Daphnia, present in a wide range of water bodies, are an important component of freshwater ecosystems. In contrast to their high dispersal capacity through diapausing eggs carried by waterfowl, Daphnia often exhibit strong population genetic differentiation. Here, to test for common patterns in the population genetic structure of a widespread Holarctic species, D. galeata, we genotyped two sets of populations collected from geographically distant areas: across 13 lakes in Eastern China and 14 lakes in Central Europe. The majority of these populations were genotyped at two types of markers: a mitochondrial gene (for 12S rRNA) and 15 nuclear microsatellite loci.The zooplanktonic cladocerans D. galeata, with distinct haplotype compositions in the two study regions but one widely distributed haplotype shared between several of the Chinese as well as European populations. At microsatellite markers, clear separation was observed at both large (between China and Europe) and small (within Europe) geographical scales, as demonstrated by Factorial Correspondence Analyses, Bayesian assignment and a clustering method based on genetic distances. Genetic diversity was comparable between the sets of Chinese and European D. galeata populations for both types of markers. Interestingly, we observed a significant association between genetic distance and geographical distance for D. galeata populations in China but not in Europe.Mitochondrial DNA demonstrated relatively shallow divergence within D. galeata across wide expanses of the Palaearctic, with one mtDNA lineage of D. galeata successfully establishing over large distances. Despite a clear differentiation of Chinese and European D. galeata at a nuclear level, the pattern of genetic variation is nevertheless similar between both regions. Overall, our findings provide insights into the genetic population structure of a cladoceran species with extremely wide geographical range.Our results indicate relatively recent spread of The online version of this article (10.1186/s12862-018-1256-4) contains supplementary material, which is available to authorized users. A major challenge in our understanding of biodiversity is why certain species have a wide distribution, spanning several biogeographical regions, while others have much more restricted ranges or are endemic. The cosmopolitanism view  holds thDaphnia galeata G. O. Sars, 1863, to improve our understanding of its dispersal and genetic diversity over small and large geographic scales. Daphnia (Anomopoda: Daphniidae) are important components of freshwater ecosystems, being grazers of phytoplankton as well as a key prey item of planktivorous fish [Daphnia are cyclical parthenogens; females mostly reproduce clonally by giving birth to parthenogenetic daughters. In unfavourable environments, however, Daphnia switch to the production of males and sexual eggs which require fertilization, and then enter diapause in a gastrula stage [Daphnia galeata, a member of the D. longispina complex , has a ised in: , being cised in: –16). Thiwl e.g. , 18). Su. SuDaphnes .D. galeata populations across Holarctic continents, using both a mitochondrial DNA (mtDNA) marker and a conservative nuclear marker (i.e. HSP90). One major mtDNA type of D. galeata was detected throughout the Holarctic with some haplotypes shared over long distances, even between Europe and North America. At the nuclear marker level, however, New and Old World D. galeata were clearly differentiated [D. dentifera to D. galeata in the New World. However, populations from China, where D. galeata is a very common zooplankter [D. galeata across China revealed low mtDNA variation; a common D. galeata haplotype (as resolved by sequencing of 12S rRNA and cytochrome c oxidase subunit I) was widespread across China, Japan, Siberia and the Western Palaearctic [D. galeata populations even on a relatively small geographical scale .D. longispina complex, D. galeata [D. galeata populations  using two types of markers, a mitochondrial gene (12S rDNA region) and 15 nuclear microsatellite loci. In the case of mtDNA, we expected pronounced divergence of D. galeata between the two study regions, but low variation within them, as mitochondrial DNA usually has low resolution to detect subtle within-species population differentiation . When apossible .Daphnia galeata localities were analysed in this study . Among D. galeata populations were processed for analyses of microsatellites. The individual DNA extractions followed the protocol reported in [D. galeata genotype (G100 from Europe) was used as a positive control to adjust for small differences in fragment length of identical alleles among runs. Genotypes were scored using GeneMapper version 3.7 (Applied Biosystems), and the alleles at each locus were defined by their fragment length (in base pairs). About 40 adult D. galeata females were genotyped from each Chinese population . Re. ReD. gaex  wer species , 24  and their interspecific hybrids, were included as reference taxa in FCA  were analysed separately. New Hybrids then assigned the newly analysed individuals together with reference individuals into six possible classes . The analysis was run for 106 iterations after a burn-in of length 106, and the probability threshold for taxon identification was set to 90%. Only individuals with pure D. galeata assignments were used further. We applied Factorial Correspondence Analysis (FCA) on all 25  used in . Forty-n used in ). Then,  used in  was applD. galeata populations between and within regions, a similar FCA was run as described above, but without the reference genotypes. In order to identify genetically homogenous groups across all 25 D. galeata populations, a Bayesian algorithm was applied in STRUCTURE V2.3.4 [K groups. For each tested value of K (i.e. 1 to 25), ten independent runs were performed and, for each run, 105 iterations were carried out after a burn-in period of 105 iterations. The most likely K was determined by the distribution of ΔK, following the methods of Evanno et al. [K: 1 to 13) and 12 European (K: 1 to 12) D. galeata populations, respectively. Next, the genetic similarity among populations from different regions was estimated by constructing an Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) dendrogram based on Nei’s genetic distances [D. galeata populations into the following components: 1) between regions , 2) among populations within regions, and 3) among individuals within populations. To estimate genetic differentiation among D. galeata populations for each region, the fixation index FST was calculated in Arlequin 3.11 with 104 permutations. Finally, the correlation between pairwise geographical distance and pairwise FST was computed using a Mantel test , in FSTAT v.2.9.3.2 [D. galeata from VIR and ZEL . The le.2.9.3.2 . D. galefunction .Daphnia communities in China , they ah) and nucleotide diversity (π) for the 12S sequences were calculated in DnaSP Version 5 [t test . The unique haplotypes were identified in Arlequin, and then aligned together with 12S reference sequences of the D. longispina complex, including D. galeata from Asia (Japan) and Europe . FCA of these D. galeata individuals (without reference genotypes) revealed two main clusters clearly separating Chinese and European populations . There was evidence of genetic isolation by distance for Chinese D. galeata populations , but not for European ones . No MLGs were shared among populations on continental or on local scales.The AMOVA showed that most of the variation in the microsatellite dataset was explained by a within-population component (48%) while another significant component was between regions (33%), followed by within-region variation  were assigned to ata Fig. ; among tDaphnia species, D. galeata. Specifically, multiple populations of D. galeata, sampled from two remote geographical regions , were screened using mitochondrial (12S) and nuclear markers (microsatellites). One of seventeen mitochondrial haplotypes described here was shared between European and Chinese populations. This haplotype (CES) was also widespread within both regions, having been detected in eight out of eleven studied European populations and four out of thirteen Chinese populations. The network analysis  and nine remaining populations. The separation of these three populations was well supported by FCA, clustering method based on genetic distances  and UPGMA analysis. The clear genetic separation between Chinese and European populations could be explained by the large geographical isolation between these two parts of the Palaearctic. However, the geographical position of localities of D. galeata populations within Europe was not associated with their genetic similarity, suggesting gene flow occurs within regions. A genetic separation of VIR, ZEL and GOSL from other D. galeata populations in Europe might have resulted from introgression of D. cucullata to D. galeata. In all three localities, we detected D. cucullata . This suggests that the introgression of the mitochondrial genome occurs after initial hybridization, as observed in other animals , whereangispina , 48, 49.ls e.g. , 51)..D. galeaD. galeata populations from China (but not from Europe). One explanation for the discrepancy could be that studied European D. galeata habitats are ecologically more variable than the mostly eutrophic Chinese lakes [D. longispina complex assemblages [D. longispina complex where mixed results were reported. Specifically, a significant association between genetic distance  and geographical distance was detected for other taxa of the D. longispina complex [D. longispina, no signal for genetic isolation by distance was observed [Interestingly, there was a significant association between genetic distance and geographical distance for se lakes . In addiemblages . Because complex  but in aobserved .D. galeata populations investigated here, regardless of their origin, showed high relative clonal richness values (as assessed by microsatellites), likely a result of frequent sexual reproduction, which recreates genotypic variation. High clonal richness of D. galeata populations was also observed previously . Howeveelection . Many poelection , may surelection , 57.D. galeata populations from two regions, i.e., Eastern China and Central Europe, were found to be clearly separated from each other, which was confirmed by three types of analyses . This principal finding was supported by mitochondrial DNA data; however, we detected in both regions a common and widespread mtDNA haplotype that was shared across the whole Palaearctic. Our finding calls for further studies to investigate the colonization history of D. galeata, and zooplankton in general, in China and adjacent regions.A high number of polymorphic nuclear loci and their codominant inheritance often provide fine resolution in detecting genetic structure . Here, bAdditional file 1:Table S1. Reference sequences of the D. longispina complex used in phylogenetic analyses. (DOCX 15 kb)Additional file 2:Figure S1. UPGMA clustering of D. galeata populations sampled from Eastern China and Central Europe based on microsatellite polymorphism at 15 loci. For lake abbreviations see Table


Anopheles funestus complex in malaria transmission in sub-Saharan Africa, sibling species have rarely been identified in the past and their vectoring potential remains understudied.Most malaria vectors belong to species complexes. Sibling species often exhibit divergent behaviors dictating the measures that can be deployed effectively in their control. Despite the importance of the An. funestus (senso lato) specimens from 21 sites in Kenya, covering the major malaria endemic areas including western, central and coastal areas. Indoor and outdoor collection tools were used targeting host-seeking and resting mosquitoes. The identity of sibling species, infection with malaria Plasmodium parasites, and the host blood meal sources of engorged specimens were analyzed using PCR-based and sequencing methods.We analyzed 1149 wild-caught Anopheles funestus (59.8%) and Anopheles rivulorum (32.4%) among the 1062 successfully amplified specimens of the An. funestus complex. Proportionally, An. funestus dominated in indoor collections whilst An. rivulorum dominated in outdoor collections. Other species identified were Anopheles leesoni (4.6%), Anopheles parensis (2.4%), Anopheles vaneedeni (0.1%) and for the first time in Kenya, Anopheles longipalpis C (0.7%). Anopheles funestus had an overall Plasmodium infection rate of 9.7% (62/636), predominantly Plasmodium falciparum (59), with two infected with Plasmodium ovale and one with Plasmodium malariae. There was no difference in the infection rate between indoor and outdoor collections. Out of 344 An. rivulorum, only one carried P. falciparum. We also detected P. falciparum infection in two non-blood-fed An. longipalpis C (2/7) which is the first record for this species in Kenya. The mean human blood indices for An. funestus and An. rivulorum were 68% (93/136) and 64% (45/70), respectively, with feeding tendencies on a broad host range including humans and domestic animals such as cow, goat, sheep, chicken and pig.The most abundant sibling species collected in all study sites were Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination.The online version of this article (10.1186/s13071-018-3171-3) contains supplementary material, which is available to authorized users. Malaria remains the most important vector-borne disease in tropical countries of the world, especially in sub-Saharan Africa (SSA). The scale-up of long lasting insecticidal net (LLIN) distribution has contributed to a decline in malaria transmission, number of cases and mortality within the last decade , promptiAnopheles gambiae and Anopheles funestus , two shared 100% identity to P. ovale  and one shared 100% identity with Plasmodium malariae . These Plasmodium species identifications are supported by the sequence alignment  and/or ITS2 rDNA (267–763 nt) PCR product sequences of specimens found positive for Plasmodium infection, 62 shared 100% identity with An. funestus , one shared 100% identity with An. rivulorum [GenBank: KR014839 (cox1)] and two shared 100% identity with An. longipalpis C . The mosquito specimen identifications are further supported by ML phylogeny  were deposited in the GenBank database under the accession numbers , MH547443  and MH547444 . Furthermore, selected sequences of Plasmodium-positive specimens were deposited in GenBank .Selected sequences of P. falciparum, P. ovale and P. malariae, respectively, were associated with An. funestus. This translated to a Plasmodium infection rate (IR) of 9.7% (62/636). One P. falciprum infection was associated with An. rivulorum  and two with An. longipalpis C . The two Plasmodium infected An. longipalpis C were collected indoors (2/7) from Kamnarok (Baringo County), while the infective An. rivulorum was recorded outdoors (1/253) in Jaribuni from coastal Kenya. Plasmodium falciparum infections, especially in An. funestus, occurred in diverse sites: P. ovale from West Alego and Bunyala, Plasmodium malariae from Samia, western Kenya  and 64.3% (45/70) for An. rivulorum. Human feeds were also encountered for An. leesoni and An. parensis. The two blood-fed An. longipalpis C specimens had each fed on cow and goat. Mixed feeding on humans and other animal hosts and was observed for An. funestus and An. rivulorum, both of which displayed a feeding tendency on a broad host range . Furthesmission . HoweverAn. funestus group than previously documented in Kenya. Furthermore, sympatric occurrence of more than three sibling species in a locality as observed in this study, highlights the importance of integrating molecular approaches in surveillance efforts for adequate assessment of the impact of interventions on vector populations [An. funestus group members.We uncovered a higher species richness among the ulations . A lack Plasmodium infection rates in An. funestus ranging from 3.5% in Bunyala to 50% in Lwanya-Bumala  at large in East Africa [Residual transmission has emerged as a major threat to effective malaria control, as key vectors alter their phenotypes to render current proven vector control interventions such as LLINs less effective. Examples include exhibition of behavioral plasticity in resting and feeding patterns. We found a rather low human blood index 64.8%) for 4.8% for  species , 31. Alt species  followint Africa , which cSpecies records are based on sampling efforts, which in our study was limited in scope (i.e. intensity and duration of trapping in each location) and targeted the rainy seasons only. Seasonal occurrence including species succession has been shown to influence changes in sibling species composition . StudiesOur findings underscore the need for improved surveillance of vector populations employing molecular techniques including sequencing to improve malaria vector control and working towards malaria elimination. This is essential for fine-tuning control strategies in the event of changing local conditions  and for An. funestus group than previously documented in Kenya. Notably is the occurrence of An. longipalpis C and for the first time, naturally infected with Plasmodium falciparum. Anopheles funestus remains the main vector species in the Funestus group in Kenya which was found to exhibit a rather low human blood index. Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination.Our findings have revealed a higher species richness among the Additional file 1:Figure S1. Sequence alignment of 162 nucleotide (nt) Plasmodium ncMS sequences corresponding to nucleotide positions 5759–5920 of GenBank accession CP017005. Study sequences are highlighted in bold and GenBank accession numbers and species identifications are indicated for each ncMS gene sequence. In sequence alignment, red = A, green = T, yellow = G, blue = C. grey = nt identity with first sequence. (TIF 503 kb)


Here, we present the complete coding sequences of two tilapia lake virus (TiLV) isolates recovered during an investigation of a mortality event in farmed Nile tilapia in the United States. Phylogenetic analysis supported the isolates as each other’s closest relatives and members of a clade of Thai TiLV strains. Here, we present the complete coding sequences of two tilapia lake virus (TiLV) isolates recovered during an investigation of a mortality event in farmed Nile tilapia in the United States. Phylogenetic analysis supported the isolates as each other’s closest relatives and members of a clade of Thai TiLV strains. Tilapinevirus and family Amnoonviridae (De novo assembly of the paired-end reads was performed in SPAdes v3.10.0 with default parameters (http://exon.gatech.edu/GeneMark/genemarks.cgi) (Tilapia lake virus (TiLV), member of the recently established genus nviridae , is a glnviridae . The TiLnviridae . Here, wnviridae . Viral gnviridae . In totarameters . BLASTN rameters . The totrameters  and visurameters . Putativrks.cgi) , restricrks.cgi) , based orks.cgi)   under accession no. SRX6878421 and SRX6878422, respectively.Complete coding sequences and raw sequence data of TiLV WVL19031-01A and WVL19054 have been deposited in NCBI GenBank under accession no.


LMNA) gene codes for nuclear intermediate filaments constitutive of the nuclear lamina. LMNA has 12 exons and alternative splicing of exon 10 results in two major isoforms—lamins A and C. Mutations found throughout the LMNA gene cause a group of diseases collectively known as laminopathies, of which the type, diversity, penetrance and severity of phenotypes can vary from one individual to the other, even between individuals carrying the same mutation. The majority of the laminopathies affect cardiac and/or skeletal muscles. The underlying molecular mechanisms contributing to such tissue-specific phenotypes caused by mutations in a ubiquitously expressed gene are not yet well elucidated. This review will explore the different phenotypes observed in established models of striated muscle laminopathies and their respective contributions to advancing our understanding of cardiac and skeletal muscle-related laminopathies. Potential future directions for developing effective treatments for patients with lamin A/C mutation-associated cardiac and/or skeletal muscle conditions will be discussed.The lamin A/C ( LMNA cause primary  laminopathies, while secondary laminopathies are caused by mutations in B-type lamins, proteins involved in prelamin A maturation, or lamin binding partners. In addition, Lamin A/C mutations are also associated with arrhythmogenic right ventricular cardiomyopathy and hypertrophic cardiomyopathy [LMNA gene (approximately 24 kb) is located on chromosome 1q22 and has 12 exons. It is expressed in most differentiated cells, with the expression in mice starting during early embryogenesis (8th day post-implantation) [CAAX motif [AAX a.a in the motif, then the methylation of the farnesylated cysteine, and lastly by the cleavage of the last 15 a.a (including the cysteine that was both methylated and farnesylated), resulting in a 646 a.a-long mature lamin A [Laminopathies consist of a variety of diseases that affect different tissue types ranging from cardiac muscle ), skeletal muscles ), adipose tissues ) to nervous system ) or affect several systems ). Mutations in the nuclear A-type lamins coding gene myopathy ,4,5. Thentation) ,7. Alterntation) . Lamins AX motif . This is lamin A . The oth lamin A ,11,12. T lamin A . The A-t lamin A . Example lamin A ,17,18.LMNA mutation can sometimes lead to different phenotypes and mutations found throughout the ubiquitously expressed gene can lead to tissue specific phenotypes is not yet well elucidated. However, based on the current data, three hypotheses seek to explain this diversity in phenotypes. The first suggests that the accumulation of misprocessed/unprocessed lamin A is toxic to cells, as in HGPS [The mechanism(s) by which the same  in HGPS ,20. The  in HGPS . Similar in HGPS ,26,27,28 in HGPS ,34,35,36LMNA mutations accounts for up to at least one-third of reported genotyped families with DCM, and presents with dilation of the left ventricle, thinning of the ventricular wall and conduction defects [LMNA-related DCM also tend to have more severe phenotype and poorer prognosis with a recent study reporting 19% required heart transplants [LMNA-related congenital muscular dystrophy (L-CMD) and EDMD are skeletal muscle conditions caused by LMNA mutations with overlapping clinical phenotypes, which include wasting of the upper arm and lower front leg muscles [LMNA mutations, also affects the skeletal and cardiac muscles [LMNA as a causal gene in EDMD in 1999, massive efforts involving both cellular and animal models have since been undertaken to understand the underlying mechanisms in cardiac and skeletal muscle conditions caused by mutations in LMNA [LMNA mutation-associated conditions will also be discussed. The majority of reported cases of laminopathies (79%) affect the cardiac and/or skeletal muscles and are predominantly autosomal dominant . DCM ass defects . Patientnsplants ,40. LMNA muscles . However muscles . 1B Limb muscles . It is c muscles . With th in LMNA ,44. ThusCellular models facilitate the characterization of the striated muscle laminopathy phenotype at both the cellular and nuclear level, and the identification of potential molecular markers for the conditions. This section gives an overview of the results obtained using patients’ and mice models’ cells and various cell lines.LMNA null cells presented with misshapen nuclei and abnormal localization of nuclear proteins such as lamin B1, B2, emerin, and syne1 [LMNA in HeLa cells, however, did not affect cell growth but indeed resulted in emerin mislocalization in the endoplasmic reticulum, similar to what was seen in the LMNA null fibroblasts [LMNA mutations, mutant lamins A and C were transfected in various cell lines including the P19 embryonic carcinoma stem cells, which do not have endogenous lamin A/C [LMNA, emerin was either clearly mislocalized in some studied DCM or EDMD mutant lamin A/C or normally expressed and distributed [LMNA mutations affecting the rod domain of the protein and causing muscular laminopathies also presented with aggregates of lamin A/C proteins, particularly lamin C, in the nucleoplasm of transfected cells [A rare lamin A/C null case in humans was discovered from a deceased newborn who was homozygous for the nonsense p.Y259X mutation . This vand syne1 . B-type nd syne1 . Moreovend syne1 . RNAi knroblasts ,48. To damin A/C ,49. The tributed ,49,50. Ned cells . Specified cells . Moreoveed cells . All of LMNA mutations [LmnaLCO) (discussed in the later part of the review) displayed normal emerin localization [LMNA mutations can result in the inability of the A-type lamins to properly connect to the nuclear envelope and can negatively impact their interaction with their binding partners. To further explore the effects of LMNA mutations on the interaction between A-type lamins and their binding partners, studies involving mutations found in regions known to interact with lamin A/C partners were performed.Transient transfection in HeLa cells of the p.E203G lamin A variant, which is associated with DCM with conduction defects (DCM-CD), resulted in lamin aggregates and the absence of the lamin A variant in the nuclear periphery . Howeverutations . Furtherlization . These rLMNA gene affect sites that are known to interact with various proteins. An example of one of these mutations—the substitution of C with T at nucleotide position 1444, producing the p.R482W lamin A/C variant and causing both FPLD and LGMD in some patients—is located in a region known to bind actin and/or influence SREBF1 interaction [LmnaΔ8–11 mice, which primarily exhibited striated muscle laminopathy, also notably lacked subcutaneous fat and presented with some metabolic parameter abnormalities [Mutations found throughout the eraction ,58,59,60eraction . This comalities ,62. To fLMNA mutations, the structural stability and dynamics of various lamin A/C variants were studied. Gilchrist et al. transfected epitope-tagged WT lamin A or lamin A variants in HT1080 cells, a human fibrosarcoma cell line [LMNA-expressing cells. As a consequence of the change in lamin A/C structure caused by ell line . The lamell line . The traell line . Fluoresell line . Howeverell line . FRAP exell line . Fluoresell line . Such inell line ,63. To dLMNA mutations. A cardiac sample from the p.D192G DCM patient presented with immense nuclear aberrations including rupture of nuclear envelope, chromatin disorganization and mislocalization of the mitochondria in the nucleoplasm in about 30% of the cardiomyocytes [LmnaΔK32/+ DCM mice model (discussed later part in the review) [LMNA mutations displayed a loss of nuclear stiffness [LmnaΔ8–11 mice (discussed later in the review) also presented with reduced nuclear stiffness and a notable increase in nuclear deformity when strain was applied to the cells compared to WT [LmnaΔ8–11 mice [LMNA cDNA  causing DCM with or without conduction defects in neonatal rat ventricular myocytes, resulted in nuclear morphology defects including blebbing [LMNA-expressing cells are stiffer, particularly the p.D192G variant, compared to the controls [LMNA mutations that were not associated with striated muscle laminopathies did not result in nuclear deformability [Putative alterations of the nucleoskeleton and/or the cytoskeleton were examined by assessing their mechanical properties in the presence of myocytes ,64. Simi review) . Furthered to WT ,32,33. B–11 mice . Viral tblebbing . Analysecontrols . On the mability ,38,54. TLmnaΔ8–11 MEFs, a significant decrease in cytoskeletal stiffness, cytoplasmic elasticity and viscosity was observed in the LmnaΔ8–11 fibroblasts compared to WT cells, indicating that lamin A/C regulates the cytoskeleton plasticity [LMNA showed disorganized actin and lower levels of actin staining compared to WT and uninfected controls [LmnaΔ8–11 myocytes displayed contractile dysfunction [LMNA-related DCM, are thought to interact, and the inhibition of p38 restored the mutant actin and mechanical phenotypes back to WT [LMNA mutant cells have been further explored using L-CMD patients’ myoblasts cultured on matrix stiffness close to that of muscle (12 kPa) [LMNA-mutated myoblasts showed aberrant activation of regulators of the mechano-response (including yes-associated protein involved in cell adaptation to its microenvironment and formins known to affect actin polymerisation and depolymerisation in a force-sensitive manner) [LMNA impair the mechanical capacity and integrity of both the nucleus and the whole cell by the disruption of the cytoskeleton, thus resulting in defects in mechano-transduction signalling. To determine if these structural changes also affect the post-translational modification status of lamin A/C, experiments looking at such were undertaken.Moreover, although no overt differences were detected between the cytoskeletal architecture of WT and asticity ,32. Evenasticity . Neonatacontrols . They alcontrols . Accordifunction . Actin ack to WT ,67,68. D(12 kPa) ,36. Muta(12 kPa) ,36. Furt manner) ,36. Inte manner) ,70. RemoLMNA mutations located in the Ig fold in the protein C-terminus [LMNA-related myopathies.As discussed in the previous paragraphs, many of the disease-causing mutations result in alteration of the lamin A/C protein structure as well as the nuclear envelope properties. Therefore, would such changes in conformation also result in aberrant post-translational protein modifications? A-type lamins are known to be phosphorylated and the phosphorylation status may influence nuclear deformability . Lamin Aterminus . Resolveterminus ,74. Moreterminus . In additerminus . Furtherterminus . Lower pterminus . Such reterminus . OverallLmnaH222P/H222P mice’s (discussed later in the review) primary myoblasts and in LmnaH222P/H222P mice’s skeletal muscle tissue [Lamin A has also been shown to be sumoylated by SUMO2 . Fibroble tissue . These rOverall, the data from patient cells and immortalized cell lines demonstrate the wide breadth of the effects caused by mutations in the lamin A/C gene. Nonetheless, such cellular phenotypic analyses can be further characterized using derived cells from the patients’ induced pluripotent stem cells (iPSCs). These iPSCs can be of immense benefit, particularly in cases when multiple tissue phenotypes are co-present, as there will be no need to collect multiple cell types from the patients. Some studies using laminopathy patient iPSCs are discussed in the next section.LMNA knocked-down control cells resulted in the same phenotype as the mutant cells [In primary culture, dermal fibroblasts from the p.R225X lamin A/C DCM-CD patient presented with decreased lamin A/C and profound nuclear defects including condensed heterochromatin clumps, nuclear blebs, nuclear pore complexes (NPCs) clustering, and mitochondria around the nuclear envelope . Electrint cells . Anothernt cells . Lamins nt cells . Althougnt cells . Interesnt cells . These dLMNA mutations and to study the phenotypes and tease out disease-associated pathways while minimizing confounding variables such as comorbidity, defined and controlled strains of different animal models have been used to study striated laminopathies. In order to further confirm the pathogenicity of certain Caenorhabditis elegans (C. elegans) possesses one lamin gene (lmn-1) that codes for lamin-1, the equivalent of the B-type lamins but functionally similar to both A and B type lamins [lmn-1 via RNAi resulted in abnormal embryos that are developmentally arrested and have abnormal nuclear morphology and disrupted chromatin [LmnaΔ8–11 male mice germ cells  [Lmna in the seminiferous tubules of mice resulted in sperm with abnormal heads [C. elegans were fertile and had an increased male proportion compared to WT [The worm species e lamins . Gonadalhromatin . Animalshromatin . The stehromatin . Similarffected) . Moreoveal heads . Neverthed to WT . NPCs weed to WT ,61,83,86ed to WT ,88,89. Fed to WT . Moreoveed to WT ,90. The ed to WT .lmn-1 gene of C. elegans, the Drosophila melanogaster LamC gene encoding lamin-C is also a paralogue of the lamin A/C gene in vertebrates [LamC null fruit fly and truncated Lamin-C variants fruit flies  were generated and resulted in developmental delay and prepupal death [LamC null fruit fly, localization of B-type lamins and heterochromatin was normal, but NPC clustering was observed [LamC null larvae compared to WT [LamC null flies [Similar to the tebrates . LamC nual death ,94. Larval death . On the al death . In the observed . Furthered to WT . Abnormall flies .LMNA variants in fruit fly resulted in a host of nuclear aberrations such as lamin-C aggregates and/or increased nuclear blebbing, which are similar to what were previously observed in transfected cells expressing striated muscle laminopathy mutations [Expression of a variety of EDMD and DCM utations ,93,96. Cutations . Muscle-utations ,98. Tranutations . An incrutations . The relutations . Muscle utations . This ilutations .Danio rerio (D. rerio), popularly known as the zebrafish, belongs to the osteichthyan branch, which also includes mice and humans. The zebrafish lmna is an orthologue of human LMNA. The zebrafish is an established model of cardiac diseases owing to its cardiac system’s relative comparability to its mammalian counterpart, its transparency during early development, which allows for organ observation, its high fecundity and its ability to tolerate severe cardiac dysfunction that would be lethal at the foetal level in mammals [lmna resulted in slow skeletal muscle fibres damage at 24 h post-fertilization (hpf) [lmna knockdown showed that the zebrafish lamin A/C morphants presented with blood congestion at 24 hpf, cardiac oedema, decreased heart rate, and decreased cardiac function during the period of observation (48–96 hpf) [lmna morphants presented with some form of cardiac arrhythmia including atrial fibrillation, thus closely phenocopying LMNA mutation-caused DCM disease presentation in humans [ mammals ,100. A son (hpf) . Moreoveon (hpf) . Such anon (hpf) . Decreason (hpf) . Anotherlmna EDMD variants were created by Verma and Parnaik [Aside from morphants, zebrafish mutant lamin A/C transgenic lines have also been generated to study striated muscle laminopathies. Two transgenic lines with cardiac-specific expression of GFP-tagged zebrafish  Parnaik . Homozyg Parnaik . However Parnaik . Moreove Parnaik . In addi Parnaik .Mus musculus (M. musculus) is the species most closely related to us. The mice prelamin A shares 96.4% amino acid sequence identity with human prelamin A  mice was created using a different technique, Cre-mediated recombination [LmnaΔ/Δ mice were indistinguishable from their littermates at birth [LmnaΔ/Δ mice weighed about half as much as their siblings and died by 16‒18 days of age [LmnaΔ/Δ mice showed significantly smaller muscle fibres compared to WT mice [LmnaGT−/− mice [Another bination . The Lmnat birth . Howevers of age . Haemato WT mice . These c−/− mice ,104. In LmnaΔ8–11 mice expressing a truncated lamin A that lacks its C-terminus [Lmna gene [LmnaΔ8–11 mice were comparable at birth to their heterozygous and WT siblings [LmnaΔ8–11 mice compared to WT [LmnaΔ8–11 mice showed dystrophy, although no significant increase in creatine kinase was observed [LmnaΔ8–11 mice for lamina-associated polypeptide 2 (LAP2), Nup153 (component of NPCs) and lamin B showed misshapen nuclei and mislocalization of proteins such as lamin B and emerin, mimicking what was observed in the lamin A/C null cells of the child homozygous for the p.Y259X lamin A/C variant [A very well characterized model is the terminus ,105. It mna gene . The Lmnsiblings . Howeversiblings . In addisiblings ,62. Moreobserved . Lamin Aobserved . This suobserved . Furtherobserved ,106. How variant ,61. LmnaΔ8–11/+) displayed late-onset cardiomyopathy (with or without CD) with no skeletal muscle defect [n = 18) of LmnaΔ8–11/+ mice exhibited rhythm or conduction abnormalities compared to none in WT mice (n = 17) [On the other hand, heterozygous mice (e defect . At 10 w(n = 17) . Histoch(n = 17) . Moreove(n = 17) . However(n = 17) ,107.LmnaΔ8–11 and LmnaΔ8–11/+ mice models that express a truncated lamin A, this next mice model expresses non-farnesylated prelamin A only (LmnanPLAO/nPLAO) [LmnanPLAO/nPLAO mice presented with and died because of dilated cardiomyopathy [LmnanPLAO/nPLAO mice had increased nucleoplasmic prelamin A localization and nuclear blebs, but there was no significant change in the nuclear stiffness [LmnanPLAO/nPLAO MEFs compared to WT [LmnanPLAO/nPLAO mice died prematurely  [Myh6) expression and increased myosin heavy-chain beta (Myh7) expression [As opposed to the O/nPLAO) ,107,108.ed to WT . Overallpression ,110,111.pression .LmnanPLAO/nPLAO mice, mature lamin-A-only-expressing mice (LmnaLAO) were normal, healthy, did not present any discernible pathological phenotype and had comparable survival (>24 months) to WT mice [LmnaLAO mice compared to WT [LmnaLAO mice [In contrast to the  WT mice . Lamin A WT mice . Howevered to WT . Closer LAO mice . This suLAO mice . LmnaLCO) was comparable to WT with no overt physiological and cellular defects  over two years of observation [LmnaLCO embryos also showed that lamin C, emerin and LAP2 localization was normal and that there was no significant alteration in nuclear lamina structure [LmnaLCO cells when compared to WT cells [A mouse model that lacks lamin A but expresses lamin C only  had a mild delay in weight gain beginning at four weeks but appeared to be otherwise healthy until an acute period of deterioration followed by death at 12–14 weeks after birth [LmnaN195K/N195K mice but mild muscular degeneration and cardiac chamber dilatation coupled with thinning of the chamber walls were noted in cardiac tissues [LmnaN195K/N195K mice presented with a significant decrease in fractional shortening compared to their siblings at eight weeks, and the condition worsened with age [+) current in the homozygous mice compared to WT [LmnaN195K/N195K mice [LMNA mutations impact the physiology of the entire cell, including the plasma membrane organization, through their effect on an intermediate filament protein like desmin. Mis-expression of the transcription factor Hf1b/Sp4, which is important in cardiac cell differentiation, particularly for the development of the cardiac conduction system, was also noted [LMNA-related DCM-CD.The p.N195K lamin A/C variant is associated with DCM-CD in humans . The homer birth . The homer birth . The heter birth . No hall tissues . In line tissues . Furtherwith age . Close mwith age . Characted to WT . Primaryed to WT . The mus95K mice . In addi95K mice . These tso noted ,114. ThiLmnaE82K) [LMNA mutation identified in a Chinese family with DCM-CD (atrioventricular block) [Another model for DCM-CD is transgenic mice with cardiac-specific expression of the p.E82K lamin A/C variant (mnaE82K) . This var block) ,117. Trar block) . A portir block) . Gross mr block) . Cardiacr block) . Moreover block) ,118. Ther block) . In addir block) . Thus, tr block) .To model another type of striated muscle laminopathy, EDMD, which also oftentimes co-present with a cardiac phenotype, a transgenic mouse with cardiac-specific expression of the p.M371K lamin A variant causing EDMD in humans was created ,119. ThiLMNA mutation substituting A to C at nucleotide position 665, resulting in the lamin A/C protein p.H222P variant, was expressed in mice [LmnaH222P/H222P) presented with skeletal and cardiac phenotypes mimicking the human disease presentation. All homozygous mice died at 13 months [LmnaH222P/H222P mice compared to their heterozygous and WT siblings [LMNA mutations [LMNA-related DCM may depend on sex hormone levels and inhibition of the androgen receptor‒SRF‒FHL2 complex may provide a potential therapeutic benefit [LmnaH222P/H222P mice cardiac and/or skeletal muscle samples [LmnaH222P/H222P mice [LmnaH222P/H222P mice implicated the AKT/mTor and WNT/β-catenin pathways [LmnaH222P/H222P mice and the inhibition of mTor improved autophagy in the p.H222P mice hearts [LmnaH222P/H222P mice as seen in the LmnaN195K/N195K mice [To model another EDMD laminopathy associated with arrhythmia, the  in mice . The hom3 months . Analyse3 months . Locomot3 months . A signisiblings . These psiblings . Moreovesiblings ,120,121. samples ,122. Inh22P mice ,125,126.22P mice . Furtherpathways ,128,129.pathways ,128,129.95K mice ,129. How95K mice . TherefoLmnaΔK32/ΔK32), which results in severe EDMD or L-CMD in patients, was generated [LmnaΔK32/ΔK32 mice presented with major growth retardation and died three weeks after birth due to severe metabolic deficiencies associated with the repression of SREBF-1 [C. elegans, this lamin A/C protein variant is present in low amounts, suggesting its instability compared to the WT lamins [LmnaΔK32/+) progressively developed DCM and died at between 35 and 70 weeks of age [Nppb, Myh7) was noted at 10 weeks (a time point that precedes notable phenotype) [LmnaΔK32/+ mice was caused by the impairment of the ubiquitin-proteasome system [LmnaΔK32/+ mice [LmnaΔK32/+ mice, which eventually developed cardiomyopathy. A third EDMD mice model expressing another variant, the truncated p.K32del lamin A/C variant  . Immunosenotype) . In the enotype) . The rese system . This sy2/+ mice . Thus, tLmnaL530P/L530P) started presenting phenotype reminiscent of HGPS at 4–6 days old and died within 4–5 weeks [LmnaL530P/L530P mice [LmnaL530P/L530P MEFs as well as a decrease in nuclear lamin A, and a mislocalization of lamin C to the cytoplasm instead of in the nucleus [However, not all mice models faithfully mimic the human phenotype. The p.L530P lamin A/C variant was discovered in a patient with autosomal dominant EDMD . Heteroz–5 weeks . Mild to30P mice . Further30P mice . At the  nucleus . Neverth nucleus ,131.LMNA mutations. Although LMNA mutations are known to cause distinct laminopathy disorders in humans, there is a large spectrum of sometimes contradictory findings obtained in cellular and animal models. One should not underestimate the fact that most mechanistic outcomes stem from models with inherent pitfalls. Differences in results between cell lines can be attributed to various reasons including the type of cell line used, the plasmid used for transfection , the presence or absence of a C or N termini reporter tag of various sizes, the duration of transient transfection, and which lamin (A or C) is being transfected [The evidence thus far demonstrates the wide array and importance of the presence and function of A-type lamins in maintaining cellular processes, as indicated by the diversity of diseases caused by nsfected ,50,53. Snsfected ,53. Trannsfected . Moreovensfected . In addinsfected . Moreovensfected ,133. FurLmnaΔ8–11 mice, which were initially deemed to be null but later proven to express a C-terminal truncated lamin A, and the real Lmna null—the Lmna KO mice (LmnaGT−/−) created using gene trap technology, in which no lamin A/C transcript or protein was detectable [Although cell line models are more manageable to handle and cheaper than in vivo models, both cellular and animal models are complementary when modelling a complex human disorder. The animal models enable not only the identification of the molecular mechanisms affected by the variant protein, but also frame the results in the context of a living organism as a whole with the opportunity to further probe the phenotype and molecular implications in specific and relevant organs/tissues in real time and/or post-mortem, as well as to eventually test for treatments. Differences or inconsistencies between the phenotypes of animal models of the same species can be a consequence of the respective techniques used. Such is the case in tectable ,103,105.tectable .LmnaGT−/− mice; LmnaΔ8–11 and LmnaΔ8–11/+ mice; LmnaΔK32/ΔK32 and LmnaΔK32/+ mice) [LmnaL530P/L530P mice, see above). Furthermore, in humans, not all LMNA mutation carriers actually present a phenotype. Some individuals bearing the same mutation as their affected family members remain asymptomatic. This is not usually the case in the published animal models . Nevertheless, the fact that animal models do not perfectly recapitulate human phenotype can be partly attributed to the genetic modification used (knockdown versus knock-out or transgenesis or knock-in), species differences and external variables. Neither a knock-down (transient) nor a knock-out (permanent) animal model that seeks to significantly lower or completely abolish gene function is a blanket representative of the human disease context because most patients possess pathogenic mutations that do not abrogate LMNA gene function but instead result in the production of variant lamin A/C proteins. Moreover, although transgenesis enables tissue-specific expression of mutant transgenes, the results are influenced by the fact that endogenous WT lamin A/C is still being expressed in the animals. The insertion sites, number of insertions and expression levels of transgene are less defined and are usually not consistent between lines. In addition, in some studies, the phenotype in the transgenics becomes less overt or disappears after a few generations [C. elegans lamin-1 to 96.4% in M. musculus. Another example of species difference is with zebrafish, which underwent genome duplication, resulting in functional duplicates of orthologues for some human genes. Though the LMNA gene has one identified orthologue in zebrafish, SYNE1, which codes for the lamin A/C interacting partner Syne1, has two orthologues in zebrafish, syne1a and syne1b. In terms of anatomy, fruit flies, for instance, have a tubal heart and zebrafish only have two cardiac chambers. Thus, these species differences could contribute to the differences in phenotype between animal models and the actual human disease presentation. Other variables that can influence the phenotype also include the gene pool, environmental conditions, co-morbidities and technology. Humans have vast genetic diversity compared to that of laboratory animals. Co-morbidities include hypertension, myocarditis, autosomal disorders, diabetes, obesity, high cholesterol, inactivity, as well as aging and treatments such as chemotherapy. The effects of the environment/lifestyle on the organism are also important, particularly since for each human being the experience is unique, while lab animals live in unnatural and controlled standard lab settings. Lastly, the lag in our capacity to identify and/or characterize phenotypes in human and animal models limits our understanding of the scale and intricacies of the disease. On the other hand, despite the immense progress afforded by both cellular and animal models, there are still differences in phenotypes between the actual human disease presentation and the models. For instance, both knock-out/knock-in mice models and transgenic zebrafish primarily presented a phenotype at the homozygous state, while in humans the conditions are predominantly autosomal dominant ,108,115./+ mice) ,107,130.erations . Knock-iLMNA mutations is the nuclear membrane disturbance(s) that compromises the structure, integrity, shape and/or size of the nucleus [LMNA mutations exert their effects on a recessive mode. An example is the LMNA mutation resulting in the p.H222Y variant that caused severe EDMD for the homozygous patient, while both his heterozygous parents were asymptomatic [Altogether, the insights learned from both cellular and animal models so far suggest that there is no one hypothesis that can completely explain the mechanisms underlying striated muscle laminopathies. However, the gene and/or mechanical hypotheses seem to provide an explanation for the cardiac and/or skeletal muscles phenotypes in most cases. The overlap in phenotypes between humans and models, particularly the phenotypic features common to more than one model, highlights the important and conserved pathways involved in the pathogenesis of striated muscle laminopathies.  nucleus ,90,91,96ptomatic . Anotherptomatic ,139,140.LMNA mutations in more relevant cellular models would usher an improved model that can better mimic what is found in patient cells. The use of iPSCs from patients also allows for a superior simulation of the cell type and tissue-specific conditions. Moreover, since iPSCs are patient-specific, derived cells can be used to conduct personalized drug screens, therefore facilitating a personalized medicine approach to treating the disease. In terms of vertebrate in vivo models, the zebrafish, with its quick, transparent development and high fecundity, allows for mass drug screening. In addition, the determination of specific molecular signatures prior to disease onset is now more feasible, thus eventually enabling clinicians to improve predictive diagnostic capabilities that may allow for prophylactic treatments to either prevent or delay disease onset and attenuate the severity of the disease. Lastly, as evidenced by the progress in the field arising from results obtained from animal models , a clinical trial involving a p38 inhibitor (ARRY-371797) to treat patients with DCM caused by LMNA mutations is in progress. Phase 2 results showed improved functional capacity  and cardiac function with mild to moderate adverse effects , thus raising our hopes that it can be used to treat laminopathies in the future [With the advent of the CRISPR/Cas9 technology, genetic manipulation has become more accessible and more precise; thus, the modelling of human diseases can be even further improved. Creating knock-in e future .


Rosa section Synstylae (Rosaceae) are mainly distributed in East Asia, and represent recently diverged lineages within the genus. Over decades, inferring phylogenetic relationships within section Synstylae have been exceptional challenges, due to short branch lengths and low support values. Of approximately 36 species in the section Synstylae, Rosa multiflora, Rosa luciae and Rosa maximowicziana are widely distributed in the Sino-Japanese floristic region. In this study, we assembled chloroplast genomes of these three species to compare the genomic features within section Synstylae, and to compare with other infrageneric groups. We found that three Rosa sect. Synstylae species had lost infA genes with pseudogenization, and they were almost identical to each other. Two protein-coding gene regions (ndhF and ycf1) and five non-coding regions  were identified as being highly informative markers. Within three section Synstylae chloroplast genomes, 85 simple sequence repeat (SSR) motifs were detected, of which at least 13 motifs were identified to be effective markers. The phylogenetic relationships of R. multiflora, R. luciae and R. maximowicziana could not be resolved, even with chloroplast genome-wide data. This study reveals the chloroplast genomic data of Rosa sect. Synstylae, and it provides valuable markers for DNA barcoding and phylogenetic analyses for further studies.Species belonging to  Rosa L. (Rosaceae) comprises approximately 125–200 species, distributed through the temperate and subtropical regions of the Northern hemisphere [Rosa is divided into four subgenera , and the subgenus Rosa is further divided into 10 sections  [Rosa section Synstylae DC. comprises approximately 36 species, which are well-circumscribed with the distinctive synapomorphic characteristic of connate styles into an exserted column [Chinenses has been suggested, from several previous studies [Rosa sect. Synstylae species are mostly distributed in East Asia, of which Rosa multiflora Thunb. and Rosa luciae Franch. & Rochebr. ex Crép (syn. Rosa wichurana Crép) are the most well-known species, as ones of the wild ancestors of cultivated roses [Rosa sect. Synstylae species are known to be recently diverged, based on poor resolutions and low support values from many previous molecular phylogenetic studies [Synstylae did not resolve the phylogenetic relationships with several phylogenetic problems, such as species delimitation failure, poor resolutions, and low support values, and inconsistency among gene trees [Synstylae is incomplete reproductive barriers among congeneric species [Rosa has been notoriously difficult, due to low sequence divergence level and hybridization [The genus misphere . In the cteatae) ,5. The R studies ,8. Rosa ed roses . Rosa se studies ,8,10,11. species . Indeed,dization . Chloroplasts are the photosynthetic organelles in plant cells, and the chloroplast genomes contain various essential genes for carbon fixation and metabolite synthesis. Most chloroplast genomes range from 120 to 170 kb in size, and have the quadripartite structure, in which two inverted repeats (IRs) are separated by a large single-copy region (LSC) and a small single-copy region (SSC) . Their gRosa sect. Synstylae in this study, i.e., R. multiflora, R. luciae, and Rosa maximowicziana Regel, are widely distributed in the Eastern Sino-Japanese floristic region, and each species represents different floristic elements. Rosa multiflora is found in the Liaoning–Shandong flora, while R. luciae and R. maximowicziana occur in the Southern Chinese flora and the Amur flora, respectively (R. maximowicziana and R. multiflora/R. luciae) or to overlap a large portion (between R. multiflora and R. luciae), these species are distinct in the ecological, morphological, or phenological characteristics  were sequenced to (1) understand the chloroplast genomic features of Rosa sect. Synstylae; (2) evaluate interspecific divergence among the chloroplast genomes of Rosa sect. Synstylae species; and (3) infer the phylogenetic relationship based on the chloroplast genomic data. In addition to the intra-sectional comparison chloroplast genomes of Rosa sect. Synstylae, the intra-generic comparison including chloroplast genomes of species in the different infrageneric group was conducted to obtain further insights on chloroplast genomes in genus Rosa. This study will provide the information of effective chloroplast markers to identify Rosa species, and the insight on directions of further systematic studies on Rosa sect. Synstylae. Moreover, it will be one of the good model study on the chloroplast genome evolution of very recently diverged species, which are geographically, ecologically, morphologically, or phenologically distinct.In previous studies, only several chloroplast region data were used to infer the phylogeny of ynstylae ,6,7,8,10Rosa sect. Synstylae species . Total genomic DNA was extracted using Exgene Plant SV mini kit  and sequenced using Illumina HiSeq 4000 platform  at Macrogen , generating paired-end sequence reads.Fresh young leaf samples were collected from wild individuals of three The paired-end reads of each species were assembled de novo into the draft chloroplast genome sequence contigs using Velvet 1.2.10 . AssemblNicotiana tabacum chloroplast genome (Z00044) [Rosa roxburghii chloroplast genome (NC_032038) [Rosa chloroplast gene maps were drawn with OGDRAW (OrganellarGenomeDRAW) [Rosa species were submitted to GenBank  genes were primarily identified using DOGMA  , and tra(Z00044)  and Rosa_032038)  from GenomeDRAW) . The ann GenBank .R. multiflora, R. luciae, and R. maximowicziana from this study and chloroplast genomes of two other Rosa species from other infrageneric groups in GenBank, Rosa roxburghii  [Rosa odorata var. gigantea  [Synstylae and among infrageneric groups, R. multiflora chloroplast genome was used as a reference genome in this analysis. Major variations in gene contents or features of Rosa chloroplast genomes were manually identified with Geneious [The complete chloroplast genomes of _032038)  and RosaF753637) , were coF753637) ,27 with F753637) . To compGeneious . For theGeneious .Rosa chloroplast genomes. Chloroplast genome sequences were aligned using Geneious Alignment, and the alignment was manually adjusted by using Geneious [Synstylae and within genus Rosa. In DNA polymorphism analysis, the window length was set to 800 bp, and the step size was set to 200 bp.DNA polymorphisms analysis was performed using DnaSP (DNA Sequence Polymorphism) v6  to calcuGeneious . NucleotSynstylae were identified using MISA-web (MIcroSAtellite identification tool-web) [The simple sequence repeats (SSRs) of 1-6 bp repeats in three chloroplast genomes of sect. ool-web) ,31. The Rosa species, five species including three from this study  were included. The chloroplast genome of Fragaria vesca ssp. vesca (NC_015206) [Dasiphora fruticosa (NC_036423) [Fragaria and Dasiphora are suggested to be ones of the closely related genera to Rosa [In the phylogenetic reconstruction of _015206)  and Dasi_036423)  from Gen to Rosa . The who to Rosa .Rosa chloroplast genomic phylogeny. In Markov chain Monte Carlo (MCMC), a couple of four chains were run for 2,000,000 generations . The trees were sampled in every 100 generations, and the first 25% of sampling were discarded as burn-in trees. Remaining trees were used to construct a 50% majority-rule consensus tree. Maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted to reconstruct the phylogenetic relationships. Tree reconstruction and the bootstrap for ML analysis were performed in IQ-TREE v1.6.1 . The subRosa sect. Synstylae species  were from 156,405 bp (R. maximowicziana) to 156,519 bp (R. multiflora), and the depth of coverage for chloroplast genome contigs were from 214× (R. multiflora) to 1418× (R. maximowicziana). Each chloroplast genomes of Rosa sect. Synstylae species had a typical quadripartite structure, in which a large single-copy region  and a small single-copy region  were separated by two inverted repeats   . The gen species . One hunNA genes . Among tNA genes .Rosa species in other infrageneric groups, R. roxburghii (subgenus Platyrhodon) and R. odorata var. gigantea (subgenus Rosa section Chinenses), three Rosa sect. Synstylae chloroplast genomes  from this study had smaller genome sizes and fewer genes, whereas the GC contents were identical among five species  gene was missing in R. roxburghii chloroplast genome. The other difference was that chloroplast genomes of R. roxburghii or R. odorata var. gigantea contained additional trnP(GGG) gene in the region where trnP(UGG) gene already exists. We conducted additional BLASTN and tRNAscan-SE searches on these genomes to verify whether these differences are evident. In BLASTN and tRNAscan-SE searches; however, trnG(GCC) gene was also found in R. roxburghii chloroplast genome. In the other difference, tRNAscan-SE only identified trnP(UGG) gene, not trnP(GGG) gene. Given these findings, the only difference in gene contents among the Rosa chloroplast genomes was that the Synstylae chloroplast genomes lost their infA genes by pseudogenization, while R. roxburghii and R. odorata var. gigantea chloroplast genomes contained infA genes.The interspecific comparison of the gene contents of five mutation . AccordindhF genes. In Rosa species, two kinds of mutations were found in the 3’ regions of ndhF genes. The mutation of T-insertion on the 3’ region of ndhF gene of R. maximowicziana caused the protein to terminate one amino acid early (746 amino acid residues), compared to the other Rosa species (747 amino acid residues). The other mutation was a point mutation on ndhF gene of R. roxburghii, altering phenylalanine 746 (Phe746) to leucine 746 (Leu746) (Another remarkable difference was the mutations on the 3’ region of (Leu746) .R. multiflora chloroplast genome and each of the other Rosa chloroplast genome  intergenic region. R. roxburghii and R. odorata var. gigantea had more variable sites compared to R. luciae and R. maximowicziana.In the overall interspecific chloroplast genome comparison, there was no large differences between t genome . EspeciaRosa chloroplast genomes, nucleotide diversity (Pi) was calculated using DnaSP. The average Pi value for five Rosa chloroplast genomes was 0.00154, and the average Pi value for the Synstylae chloroplast genomes was 0.00003. IR regions showed lower nucleotide diversity than single copy regions. While a comparison of three Synstylae chloroplast genomes was not informative, due to very few variations, a comparison of five Rosa chloroplast genome identified several variation hotspots (psbI-trnS-trnG region (Pi = 0.01313), followed by four intergenic regions , and one gene region (ycf1).To estimate the genetic distance among hotspots . Six regSynstylae chloroplast genomes, including two compound repeat regions  of 100% in ML, and a posterior probability (PP) of 1.00 in BI. In the chloroplast genome phylogeny, Rosa odorata var. gigantea was inferred to have a closer relationship with three Rosa sect. Synstylae species than R. roxburghii (100% BS and 1.00 PP support). Within the section Synstylae, however, no phylogenetic relationship was inferred, due to short branch lengths and poor support values.Maximum Likelihood and BI analyses showed identical tree topologies with high support values . Rosa seRosa sect. Synstylae  were de novo assembled for the first time in this study. The Synstylae chloroplast genomes were marginally different at best. Not surprisingly, more differences were found between the Synstylae chloroplast genomes, and two other chloroplast genomes of R. roxburghii (subg. Platyrhodon) and R. odorata var. gigantea (subg. Rosa sect. Chinenses). The major difference was that the Synstylae chloroplast genomes had lost their infA gene by pseudogenization , which has been one of major hurdles for the phylogenetic reconstruction in genus Rosa [Three chloroplast genomes of nization . Indepenm groups , but it nus Rosa . Synstylae chloroplast genomes had only few variations and no genomic structural differences, several conservative protein-coding regions could be significant candidates for the chloroplast barcoding regions. The ndhF gene could be proposed to be one of those, as a frameshift mutation on the 3’ end of ndhF gene was identified in R. maximowicziana, which caused early termination of translation. This region may be able to be used in the higher taxon, genus Rosa, as a missense mutation occurred in the 3’ ndhF of R. roxburghii. Another candidate is ycf1 gene  are expected to be highly useful genetic markers in genus Rosa, confirming that these regions have been suggested as good markers for DNA barcoding and phylogenetic analyses in Rosaceae [ndhF gene is known to have a higher substitution rate, and its 3’ region was suggested to be phylogenetically useful, because it shows a high indel frequency, a high AT content and a low homoplasy rate [Rubus subg. Idaeobatus, which is in the same subfamily with genus Rosa (subfamily Rosoideae), the 3’ regions of ndhF have shown a wide range of indel variations, resulting in frameshift mutations and alterations on the ndhF transcription termination [Rosa, given that they are the protein-coding genes. Because the Synstylae chloroplast genomes are highly conserved among species, chloroplast SSRs may provide valuable information for genotyping in the intra- and/or inter-specific level. For SSR genotyping in Synstylae species, 13 mononucleotide SSR regions were polymorphic among the species in this study (except for a deletion in the rps4-trnT region of R. maximowicziana). Among those, two regions (psbH-rpoA and rpl14-rps3 intergenic regions) could differentiate each accession of the Synstylae chloroplast genomes (Because the e tic214 ), becaus hotspot , showingRosaceae ,41,42,43asy rate . Indeed, genomes . Rosa sect. Synstylae species have been inferred to be very recently diverged, suggesting that R. multiflora and R. luciae have diverged from their common ancestor ca. 1.6 MYA, based on the chloroplast data [R. maximowicziana showed two distinctive variations in its chloroplast genome  intergenic region), the Synstylae chloroplast genomes were almost identical, showing 0.00003 Pi value among three different genomes (Synstylae species with lower diversities and shorter branch lengths than previous studies suggested [Synstylae revealed a polytomy, due to the extremely short branch lengths among three species, which suggest marginal phylogenetic differences among them (Synstylae species. The close phylogenetic relationships of three Synstylae species may suggest that the evolutionary time was not sufficient for their chloroplast genomes to be distinctly diverged, and/or that they had interspecific genetic introgressions among the Synstylae species. Indeed, the incomplete lineage sorting and genetic introgression have been suggested in phylogenetic analyses with chloroplast data of Synstylae species [Synstylae clade, chloroplast capture events may occur, which would obscure inferring the species phylogeny. For more phylogenetic resolutions and systematic implications, additional phylogenetic analyses with nuclear markers would be required, because a nuclear genome phylogeny can be different from a chloroplast genome phylogeny [Rosa multiflora and R. luciae [Synstylae species samples were collected in the Korean Peninsula, while they are distributed in the wide range of the Eastern Sino-Japanese floristic region. This sampling bias could affect the chloroplast genome comparative analyses. To verify the existence of various distinct chloroplast haplotypes of R. multiflora and R. luciae, further phylogeographic and/or population genetic studies, including many accessions from representative sites in the Eastern Sino-Japanese floristic region will shed new light on the evolution of the Synstylae species. If there are various distinct chloroplast haplotypes in the section Synstylae, and if the haplotype differentiations are found along the geographic gradients, but not along the species boundaries, it is more plausible that the genetic introgressions have occurred, rather than that the chloroplast genome divergence was not sufficient. Several studies found the haplotype sharing in some taxa in which species are closely related, but defined by ecological or morphological differences [Synstylae species in this study, these species also might share the chloroplast genomes beyond the species boundaries. Therefore, Rosa sect. Synstylae, including R. multiflora, R. luciae, and R. maximowicziana, can be another model taxonomic group for studying the chloroplast genome evolution of closely related species, which are defined by ecological or morphological differences, but shared the plastomes by the different species.ast data . Though  genomes . The comuggested ,5,7. Theong them . Given thylogeny . Because. luciae ,10, thesferences ,47,48. G


Clostridium limosum can be found in soil and the intestinal tract of animals. In 2014, C. limosum was isolated from a suspected blackleg outbreak in cattle in Schleswig-Holstein, Germany. We present a complete genome sequence of a C. limosum strain represented by a circular chromosome and three plasmids. Clostridium limosum can be found in soil and the intestinal tract of animals. In 2014, C. limosum was isolated from a suspected blackleg outbreak in cattle in Schleswig-Holstein, Germany. We present a complete genome sequence of a C. limosum strain represented by a circular chromosome and three plasmids. Clostridium is a genus of Gram-positive anaerobic bacteria within the phylum Firmicutes. The genus includes around 30 species that can cause clinical diseases in humans and animals, including birds. Clostridium limosum is a species that has received little attention in terms of its disease occurrence, prevalence, and virulence factors. The bacterium was found in various environments, including different animal and bird species  revealed homology to C. limosum (Hathewaya limosa Lawson and Rainey 2016 [NR_104825).An initial categorization of the bacterial species using partial 16S rRNA gene sequencing  followedney 2016 ) strain C. limosum isolate (14S0207) was cultured in 3 ml Selzer broth (The er broth  under ante 14S020 was culthttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Briefly, reads were trimmed using BBDuk  sequencing technology (MiSeq system) together with the Nextera XT library preparation protocol  was performed at the Institute of Bacterial Infections and Zoonoses in Jena, Germany. A total of 1,067,720 reads were received. The raw reads were checked for quality before and after read trimming using FastQC version 0.11.7 . The overlapping regions of circular sequences were determined using Gepard software version 1.40 with default parameters  with default parameters  implemenrameters . For therameters . The cirrameters . As a reC3495_10690, encoding 52 amino acids), a bacteriocin, and a virulence factor of group A Streptococcus known to possess hemolytic/cytolytic activity , CP026601 (plasmid 1), CP026602 (plasmid 2), and CP026603 (plasmid 3). The raw sequence data are available under SRA accession numbers SRR9822081 (PacBio RS II) and SRR9822082 (Illumina MiSeq). The associated BioProject and BioSample accession numbers are PRJNA432648 and SAMN08456352, respectively. The versions described in this paper are the first versions.This whole-genome sequencing project has been deposited at DDBJ/EMBL/GenBank under the accession numbers


Yersinia pestis. Transmitted by fleas from rodent reservoirs, Y. pestis emerged <6000 years ago from an enteric bacterial ancestor through events of gene gain and genome reduction. It is a highly remarkable model for the understanding of pathogenic bacteria evolution, and a major concern for public health as highlighted by recent human outbreaks. A complex set of virulence determinants, including the Yersinia outer-membrane proteins (Yops), the broad-range protease Pla, pathogen-associated molecular patterns (PAMPs), and iron capture systems play critical roles in the molecular strategies that Y. pestis employs to subvert the human immune system, allowing unrestricted bacterial replication in lymph nodes (bubonic plague) and in lungs (pneumonic plague). Some of these immunogenic proteins as well as the capsular antigen F1 are exploited for diagnostic purposes, which are critical in the context of the rapid onset of death in the absence of antibiotic treatment (less than a week for bubonic plague and <48 h for pneumonic plague). Here, we review recent research advances on Y. pestis evolution, virulence factor function, bacterial strategies to subvert mammalian innate immune responses, vaccination, and problems associated with pneumonic plague diagnosis.Plague is a vector-borne disease caused by  Yersinia pestis [Plague is a vector-borne illness transmitted by fleas to a variety of wildlife rodents, which represent natural reservoirs for the disease in a wide range of habitats around the world . The etia pestis , discovea pestis . Plague a pestis . In the a pestis , and reca pestis , China [a pestis , Democraa pestis , and Mada pestis , 10 remiY. pestis is highly similar on a genomic level to the enteric pathogen Y. pseudotuberculosis; however, a series of gene gain and gene loss events have led to the appearance of markedly different mechanisms of disease as well as niche preference and lifestyle [Y. pestis displays a quite unique set of virulence factors that allow successful infection of fleas and subversion of immune responses in mammalian hosts, leading to rapid host death in the absence of adequate treatment. In this article, we review recent advances in plague research, particularly in the fields of evolution, virulence determinants, and subversion of mammalian immune responses, as well as an overview of the critical aspects of vaccination and pneumonic plague diagnosis. For other aspects of Y. pestis infection, such as Y. pestis adaptation to the flea, see, e.g., the review by Hinnebusch et al. [ifestyle . Y. pesth et al. .Y. pestis phylogeny are emerging from ancient DNA (aDNA) paleo-genomic studies, which have been positively impacted by the development of high-throughput, “next-generation” DNA sequencing (NGS) technologies, together with an impressive decline in sequencing costs. The recovery of Y. pestis DNA from the teeth of prehistoric individuals has uncovered the branching of independent lineages throughout Eurasia in the Neolithic, and allowed to accurately estimate that Y. pestis diverged from Y. pseudotuberculosis around 5700–6000 years ago (ya) [pde3 [Insights into ago (ya) –16. Thisa) [pde3 , 17.Y. pestis [Y. pestis lineages persisting today (0.PE7 and 0.PE2), and to two extinct Neolithic and Bronze Age lineages  [ureD, rcsA, flhD, and pde2 as well as to lack the Yersinia murine toxin encoded by the ymt gene , which has been associated with reduced bacterial dissemination in mammals [Whole-genome-based phylogenies have defined a five-branch population structure for . pestis , followictively) . These lene Fig. . The acqFig. ymt  and inacby fleas . Interes mammals . This su mammals , 15.Fig.microtus) and the diverse lineages that led to all known plague pandemics [ymt and had the derived, inactive forms of ureD, rcsA, flhD, and pde2, suggesting that the flea-mediated transmission mechanism causing bubonic plague evolved as early as 5000 ya [Y. pestis among Neolithic human remains indicates that this pathogen was prevalent and virulent, hypothesizing the existence of prehistoric plague pandemics [Y. pestis lineage that led to the historically recorded pandemics remains unclear.A second expansion event around 4000 ya gave rise to an extinct Bronze Age lineage (RT5) and to lineages persisting to the present day: 0.PE4  [big bang polytomy that gave rise to most of the extant strain diversity [Paleo-genomic studies also provided unambiguous evidence confirming the presence of nturies) , 22 and nturies) , 24. Thenturies) . In contiversity , 26, incY. pestis genomes suggested that different bacterial clones were responsible for the second pandemic [Y. pestis diversity during the second pandemic and propose a different scenario, in which plague may have established in natural rodent-based foci within Europe that evolved and persisted until the 18th century, spreading eastward back to Central and East Asia to become the source of the contemporary epidemics [Y. pestis genomes, and infers different waves of introduction of plague to Western Europe during medieval times [Several scenarios have been proposed to explain the transmission dynamics and persistence of plague in Europe after its introduction from Central/Eastern Asia during the Black Death. Initial paleo-genomic analyses using medieval pandemic , proposipandemic  and sprepandemic . Other spidemics , 30. A ral times . Both loal times , which cal times .Y. pestis spread [Y. pestis. In light of the recent genotyping of 359 modern Y. pestis strains from the natural plague foci in the former Soviet Union countries, an update of the intraspecific taxonomy was proposed [pestis, which includes the vast majority of virulent lineages that participated in the historically recorded pandemics ; as well as six other clusters, including ssp. caucasica—0.PE2, ssp. angolica—0.PE3, ssp. central asiatica—0.PE4, spp. tibetica—0.PE7, ssp. ulegeica—0.PE5, and ssp. qinghaica—0.PE10.The third pandemic originated around 1855 in the Chinese province of Yunnan with the emergence of the biovar Orientalis strains, which belong to the 1.ORI branch. From this origin, plague spread worldwide via steamships carrying infected rats and is nowadays present in Asia, Africa, and America . The tras spread , 33–35. proposed  Y. pestis complex pathogenesis, which relies on a finely regulated arsenal of virulence determinants [Yersinia outer-membrane proteins (Yops), which constitute an array of effectors directly translocated into host cells through a type 3 secretion system (T3SS) [Yersinia virulence plasmid pYV/pCD1, which is also present in the enteric pathogens Y. pseudotuberculosis and Y. enterocolitica. As previously mentioned, the emergence of Y. pestis from Y. pseudotuberculosis is associated with the acquisition of the pFra and pPla plasmids, which encode critical virulence factors (see below), as well as major gene loss  that had a major impact in adaptation to the flea vector [Plague prevention requires a complete understanding of rminants . Among tm (T3SS) –39. The a vector . Here, wY. pseudotuberculosis or Y. enterocolitica, and remains to be investigated in Y. pestis.Yops have been shown to play pleiotropic roles during infection: they inhibit Rho GTPases and disrupt the actin cytoskeleton in order to inhibit phagocytosis, they downregulate the production of pro-inflammatory cytokines, and they induce cell death by multiple sophisticated mechanisms  Fig. 2)2)  was identified as required for bacterial intracellular survival during the early stages of bubonic plague [Y. pestis pathogenesis for the type 6 secretion system (T6SS) component VasK and the ribose transport system ATP-binding protein RbsA [o oxidase CyoABCDE, and the multifunctional Tol-Pal system proteins Ypo1119–1120 [Recently, novel virulence factors have been identified using a “per-pool” mutant screening method , as wellc plague . Besidesc plague . Using Sc plague . A seconein RbsA . Additioein RbsA . In the 119–1120 . FurtherY. pestis secretes the siderophore yersiniabactin (Ybt), which has been shown to be essential for bubonic and pneumonic plague [Y. pestis. Together with Ybt, the ABC transporter ZnuABC and YbtX, a member of the major facilitator superfamily, contribute to infection in murine models of plague [Y. pestis [A critical property of bacterial pathogens is their capacity to overcome nutritional immunity, i.e., host sequestration of essential metals such as iron and zinc , a mechac plague . In addic plague . Interesf plague –71. YbtX. pestis . The rolY. pestis infection, either through a flea bite or through contamination via respiratory droplets, is the brutal transition from an absence of immune response and clinical symptoms, to a bursting inflammation and fatal sepsis with abundant bacteria in the body. This lag period has been termed “pre-inflammatory phase” and benefits Y. pestis, which migrates toward lymph nodes (bubonic plague) or lungs (pneumonic plague) to multiply silently. This immune evasion is exerted by several mechanisms that include the T3SS-mediated neutralization of immune cells (see the previous section), the absence of detectable pathogen-associated molecular patterns (PAMPs [One of the most surprising features of s (PAMPs ), and moY. pestis is eliminated effectively when phagocytosed by neutrophils, but survives and replicates when phagocytosed by macrophages. It has now been shown that Y. pestis grown at flea temperature (close to that of the host skin) at which the T3SS is not yet upregulated, can survive and replicate within human neutrophils [Y. pestis can also transit from this first niche to a second one when macrophages phagocytose infected neutrophils in a cleaning process named efferocytosis [Y. pestis can survive in autophagosomes [Y. pestis is also capable of inhibiting phagosomal maturation by perturbing the host endosomal recycling pathway, allowing bacterial intracellular replication [Y. pestis to reduce innate immunity mechanisms and multiply, meanwhile upregulating its T3SS. Then, in target organs, Y. pestis would preferentially inject Yops in neutrophils by specifically recognizing their surface complement receptor 3 [Neutrophils are a very early line of innate defense and inflammation at infection sites. It was commonly accepted that trophils . Y. pestocytosis . Within agosomes , and it lication , 76. Efflication , a cytokceptor 3  and resiceptor 3  and the ceptor 3 .Y. pestis disseminates from skin flea injection sites to lymph nodes was unknown and it was suspected that phagocytes  could carry the bacteria. This scenario has been challenged by recent observations in murine models of infection, suggesting that the Y. pestis spread from the skin is very rapidly achieved [How achieved  in the aachieved , suggestachieved . Howeverachieved .Y. pestis [Y. pseudotuberculosis infection models, it has been suggested that the T3SS effector YopJ induces macrophage apoptosis [Y. pestis virulence [Y. pestis dissemination from lymph nodes during the pre-inflammatory phase: cell membrane rupture leads to bacterial release and subsequent capture by phagocytes chemotactically attracted by S1P [Y. pestis node-to-node trafficking [Y. pseudotuberculosis redirects apoptosis to caspase-1-dependent inflammatory pyroptosis in pre-activated cells [Y. pestis-induced pyroptosis results from YopJ blockade of kinase TAK1, which activates caspase-8 and pore-forming gasdermin D [Y. pestis could induce necroptosis also requires clarification, as caspase-8 cleavage, the signal for Y. pestis-induced cell death [Yersinia infection [Within the infected lymph nodes, several pathways of cell death have been proposed to be activated by . pestis . Applyinpoptosis , a noninpoptosis . Howeverirulence . Necroptd by S1P . Bacterifficking . Of noteed cells , and Y. dermin D . Paradoxdermin D , 51. Howll death , has beell death . Interesnfection .Y. pestis growth [Y. pestis infecting lungs via aerosols that initially grew at 37 °C in the contaminating patient’s lungs [Despite being caused by the same pathogen, pneumonic plague differs from bubonic plague by the mode of infection (aerosols versus fleas), the target tissue (lungs versus skin/lymph nodes), and ultimately pathophysiology and lethality. The pre-inflammatory phase during pneumonic plague generates a permissive environment that allows s growth . Residens growth . Inflamms growth . Therefos growth , 96. In s growth . In thiss growth . An impo’s lungs , have th’s lungs . In consY. pestis virulence [Y. pestis infection process. In mouse lungs, a cytokine-induced recruitment of neutrophils reduced bacterial counts and improved survival from pneumonic plague [The existence of a pre-inflammatory phase during plague suggested that intervention to trigger a faster response of innate immunity could possibly counteract infection, as first indicated by an earlier report that inflammation resulting from a strong lipopolysaccharide response overcame irulence . Two recc plague . In a moc plague .γ [Y. pseudotuberculosis [Y. pestis NIIEG strain induces a Th17 polarization in humans [Y. pseudotuberculosis oral plague vaccine [Innate immunity mechanisms are amplified when adaptive immunity is effective, such as after vaccination (see next section), and contribute to protection. In that regard, several recent observations point to a role for IL-17 in both wings of this response. During the innate response, IL-17 acts by inducing production of antimicrobial peptides and attracting neutrophils , as wellγ . IL-17-pγ . Surprisrculosis . In agren humans . Moreove vaccine . TherefoY. pestis formerly produced in the United States and Australia, which was discontinued because of its short-lasting protection and low efficiency. The live Y. pestis EV vaccine previously used with benefit in Madagascar, and still used in Asia and Russia, was never licensed in occidental countries. Most current efforts are focused on vaccines against pneumonic plague, considering Y. pestis as a potential biowarfare agent. The most advanced candidates are molecular vaccines developed by the United States, the United Kingdom, and China governmental agencies, and are patented but not yet licensed. They combine Y. pestis F1 and V antigens and are called rF1-V, RypVax, and SV1, respectively. F1 is the main protein component of the pseudocapsule, and is a dispensable virulence factor. The V antigen (LcrV) is an essential part of the T3SS. Two doses of these F1/V vaccines provide 100% protection to mice against bubonic and pneumonic plague [Cynomolgus macaques could be protected against aerosolized Y. pestis, but not African green monkeys [Despite considerable progress in prevention and cure, natural foci still exist in Africa, Asia, and America, and plague is endemic in more than 25 countries worldwide, which would benefit from vaccination. No new vaccine has recently been licensed in Europe or in the United States to replace the killed whole-cell c plague –111. How monkeys . Therefo monkeys . rF1-V i monkeys .Y. pestis strains attenuated by genetic engineering are immunogenic and confer protection, but none was brought to the clinical evaluation phases. Another molecular vaccine, patented by the University of Chicago and called V10, is composed of a shorter V antigen only, and provided 100% protection against bubonic and pneumonic plague to mice and Cynomolgus macaques [Salmonella as a vector. However, these vaccines did not progress to clinical trials. A live Y. pseudotuberculosis may also be a valuable vaccine against plague [Y. pseudotuberculosis strain producing the F1 pseudocapsule, named VTnF1 [Y. pestis, VTnF1 appears as a very promising plague vaccine candidate.Several other vaccine candidates have been proposed. Many macaques . Live vet plague , 117. Weed VTnF1 . BecauseAs previously mentioned, pneumonic plague is transmitted from person to person through respiratory droplets. After an incubation period of 24–96 h, the disease progresses rapidly and is nearly always fatal in a few days in the absence of an early antimicrobial treatment. From August to November 2017, Madagascar experienced an unprecedentedly large pneumonic plague outbreak with multiple foci, including two main urban areas: Antananarivo, the capital, and Toamasina, the main seaport . A totalY. pestis [Screening of the suspected patients was based on epidemiological and clinical criteria, as described in the case definition provided by the World Health Organization (WHO)  and biol. pestis , is a prpla, caf1, inv, and yopM genes [pla gene is located on the pPla/pPCP1 plasmid, which is present in 150–200 copies per bacterium [pla in the sputum [pla may also be found in other Enterobacteriaceae such as Citrobacter koseri and Escherichia coli [caf1 gene is located on the pFra/pMT1 plasmid, which is considered specific to Y. pestis, but it is present in only about two copies per bacterium [pla and caf1 has been described [yopM gene, located on the Yersinia virulence plasmid pYV/pCD1, is present in about four copies per bacterium, but pYV may also be found in Y. pseudotuberculosis and pathogenic Y. enterocolitica strains. The inv gene is a chromosomal gene present in Y. pestis and Y. pseudotuberculosis; an insertion in Y. pestis made it larger [Y. pseudotuberculosis (400 bp) compared with Y. pestis (1100 bp). During the 2017 pneumonic plague outbreak in Madagascar, the strategy chosen to detect Y. pestis DNA was first to test the samples by a multiplex real-time PCR targeting pla and caf1, and to confirm the uncertain cases by a conventional PCR targeting pla, caf1, inv1100bp, and yopM. It is worth mentioning that the chromosomal gene ypo2088 is specific to Y. pestis and spiked expectorations have been successfully tested by real-time PCR targeting this gene [Molecular biology tests have been developed in order to reduce the delay and increase the sensitivity of diagnostics Fig. . ConventpM genes , 122 redacterium , resultie sputum  displayshia coli  and thesacterium . A multiescribed  but it wt larger  and a cohis gene . Moleculhis gene . Assays his gene , but theY. pestis remains the gold standard. Although Y. pestis can grow on usual culture media, the use of a selective agar supplemented in cefsulodin–irgasan–novobiocin (CIN) favors the isolation of the bacterium in polymicrobial samples such as sputum. WHO recommendations are to rinse the mouth out with water prior to sample collection in order to reduce the contaminations by the oral flora [Y. pestis-specific phage lysis. Automated identification systems are more and more often used in the laboratories: they are fast and can efficiently identify a large variety of bacteria; however, misidentifications of Y. pestis have been reported [Among the biological diagnostic tests, microbial isolation of al flora . After 2reported ; therefoY. pestis is a remarkable model to study infection and the evolution of bacterial pathogenicity. The recent improvement in high-end technologies, allowing whole-genome sequencing and more resolution in vitro as well as in vivo microscopy, has directly benefitted the understanding of various aspects of plague, including bacterial phylogeny, pandemics history, pathogenesis mechanisms, and subversion of immune responses.Due to its exceptional virulence, its multiple modes of transmission and pathogenesis, its outstanding skills to escape host molecular and cellular immunity mechanisms, and the indelible mark it has left on human societies in the past, Y. pestis in the environment remains to be fully explored, as novel potential reservoirs are discovered [Y. pestis have been inferred from comparisons with other pathogenic Yersiniae, including the enteric pathogens Y. pseudotuberculosis and Y. enterocolitica, which might bias our understanding of plague. More studies using fully virulent Y. pestis strains are required to completely grasp the physiopathology of the disease. The bacillus discovered 125 years ago by Alexandre Yersin has still a lot of secrets to reveal.However, several aspects of plague natural history are still incompletely understood. The ecology of scovered , 134. So