Borrelia miyamotoi infection occurs in the far-western United States, we tested archived sera from northwestern California residents for antibodies to this emerging relapsing fever spirochete. These residents frequently were exposed to I. pacificus ticks in a region where B. miyamotoi tick infection has been reported. We used a two-step B. miyamotoi rGlpQ assay and a B. miyamotoi whole-cell lysate (WCL) assay to detect B. miyamotoi antibody. We also employed Borrelia hermsii and Borrelia burgdorferi WCL assays to examine if these Borrelia induce cross reacting antibody to B. miyamotoi. Sera were collected from 101 residents in each of two consecutive years. The sera of 12 and 14 residents in years one and two, respectively, were B. miyamotoi rGlpQ seroreactive. Sufficient sera were available to test 15 of the 26 seropositive samples using B. miyamotoi and B. hermsii WCL assays. Two residents in year one and seven residents in year two were seroreactive to both Borrelia antigens. Although discernible differences in seroreactivity were evident between the B. miyamotoi and B. hermsii WCL assays, infection with one or the other could not be determined with certainty. Sera from two Borrelia burgdorferi /B. miyamotoi seropositive subjects reacted strongly against B. miyamotoi and B. hermsii WCL antigens. Ecological, epidemiological, and clinical data implicated B. miyamotoi as the probable cause of infection among those whose sera reacted against both antigens. Our findings suggest that human B. miyamotoi infection occurs in northern California and that B. hermsii and B. burgdorferi infections produce antibodies that cross-react with B. miyamotoi antigens. Health care professionals in the far-western United States should be aware that B. miyamotoi disease may occur throughout the geographic distribution of I. pacificus and that improved relapsing fever group spirochete antibody assays are urgently needed.To determine whether human  Borrelia miyamotoi is a relapsing fever-group spirochete that was discovered in Ixodes persulcatus ticks in Japan more than 20 years ago and later determined to cause clinical illness in humans   –19. SeroB. burgdorferi seroreactivity as part of an inter- and intra-laboratory comparative study [B. miyamotoi testing. In the initial serosurvey [Serum samples were obtained in 1988 and 1989 from 101 residents of a community at high risk for Lyme disease (CHR) located in the Ukiah area of southern Mendocino County –19. Thesve study . Since tB. miyamotoi glycerophosphodiester phosphodiesterase antigen (rGlpQ) using an IgG ELISA [B. miyamotoi antibody. As positive controls, we used sera from patients who were confirmed by PCR or serology to have B. miyamotoi infection.Serum samples were diluted 1:320 and tested for IgG antibodies against recombinant gG ELISA . As a neB. miyamotoi seropositive if both ELISA IgG and Western blot IgG tests yielded positive results.Serum samples that were equivocal or positive by ELISA were retested for antibodies against rGlpQ using a Western blot IgG antibody assay . NitroceB. miyamotoi and B. hermsii WCL antigens in Western blot assays [B. miyamotoi and B. hermsii set of blots, we included serum from a subject who resides in a non-Lyme disease endemic area and had no history of prior B. miyamotoi infection (negative control). For both sets of blots, we also included serum from a subject who had PCR-confirmed B. miyamotoi infection (positive control) and from another subject who had confirmed B. hermsii infection (positive control). Two of us (PJK and SN) independently counted the number of bands for the positive and negative control sera and the CHR sera that were tested using B. miyamotoi and B. hermsii whole lysate assays. A seronegative serum was defined as one with the same or fewer bands as the negative control sera. A seroreactive serum was defined as one that had one or more bands than the negative control. Seroreactive sera were further classified into “moderately reactive” if they had one or more bands than the negative control but fewer bands than the positive control or “strongly reactive” if they had at least as many bands as the positive control.Serum samples diluted 1:100 were assayed for antibodies against t assays . For botB. burgdorferi seropositive) and no history of B. miyamotoi infection, and the same B. miyamotoi seropositive and seronegative controls as described above were diluted 1:100 and assayed for antibodies against B. miyamotoi and B. burgdorferi. We used WCL antigens prepared from in vitro cultivated B. miyamotoi or B. burgdorferi in separate Western blot assays [Serum samples from patients with physician diagnosed Lyme disease (erythema migrans rash and t assays –23.B. miyamotoi serologic testing. The mean age of the 101 subjects was 32 years (range 4 to 52 years) and participants included similar percentages of females (51%) and males (49%). Using the two-step ELISA-Western blot rGlpQ assay, we found that sera from 12 subjects in 1988 and 14 subjects in 1989 reacted against B. miyamotoi rGlpQ antigen , Lyme disease alone (erythema migrans rash and B. burgdorferi seropositive using the standard ELISA-Western blot assay), and neither infection (negative control) using a B. burgdorferi WCL assay and a B. miyamotoi WCL assay using a different B. miyamotoi WCL assay than that described above. The sera from patients with Lyme disease were reactive against B. burgdorferi WCL and against several B. miyamotoi bands in the B. miyamotoi WCL assay  who were seropositive for ive sera . To deteCL assay  but did ot assay . The serCL assay .B. miyamotoi GlpQ and WCL antigens and B. hermsii WCL antigen were much more likely to have been infected with B. miyamotoi than either B. hermsii or Borrelia parkeri, two other relapsing fever spirochetes present in northern California. B. miyamotoi has been detected in ticks from Mendocino County but not B. hermsii or B. parkeri. Indeed, human cases caused by the latter two spirochetes are acquired distant from the CHR community and the incidence of human infection with them is either low (B. hermsii) or rare (B. parkeri) statewide. A high percentage (78%) of CHR residents reported tick bite, which is more consistent with hard-bodied than soft-bodied ticks. The latter feed for less than an hour at night and are very seldom noticed in people who suffer soft tick-borne relapsing fever. One or more relapsing fever episodes typically occur in patients infected with all soft tick-borne relapsing fever infections but with only 10 percent or fewer people experiencing B. miyamotoi infection. No relapsing fever episodes were reported by CHR residents in 1988 and 1989.Ecological and epidemiological evidence indicates that CHR subjects whose sera reacted against B. miyamotoi infection. The CHR residents were an ideal group to screen because B. miyamotoi-infected I. pacificus ticks have been identified from nearby areas in Mendocino County. Moreover, the members of this rural community experienced extraordinarily high tick exposure rates that far exceed published exposure rates from the Northeastern United States [B. miyamotoi GlpQ and WCL antigens. Although these residents were also seroreactive to B. hermsii antigens, this was likely a result of B. miyamotoi cross reacting antibody, rather than previous B. hermsii infection. Human B. hermsii infection is infrequently reported in mountainous areas far to the east of Mendocino County and none of the residents reported having experienced relapsing fever episodes that are characteristic of B. hermsii during the two-year study period. Although we found that B. burgdorferi, like B. hermsii, can elicit cross-reacting antibody against B. miyamotoi antigens, none of our putative B. miyamotoi seropositive CHR residents could have had Lyme disease alone because they reacted against B. miyamotoi GlpQ antigen and B. burgdorferi does not produce GlpQ [B. miyamotoi infection occurs in the Far West and that B. miyamotoi, B. hermsii and B. burgdorferi spirochetes share many proteins that elicit cross reacting antibodies, which complicate the serodiagnosis of B. miyamotoi and other relapsing fever infections.Our principal aim was to determine whether residents of the far-western United States are at risk of human d States , 24, 25.uce GlpQ . In sum,B. miyamotoi infection in northern California. We found that between 2 and 7 of the 101 CHR residents were seroreactive against B. miyamotoi GlpQ and WCL antigen in 1988 and 1989, respectively. This seropostitivity range approximates the 0.5% to 15% B. miyamotoi infection range that has been found in I. pacificus ticks in Northern California [I. pacificus and its manifold vertebrate and reservoir hosts abound [I. pacificus saliva antibodies [B. miyamotoi transmission to humans at the CHR include the prolonged activity periods of I. pacificus nymphs and adults (compared to the lesser year-round activity periods of I. scapularis in the Northeast) that collectively span most of the year, as well as the occurrence of transovarial transmission of B. miyamotoi in its primary Ixodes spp. tick vectors [B. hermsii or B. parkeri. Neither of these two soft tick-borne relapsing fever species have been detected in ticks from Mendocino County [B. hermsii infection incidence averages only 5 to 7 reported cases per year in California, and it occurs mainly in high mountains located about 500 to 1000 km to the east of the CHR [B. hermsii typically causes 1–10 febrile episodes accompanied by a viral-like illness that tends to occur in clusters among family members or friends who frequent the same mountain cabins. None of the CHR residents reported a previous diagnosis of soft tick-borne relapsing fever or episodes of a relapsing fever-like illness 1–2 years pre-entry or 1 year post-entry. B. parkeri, was incriminated in only a single human case in the distant Central Valley during the1940’s [B. coriaceae, first isolated from the soft tick Ornithodoros coriaceus in southern Mendocino County decades ago [Several ecological and epidemiological observations support the possibility of human lifornia , 14, 15.tibodies . By comptibodies , 26. Oth vectors , 28. It o County , 14, 15. the CHR , 30. B. he1940’s . Human cades ago .B. burgdorferi also can elicit cross-reacting antibody against B. miyamotoi antigens, none of our putative B. miyamotoi seropositive CHR residents could have had Lyme disease alone because they reacted against B. miyamotoi GlpQ antigen and B. burgdorferi does not produce GlpQ [B. miyamotoi WCL antigen of all the B. miyamotoi/ B. hermsii seropositive CHR sera were in the two residents who also reported previous Lyme disease. The intense reactivity to B. miyamotoi and B. hermsii WCL antigens is likely due in part to B. burgdorferi cross-reacting antibody [B. miyamotoi-infected patients cross react to B. burgdorferi C6 peptide [B. miyamotoi/B. herrmsii WCL antigens also may have resulted from previous Haemophilus influenza or certain other gram-negative bacterial infections that produce GlpQ. The genetic difference between the GlpQ of these pathogens and B. miyamotoi is so wide, however, that significant cross-reactivity is unlikely [Although we found that uce GlpQ . Interesantibody . Recent  peptide , 34. Crounlikely , 20.B. miyamotoi DNA was detected in sera from CHR subjects, although these samples were obtained a few weeks to months after acute illness was likely to have occurred. The positive control sera used for the B. miyamotoi set of blots in B. burgdorferi antibody testing of the CHR residents was carried out before the development of the current two-tiered assay but the IFA assay that was used was determined to be highly sensitive and specific [B. miyamotoi and B. hermsii antibody testing, but this is unlikely to have reduced antibody concentration.Our study was subject to several limitations. We enrolled a relatively small number of subjects and controls. No PCR-amplifiable specific , 35. FinB. miyamotoi occasionally infects residents of Mendocino County in northwestern California. The presence of two soft-tick borne borreliae in California that either occasionally (B. hermsii) or rarely (B. parkeri) infect people in other counties, plus the occurrence of a third species (B. coriaceae) of uncertain human significance, precludes absolute confirmation that the seroreactive CHR residents were infected with B. miyamotoi. Our findings also highlight the pressing need to develop a serodiagnostic assay capable of differentiating all members of the relapsing fever group.Taken together, our serological, ecological and epidemiological data provide presumptive evidence that S1 Table(DOCX)Click here for additional data file.


The present study was undertaken to determine bacterial load as well as characterize bacterial flora of ready to eat (RTE) betel leaf sold at local markets in Mymensingh city.A total of 25 RTE betel leaf samples were collected from five local markets such as Kamal-Ranjit (KR) market, Shesh more, Kewatkhali, Jobber more, and Ganginar par.10 mean colony forming unit±standard deviation/ml) was 7.58±0.04 for KR market, 7.72±0.06 for Shesh more, 7.62±0.04 for Kewatkhali, 7.40±0.03 for Jobber more, and 7.60±0.06 for Ganginar par. A total of 98 bacterial isolates belong to five genera  were identified. The prevalence of E. coli was 17.34%, Salmonella spp. was 25.51%, Vibrio spp. was 19.39%, Bacillus spp. was 18.37%, and Staphylococcus spp. was 19.39%. Antibiotic sensitivity test showed that all isolates were sensitive to two antibiotics such as ciprofloxacin and gentamicin. Four isolates  were resistant to two antibiotics . Antibiogram profile of bacterial isolates of betel leaf suggests that they were multidrug resistance.Total viable count of bacteria in betel leaf (logData of this study indicate that betel leaf sold at local market harbors multidrug resistance food-borne bacteria which might cause public health hazards if these antibiotic resistant transfer to human through food chain. Piperaceae [Piper betle [Betel leaf commonly known as paan belongs to the family peraceae , and theer betle . Paan iser betle . In BangSalmonella in Bangladeshi betel leaves which was harmful for health and caused diarrhea and vomiting and other related serious illness to the people who consumed it, and ultimately, the EU stopped the importation of betel leaf from Bangladesh [Salmonella contamination in betel leaf [Bangladesh earns US$ 8 million per year by exporting betel leaf to European country which contributes a great to our national economy . Howeverngladesh . Export tel leaf . ResearcThe aim of this study was to determine bacterial load as well as to isolate and identify bacteria from betel leaves collected from different local markets of Mymensingh.The experiment was approved by Institutional Ethical Committee, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.A total of 25 betel leaf samples were collected from five different local markets such as Kamal-Ranjit (KR) market, Shesh more, Kewatkhali, Jobber more, and Ganginar par in Mymensingh . IndividThe betel leaf samples in polythene bag were washed with sterile phosphate-buffered saline (PBS). One betel leaf was washed with 20 ml of sterile PBS. A 10-fold serial dilution of the washed samples was prepared in nutrient broth. Five betel leaf samples from each market were washed and analyzed for isolation of bacteria.−1 to 10−7) was transferred and spreaded onto plate count agar and incubated at 37°C for 24-48 h. The number of colonies (30-300) in a particular dilution was multiplied by the dilution factor to calculate TVC which was expressed as mean log10±standard deviation colony-forming unit (CFU)/ml.A total of 0.5 ml 10-fold diluted sample  agar for Salmonella spp., thiosulfate-citrate-bile salts-sucrose (TCBS) agar for Vibrio spp., blood agar for Bacillus spp., and mannitol salt (MS) agar for Staphylococcus spp. and incubated at 37°C for 24 h. Single colony was further subcultured until pure culture was obtained. Identification of bacteria was performed on the basis of colony morphology; Gram’s staining reaction, motility test, and biochemical tests.Isolation and identification of bacteria were performed according to the method described by Carter . WashingE. coli was mixed with 100 µl of distilled water which was boiled for 10 min then immediately kept on ice for cold shock. Finally, centrifugation was done at 10,000 rpm for 10 min. The supernatant was collected and used as DNA template for PCR.A pure bacterial colony of E. coli using previously published primers [A genus-specific PCR was performed to amplify 16S rRNA of  primers . The lisFive isolates randomly selected from five genera were tested for antimicrobial drug susceptibility against five commonly used antibiotics such as ampicillin, chloramphenicol, ciprofloxacin, gentamicin, and cephalexin by disc diffusion or Kirby-Bauer method . AntimicThe results of TVC of bacteria of betel leaf sold at local markets were analyzed for statistical significance using Duncan’s multiple range test . A p<0.05 was considered to be statistically significant.The TVC of betel leaf samples collected from different markets is presented in E. coli, Salmonella spp., Vibrio spp., Bacillus spp., and Staphylococcus spp. were isolated from 25 betel leaf samples  and Voges–Proskauer (VP) are listed in E. coli fermented all five basic sugars such as dextrose, maltose, lactose, sucrose, and mannitol and produced acid and gas. Salmonella spp. fermented dextrose, maltose, and mannitol and produced only acid, whereas lactose and sucrose were negative. Vibrio spp. fermented dextrose, maltose, sucrose, and mannitol and produced acid, whereas lactose was negative. Bacillus spp. fermented dextrose, sucrose, and mannitol and produced acid and gas. Bacillus spp. also fermented maltose and lactose and produced only acid. Staphylococcus spp. fermented all sugars and produced acid.In sugar fermentation test, E. coli were MR and Indole positive but VP and catalase negative; Salmonella was MR positive but VP, indole, and catalase negative; Vibrio was MR, indole, and catalase positive but VP negative; and Bacillus was MR and indole positive but VP and catalase negative.All isolates of E. coli was 17.34% (17 of 98), Salmonella spp. was 25.51% (25 of 98), Vibrio spp. was 19.39% (19 of 98), Bacillus spp. was 18.37% (18 of 98), and Staphylococcus spp. was 19.39 (19 of 98). The overall prevalence of E. coli, Salmonella spp., Vibrio spp., Bacillus spp., and Staphylococcus spp. in betel leaf samples is presented in The prevalence of E. coli isolates was used in the PCR assay. PCR primers targeting 16S rRNA of E. coli amplified 704 bp fragments of DNA confirming the identity of E. coli  was subjected to antibiotic sensitivity test against five commonly used antibiotics such as ciprofloxacin, ampicillin, cephalexin, gentamicin, and chloramphenicol. The zone of inhibition of five antibiotics against tested bacteria is shown in One isolate randomly selected from each genus E. coli, Salmonella sp., Vibrio sp., Bacillus sp., and Staphylococcus sp. against antibiotics is presented in Summary of antibiogram profile of Consumption of ready-to-eat (RTE) food items including betel leaf with food-borne bacteria is an important obstacle in international trade as well as public health issue worldwide. The present research work was undertaken to measure bacterial loads and identification of bacteria in betel leaf sold at local markets in Mymensingh city.In this study, TVC of bacteria in betel leaf sample sold at different local markets ranged from log 7.403 CFU/ml to log 7.718 CFU/ml which was higher than the permissible limit (10-100 CFU/ml). Higher TVC recorded in this study might be resulted from lack of hygienic measures during cultivation, harvesting, transportation, and selling of betel leaf. A study conducted in restaurant salad vegetables in Chittagong city recorded 5.20-6.87 log CFU/g TVC . In NigeIn this study, the highest TVC of betel leaf was found at Shesh more (log 7.718 CFU/ml) and the lowest TVC was recorded at Jobber more (log 7.403 CFU/ml). The variation of TVC in betel leaf sold at local markets is statistically significant (p<0.05). This higher TVC of betel leaf might be due to unsanitary environment, use of polluted water to wash betel leaf, and unclean utensil used to storage betel leaf.Salmonella spp. was the highest (25.52%), and E. coli prevalence was the lowest (17.34%). Fakruddin et al. (2017) found that 77% betel leaf sample collected from different markets of Dhaka city was found to be contaminated with Salmonella spp. [Xanthomonas campestris pv. Betticola bacteria from diseased betel leaves. In a separate study conducted by Sharmin [E. coli, Salmonella spp., Vibrio spp., and Bacillus spp. from RTE salad vegetables sold at different local markets in Mymensingh city. E. coli, Salmonella spp., Vibrio spp., Bacillus spp., and Staphylococcus spp. are some of food-borne pathogen associated with food-borne infection and intoxication. Foods can be contaminated at many points in the food preparation, storage, and handling process. In this study, betel leaves might be contaminated with bacteria due to the use of dirty water for washing, handling of betel leaves with unclean hands, and use of unclean utensil or cutting board when preparing RTE betel leaves.In this study, five different genera of bacteria were isolated from betel leaf. The prevalence of lla spp. . A studylla spp. . Mishra lla spp.  isolated Sharmin , isolateE. coli from samples. Colony characteristics of E. coli observed in EMB were similar to the findings of Sharada et al. [E. coli was Gram-negative short rod arranged in single or paired and motile. Thomas et al. [E. coli. The identified bacteria were reconfirmed by sugar fermentation and other biochemical tests which were found similar with the findings of Thomas et al. [In this study, various selective media were used for isolation of a et al. . Morphols et al.  found sis et al. .Vibrio spp. on TCBS agar plate were similar to the findings of Khan et al. [Vibrio spp. fermented dextrose, maltose, mannitol, and sucrose with the production of acid but gave a negative result to VP test [In this study, the colony characteristics of n et al. . In Gramn et al. , 21. In Salmonella spp. on XLD agar plate were opaque, translucent with black centers which were similar to the findings of Cheesbrough [Salmonella spp. exhibited short rods, Gram-negative, single, or paired in arrangement. Similar findings were also reported by Buxton and Fraser [Salmonella spp. in the present study showed similarities with the findings of Cowan [Bacillus spp. recorded in this study are in agreement with the findings of Merchant and Packer [Bacillus spp. fermented five basic sugars with the production of acid [In this study, the colonies of esbrough . In Gramd Fraser . Sugar fof Cowan . Morphold Packer . Bacillu of acid .Staphylococcus spp. observed in MS agar were similar to the findings of Chatterjee et al. [Staphylococcus spp. isolates in this study fermented glucose, maltose, lactose, sucrose, and mannitol fermentation with only acid production. These findings are in close agreement with the findings of Chatterjee et al. [Colony characteristics of e et al.  who recoe et al. .E. coli, Salmonella spp., Vibrio spp., Bacillus spp., and Staphylococcus spp. exhibited multidrug-resistant profile. Salmonella spp., in this study, showed resistant against three antibiotics. Singh et al. [Salmonella from RTE betel leaves in North Indian cities. In this study, Salmonella spp. was found sensitive to ciprofloxacin and resistant to chloramphenicol. Salmonella spp. isolated from betel leaves was found sensitive to both ciprofloxacin and chloramphenicol [E. coli [In this study, antibiotic sensitivity assay showed that all bacterial isolates were resistant to ampicillin. Four isolates such as h et al.  isolatedphenicol . Indiscr[E. coli .Data of this study suggest that betel leaves sold at local markets of Mymensingh city harbor multidrug-resistant bacteria which underscore the need of implementation of hygienic practices during production, harvesting, transportation, storage, selling, and preparation of betel leaves to safeguard public health.MMK and MAI designed the study. MMH and MARS collected and processed the samples for isolation and identification of bacteria. MMH, MARS, and RAR were done PCR and electrophoresis. MMH and MMK interpreted the results and analyzed the data. MMH, MAI, and MMK prepared the manuscript. All authors read and approved the final manuscript.


Eucommia ulmoides (E. ulmoides), the sole species of Eucommiaceae with high importance of medicinal and industrial values, is a Tertiary relic plant that is endemic to China. However, the population genetics study of E. ulmoides lags far behind largely due to the scarcity of genomic data. In this study, one complete chloroplast (cp) genome of E. ulmoides was generated via the genome skimming approach and compared to another available E. ulmoides cp genome comprehensively at the genome scale. We found that the structure of the cp genome in E. ulmoides was highly consistent with genome size variation which might result from DNA repeat variations in the two E. ulmoides cp genomes. Heterogeneous sequence divergence patterns were revealed in different regions of the E. ulmoides cp genomes, with most (59 out of 75) of the detected SNPs (single nucleotide polymorphisms) located in the gene regions, whereas most (50 out of 80) of the indels (insertions/deletions) were distributed in the intergenic spacers. In addition, we also found that all the 40 putative coding-region-located SNPs were synonymous mutations. A total of 71 polymorphic cpDNA fragments were further identified, among which 20 loci were selected as potential molecular markers for subsequent population genetics studies of E. ulmoides. Moreover, eight polymorphic cpSSR loci were also developed. The sister relationship between E. ulmoides and Aucuba japonica in Garryales was also confirmed based on the cp phylogenomic analyses. Overall, this study will shed new light on the conservation genomics of this endangered plant in the future. Unfor extinct . Many pl extinct ,5,6. ConEucommia ulmoides Oliver, a dioecious woody plant endemic to China, is the sole species in the family Eucommiaceae [E. ulmoides has been widely cultivated and used as a herbal drug to reduce blood pressure and strengthen the body in central and southern China for at least 2000 years [E. ulmoides is also well-known as a “hardy rubber” tree that produces trans-polyisoprene rubber  in the leaves, bark, and pericarp [Eucommia fossils occurred widely across the Northern Hemisphere from the Palaeocene onwards [E. ulmoides is a representative model of Tertiary relict species, i.e., living from Tertiary to present. However, E. ulmoides may have been extinct in the wild and already listed in the Red List of Endangered Plant Species in China probably due to exhaustive human exploration [mmiaceae . E. ulmo00 years ,9. E. ulpericarp ,11. It h onwards , which iloration ,14. TherE. ulmoides have mainly focused on the morphological variation and the natural products [E. ulmoides [E. ulmoides [E. ulmoides is relatively low, with limited population genetics information. A new and promising marker type i.e., Single Nucleotide Polymorphism (SNP) has gained high popularity during the last two decades [Pinus ponderosa Douglas ex Lawson [Sciadopitys verticillata (Thunb.) Siebold and Zucc [To date, studies on products ,15. Moleproducts ,17. Receulmoides ,19. Amplulmoides ,21. The ulmoides ,23,24. N decades ,26. With decades ,28. Usinx Lawson , and Sciand Zucc .E. ulmoides with a length of 163,341 bp. Clearly, the availability of additional sequenced cp genomes from E. ulmoides would aid our understanding of the cp genome-wide variation at the individual level. Through comparative genomic analysis, polymorphic cpDNA loci with plentiful SNPs and indels i.e., nucleotide insertions and deletions can also be detected, which would be useful for further population genetics studies of E. ulmoides.Chloroplast (cp) DNA sequences have been extensively used in the studies of plant population genetics and molecular phylogenetics ,32,33. TE. ulmoides using the genome skimming approach. By comparing the cp genome generated in this study and the one published previously [E. ulmoides show structural rearrangements; (2) reveal the divergence pattern of the cp genome in E. ulmoides; (3) identify highly variable cp genome-wide markers for subsequent population genetics studies of E. ulmoides.Genome skimming is currently one of the most economical techniques to obtain plastome sequences , througheviously , our maiE. ulmoides with high genome coverage (>180×) and there is no difference between the two assembled sequences, suggesting a high-quality cp genome map was achieved. The final cp genome size was determined to be 163,586 bp, similar to the previously published one (KU204775) (E. ulmoides cp genome (KU204775). We have deposited the newly sequenced E. ulmoides cp genome in GenBank with accession number MF766010. The genome skimming sequencing reads have also been deposited in the Sequence Read Archive (SRA) with the accession number PRJNA399774.About 20 million clean reads (4.72 Gb data) were generated from genome skimming sequencing. Two assembly methods (CLC Genomics Workbench and SPAdes software) both obtained the complete cp genome of U204775) . The numE. ulmoides is highly conserved and perfectly syntenic (E. ulmoides cp genome. We found 15 insertions with more than ten nucleotides (11–111 bp) in the two cp genomes  were found in the gene sequences, including 31 protein-coding genes and one tRNA gene. In contrast, indels were mainly (50 out of 80) distributed in the intergenic spacers , we found that all the mutations were located in the intron regions. In all, 40 SNPs and 14 indels occurred in the plastid-coding sequence (CDS) regions.A total of 155 mutational events, including 75 nucleotide substitutions (SNPs) and 80 nucleotide indels (insertions and deletions), were detected within 71 loci of the cp genome in ulmoides . There w spacers . Upon fuatpF-atpA, rps18-rpl33, psaJ, infA and rpl32 had variations exceeding 1.00%. Considering the relatively high percentage of variability and convenience for primer design in PCR (Polymerase Chain Reaction) and sequencing experiments, we chose 20 highly variable loci with length of 200–1500 bp as potential molecular markers for subsequent population genetic studies  as reference revealed a total of 75 SNPs. This result of SNP occurrence was consistent with the aforementioned molecular marker analysis , which was subsequently sister to Aucuba japonica Thunb. in the Garryales clade with high statistical support values (100/100/1.00). Garryales, Gentianales and Solanales formed as the highly supported lamiids lineage (100/100/1.00), resolved as ). Asterales and Dipsacales formed a highly supported clade, campanulids (100/100/1.00), which was sister to lamiids (100/100/1.00) in asterids. Ericales was resolved as the basal most group in the asterids lineage (100/100/1.00). Brassicales, Malvales, Myrtales and Sapindales clustered in the malvids clade (95/98/1.00) with resolution as , Sapindales), Myrtales). Fabales, Malpighiales and Rosales were located in the fabids lineage (98/100/1.00), which was a sister clade to malvids in rosdis.The final length of the supermatrix dataset contained 96,894 unambiguously aligned nucleotide characters. Three methods produced a congruent phylogenetic tree, shown in E. ulmoides in terms of the contained genes and coding regions in the cp genomes  in this study is 245 bp larger than that of the previously reported one , psbD-trnT(GGU), rps12-rpl20 and accD-trnM(CAU) have been detected in the study of Wang et al. [E. ulmoides cp genome may contribute to the cp genome size variation.It is noteworthy that the newly sequenced cp genome of ,341 bp, )  that varied the most quickly, all having variations exceeding 1.2% . The rebination ,60 may lE. ulmoides [E. ulmoides. The relationships among natural and cultivated populations of E. ulmoides are elusive at present [circa 200 to 1500 bp using Sanger sequencing method [E. ulmoides. These selected plastid genome-wide loci would genetically be informative for uncovering the genetic relationships among the natural and cultivated E. ulmoides populations.The genetic markers of SSR, AFLP and SRAP have been developed and used for the population genetics studies of ulmoides ,20,21. Hulmoides ,24 may p present ,24. SNP  present ,62. High present  and temp present . Given tg method , we thusE. ulmoides here would be potential genetic markers to facilitate subsequent population genetics studies in the future.Additionally, 27 cpSSR loci identified by Wang et al.  were alsE. ulmoides cp genome was further validated via phylogenomic analyses using 34 complete plastomes from 10 major lineages of angiosperms. The resulting phylogenomic tree highly supported the clade of two E. ulmoides cp genomes  in the updated APG IV system [E. ulmoides and A. japonica are both woody and have unisexual flowers in separate individuals, which seem to be morphological synapomorphies for the order Garryales [E. ulmoides and A. japonica were also detected, suggesting a high similarity between the two species at the molecular level. Garryales was shown to be closely related to the clade of  in lamiids, in line with the APG IV system [The newly obtained  genomes , confirmar genes  and 36 par genes , supportV system . E. ulmoarryales . An averV system .E. ulmoides among the flowering plants [All 20 sampled orders were highly supported to be monophyletic separately , agreeing plants .E. ulmoides growing in the Arboretum of Northwest Agricuture and Forest University in Yangling, Shanxi, China, in April 2015. After collection, the leaves were immediately immersed in liquid nitrogen and then stored at −80 °C until use. The voucher specimen of this tree was deposited at the Trees Herbarium of Northwest A and F University with accession number ZXZ15027.Fresh healthy leaves were collected from an adult male individual of circa 500 bp were constructed from fragmented genomic DNA based on standard Illumina protocols . Prepared library was then sequenced for PE 100 bp read length on the Illumina HiSeq 2000 platform at the Beijing Genomics Institute (BGI) in Shenzhen, China.Total genomic DNA was extracted by the CTAB method . Paired-E. ulmoides cp genome. (1) The cp genome was de novo assembled using the CLC Genomics Workbench v7.5 software  based on the clean reads. After discarding contigs with length <300 bp and sequences with coverage <50, the remaining contigs were searched against the available cp genome of E. ulmoides (GenBank accession number KU204775) that used as the reference by BLAST (http://blast.ncbi.nlm.nih.gov/) with e-value <10−5. Aligned contigs with ≥90% similarity and query coverage were determined as cpDNA sequences and ordered according to the reference genome. Small gaps were filled using PE clean reads as conducted in Wang et al. [E. ulmoides to determine the proportion of cpDNA using Bowtie v2.3.1 program [Fastq format PE reads were supplied with adaptor sequences removed. Poor quality reads with phred scores lower than 20 for more than 10% of their bases were also removed. Two independent methods were used to assemble the g et al. . (2) The program  with a mDOGMA software  was usedE. ulmoides (accession number: KU204775) was downloaded from GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). This genome was aligned with the E. ulmoides cp genome described herein, using MAFFT program [E. ulmoides cp genomes.The previously published cp genome of  program  and MAUV program , respect program . Moreove program , we examE. ulmoides cp genomes were analyzed to identify molecular markers that can be selected in subsequent population genetic studies. We firstly extracted both the genic and intergenic DNA fragments in each cp genome using the “Extract Sequences” option in DOGMA [http://www.drive5.com/muscle/) [The two in DOGMA . Then thmuscle/)  implemenmuscle/)  with defE. ulmoides cp genome were detected by MISA perl script (http://pgrc.ipk-gatersleben.de/misa/). The parameter of minimum repeat unit was set as 10 for mono-, 6 for di-, and 5 for tri-, tetra-, penta-, and hexanucleotide SSRs [E. ulmoides.Polymorphic cpSSR loci were further mined by genome comparison. Firstly, SSRs in the newly sequenced ide SSRs . Then, aide SSRs  to develE. ulmoides cp genome (KU204775) [http://broadinstitute.github.io/picard/) and samtools v0.1.18 [E. ulmoides cp genome caused amino acid substitution on protein level, we firstly translated each protein-coding gene into amino acids in Geneious v9.0 [To confirm the SNPs identified by the aforementioned cp genome alignment, we here mapped the genome skimming clean reads generated in this study to the previously published U204775)  for SNP  v0.1.18  were app v0.1.18  was furtous v9.0 . Then thous v9.0 . The mutE. ulmoides cp genome. 34 plastomes representing 10 major lineages of angiosperms , maximum likelihood (ML), and Bayesian inference (BI) were used for phylogenetic reconstruction. We performed parsimony heuristic tree searches in PAUP v4.0b10  with parE. ulmoides using the genome skimming approach. Through comprehensive genome-wide comparative analyses we found that the cp genomes within E. ulmoides were highly conserved in terms of structure and content. Nevertheless, obviously heterogeneous sequence divergences were revealed in different regions of the E. ulmoides cp genome. A total of 20 polymorphic DNA fragments and eight SSR loci have been identified as potential cpDNA markers for subsequent population genetics studies of this tree species. The phylogenetic placement of E. ulmoides in angiosperms was robustly resolved as well based on the cp genomes data, strongly supporting the sister relationship between E. ulmoides and A. japonica in the asterids lineage. The data presented here will aid further conservation genomic studies and facilitate the development of plastid genetic engineering for E. ulmoides.In summary, in the present study we generated one complete cp genome of


Immune checkpoint inhibitors (ICIs) therapy is a novel strategy for cancer treatments in recent years. However, it was observed that most patients treated with ICIs could not get benefit from the therapy, which led to the limitation of clinical application. Motivated by potent and durable efficacy of ICIs, oncologists endeavor to explore the mechanisms of resistance to ICIs and increase the drug sensitivity. It is known that heterogeneity of gut microbiome in populations may result in different outcomes of therapy. In xenograft model, bacteria in gut have been proved as a crucial factor regulating immunotherapy efficacy. And the similar phenomenon was obtained in patients. In this review, we summarized relevant advancements about gut microbiome and ICIs. Furthermore, we focused on modulatory function of gut microbiome in ICIs therapy and possible antitumor mechanism of specific commensals in ICIs treatment. We propose that gut microbiome is an important predictive factor, and manipulation of gut microbiome is feasible to elevate response rate in ICIs therapy. Various bacteria populating in mammal gastrointestinal tract are an indispensable part in intestine ecosystem and play a pivotal role in gut barrier . CommensNotably, this influence on host immune system even affects the efficacy of some agents, although the exact mechanism is unknown. Immune checkpoint inhibitors (ICIs), known as the novel immunotherapy agents, take significant and durable curative effects on advanced hematological and solid malignancies –15. SimuNevertheless, the majority of patients shows primary or acquired resistance during treatment (up to 60–70% in melanoma and even higher rate in other cancers), which limits the clinical application of ICIs . BecauseLachnospiraceae, Ruminococcaceae, and Butyrate producing bacterium L2-21, upregulate butyrate level in lumen which is responsible for intestinal epithelial cells renewal. Specific commensal bacteria are also correlated with outcompeting pathogens and the synthesis of essential vitamins for human, such as vitamin B and vitamin K [Enterotoxigenic Bacteroides fragilis, undermines the integrity of gut barrier and increases the chance of immune tissue’s exposure to bacteria and metabolites, which in turn activate inflammation responses and increase colorectal cancer risk [Symbiotic commensals in human gastrointestinal tract play a vital role in sustaining host homeostasis and health. Symbiotic commensals participate in many physiological functions. As a part of digestive system, symbiotic commensals promote process of saccharide digestion and absorption by transforming polysaccharides into disaccharides and monosaccharides , 23. By itamin K , 25. Theitamin K . It is aitamin K . Besidesitamin K . Pathogeitamin K . Metallocer risk .Clostridiales, suppress immune response by inducing Tregs differentiation and IL-10 production in intestine and extra-intestine [B. fragilis has the ability to correct immune deficiencies  in germ-free mice [E. hirae induces pathogenic Th17 (pTh17) cells response and increases cytotoxic T cells/Tregs ratio in extra-intestinal tissue, while B. intestinihominis enhances systemic Tc1 and Th1 response [Notably, resident commensal bacteria modulate host immune system by cross-talk with epithelial cells and lymphoid structures . It has ntestine . Apart free mice . E. hiraresponse . Howeverresponse . In mousresponse . Even thresponse .Due to the advancement of sequencing technology, especially the appearance of Next-Generation Sequencing (NGS) technology, it is available to analyze composition of microbiota. Bacterial 16S rRNA sequencing and metagenomic shotgun sequencing have been widely applied for taxonomic assignment. Bacterial 16S rRNA sequencing provides a convenient access to analyze the microbiota . BecauseICIs, including CTLA-4 and PD-1/PD-L1, are the monoclonal antibodies to specific receptors on cell membrane and aim to block the signaling pathways which negatively modulate the immune system. ICIs restore the exhausted T cells and activate the immune system to promote destruction of tumor cells through blocking related signaling pathways mentioned above. PD-1 is the most important immunotherapy target, expressed on tumor infiltrating lymphocytes (TILs) and other immune cells . PD-1 is+ CD25+ Foxp3+ regulatory T cells, and it is upregulated transiently in activated conventional T cells [CTLA-4 receptor is another target for immunotherapy, similarly to PD-1/PD-L1 signaling pathway, negatively regulating immune system. CTLA-4 is constitutively expressed in CD4 T cells . Sharing T cells . Through T cells , 46. Bes T cells . CTLA-4  T cells . In tumo T cells . Apart f T cells . And SHP T cells . Further T cells . By mann T cells . ICIs in T cells –52. By m T cells , 54.2A receptor and adenosinergic pathway which consists of CD39 and CD73, participate in angiogenesis, metastasis, and immune suppression [The clinical trial Keynote 006 NCT01866319), involving 843 patients with advanced melanoma, showed that the patients receiving pembrolizumab treatment had response rates ranging from 33.7% (10 mg/kg every 2 weeks) to 32.9% (10 mg/kg every 3 weeks), while patients receiving ipilimumab (3 mg/kg every 3 weeks) had a worse response rate of 11.9% . Besides, involvipression –66. Morepression .Bifidobacterium on PD-1 blockade treatment, using two strains mouse models (JAX/TAC) bearing B16.SIY melanoma [+ T cells and more potent tumor-specific immune response were observed in JAX, and the difference was abrogated by cohousing. Besides, researchers found that transferring fecal microbiome from JAX to TAC could elevate specific tumor lymphocytes and suppress tumor growth. Interestingly, in TAC, just fecal microbiome transfer from JAX could inhibit tumor growth in the same degree with PD-1 blockade treatment, and it had synergetic effect with PD-1 blockade treatment in promoting tumor regression [Bifidobacterium abundance was related to tumor specific immune cytotoxicity, and the abundance increased over 400-fold after fecal microbiome transplantation in TAC [Bifidobacterium operational taxonomic units (OTUs) were identified to be similar with Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium adolescentis in 99% identity  [Distinguished from cytotoxic therapies, ICIs mediates tumor regression via enhanced host immune activation. Some studies revealed the shift in gut microbiome composition influencing ICIs efficacy. As early as 2015, researchers noticed the relationship between gut symbiotic bacteria and PD-1 blockade. Ayelet Sivan et al. explored the influence of melanoma . The subgression . Gut micn in TAC . By 16S ls (DCs) .Table 1MClostridiales/Ruminococcaceae were prone to respond to PD-1 blockade effectively, contrary to ones enriched in Bacteroidales  caught researchers’ attention [. Patients with high abundance of Faecalibacterium had longer PFS (p = 0.03) and advantage in hazard ratio  in comparison with patients with low abundance of Faecalibacterium [+ T cells was positively related to abundance of Faecalibacterium genus, contrary to Bacteroidales order [Faecalibacterium, Clostridiales, and Ruminococcaceae had more effector T cells, while patients with overrepresentation of Bacteroidales had more Tregs and myeloid-derived suppressor cells [Faecalibacterium [. The fecal microbiome transplantation in mice supported the conclusions aforementioned [Then, two studies involving some advanced tumor patients for further exploration of relationship between gut microbiome and PD-1 blockade were carried out subsequently in 2017. Gopalakrishnan V et al. analyzed differences of gut microbiome diversity and composition between responding individuals (R) and non-responding individuals (NR) . Throughttention . Patientacterium . Besideses order . In perior cells . Multiplacterium . The fecentioned .Akkermansia muciniphila was the most significant factor related to potent response  and better clinical outcome  was accompanied with the decrease of Bacteroidales and Burkholderiales  [Bacteroidales [However, in 2017, another trial focusing on the relationship between gut microbiome at baseline and clinical benefit was conducted in metastatic melanoma individuals, and the result was conflicted with conclusion of Marie Vétizou. Inconsistent with the conclusion of trial in mouse models, the study found that both  and OS) . Contrar and OS) . Given t and OS) , 12, 73. and OS) . Apart froidales . Notablyroidales . Given troidales . And furGut microbiome has been verified to participate in oncogenesis as well as immune surveillance suppressing malignant transformation –77. By iFaecalibacterium prausnitzii and Clostridia class induce Tregs differentiation and suppress inflammation [Faecalibacterium and other species of Firmicutes had lower proportion of systematic inflammation lymphocytes at baseline. On the contrary, patients enriched in Bacteroides had increased systematic inflammation biomarkers in peripheral blood at baseline, accompanied with decreased colitis risk and clinical benefit [Bacteroides led to the decreased colitis incidence risk [Owing to the predominant expression of CTLA-4 in Tregs, the status of Tregs at baseline is pivotal to determine CTLA-4 blockade efficacy, which is distinguished from PD-1 blockade. Tregs play a crucial role in modulating immune response and correlate to the failure of immune co-stimulation process. Some specific bacteria species and metabolites have been confirmed to participate in Tregs differentiation. Bacteria including ammation , 89. Gutammation , 90. Bes benefit . Presuma benefit . Increas benefit . The connce risk .Increased CTLA-4 level at baseline helps tumor cells escape from immune surveillance, but it increases the sensitivity to CTLA-4 blockade simultaneously. This factor is more crucial for CTLA-4 blockade in comparison with PD-1 blockade, because of greater depletion of Tregs resulting from CTLA-4 blockade . PD-1 blBifidobacterium has been confirmed to upregulate gene transcription of DCs, associated with interaction of cytokines, maturation of DCs, and activation of lymphocytes [+ T cells and enhanced lymphocytes function have synergetic effects with ICIs, contributing to tumor regression.Enhanced function of DCs is generally believed as an important manner to promote T cell activation. phocytes . The shiphocytes . Besidesphocytes . IncreasAkkermansia muciniphila and Enterococcus hirae in gut is related with emergence of CD4+ central memory T cell (TCM) in tumor bed, tumor draining lymph code, and mesenteric lymph code [CM expresses chemokine receptor CXCR3 and/or CCR9. CXCR3 and CCR9/CCL25 axis have been verified to be related with prolonged PFS and OS in patients with some advanced tumors [Colonization of mph code . Intrigud tumors , 94. CXCd tumors . PresumaAkkermansia muciniphila and Enterococcus hirae are prone to having longer PFS [Enterococcus hirae, to secondary immune organs even tumor bed by impaired gut barrier may influence systemic inflammation [ICIs undermine gut immune tolerance, accompanied with response targeting these commensal bacteria. Patients with memory T cells response targeting nger PFS . Though nger PFS , 96. Intammation . Besidesammation , 78. ResExcept for mechanism mentioned above, some other factors are speculated to involve in ICIs treatment. Some bacterial metabolites, such as SCFAs, can be utilized by intestinal epithelial cells as source of energy , 98. SCFThe cancer-immune checkpoint set point model could summarize pathways involving in ICIs treatments, which could be understood as the threshold to overcome for immune response to neoantigen . In thisCancer immunotherapy includes the use of antibodies, lymphocytes, and cytokines , 102. ICTranslation of the findings in mouse model into clinical trial has a long way to go. First of all, identification of bacteria with modulatory ability needs a great quantity of data. Besides, modifying gut microbiota of patient is another obstacle to overcome in clinical trial after identification of favorable microbial communities. Just as widely applied in mouse model experiments, fecal microbiome transplantation is a promising method and other methods are worthwhile to try . Further


This article reviews potential molecular targets and associated novel agents for the treatment of advanced SCC in the era of personalized medicine. Elements of various pathways including  No predromising . Conversromising , 36. Des results –57. To d results .FGFR pathway have also been frequently reported in various malignancies including SCC of the lung , has been investigated for the treatment of NSCLC. Pitini et al. reported a case of a patient with DDR2 mutated SCC who presented a nearly complete response following treatment with dasatinib for a concurrent CML (EGFR mutated adenocarcinoma and one with SCC (Discoidin domain receptor tyrosine kinase 2 is a widely expressed receptor tyrosine kinase (RTK) in normal cells that is activated through its interaction with various types of extracellular matrix protein collagen. Once activated by ligand binding and phosphorylation, DDR2 has been shown to promote various cellular functions such as migration, differentiation, proliferation, and survival . This RT% of SCC , 22. Furll lines . Dasatinrent CML . In a phrent CML . Its usewith SCC . It is hMET is disrupted in various cancers including NSCLC .The proto-oncogene ng NSCLC . It encon kinase . Variousrcinomas . Once uprcinomas . Clinicarognosis . Cells hrognosis , 82. Sevrognosis –53. For rognosis . Moreoverognosis . Tivantirognosis . In subgistology . FinallyIn recent years, immunotherapy agents have elicited great interest for the treatment of several tumor types. Various immune checkpoint inhibitors including antibodies directed against cytotoxic T-lymphocyte associated protein 4, programmed cell death protein 1 (PD-1), and programmed death ligand-1 (PD-L1) are under investigation or approved for clinical practice, revolutionizing the approach to lung cancer treatment. Patients diagnosed with SCC in particular have benefited from these advancements, as alternative treatments are sparse, and they have higher mutation burden, which may be associated with benefit. Agents such as nivolumab, pembrolizumab, and atezolizumab have demonstrated improvement in survival outcomes in the second-line setting including in SCC –87. FurtSCC represents complex tumors with alterations in various interacting pathways . DespiteSS researched data for review topic, drafted manuscript, and edited manuscript revisions. NL researched data for review topic and edited manuscript.SS: none, NL: none known .


Plasmodium vivax and Plasmodium ovale malarias and therefore prevents their relapse . It is also the only generally available antimalarial that rapidly sterilises mature P. falciparum gametocytes. Radical cure requires extended courses of primaquine , whereas transmissibility reduction in falciparum malaria requires a single dose . The main adverse effect of primaquine is dose-dependent haemolysis in glucose 6-phosphate dehydrogenase (G6PD) deficiency, the most common human enzymopathy. X-linked mutations conferring varying degrees of G6PD deficiency are prevalent throughout malaria-endemic regions. Phenotypic screening tests usually detect <30% of normal G6PD activity, identifying nearly all male hemizygotes and female homozygotes and some heterozygotes. Unfortunately, G6PD deficiency screening is usually unavailable at point of care, and, as a consequence, radical cure is greatly underused. Both haemolytic risk  and relapse rates vary, so there has been considerable uncertainty in both policies and practices related to G6PD deficiency testing and use of primaquine for radical cure. Review of available information on the prevalence and severity of G6PD variants together with countries’ policies for the use of primaquine and G6PD deficiency testing confirms a wide range of practices. There remains lack of consensus on the requirement for G6PD deficiency testing before prescribing primaquine radical cure regimens. Despite substantially lower haemolytic risks, implementation of SLD primaquine as a P. falciparum gametocytocide also varies. In Africa, a few countries have recently adopted SLD primaquine, yet many with areas of low seasonal transmission do not use primaquine as an antimalarial at all. Most countries that recommended the higher 0.75 mg/kg single primaquine dose for falciparum malaria  have not changed their recommendation. Some vivax malaria–endemic countries where G6PD deficiency testing is generally unavailable have adopted the once-weekly radical cure regimen (0.75 mg/kg/week for 8 weeks), known to be safer in less severe G6PD deficiency variants. There is substantial room for improvement in radical cure policies and practices.Primaquine is the only available antimalarial drug that kills dormant liver stages of  Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most prevalent enzyme deficiency in the world. An estimated 400 million people are affected (reviewed in –5). WithAs G6PD deficiency is X-linked, males are hemizygous and either phenotypically normal or deficient  . HomozygPlasmodium vivax and P. ovale), and rapid gametocyte sterilising  activities  between the late 1940s and the early 1960s –14. HaemP. vivax ..29].P. falciparum gametocytocide. The exceptions are Ethiopia, where single low dose (SLD 0.25 mg/kg) is now accepted although not yet widely implemented, and Madagascar, Botswana, Eritrea, Swaziland, Zimbabwe, Mauritania, which recently included single-dose primaquine as policy (2015), and Comoros, Mayotte, Namibia, Sao Tome and Principe, Algeria, and Cabo Verde  to include primaquine for radical cure (0.25 mg/kg for 14 days), but Botswana, Namibia, and Madagascar do not require G6PD deficiency testing before primaquine, while Mauritania, Mayotte, Sao Tomé and Principe, and Cabo Verde do , and policy is not to use primaquine as a bo Verde . PrimaquVerde do .The enormous populations of this large region host a heterogeneous array of G6PD variants . While tP. falciparum, leading to ACT failure in 5 countries of the Greater Mekong subregion  [Most countries in Asia have low seasonal transmission, and in recent years, many have set ambitious targets for malaria elimination within the next decade. The emergence and recent spread of artemisinin-resistant Vietnam) , threateRadical cure primaquine treatment policies vary across the Asian region. Some countries state a requirement for G6PD deficiency testing before primaquine administration , although in practice testing is often not available. Myanmar acknowledges the lack of access to testing at village level, where most malaria cases are diagnosed, and has recently adopted a policy of weekly primaquine administration for 8 weeks for all patients treated by community malaria volunteers. Iran also recommends this regimen . While iP. vivax in East Asia and Oceania, most countries there still recommend the lower 0.25 mg/kg/day dose except for Malaysia, Myanmar, and the Philippines (Although WHO has recommended the higher daily dose of primaquine (0.5 mg/kg/day) for radical cure of lippines .Sri Lanka is currently in the prevention-of-reintroduction phase after having successfully eliminated malaria  . China, P. falciparum gametocytocide . Paraguay has applied to be certified as malaria-free, and Argentina is in the prevention-of-reintroduction phase. In sad contrast, malaria has resurged in Venezuela as the public health services there have weakened. The majority of malaria cases in the Americas come from the Amazon region . There icytocide , mostly P. vivax is estimated to have caused approximately 69% of the malaria cases in the Americas in 2015 [P. vivax malaria as policy. Primaquine 0.25 mg/kg for 14 days is given with chloroquine except in Brazil, Bolivia, Peru, Mexico, Nicaragua, and Ecuador, where the same total primaquine dose is recommended but given over 7 days (0.5 mg/kg/day for 7 days)  . In MexiP. falciparum gametocytocide, the risk assessment is relatively easy, whilst the assessment of benefit is more difficult. The currently recommended primaquine SLD is 2–3 times lower than the 0.75 mg/kg dose recommended previously and 14–28 times lower than the total radical curative dose. In large studies of mass treatment conducted in areas where the G6PD Mahidol variant predominated, SLD primaquine was not associated with clinically significant haemolysis [P. falciparum gametocytocide. However, as malaria transmission is reduced actively in an elimination programme, every transmission source must be removed. There is no clear explanation of why SLD primaquine is not deployed in all countries aiming for elimination. However, once the SLD primaquine policy is adopted, every effort should be made to ensure high coverage. Optimised age-based dosing regimens for SLD primaquine are recommended, based on anthropometric data for specific populations [There is substantial variation in national policies for the use of primaquine, even among countries with similar patterns of malaria epidemiology, drug resistance, and G6PD deficiency prevalence. This variation is amplified by the disconnect between policy and practice resulting from divergent opinions on risks and benefits. In many cases in this study, respondents confirmed discordance between the national recommendation and the current practice, invariably leading to primaquine underuse. For single-dose primaquine as a emolysis . Given temolysis . G6PD deulations , 44. An ulations , 46.Use of primaquine in radical curative regimens carries much greater risks of haemolysis in G6PDd patients, but it also provides substantial benefits to treated patients . The risP. vivax or P. ovale malaria who are G6PDd, WHO recommends they should not receive daily primaquine. Instead, these patients may receive once weekly primaquine 0.75 mg/kg for 8 weeks to prevent relapses, provided they are under close medical supervision for signs and symptoms of acute haemolytic anaemia during the first 3 weeks of treatment and they have access to health facilities with capacity for safe blood transfusion [Where relapse rates are high , effective radical cure more than halves the incidence of vivax malaria. The benefit is substantial. If G6PD deficiency testing is unavailable, the correct approach to patient management is unclear as it depends on the prevalence and severity of G6PD deficiency in the patient’s original population, the degree of anaemia, and the availability of blood transfusion in case of severe haemolysis, balanced against the probability of relapse and its pathological consequences and the likely dose regimen required for radical cure. More could be done to improve the safety of primaquine administration through better packaging and presentation of the drug as for Coartem (artemether-lumefantrine) , which insfusion .P. vivax relapse risks and the prevalence and severity of G6PD deficiency, but this review suggests there is plenty of room for improvement. The gap between policy and practice is also too wide, and primaquine is underused. Informed review of risks and benefits should help to harmonise treatment policies. As is often the case in malaria, the strength of opinion seems inversely proportional to the quality and quantity of the evidence, so more information is needed on the safety of radical cure regimens in the more severe G6PD deficiency variants to inform policies and practices. Measuring adherence accurately is very difficult, but as it is such a critical determinant of therapeutic response; it deserves further behavioural, pharmacokinetic, and epidemiological studies to try and ensure people do adhere to current and future regimens. Widescale availability of point-of-care G6PD deficiency testing would undoubtedly improve prescribing safety, although the current 30% activity threshold will not identify the majority of female heterozygotes. Safer primaquine regimens could be developed. A new 8-aminoquinoline tafenoquine will be introduced soon. This provides radical cure in a single dose, but as it should not be given to patients with less than 70% of normal G6PD activity, it will require deployment of a new G6PD deficiency test. This will still leave a significant minority of patients (particularly women of childbearing age) requiring a safe primaquine regimen. Policies and practices both need frequent review.If vivax malaria is to be eliminated, use of radical curative treatment will have to increase. Some heterogeneity in policies for radical cure of vivax malaria is understandable, given the geographic variation in Plasmodium falciparum, causes dose-dependent haemolysis in G6PD-deficient individuals.Glucose 6-phosphate dehydrogenase (G6PD) deficiency, the most common human enzymopathy, is prevalent in tropical and subtropical areas where malaria is or was endemic. Primaquine, the only drug licensed for radical cure of vivax and ovale malaria, and the only licenced antimalarial with specific gametocytocidal activity against Primaquine is underused (reflected in both policies and practices) because of low availability of G6PD deficiency testing, poor adherence to radical cure regimens, lack of paediatric formulations, and concerns about toxicity.The single low dose of 0.25 mg/kg of primaquine recommended by WHO in 2012 to be added to falciparum malaria treatment to reduce transmission in low-transmission settings is safe but has not been adopted by many countries, particularly in Africa.A stronger commitment to implement primaquine treatment by malaria control programmes is needed. For current regimens recommended for radical cure of vivax and ovale malaria, this is unlikely to happen without expanded access to G6PD deficiency testing, patient education on toxicity warning signs, and improved dosage forms for children.Beutler E. The hemolytic effect of primaquine and related compounds: a review. Blood. 1959 Feb;14(2):103–39. PubMed PMID: 13618370.Piasmodium vivax by intermittent regimens of drug administration: a preliminary report. Bull World Health Organ. 1960;22:621–31. PubMed PMID: 13793053; PubMed Central PMCID: PMC2555355.Alving AS, Johnson CF, Tarlov AR, Brewer GJ, Kellermeyer RW, Carson PE. Mitigation of the haemolytic effect of primaquine and enhancement of its action against exoerythrocytic forms of the Chesson strain of 10.1111/bjh.12665. Epub 2013 Dec 28. Review. PubMed PMID: 24372186; PubMed Central PMCID: PMC4153881Luzzatto L, Seneca E. G6PD deficiency: a classic example of pharmacogenetics with on-going clinical implications. Br J Haematol. 2014 Feb;164(4):469–80. doi: 10.1371/journal.pmed.1001339. Epub 2012 Nov 13. PubMed PMID: 23152723; PubMed Central PMCID: PMC3496665.Howes RE, Piel FB, Patil AP, Nyangiri OA, Gething PW, Dewi M, Hogg MM, Battle KE, Padilla CD, Baird JK, Hay SI. G6PD deficiency prevalence and estimates of affected populations in malaria endemic countries: a geostatistical model-based map. PLoS Med. 2012;9(11):e1001339. doi: Plasmodium falciparum gametocytocide in populations where G6PD deficiency is common. Malar J. 2012 Dec 14;11:418. doi: 10.1186/1475-2875-11-418. Review. PubMed PMID: 23237606; PubMed Central PMCID: PMC3546849.White NJ, Qiao LG, Qi G, Luzzatto L. Rationale for recommending a lower dose of primaquine as a


Chlamydia suis is an important, globally distributed, highly prevalent and diverse obligate intracellular pathogen infecting pigs. To investigate the prevalence and genetic diversity of C. suis in China, 2,137 nasal, conjunctival, and rectal swabs as well as whole blood and lung samples of pigs were collected in 19 regions from ten provinces of China in this study.C. suis following screening by Chlamydia spp. 23S rRNA-based FRET-PCR and high-resolution melting curve analysis and confirmatory sequencing. For C. suis-positive samples, 33.3 % of whole blood and 62.5% of rectal swabs were found to be positive for the C. suis tetR(C) gene, while 13.3% of whole blood and 87.0% of rectal swabs were positive for the C. suis tet(C) gene. Phylogenetic comparison of partial C. suis ompA gene sequences revealed significant genetic diversity in the C. suis strains. This genetic diversity was confirmed by C. suis-specific multilocus sequence typing (MLST), which identified 26 novel sequence types among 27 examined strains. Tanglegrams based on MLST and ompA sequences provided evidence of C. suis recombination amongst the strains analyzed.We report an overall positivity of 62.4%  of C. suis strains are exceedingly prevalent in pigs. As it stands, the potential pathogenic effect of C. suis on pig health and production of C. suis remains unclear and will be the subject of further investigations. Further study is also required to address the transmission of C. suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans.Genetically highly diverse The online version of this article (10.1186/s12917-017-1295-x) contains supplementary material, which is available to authorized users. Chlamydia are the etiological agents of chlamydiosis in wild and domestic birds, mammals and humans  per ml whole blood), 105.34±0.16 per rectal swab; 104.39±0.14 per conjunctival swab, and 104.37±0.14 per nasal swab  and weaned pigs , which were significantly higher than in suckling pigs  and sows . Interestingly, the average copy number of C. suis was significantly higher in weaned pigs  than in sows (103.11±0.09 per swab), finisher pigs (103.09±0.07 per swab) and suckling pigs (102.94±0.05 per swab)  of whole blood samples from Yangzhou in Jiangsu, 39/45 (86.7%) of rectal swabs from Kunming in Yunnan and 8/9 (88.9%) of rectal swabs from Shaoxing in Zhejiang province contained the tet(C) gene while 15/45 (33.3%) of whole blood samples and 5/8 (62.5%) of rectal swabs samples were found to be positive for the tetR(C) gene while 6 of 45 (13.3%) whole blood samples and 47 of 54 (87.0%) rectal swabs samples contained the tet(C) gene. We also tested the presence of tet(C) genes in C. suis-negative samples in this study. For C. suis-negative samples, no  whole blood samples from Yangzhou in Jiangsu and 19/38 (50%) of 53 (54.7%) rectal swabs from Kunming in Yunnan and 10/15 (66.7%) of rectal swabs from Shaoxing in Zhejiang province contained the tet(C) gene. Both C. suis tet(C) and tetR(C) were detected in samples from pigs in 3 provinces sampled in this study. In a word, the tet(C) positivity in C. suis-positive samples was significantly higher than in C. suis-negative ones for whole blood from Jiangsu  and rectal swabs from Yunnan and Zhejiang .For ompA sequences encompassing the variable domain 1 and 2 (VD1-2) from 108 pig C. suis strains  deposited from Japan. Fine-detailed epidemiological analysis of these sequences further revealed that the same ompA sequences could be found in different cities and provinces, suggesting that some strains were more widely distributed than other strains.We obtained 77 distinct partial n Figure . While cC. suis strains detected using a C. suis-specific MLST scheme. The MLST of the 27 global C. suis strains confirmed high genetic diversity for this pathogen, identifying 26 novel STs among 27 strains (0.96 ST/strain). Sequence analysis of individual as well as concatenated C. suis MLST gene fragments from this study confirmed that the C. suis HK genes are conserved and under purifying selection with dN/dS ratios < 1, yet with high haplotype diversity. The number of mutations was comparable between the C. suis HK alleles, ranging from 29 SNPs in gidA to 12 SNPs in gatA, with most resulting mainly in synonymous substitutions  related STs obtained from various anatomical sites of different hosts  closely related STs obtained from the same anatomical site from different hosts .The phylogenetic analyses further resolved the on-farm epidemiology of Chinese C. suis STs cluster analyses and minimum spanning tree also confirmed high ST diversity   Fig. . As obseompA alignments using DualBrothers. Based on observed break-points  SPF pigs were C. suis-positive.The present study investigated the prevalence of  culture , 31, 43.C. suis in blood samples. For paired samples from the same host, nasal, conjunctival and rectal swabs were C. suis-positive when the whole blood samples were also C. suis-positive. Furthermore, 6/23 lung samples were C. suis-positive, suggesting that systemic infection is a common outcome of the haemtogenous spread of the bacteria.Our study was the first to show the presence of C. suis was prevalent in pigs at all stages of their production cycle.In this study, generally, the infection rate of sows was significantly lower when compared with suckling, weaned and finisher pigs Fig. . These oC. suis was found to be the most common species in pigs [While  in pigs , this or in pigs –47 and c in pigs , 49, and in pigs , nasal s in pigs , lung [5 in pigs  and the  in pigs .C. suis DNA in rectal swabs was significantly higher when compared to those taken from nose and eyes, with whole blood samples having the lowest C. suis DNA incidence. This result is most likely due to the gastrointestinal tract being the primary site infection and chlamydial replication. Alternatively, it is also possible that pigs can swallow C. suis eliminated from the respiratory tract by mucociliary clearance [C. suis DNA in rectal, nasal, conjunctival swab samples and whole blood samples was significantly higher for weaned pigs when compared with sows. Data of the present study may suggest the potential C. suis transmission via a fecal-oral route. Besides the fecal-oral route as a potential route of transmission, the transmission may presumably also occur via exchange of body fluids, particularly secretions from the conjunctiva and nose, which may also lead to aerosol formation [C suis shed in boar semen could cross current biosecurity barriers, meaning vertical transmission may also need to be considered a route of transmission.In terms of transmission, in this study, the prevalence of learance . Furtherormation . Hamonicormation  recommenC. suis infections in pigs are growing in the USA, Italy, Cyprus, Germany, Israel, Switzerland and Belgium [tet(C) gene was detected in clinical samples from 3 provinces, which suggests that the presence of the tet(C) gene in C. suis is common in the Chinese domestic pig population. Selective pressure from tetracycline as drug may be responsible for recent bottlenecks in C. suis populations [C. suis isolates. However, we cannot exclude that the tet(C) gene of other bacteria was detected, and 54.7% C. suis-negative rectal swab samples was found to be tet(C) positive in this study. For both whole blood and rectal swabs samples in this study, the tet(C) positivity of C. suis-positive samples were significantly higher than C. suis-negative samples, suggesting these C. suis strains contained tet(C) gene. This may be associated with the inclusion of 10-50 gram oxytetracycline in every 1,000 kilogram pig feed in the farms examined and in the Chinese pig industry. Given the apparently prevalence of this tet(C) positivity in C. suis, it is concerning to note that, Suchland et al. [in vitro horizontal transfer of tetracycline resistance from C. suis to clinical strains of C. trachomatis, an important human pathogen. As such, C. suis may transfer tetracycline resistance to other chlamydial species, through mixed infections of livestock or farmers. Therefore, for the Chinese pig industry and public health, effective preventive action, such as probiotics or vaccines, should be promoted to counteract it. Targeted surveillance of tetracycline-resistant C. suis strains may also be warranted to curb the spread of these unusual chlamydial strains.Since 1998, the number of reports of TcR  Belgium , 22, 55.ulations . In thisd et al.  previousompA VD1-2 gene fragment of the C. suis strains is highly polymorphic  are compared with 50 other C. suis sequences deposited in GenBank from six countries: Germany, Switzerland, Italy, USA, Japan and China (in black font). Branch lengths are measured in nucleotide substitutions and numbers show branching percentages in bootstrap replicates. Scale bar represents the percent sequence diversity. (PDF 376 kb)Mid-point rooted NJ phylogeny of ompA variable domains 1-2. A 489 bp region encompassing ompA VD1-2 sequences of 77 porcine Additional file 2:ompA fragment alignment of 26 Chinese C. suis strains. The top plot shows marginal posterior probabilities of the four most probable tree topologies, where break and change points of topologies are indicative of recombination. The last two plots show 95% Bayesian confidence interval (shaded in green) of the Kappa transition/transversion ratio, and average divergence Mu. (PDF 141 kb)DualBrother recombination detection using the 489bp Additional file 3:DualBrother recombination detection using the 489bp ompA fragment alignment of all 40 C. suis strains used in this study. The top plot shows marginal posterior probabilities of the four most probable tree topologies, where break and change points of topologies are indicative of recombination. The last two plots show 95% Bayesian confidence interval (shaded in green) of the Kappa transition/transversion ratio, and average divergence Mu. (PDF 157 kb)Additional file 4:C. suis strains used in this study. The top plot shows marginal posterior probabilities of the four most probable tree topologies, where break and change points of topologies are indicative of recombination. The last two plots show 95% Bayesian confidence interval (shaded in green) of the Kappa transition/transversion ratio, and average divergence Mu. (PDF 143 kb)DualBrother recombination detection using the concatenated 3,080 bp MLST alignment of all 40


Physcomitrella patens is an interesting organism to explore the adaptation mechanisms developed in the evolution of plant defenses to microbes. Cellular and biochemical approaches, gene expression profiles, and functional analysis of genes by targeted gene disruption have revealed that several defense mechanisms against microbial pathogens are conserved between mosses and flowering plants. P. patens perceives pathogen associated molecular patterns by plasma membrane receptor(s) and transduces the signal through a MAP kinase (MAPK) cascade leading to the activation of cell wall associated defenses and expression of genes that encode proteins with different roles in plant resistance. After pathogen assault, P. patens also activates the production of ROS, induces a HR-like reaction and increases levels of some hormones. Furthermore, alternative metabolic pathways are present in P. patens leading to the production of a distinct metabolic scenario than flowering plants that could contribute to defense. P. patens has acquired genes by horizontal transfer from prokaryotes and fungi, and some of them could represent adaptive benefits for resistance to biotic stress. In this review, the current knowledge related to the evolution of plant defense responses against pathogens will be discussed, focusing on the latest advances made in the model plant P. patens.Bryophytes, including mosses, liverworts and hornworts are early land plants that have evolved key adaptation mechanisms to cope with abiotic stresses and microorganisms. Microbial symbioses facilitated plant colonization of land by enhancing nutrient uptake leading to improved plant growth and fitness. In addition, early land plants acquired novel defense mechanisms to protect plant tissues from pre-existing microbial pathogens. Due to its evolutionary stage linking unicellular green algae to vascular plants, the non-vascular moss  This review summarizes the current knowledge on the adaptation mechanisms and the evolution of plant defenses against pathogens, focusing on the latest advances made in the model plant P. patens.Bryophytes are also colonized by microbial pathogens, including fungi, bacteria, oomycetes and viruses that cause disease evidenced by necrosis and tissue maceration . Among bescribed . The seqribed , induction of a hypersensitive-like response (HR), cell wall reinforcement, changes in hormones levels and expression of defense genes . Angiosperms have pattern recognition receptors (PRRs) at the plasma membrane to detect conserved pathogen-associated molecular patterns (PAMPs), and induce a defense response called PAMP-triggered immunity (PTI), providing protection against non-host pathogens (R) genes to detect directly or indirectly the effector proteins leading to effector-triggered immunity (ETI), which is highly specific and often accompanied by a HR and systemic acquired resistance (SAR). Up to date, several PAMPs and the corresponding PRRs have been identified in angiosperms. In Arabidopsis thaliana , the receptors FLS2 (for flagellin-sensing 2), EFR1 (for Elongation factor tu receptor 1), and LYK1/CERK1 (for LysM-containing receptor-like kinase1/chitin elicitor receptor kinase1), recognize bacterial flagellin, the elongation factor Tu, and the fungal chitin, respectively . Mutation in moss PpCERK1 leads to reduced defenses, including less expression of defense genes and less incorporation of phenolic compounds associated to cell wall defenses, showing that PTI is an ancient plant defense response  proteins are also linked to plant-pathogen interactions, and orthologs have been found in P. patens, the liverwort Marchantia polymorpha (M. polymorpha) and the primitive vascular plant Selaginella moellendorffii (S. moellendorffii)  is phosphorylated upon treatment with flg22, and it is speculated that it may act as a co-receptor of FLS2  as well as homologous proteins to the corresponding interacting proteins (PpXB3s) (P. patens could have an OsXa21-like RLK-type sensing system to detect molecules from pathogens (Other receptor-like kinases (RLKs) such as dorffii) . In A. tinvolved . In A. t of FLS2 . CrRLK1L of FLS2 . Interes of FLS2 . Other p of FLS2 . Consistnhardtii . P. pate(PpXB3s) . Some ofathogens .P. patens, including kinase-Nucleotide Binding Site (NBS)-Leucine Rich Repeat (LRR) receptors and Toll/interleukin-1 like Receptor (TIR)-NBS-LRR  have been identified in -NBS-LRR . Since a-NBS-LRR , R-like . patens . Further2+) is a second messenger essential in both angiosperm PTI and ETI responses leading to ROS and nitric oxide production, and induction of plant defense genes  and MAPK pathways modulate plant biotic stress signaling  are activated by PRRs  are components of an immune pathway in P. patens, and are required for defense responses to the fungal chitin , NADPH-oxidase (PpNOX), and increased peroxidase (PpPrx34) activity  containing elicitors such as harpin and plant cell wall degrading enzymes secreted by this bacteria . Although the precise role of chloroplast repositioning is at present unknown, plastidic ROS sources as well as the production of specific chloroplastic molecules could be part of the defense response to biotic stress. Interestingly, Nicotiana benthamiana chloroplasts send out dynamic tubular extensions called stromules during plant defense responses, which form connections with the nucleus  infection (Chloroplasts are a rich source of ROS and these organelles are repositioned in  nucleus . These snfection . Actin cnfection . FurtherP. patens cells infected or treated with elicitors of HR-inducing pathogens such as Botrytis cinerea (B. cinerea), Colletotrichum gloeosporioides (C. gloeosporioides), and P.c. carotovorum, induce a HR-like reaction  develop spreading necrosis upon Alternaria brassicicola infection, and fungal toxin fumonisin B1 treatment , suggesting that SA is involved in moss defense (P. patens colonies increases resistance to P. wasabiae (Amblystegium serpens activates a SAR-like response upon inoculation with Pythium irregulare (P. irregulare) or treatment with β-1,3 glucan increasing resistance against subsequent P. irregulare infections , jasmonic acid (JA), and ethylene have a central role in defense against pathogens in angiosperm. Additionally, other hormones such as abscisic acid (ABA), auxins, cytokinins, gibberellins, and brassinosteroids have emerged as regulators in plant immunity . These h. patens . Studieshe plant . NPR3 anhe plant . SA leve defense . Consistwasabiae . Furtherfections . This fiP. patens encode the orthologs of all JA signaling components, although moss and liverworts do not produce the oxygenated fatty acid (oxylipin) JA  and B. cinerea  JA . The pre cinerea . Similared genes . Surprisectively . Furtherd plants . Since gresponse . Interesana COI1 . The simana COI1 . Jasmonaana COI1 .P. patens plants infected with B. cinerea show a small increase of ABA, however, it cannot be discarded that it is produced by the pathogen  involved in the production of oxylipins, an intracellular pathogenesis-related protein, a N-hydroxycinnamoyl/benzoyl transferase involved in the production of phytoalexin, a hydroxyproline-rich glycoprotein and a proline-rich protein involved in cell wall reinforcement, an ascorbate peroxidase and a peroxiredoxin, suggesting that ABA could be involved in moss defense against pathogens   , althougresponse , suggestresponse . Until pectively , and treectively . In addireatment . Similarse genes . FurtherP. patens and M. polymorpha transcriptomes orthologs of all the signaling and transport core components were found, except for the liverwort Auxin-Binding Protein1, probably due to low coverage of the transcriptome  which has both ent-diphosphate synthase and ent-KS activities  catalyzes a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene, which is a precursor for phytoalexin momilactones  and Apiospora montagnei  and C20-fatty acids (20:4 arachidonic and 20:5 eicosapentaenoic acids) as substrates yielding a broader range of oxylipins than angiosperms   . These epha-DOX) . Oxilipiotovorum .P. patens after P.c. carotovorum elicitor treatment or pathogen infection, including PR-1, PR-10 and PR-3 (chitinase)  . Moreoveitinase) . Transcritinase) .It was r defense . Moss liathogens . PpPR-10athogens , howeverathogens . Other pathogens , which cathogens .P. patens and associated to defense were acquired by horizontal gene transfer, providing an adaptive advantage that probably played a role in defense against microbial colonization in basal land plants. Examples include the bifunctional CPS/KS enzyme, which probably came from fungi by horizontal gene transfer, and unifunctional CPS and KS evolved gradually in angiosperms (P. patens (P. patens by horizontal transfer from bacteria and fungi are directly or indirectly related to plant defense responses and stress tolerance. A glutamate-cysteine ligase acquired from bacteria is involved in glutathione formation, which is important for plant disease resistance (P. patens to better adapt to pathogen infection as well as to beneficial microbes. P. patens has 13% orphan genes (P.c. carotovorum elicitor-treated P. patens tissues (P. patens during abiotic stresses, species- or lineage-specific genes could have played an important role in the acquisition of abiotic stress tolerance in mosses (Several genes present in iosperms . PAL wasiosperms , and the. patens . The acq. patens . Severalsistance . Other asistance . These aan genes , and som tissues , suggestn mosses . FurtherP. patens adapted cellular and molecular responses to interact with beneficial microbes and to cope with microbial pathogens during their life cycle. Comparative studies between beneficial and pathogenic interactions open the possibility to gain insights into the molecular mechanisms involved during coevolution, and how basal land plants have evolved the ability to distinguish between these different types of microbes. Further research is needed to identify and characterize more moss PRRs and possible targets of pathogen effector proteins that inhibit PTI or ETI mediated signaling in bryophytes. Studies conducted so far have shown that several defense mechanisms against pathogen infection are conserved among moss and angiosperms. Some of these mechanisms emerged when plants colonized land, probably influencing fitness and conferring adaptive advantages to biotic stressors. Certain precursor stages of defense mechanisms were already present in the algal ancestor of plants, while several moss genes associated to defense were acquired with terrestrialization, either via horizontal gene transfer from microbes or by duplication events and probably further diversification and functional specialization. The expansion of metabolic gene families, the production of novel metabolites, and the presence of orphan genes with possible roles in P. patens defense, suggest that 470 million years of divergent evolution lead to the development of alternative defense strategies for survival in extant moss and angiosperms. The efficient transient transformation and generation of P. patens knockouts in several genes using CRISPR-Cas9 open new perspectives for functional analysis of gene families that participate in moss defense (As early land-growing plants, bryophytes like  defense . FurtherIPDL conceived the review and wrote the manuscript. MM participated in the discussions and helped to draft and write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer EO and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Andrias davidianus, the evolution analysis was carried out among several species transcriptome data. The transcriptome data of Notophthalmus viridescens, Xenopus tropicalis, Cynops pyrrhogaster, Hynobius chinensis and A. davidianus were obtained from the Genbank and reassembled except Xenopus tropicalis. The BLAST search of transcriptome data obtained 1244 single-copy orthologous genes among five species. A phylogenetic tree showed A. davidianus to have the closest relationship to H. chinensis. Fourteen positively selected genes were detected in A. davidianus and N. vridescens group and fifteen in A. davidianus and H. chinensis group. Five genes were shared in the both groups which involved in the immune system, suggesting that A. davidianus adaptation to an aquatic and cave environment required rapid evolution of the immune system compared to N. viridescens and H. chinensis.To search the evidence of molecular evolution mechanism for aquatic and cave habitat in The online version of this article (10.1186/s41065-018-0056-6) contains supplementary material, which is available to authorized users. Amphibians played an important role as a transitional group linking aquatic to terrestrial in the evolution of vertebrates . To elucAndrias davidianus is a typical urodele, and an important species both as a biological resource and with respect to its value as a living fossil [A. davidianus adaptation to its aquatic life history and to a cave habitat, transcriptome data of other amphibian species were obtained from GenBank, and comparative transcriptome analysis was carried out to detect genes positively selected for in evolution.The Chinese giant salamander g fossil . The speTotal RNA was extracted from five ovaries and testes using Trizol reagent  according manufacturer’s instructions and treated with RNase-free DNase I  to remove the genomic DNA, respectively. After RNA quality and quantity test, RNA was broken into short fragment, and first-strand cDNA was synthesized, and then the sequencing adapter was added. The cDNA libraries were constructed and sequenced on the Illumina sequencing platform (Illumina HiSeq™ 2500). All raw reads, low quality sequences, and reads containing adaptor sequences were removed, and the clean reads were obtained.A. davidianus were provided by my lab. To expand data of A. davidianus, transcriptome data of skin (SRX729810) and spleen (SRX729743) were obtained from NCBI database and reassembled with the gonad transcriptome data. Transcriptome data of N. viridescens from heart, lens, brain, eye, liver, lung, spleen, kidney, testis, and ovary (ERR108189), C. pyrrhogaster lens and neural retina (SRR1051839), H. chinensis whole body (SRR1042328) and X. tropicalis from genome sequencing (GCA_000004195) were also obtained. The unigenes were reassembled from the downloaded raw reads, except for X. tropicalis. The numbers of unigenes for each species is given in Table Two gonad transcriptome data (SRR3308418 and SRR3308420) of kin SRX72810 and sForm the orthologous gene, only one orthologous gene in other species was classed as single-copy orthologous by PERL package , 13. TheN. viridescens, X. tropicalis, C. pyrrhogaster, H. chinensis, and A. davidianus, and transcriptome data were downloaded and reassembled  and non-synonymous (Ka) substitutions per site were observed  and non-synonymous substitution rates (Ka) were calculated according to the phylogenetic tree by PAML software [Erythroculter ilishaeformis and zebrafish Danio rerio, in which 38 candidate genes exhibited signs of positive selection with dN/dS ratios > 0.5 [A. davidianus/H. chinensis and A. davidianus/N. viridescens groups. Andrias davidianus is aquatic and inhabiting subterranean rivers and caves while N. viridescens and H. chinensis are mainly terrestrial and only special stage in water.To elucidate the phylogenetic evolution of software , 25, witos > 0.5 . Five geos > 0.5 –29 [cystA. davidianus immune system should show more rapid mutations, as was confirmed in our investigation. Due to lack of full-length according to the transcriptome sequencing, many gene relevant to positive selection was omitted and Ka/Ks ratio was decreased from normal level [Aquatic and cave dwelling organisms generally encounter more bacteria than do terrestrial animals. Thus, the al level . FurtherAdditional file 1:Figure S1. Phylogenetic tree of selected species based on 1244 single-copy orthologous genes. (TIFF 212 kb)Additional file 2:Table S1. Orthologs gene under positive selection among species. (DOCX 18 kb)