of 165 kb. In total, 24,222 protein-coding genes were conservatively predicted as present in the T. tridentatus genome, revealing 98.7% completeness with 1052 BUSCOs.Our draft assembly of T. tridentatus genome included phylogeny analysis and divergence time dating using newly published Chelicerata species, as well as selective analysis of Hox genes, innate immunity-related genes, coagulation factors and antimicrobial peptides. We found that the two horseshoe crabs T. tridentatus and L. polyphemus had the most orthologues shared among two species, and were enriched for the immune-related JAK-STAT signalling pathway. Furthermore, two clusters of Hox genes were predicted in both assemblies, suggesting that at least one round of whole-genome duplication occurred in both T. tridentatus and L. polyphemus. The innate immunity gene investigation showed that conserved pattern recognition receptor gene families and several signal transduction pathway genes involved in IMD and JAK-STAT exhibit a certain degree of expansion in both horseshoe crab genomes. Moreover, all of the antibacterial peptides reported in previous studies, as well as their effective antimicrobial sites, have also been identified in the T. tridentatus genome. Beyond that, according to our analysis, intact coagulation cascade-related genes were identified in the T. tridentatus genome, with high numbers of total gene counts. Apart from the abovementioned results, there may remain other aspects of horseshoe crabs that may be greatly conducive to their adaptive advantages in marine environments through their long evolutionary history that need to be studied further and established in future studies.Further analysis of the T. tridentatus is endangered according to the Red List (https://www.iucnredlist.org/species/21309/149768986). The use of the T. tridentatus species collected in this study was to separate 0.1 g leg skeleton muscle tissue for DNA extraction. This study was reviewed and approved by the Animal Care and Use Committee of Minjiang University. All the required collection and permits had been obtained before any work was started. Only one T. tridentatus was purchased in the aquatic market in Xiamen, Fujian Province, China and was released after 0.1 g leg muscle collection. Total genomic DNA was extracted using an E.Z.N.A.® Insect DNA Extraction Kit  at the State Key Laboratory of Genetic Engineering in Fudan University, Shanghai, China. The genomic research about T. tridentatus covers the evolution history of this endangered species, and may reveal genomic characteristics that limit their survival, which could provide a basis for the species protection.T. tridentatus was estimated using online scripts (https://github.com/josephryan/estimate_genome_size.pl) from a k-mer distribution generated by jellyfish. Contigs were assembled using Velvet [The Illumina TruSeq Nano DNA Library Prep Kit  was used to construct the 400-bp and 800-bp paired-end sequencing libraries. Long mate-pair libraries featuring 3-kb inserts were prepared using a CHGC kit . The Nextera MP Sample Prep Kit  was used to build long mate-pair libraries with 8- and 12-kb insert sizes. Whole-genome sequencing was performed on the Illumina HiSeq 2500 and HiSeq X Ten platforms generating 250-bp and 150-bp paired-end reads, respectively. Five libraries of nominal insert sizes, including 400 bp, 800 bp, 3- kb, 8- kb and 12- kb, were sequenced at expected genome coverages of 60×, 30×, 7×, 7× and 20×, respectively. The generated clean reads from the 400 bp, 800 bp and 3- kb insert size libraries were used for the estimation of the genome size via JELLYFISH v1.116  with a kg Velvet  with lonT. tridentatus genome assembly automatic gene prediction was established using AUGUSTUS (Version 3.3) [− 5. The three annotation results were combined as the annotation of the predicted genes. The benchmarking sets of universal single-copy orthologues (BUSCOs) [T. tridentatus and L. polyphemus were first analysed using RepeatMasker (version open-4.0.5) [ion 3.3) . The preion 3.3)  and Inteion 3.3)  with an (BUSCOs)  were usen-4.0.5)  with mern-4.0.5)  and RepBn-4.0.5) . RepeatMn-4.0.5)  was usedT. tridentatus were downloaded from the NCBI SRA database (accession number SRX330201) [RNA-seq raw data obtained from the embryonic sample at Stage 21 (the hatch-out stage) of X330201)  with defX330201) .L. polyphemus submitted by Washington University were downloaded from NCBI with RefSeq ID 2304488, accession GCF_000517525.1. The non-redundant protein sequences in L. polyphemus were selected by sorting the protein scaffold positions and filtering out overlapped proteins. Protein sequences of other 11 Chelicerata species, including three Araneae , seven Acari , and one scorpion (Centruroides sculpturatus) were downloaded from the GenBank database. Additional outgroup protein sequences from the centipede Strigamia maritima were downloaded from the UniProt database. OrthoMCL [L. polyphemus and T. tridentatus and the single-copy gene set for the 13 Chelicerata species and centipede with the KEGG database [The published genome assembly, coding sequences and protein sequences for the Atlantic horseshoe crab OrthoMCL  and MaxiOrthoMCL , with ceOrthoMCL  was useddatabase .Tachypleus tridentatus, Carcinoscorpius rotundicauda and Limulus polyphemus), one scorpion (Mesobuthus gibbosus) and three Araneae . The 13 mitochondrial coding sequences for T. tridentatus collected in Korea were used to query for identical genes in our T. tridentatus genome and transcriptome assembly using blastn . Alignment of sequences with identities of at least 97% was considered to be the counterparts of mitochondrial coding sequences in T. tridentatus identified in this study. Multiple sequence alignments of concatenated mitochondrial coding sequences of 7 species were processed using ClustalW v2.0.12 [Thirteen intact mitochondrial coding sequences of 7 species were downloaded from the NCBI gene database and used to build the time tree containing three horseshoe crabs  and InterPro database [Protein sequences of Hox genes, essential immune signalling-related genes and coagulation factors of species closely related to horseshoe crabs were downloaded from the NCBI protein database and used as query sequences. Blastp was then used with an e-value of 10database .Stegodyphus mimosarum, Tak1 from Xenopus tropicalis and JNK from Parasteatoda tepidariorum were chosen as outgroups for the above phylogenetic trees.MEGA (version 7.0)  was usedT. tridentatus and L. polyphemus genome assemblies as databases. Identified putative AMPs were further used as query sequences to search for corresponding transcripts. The prediction of ORFs was performed using NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) and the annotation of those ORFs was performed by blastp with the NR database.Because antimicrobial peptides (AMPs) are commonly short in length, display wide variety and have large differences in their structures and functions within species, tblastn was used to identify potential AMPs using the Additional file1: Table S1. Comparison of homeobox ANTP class genes between T. tridentatus and L. polyphemus genome.


Chronic hepatitis B liver fibrosis is significant public concern. Ganshuang granules (GSG) are used to treat liver fibrosis for a long time. The aim of this study is to synthesize related data to explore efficacy and safety of GSG as an adjuvant treatment for chronic hepatitis B liver fibrosis.Electronic database were used to identify related studies. We chose PubMed, China Knowledge Network Infrastructure, China Biomedical Database, Wan Fang Data, VIP Database, EMBASE, and Cochrane Library as retrieval tool. Two independent individuals conducted the publication selection, data extraction, data assessment. Any problems between 2 researchers will be resolved by a third reviewer through negotiation. RevMan 5.3  software will be used for data analysis.This study will systematically detect the efficacy and safety of GSG for treating chronic hepatitis B liver fibrosis.This study will provide scientific evidence to explorer whether GSG are efficacy and safety in treating chronic hepatitis B liver fibrosis.INPLASY202090027 The estimated global prevalence of chronic HBV infection was 3% to 5% in 2016, with 257 million people chronically infected. Vaccination shows effective in prevention of infection and virus carriage. With the declining prevalence of infection globally, this virus remain a serious threat in many countries and regions. Liver fibrosis is a main adverse sequelae of chronic hepatitis B hepatitis, which some patients leads to cirrhosis, even hepatocellular carcinoma. Liver fibrosis is considered to be reversible. For chronic hepatitis B liver fibrosis, the most effective treatment is antiviral therapy. Antiviral therapy can decrease the risk for developing cirrhosis and progression to cancer, even can reverse liver fibrosis. Due to the low cure rates, life-long antiviral therapy is significant for most patients. However, anti-fibrogenic agents including resveratrol, vitamin E, silymarin still lack sufficient evidence. To date, there are no biochemical drugs for liver fibrosis approved by the FDA.Chronic hepatitis B virus infection, a global distribution disease, is severe issue due to the adverse sequelae-related morbidity and mortality. Because of syndrome differentiation, treatment, and multi-target pharmacological effect, TCM demonstrates an unique advantage in treating chronic hepatitis B liver fibrosis. As a complementary and alternative medicine, TCM provide a new methods to anti- chronic hepatitis B liver fibrosis. Ganshuang granules (GSG) is a prepared prescription drug to treat liver fibrosis. This drug is composed of Dangshen (Codonopsis pilosula), Chaihu , Baishao (Paeonia lactiflora), Danggui (Angelica sinensis), Fuling (Wolfiporia extensa), Baizhu , Zhike (Hedgehog), Pugongyin (Dandelion), Huzhang (Giant knotweed), Xiakucao , Danshen , Taoren (Peach kernel), Biejia (Turtle shell). In China, GSG combined with antiviral therapy is indicated for chronic hepatitis B liver fibrosis. Recent study shows that GSG inhibits stimulation of liver stellate cells through by inhibiting mammalian target of rapamycin. Another experiment suggests that the mechanism of anti-liver fibrosis of GSG is suppressing regulatory T cells.Traditional Chinese medicine (TCM) is used for treating chronic hepatitis B liver fibrosis for decades.GSG in combination with antiviral treatment is widely used for treating chronic hepatitis B liver fibrosis in China. However, the efficacy and safety of GSG are still unclear. Herein, we performed this research to evaluate effective and safety of GSG. We hope this work will contribute to the clinical application of GSG as adjuvant treatment for chronic hepatitis B liver fibrosis.22.12.1.1Any randomized controlled trials involving efficacy and safety of GSG for treating chronic hepatitis B liver fibrosis will be included.2.1.2In this study, any subject with chronic hepatitis B liver fibrosis will be included, regardless of age and gender.2.1.3The treatment group are treated with GSG combined with entecavir. The control group are oral administration of entecavir in any dosage form .2.2The primary outcomes of this study are hyaluronic acid, type IV collagen, laminin, and pre-type-iii collagen, which are the 4 indicators of liver fibrosis. The additional outcomes of this study include ALT, AST, and HBV-DNA negative conversion rates.2.3 We conduct this study through by searching Web of Science, PubMed, China Knowledge Network Infrastructure, China Biomedical Database, Wan Fang Data, VIP Database, EMBASE, and Cochrane Library. The above database will be retrieved from its establishment to July 2020. The terms or key words will be searched singly or in varying combinations, as shown in Table This research is conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses Statement guidelines.2.4Two authors (SQ Zeng and YF Liu) independently screen the titles and abstracts to select potentially relevant manuscripts to meet the inclusion criteria. Duplicate and irrelevant articles will be removed. Any divergence will be settled by consultations. Selected papers will be reviewed by third reviewer. Two authors (SQ Zeng and YF Liu) independently check the titles and abstracts of papers to evaluate whether the papers meet the eligible criteria. All papers included will be investigated as full text. If a given paper is in doubt, the final decision will be made by a third reviewer. The content extracted from publications are author, journal, publication year, study setting; participant demographics and baseline characteristics Fig. .2.5 Every study will be assessed the risk of bias. We will divide each study into 3 grades: low risk, high risk and unclear risk. An information sheet will list the results of quality evaluation.Methodological quality of included trials will be assessed by Cochrane risk of bias tool.2.62 test. I2 < 50% or P > .10 suggests heterogeneity across studies, and a fixed-effects model will be conducted for pool analysis. If I2 > 50% or P < .10, the pooled effect sizes will be calculated by a random-effects mode. The confidence interval will be set at 95%. The publication bias will be visually assessed by funnel plots.Data analysis will be conducted by RevMan 5.3 software . Weighted mean difference (WMD) will be used for continuous outcomes. Risk ratio will be adopted for dichotomous results. Heterogeneity across studies will be checked by using I2.7If required, a subgroup analysis will be performed based on the different study characteristics, study quality, and outcome measurements.2.8Stability of merger results will be checked by sensitivity analysis. If any low-quality trials exist, we will remove it.2.9This study involves publications only and will not collect any information from patients. Thus, the ethical approval is not required.This study will be submitted on a peer-reviewed journal.3 Antiviral therapy is fundamental to clinical practice. However, potent anti-fibro genic agents are still lacking. Therefore, clinicians intend to utilized other methods to address this issue when implementing antiviral therapy. In China, TCM has been treating illnesses for a long time. In TCM theory, liver fibrosis mainly caused by blood stasis in the liver. It is believed that GSG can promotes blood circulation in the liver to slow down liver fibrosis progression. Consequently, GSG combined with antiviral therapy is often used in clinical practice for treating chronic hepatitis B liver fibrosis. This protocol is drafted to investigate efficacy and safety of GSG, which is an adjunctive drug in this illness. We expect this study can provide clinical evidence to guide the GSG usage.Chronic hepatitis B virus infection remains an urgent medical challenge worldwide. With active viral replication, this chronic infection may continue to progress, and liver fibrosis, cirrhosis, hepatocellular carcinoma may occur. Chronic hepatitis B liver fibrosis is prevalent in Asia-Pacific countries and regions.Conceptualization: Cen Jiang, Baixue Li.Data curation: Shaoqian Zeng, Yefang Liu.Formal analysis: Shaoqian Zeng, Yefang Liu.Funding acquisition: Quansheng Feng, Cen Jiang, Li Wen.Investigation: Shaoqian Zeng, Yefang Liu.Methodology: Cen Jiang, Li Wen.Project administration: Li Wen, Quansheng Feng.Resources: Baixue Li.Software: Shaoqian Zeng, Yefang Liu, Li Wen.Supervision: Baixue Li, Quansheng Feng.Validation: Quansheng Feng.Writing – original draft: Shaoqian Zeng.Writing – review & editing: Quansheng Feng.


Escherichia coli varying from avirulent to highly pathogenic strains, including the emergence of virulent hybrid microorganism. This ability also contributes to the emergence of antimicrobial resistance. These hybrid pathogenic E. coli (HyPEC) are emergent threats, such as O104:H4 from the European outbreak in 2011, aggregative adherent bacteria with the potent Shiga-toxin. Here, we briefly revisited the details of these E. coli classic and hybrid pathogens, the increase in antimicrobial resistance in the context of a genetically empowered multifaceted and versatile bug and the growing need to advance alternative therapies to fight these infections.Genetic plasticity promotes evolution and a vast diversity in  Escherichia coli (or E. coli) is a Gram-negative versatile bacterium, easily found and amenable to natural and random genetic alteration. There is a vast collection of sequenced E. coli genomes which exhibit different sizes and genomic diversity among commensal and pathogens, indicating a great assortment within the same bacterial species. They comprise of non-pathogenic bacteria that may act as commensals and belong to the normal intestinal microbiota of humans and many animals. There are also pathogenic variants, divided as diarrheagenic and extraintestinal pathogens, with different pathotypes and various natural hybrid strains  are differentiated from non-pathogenic E. coli and extraintestinal pathogenic E. coli (ExPEC). The ExPEC are classified as uropathogenic E. coli (UPEC), sepsis-causing E. coli (SEPEC) and neonatal meningitis-associated E. coli (NMEC) (E. coli (STEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely-adhering E. coli (DAEC), adherent-invasive E. coli (AIEC), and cell-detaching E. coli (CDEC) (Based on virulence factors in i (NMEC) . Recent i (CDEC)  .Although, all ExPEC and DEC pathotypes are not enough to fully classify all pathogenic E. coli has an astonishing facility to amend very well, replicate and disseminate. These features allowed the advent of novel HyPEC. Acquired virulence genes and novel functions appear from mutation, recombination and other genetic changes. All these genetic differences have increased the occurrence of novel hybrid and antimicrobial resistance among DEC and ExPEC  in Germany. This outbreak of h Africa . Emerginh Africa . Moreove to STEC .E. coli, such as STEC/ExPEC O80:H2 serotype, which caused HUS and bacteremia due the presence of stx2 and eae genes from STECs and pS88-like plasmid, described in meningitis, urosepsis and avian pathogenic strains of ExPEC , CNPq (307418/2017-0), and “Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP-PADC. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.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.


Klebsiella pneumoniae (K. pneumoniae) is a common pathogen associated with hospital and community-onset infections. This study aimed to compare the clinical and microbiological characteristics of nosocomial, healthcare-associated (HCA), and community-acquired (CA) K. pneumoniae infections.K. pneumoniae (CRKP) and ESBL-producing strains was performed by pulsed-field gel electrophoresis (PFGE).Clinical data were extracted from electronic medical records and analyzed retrospectively. Antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) production were determined for all identified strains. Carbapenemase and ESBL genes were amplified by PCR. Genotyping of carbapenem-resistant K. pneumoniae infections, 98 (25.9%) were nosocomial, 195 (51.5%) were healthcare-associated, and 86 (22.6%) were community-acquired. Hematological malignancy (OR = 4.467), and hypertension (OR = 2.08) and cerebral vascular disease (OR = 2.486) were associated with nosocomial and HCA infections respectively, when compared to CA infections. Overall, the incidence of antimicrobial resistance for the majority of agents tested was similar between nosocomial and HCA infections (P > 0.05) and both groups had a higher incidence than CA infections (P < 0.05). Moreover, 95.1% (78/82) of CRKP strains were isolated from the nosocomial and HCA groups. The blaKPC was the most prevalent carbapenemase gene among CRKP strains . ESBL-producing strains were prevalent among nosocomial (40.8%), HCA (35.9%) and CA groups (24.4%). The blaCTX-M-9-group and blaCTX-M-1-group genes were predominant in nosocomial (65.0%) and CA strains (66.7%), respectively. PFGE results showed ESBL-producing and CRKP strains were genetically diverse. Identical PFGE profiles were observed among HCA and nosocomial strains.Of 379 K. pneumoniae infections presented similar clinical features and antimicrobial resistance, and both two types of infections were different to CA infections. CRKP and ESBL-producing strains were disseminated mainly in HCA and nosocomial groups, and showed a clonal diversity. The cross transmission of CRKP was existed among HCA and nosocomial patients. This finding suggests that similar empirical therapy should be considered for patients with nosocomial and HCA K. pneumoniae infections and bacterial resistance surveillance of these infections is necessary.Nosocomial and HCA  Klebsiella pneumoniae is a threatening human pathogen that causes various diseases, including respiratory tract and urinary tract infections, blood infections, and liver abscesses [K. pneumoniae infections are limited [bscesses . Traditibscesses . Previoubscesses , 3. Alth limited , 5. InadK. pneumoniae is a significant concern to public health. The CRKP and ESBL-positive K. pneumoniae strains are the most important and challenging drug-resistant bacteria, leading to limited treatment options and high mortality [Antimicrobial resistance in ortality –8. Thus,K. pneumoniae [blaKPC, blaNDM, and blaOXA-48-like [BlaSHV, blaTEM, blaCTX-M, and blaOXA are the major ESBL genes identified to date [The production of carbapenemase is the main carbapenem-resistance mechanism in eumoniae . Carbape-48-like –12. BlaS to date , 14. The to date , 16.K. pneumoniae isolates contributes to the identification of specific clone dissemination and the source of infections, on which the prevention approach depends.Strain typing is of great significance for epidemiological surveillance. PFGE is a gold standard for the genotyping of strains. The molecular typing of K. pneumoniae infections. And the study data on antimicrobial resistance of HCA K. pneumoniae infections is scarce. Furthermore, strains isolated from different regions present their own distinct clinical and microbiological features. Therefore, this study aimed to characterize the clinical features, antimicrobial resistance and the distribution of CRKP and ESBL-positive strains among nosocomial, HCA, and CA infections caused by K. pneumoniae, and to characterize the genetic relatedness of CRKP and ESBL-positive isolates identified from the three different groups. The results from this study will be helpful in developing effective preventive and treatment strategies against K. pneumoniae infections both in hospitals and in communities.Currently, there are limited data comparing the clinical and microbiological characteristics of nosocomial, HCA, and CA K. pneumoniae infections from January 2019 to December 2019 were included in the study. K. pneumoniae strains were consecutively collected from these patients. For patients with multiple K. pneumoniae isolates, only the first isolate was analysed. During the study period, the number of admittances and outpatient treatments were about 103,630 and 1,610,618, respectively.This retrospective study was conducted at a 2300-bed tertiary hospital in Guangzhou, China. All patients presenting K. pneumoniae infection was based on previously described criteria [K. pneumoniae infection was defined as an infection that occurred 48 h after the patient’s admission. A HCA K. pneumoniae infection was defined as an infection developing within 48 h of admission in patients presenting any of the following risk factors [K. pneumoniae infection; a history of hospitalization for 2 or more days in the three months before the K. pneumoniae infection; or residence in a nursing home or long-term care facility. A CA K. pneumoniae infection was defined as an infection occurring in patients who did not fulfill the definitions of nosocomial or HCA infections.The definitions of nosocomial, HCA, and CA criteria . A nosoc factors : a histoK. pneumoniae infection.We extracted the following clinical data from electronic records for all patients: demographic characteristics, underlying disease, site of infection, immunosuppression, and mortality. Exposure to prior antimicrobial treatment was defined as any treatment received for at least 48 h in the 30 days before the K. pneumoniae strains were collected from patients during the study period. The Vitek 2 system  was used for bacterial identification. Antimicrobial susceptibility and the ESBL phenotype were determined using the Vitek 2 System following the Clinical and Laboratory Standards Institute guidelines . The antibiotics tested included ampicillin (AMP), piperacillin/tazobactam (TZP), amoxicillin/clavulanic acid (AMC), cefoperazone/sulbactam (CPS), cefazolin (CZO), cefuroxime (CXM), cefepime (FEP), ceftriaxone (CRO), ciprofloxacin (CIP), cefoxitin (FOX), ceftazidime (CAZ), imipenem (IMP), amikacin (AMK), gentamicin (GEN), ertapenem (ERP), meropenem (MEM), aztreonam (ATM), levofloxacin (LVE), trimethoprim/sulfamethoxazole (SXT), and tigecycline (TGC). The breakpoint of tigecycline was based on the standard of the United States Food and Drug Administration (FDA). Escherichia coli ATCC 25,922 and K. pneumoniae ATCC 700,603 served as quality control strains.A total of 379 K. pneumoniae (CRKP) isolates were defined as isolates that displayed resistance to one or more carbapenem agents such as meropenem, ertapenem, or imipenem [Multidrug resistant (MDR) strains were defined as strains with no susceptibility to at least three different classes of antimicrobials . Carbapeimipenem .blaKPC, blaNDM, blaGIM, blaSPM, blaIMP, blaSIM, blaVIM, blaIMI and blaOXA-48-like) in the CRKP strains and ESBLs genes  in the ESBL-producing strains [http://blast.ncbi.nlm.nih.gov).Polymerase chain reaction (PCR) was performed to detect carbapenemase genes . Briefly, the strain DNA were digested with Xbal for 2 h at 37 ℃. Electrophoresis was performed using the CHEF-DRIII system , with a running time of 19 h and a switch time of 2.16–54 s. The PFGE fingerprint patterns were uploaded to the K. pneumoniae database of the Chinese Pathogen Identification Net (http://139.9.117.189/CPIN/). A Dice coefficient-based PFGE dendrogram was constructed using the unweighted pair-group mean Analysis. Isolates were grouped into the same PFGE cluster if they shared ≥ 80% similarity.All 186 CRKP or ESBL-producing strains were subjected to PFGE following the PulseNet standardized procedure  were considered statistically significant. Multivariable logistic regression analysis was conducted to identify variables associated with nosocomial, HCA, and CA infections. Variables regarding age, underlying disease, and immunosuppression with P < 0.05 in the univariate analysis were included in the multivariable logistic regression model.Categorical variables were analyzed by Chi-square or Fisher’s exact tests . K. pneumoniae infection were included  in the analysis. The incidence of K. pneumoniae infections was approximately 3.66 per 1000 admissions in the hospital. Male patients accounted for 64.12% of K. pneumoniae infections. The overall 30-day crude mortality rate was 12.4% (47/379).In total, 379 patients with K. pneumoniae infections are presented in Table The clinical features of There were differences between the three groups regarding the infection type, with bloodstream infections being dominant in nosocomial patients (45.9%), respiratory tract infections being frequent in HCA patients (33.9%), and skin and soft tissue infections being predominant in CA patients (25.6%).Nosocomial infections and HCA infections shared similar features in terms of underlying diseases. However, CA infections differed from both nosocomial and HCA infections. In multivariate analysis, hematological malignancy (OR = 4.467) and brain tumor (OR = 7.865) were independent variables associated with nosocomial cases compared to CA cases, whereas diabetes mellitus was more frequently associated with CA cases (OR = 0.342). Cerebral vascular disease (OR = 2.486) and hypertension (OR = 2.080) were independent variables associated with HCA infections compared to CA infections.Both patients with nosocomial and HCA infections presented a higher prevalence of prior antibiotic exposure, accounting for 54.1% and 59.0%, respectively. No prior antibiotic exposure was observed in CA infection patients. Patients with nosocomial (19.4%) and HCA (12.3%) infections exhibited higher rates of 30-day mortality than patients with CA infections (4.7%).K. pneumoniae strains isolated from the three groups is summarized in Table P > 0.05), and both the strains were significantly more resistant than the CA strains (P < 0.05). The percentages of CRKP strains, ESBL-producing strains and MDR strains were similar between the nosocomial and HCA cases and were higher in both cases than in CA cases:  for CRKP strains (22.5% vs. 28.7% vs. 4.7%), ESBL-producing strains (40.8% vs. 35.9% vs. 24.4%), and MDR strains (53.1% vs. 52.3% vs. 23.3%).The distribution of the percentage of antimicrobial-resistant blaKPC , blaNDM , and blaOXA-48-like , were identified among the 82 CRKP strains. Four strains presented simultaneously blaKPC and blaNDM genes. Five isolates were negative for the screened carbapenemase genes.A total of 82 CRKP isolates were identified from the three groups. The distribution of carbapenem resistance genes of the CRKP isolates among the three groups is shown in Table blaSHV, 64.9% (85/131) for blaTEM, 51.9% (68/131) for blaCTX-M-9-group, 42.0% (55/131) for blaCTX-M-1-group  having co-existence of blaSHV, blaTEM and blaCTX-M-1-group, and these strains were more commonly identified in the CA group than in the nosocomial group . Conversely, strains possessing both blaSHV and blaCTX-M-9-group were more likely to be found in the nosocomial infection group than in the CA group .PCR showed that all nosocomial, HCA, and CA ESBL-positive strains harbored at least one of the ESBL gene groups, with positive rates of 91.6% (120/131) for , 64.9% 8/131 for , 64.9% 8/131 for bla genes. Isolate 98 carrying blaKPC showed 100% similarity to isolate 49 carrying blaNDM.All CRKP or ESBL-producing strains were subjected to PFGE for assessing the clonality of the strains. Three ESBL-producing isolates failed to be genotyped by PFGE. Fifty-six nosocomial strains were divided into 36 clusters at a cut-off of 80% similarity, with the major cluster C13 accounting for 14 ESBL-producing strains , indicating other resistance genes not investigated may mediate the carbapemase activity. The remaining four CRKP may have other resistance mechanisms contributing to carbapenem resistance. The production of ESBLs combined with mutations in or loss of the porin Ompk35 or Ompk36 can be associated with carbapenem resistance [The spread of CRKP strains has become a new public health crisis worldwide. Published studies on the distribution of CRKP isolates among nosocomial, HCA, and CA infections are scarce. The present study showed that CRKP strains were rare in the CA group, but more prevalent in both HCA and nosocomial groups. Thus, it should be of caution when use carbapenems to treat patients with HCA and nosocomial infections. In agreement with other studies, the  strains , 29. In in China –32. In asistance , 34. In K. pneumoniae infections. These data suggest that ESBL producers have disseminated to the community, which poses a challenge to resistance control. BlaCTX-M was the most frequently identified ESBL gene among ESBL producers in our study. This is accordance with the fact that CTX-M is the most common ESBL genotype in China [blaCTX-M, blaSHV and blaTEM genes among isolates from the three groups, suggesting the wide dissemination of these genes possibly via horizontal transfer in different populations. Interestingly, the CTX-M-1 variant was more frequently detected among CA strains than among nosocomial strains, whereas the CTX-M-9 variant was more common in nosocomial strains than in CA strains. This suggests that nosocomial and CA ESBL-producing strains may carry different plasmids, which carried the different subtype of CTX-M genes.This study showed that the ESBL-producing strains were prevalent both in medical and community settings. Previous studies by Zhang et al.  and Quanin China . No signK. pneumoniae among HCA patients. It suggests the healthcare-associated transmission of CRKP strains in the local area. In addition, the same PFGE profiles were observed in strains carrying different resistance gene profiles, suggesting later acquisition of the resistance genes via horizontal transfer under antibiotic selection pressure.Overall, the PFGE typing showed a high clonal diversity among the ESBL-producing and CRKP strains isolated from the three different groups, indicating the variety of infection sources acquired by the three populations. Of note, the clonal dissemination of ESBL-producing strains has occurred in the hospital as indicated by the three identical profiles observed in cluster C13. Further study is needed to investigate the origin and the route of the transmission. In cluster C82-II and cluster C82-III, the HCA strains and nosocomial strains showed the same PFGE profiles, suggesting the cross transmission of CRKP among HCA and nosocomial patients. CRKP strains could be introduced into the hospital, then spread within the institution and might cause nosocomial infections during the entry of HCA patients with CRKP infections, of which we should keep alert. Identical profiles among HCA CRKP isolates implies the clonal spread of K. pneumoniae strains from different regions, more epidemiological data on nosocomial, HCA, and CA K. pneumoniae infections are needed to better manage patients.The main finding in the present study was that the antimicrobial resistance of HCA isolates was similar in severity to nosocomial isolates. This study was conducted at a tertiary hospital in Guangzhou city, Guangdong province, where more severe patients from all over the province or neighboring provinces had been hospitalized and approximately 20% had been referred from other hospitals, which could explain the serious drug resistance in the HCA group in our study. Given distinct features of K. pneumoniae infections were similar to nosocomial infections regarding clinical features and antimicrobial resistance, and were different to CA infections. CRKP and ESBL-producing strains were mainly prevalent in patients with HCA and nosocomial infections, showing genetic diversity. Identical profiles observed in HCA and nosocomial CRKP strains suggested the transmission of CRKP among HCA and nosocomial patients. These findings indicate that empirical antimicrobial treatment for patients with HCA K. pneumoniae infections should be similar to those for patients with nosocomial infections and great attention should be paid to these infections due to the possible dissemination of antimicrobial resistance among them.HCA Additional file 1: Table S1. Primer sequences of resistant genes for K. pneumoniae.


Schistosoma haematobium and S. mansoni, which are transmitted by specific intermediate freshwater snails, Bulinus spp. for the former and Biomphalaria spp. for the latter. The two schistosomes cause either urogenital or intestinal schistosomiasis, respectively  and S. haematobium [♀]) and various hetero-specific  worm couplings [S. haematobium and S. intercalatum) or more critically as yet unknown interactions . In contrast with experimental schistosomiasis [Across Africa, whilst the continental burden of coinfection with urogenital and intestinal schistosomiasis is not formally reported, at the local level, it often is , 8. Insiouplings . Cross-souplings . As furtouplings , some hesomiasis , in whicsomiasis . This opsomiasis . Furthersomiasis .https://www.who.int/neglected_diseases/Ending-the-neglect-to-attain-the-SDGs--NTD-Roadmap.pdf?ua=1), with future disease-specific targets being set. Assumptions for successful control of S. haematobium include negligible capacity for viable genetic introgression(s) and maintenance of transmission in animal reservoirs. Seminal field-based parasitological surveys in Senegal, molecularly characterizing schistosome populations [S. haematobium and S. bovis [S. curassoni have been revealed [S. haematobium and S. bovis populations has shown no genetic admixing [S. haematobium–bovis hybrid has been recently flagged in autochthonous transmission of urogenital schistosomiasis on Corsica [Control of schistosomiasis is of global importance and features in the World Health Organization (WHO) 2021–2030 neglected tropical disease (NTD) road map  was first reported over 70 years ago [S. mansoni. The latter species could be a cryptic infection driver altering worm behaviours by promoting mate exchange, for here in Malawi, intestinal schistosomiasis is emerging and appears very recent, being first noticed two years ago, driven by first colonisation of Bi. pfeifferi within the lake [S. haematobium, S. haematobium–mattheei and bovis, and S. mansoni), driving plausible schistosome interactions, as meshed with ongoing environmental change. This includes regular or sporadic natural or manmade events such as cyclonic flooding, human migration, over-fishing, and increased pisciculture, where there have been significant, albeit largely unexplained, saltatory changes in snail fauna and schistosome-snail host ranges [Even though phenotypic suggestion of ears ago , being lears ago , 18, it the lake . With tht ranges . This clS. haematobium–mattheei and S. haematobium–bovis hybrids although each hybrid was sympatric, geographically synonymous, with S. haematobium and S. mansoni [S. haematobium eggs in urine. Egg-patent S. mansoni was also noted in several children’s stools as well as ectopic excretion in children’s urine, with the general prevalence of intestinal schistosomiasis increasing (more than 50% by urine-CCA testing), enabling coinfections to surge [S. haematobium  was 17.1% (95% CI 4.8–22.2) although atypical S. haematobium eggs or S. mansoni eggs in urine were not found [Schistosoma DNA probe, infection prevalence was much higher at 26.5% (95% CI 18.4–34.6), and, upon using urine-CCA tests, prevalence of intestinal schistosomiasis was 3.8% (95% CI 3.1–4.5) [S. mattheei?) which has not yet been subjected to molecular analyses, although a S. mattheei infection has been detected locally in B. globosus by molecular methods [From our targeted epidemiological surveys of Malawian school children, an initial genetic analysis of a selection of schistosome eggs revealed an allopatric, geographically separated, distribution of  mansoni . Egg-patto surge , 19. Insot found . Upon re methods .S. haematobium–bovis hybrid is particularly intriguing as the literature notes an absence of S. bovis greater than 10o south of the equator. This demonstrates a more itinerant nature of this hybrid form outside the known range of S. bovis [Clearly, multispecies schistosome coinfections are occurring here in Malawi with an underlying set of putative homo- and hetero-specific worm-pair coupling that enable new opportunities for known, but more importantly, as yet unknown, genetic interactions. The presence of the S. bovis . Since mS. haematobium–bovis hybrid is yet to take place, analysis of West African S. haematobium–bovis hybrids reveals several large 100 kb identical chromosomal regions, which is indicative of a single or very limited number of hybridization events given putative multiple rounds of subsequent meiosis [S. haematobium. In terms of the epidemiology of S. haematobium–bovis, in Senegal it was noted that the frequency of hybrids, by village, was not associated with the prevalence of urogenital schistosomiasis but oddly was with the prevalence of intestinal schistosomiasis [S. haematobium–mattheei hybrid, we surmise that with increasing epidemiological opportunities for multi-species coinfections, both pre-  and post-  zygotic species barriers are breaking down, allowing perhaps even broader schistosome species barriers to become eroded.Whilst genomic analysis of this  meiosis . An ancesomiasis . ReferenS. haematobium and S. mattheei combinations so far attempted, but this phenomenon did not spark increased public health vigilance even though heterosis in worm fecundity  was observed [S. haematobium group hybrids and assorted coinfection permutations in Malawi is now of fresh concern by enhancing future hybridization and disease potentials. For example, upon various plausible schistosome pairings such as S. haematobium–mattheei (♂) and S. mansoni (♀) or any back crossing of viable variants thereof could these worms with more eroded species-specific barriers now introgress? Of particular note, from experimental schistosomiasis is that viable progeny from S. mansoni (♂) and S. mattheei (♀) pairings has already been reported but were then thought, although not proven, to be parthenogenetic matriclonal lineages [S. haematobium–bovis migration, and subsequently intermingle with other populations? We should now be especially mindful of more distant species variants, particularly given ancestral genomic signatures [S. mansoni–haematobium hybrids in a migrant boy, even though the viability of their progeny was not determined [S. haematobium–mattheei hybrids, with evidence of S. mansoni infection, as detected in returned Belgium travellers from South Africa that were subjected to a more in-depth diagnostic investigation with molecular methods than usual routine practice [As the reproductive biology of the schistosome is unusual , 9, mentgnatures  as well termined . Of recepractice .It is an unfortunate situation that where schistosomiasis transmission occurs, there is typically only rudimentary parasitological surveillance and formal epidemiological inspection of animals is very rare , 25. ThiS. haematobium and/or S. mansoni infection. However, we can envisage two likely scenarios as outlined by the dashed red lines: H1—the age-profile of hybrid coinfection follows that of a “classic” monospecific infection but is reduced in amplitude in line with diminishing ova-shedding, an accepted feature of schistosomiasis—or H2—the age-profile is constant (or sporadic) across ages and does not track a “classic” monospecific infection, perhaps by being a less facultative transmission process in local snails. In H1, there is also the possibility of an epidemiological “peak-shift” where the age-profile maximum prevalence is either advanced or retarded in reference to the monospecific infection (red arrow). If so, there could be many reasons for this; for example, hybrid worms may have a faster environmental transmission dynamic  or hybrid worms live longer . A similar age-prevalence argument based upon “peak-shift” [S. haematobium and S. haematobium-S. bovis in Benin, they did not comment on the age of their cattle examined but they did observe altered shedding times of schistosomes from infected snails, noting their importance as environmental transmission drivers.To highlight, in outline, the real epidemiological implications of these knowledge gaps in the detection and transmission of hybrids, an epidemiological postulate is provided in k-shift”  highlighk-shift”  recentlyS. haematobium hybrids please stand-up!To close, for a more thorough surveillance of hybrid schistosomes in Africa, we strongly advocate a OneHealth approach. Where possible, this could include noninvasive larval sampling of schistosomes from livestock with spatial tracking of sentinel animals within mobile infected herds for real-time monitoring of infection dynamics, with better snail surveillance at well-noted animal water contact sites. Upon postmortem inspection of animals at slaughter or upon selected cull, we should also be on guard for any ectopic worms in the vasculature of either urogenital or intestinal systems as well as how ova-contaminated water from carcasses might cycle back into aquatic habitats from abattoir effluents. Without doubt, the search for emergence of further schistosome hybrid combinations in humans, alongside our growing ability to detect them, is increasing; to help us in this quest, simply put, can all


Thalamita danae (Stimpson1858), re-described as a new report, based on an integrative taxonomy approach combining 16S rRNA partial sequence of mitochondrial DNA and morphological analyses was used for the accurate identification of specimens. The morphological and molecular analysis provides the confirm evidence of T. danae in coastal waters of Pakistan. Results were confirmed by amplification of partial sequences of 16S rRNA mt-DNA gene and the sequence was searched for similarity using BLASTn , the result showed 97% sequence similarity with partial sequences of T. danae Stimpson, 1858 as obtained from gene bank. The attained sequence was submitted to gene bank after confirmation of genetic and morphological similarity. High sequence similarity with accession no: FJ152165.1 indicated, that misidentification of species does not occur. Scylla and Portunus  has worldwide distribution Hartnoll  and a diPodophthalmus, Carcinus, Scylla, Portunus, Charybdis and Thalamita). In previous studies, only three species of genus Thalamita Latreille, 1829:  reported from Pakistan. Now one more species; Thalamita danae  was identified as new report based on morphological and molecular analysis.Besides their economic and ecological significance and availability of previous taxonomic checklist Hashmi  the moleThalamita danae originated from the rocky coast of Buleji . After confirmation of species through sequence analysis, these specimens were again collected through handpicked method for morphological description and confirmation of species status. After taking the DNA sample and morphometric measurements: Carapace length (CL), Carapace width (CW), Cheliped length (Ch.L), to the nearest 0.1 mm, the sample preserved in ethanol 80% to be used for taxonomic identification. The species identified as T. danae and than verified on the basis of morphological characterstics  present in MRCC, University of Karachi.Three species of Species identification exclusively based on a partial fragment of the mt DNA 16S rRNA gene. The approximately 25 mg of tissue samples from Chela were used for the extraction of total genomic DNA by using Qiagen DNeasy Blood and Tissue Kit (Cat. no. 69504),The polymerase chain reactions (PCR) technique was used to amplify the region of the 16S rRNA gene by using the primers 16Sar forward (5′-CGC CTG TTT ATC AAA AAC AT-3′) paired with the reverse primer 16Sbr (5′-CCG GTC TGA ACT CAG ATC ACG T-3′)  and the PCR conditions applied following Lai et al. .www.ncbi.nlm.nih.gov/BLAST . Sequences were aligned using the Clustal W tool, MEGA 6, MEGA Inc., Ocheyedan, IA). DNA sequence data were analyzed through applied biosystem sequence Scanner v1.0 software  then submitted to the Genbank (accession number: KU 130124).PCR products confirmed for gene amplification through 2% agarose gels with ethidium bromide stained. Successfully amplified PCR products were purified and sequenced from the Macrogen Company  and the procured sequence was utilized for the species identification as initially searched for sequence similarity using the NCBI web link (T. danae (accession no: FJ152165.1), T. crenata (accession no: FJ152164.1), T. sima (accession no: FJ152166.1), T. admete (accession no: FJ152163.1) and T. prymna (accession no: AM410537.1) included in the evolutionary analysis as performed in MEGA6  Dana 1852c: 282; Dana, 1855: pl. 17, Figure 7.Thalamita danae  Stimpson, 1858a: 39; A. Milne-Edwards, Type locality: Karachi, Pakistan.New material: Three males, three females on 4 April 2014 and six males, two females on 16 October 2014 from Buleji  Karachi, Pakistan.Intertidal areas of rocky shore and mudflats of sand or muddy sand with stones, areas with pebbles under stones.Whole body light and dark greenish coloration with slight purplish shade.East Africa to tropical Indo-west Pacific, Australia, Taiwan, Mozambique, Red Sea, India, China, Japan, Singapore, Hong Kong, Philippines, Malaysia, Indonesia, New Caledonia, Marshall Islands, Fiji, Samoa, now from Pakistan.T. danae from the coastal waters of Pakistan compared with the collection of theThe present reported specimen of Natural History Museum London  and with neotype material of Wee and Ng  as durinThalamita danae as was destroyed  (accession no: FJ152165.1) specimen collected from Labradar, Singapore, in 23 December 1990 and deposit in University of Louisiana at Lafayette Zoological Collection (ULLZ) as a voucher no. ULLZ 4760. The specimen of T. danae confirmed from the identification keys of Yu , 90% with T. sima , 93% with T. admete  and 86% with T. prymna with the evolutionary rate of 0.01 for base substitutions. The molecular datasets consisted of 516 aligned characters. The maximum composite-likelihood (MCL) approach showed that 0.015 distance between T. danae (Pakistan) and T. danae (Singapore) and 0.017 between T. danae and T. prymna (The evolutionary history was inferred using the maximum-likelihood method based on the Kimura 2-parameter model Kimura  and the . prymna .Thalamita danae reported as new range based on morphological and molecular analysis of fresh material procured from coastal waters of Pakistan. The morphological identification described according to Poore and Ahyong (T. prymna as reported earlier (Alcock T. Prymna specifically distinct due to absence of spines on the basal antennal segment joints (Kossmann, T. prymna for species confirmation.d Ahyong  and 97% d Ahyong . The sper Alcock  but the


Phoenix dactylifera var. Khanezi. The results revealed a circular genome of 715,120 bp, having G + C content of 45.1%, containing 40 protein coding genes, 3 rRNA, and 18 rRNA genes. Evolutionary relationship analysis suggested that P. dactylifera var. Khanezi is more closely related to previously reported P. dactylifera var. Khalas.In this study, we determined the complete mitochondrial (mt) genome of  Phoenix dactylifera L., belongs to Arecaceae and is an ecologically, culturally and economically important fruit tree in North Africa and Middle East , dioecious, cross-pollinated tree that has been widely cultivated  and to infer its phylogenetic position on the basis of entire mito-genomes.The mitochondria play a vital role in plant growth and development , plants were received from the GenBank at the University of Nizwa, Nizwa Oman. Young leaves were used to extract mitochondrial DNA by using the method described previously  was used to filter and assemble the raw alumina reads. All contigs were blasted using the NCBI database (http://www.ncbi.nlm.nih.gov/) for the annotation of mitochondrial sequences. Similarly, tRNA scan-SE software (http://lowelab.ucsc.edu/tRNAscan-SE/) was used to identify tRNAs. The entire mitochondrial genome was used to determine its phylogenetic position using Maximum persimony tree with 1000 bootstrap replications . The largest part of the P. dactylifera mtDNA comprises the non-coding sequences (93.2%), which is slightly larger than the average non-coding sequences content (89.45%) in other angiosperm mt genomes (Chaw et al. P. dactelifera var. Khanezi is closely related to P. dactelifere var. Khalas (NC016740; Cocos nucifera. This study will help to understand the evolution of various date palm cultivars mitochondrial genome with related species.The mitochondrial genome of