TP53 mutations in three tumor tissues. These variations might contribute to development of the triple primary malignant tumors in the patient. The patient underwent laparoscopic feeding jejunostomy and postoperative radiotherapy for synchronous esophageal and gastric carcinomas. Then, he underwent laparoscopic-assisted resection of right-sided colonic cancer and lysis of abdominal adhesions. By the time of submitting this manuscript, the patient had been well and no sign of recurrence or metastasis had been observed. To the best of our knowledge, this case is the first one to clarify the genetic abnormalities of triple primary cancers of esophagus, stomach and colon in a Chinese patient. It may contribute to understanding the molecular pathogenesis of multiple primary digestive malignancies and providing valuable treatment strategies for the similar patients in the future.The incidence of multiple primary malignant tumors (MPMTs) has increased greatly with the progress of tumor diagnosis and therapy technology. However, triple primary cancer is still very rare, and its genetic change is not clear yet. This case report described a 70-year-old Chinese male patient with triple primary cancers of the esophagus, stomach and right-sided colon. Pathological examination confirmed that each malignant tumor developed independently. Next-generation sequencing (NGS) using a 599-gene panel revealed five  With the development of tumor diagnosis and treatment technology, the incidence of multiple primary malignant tumors (MPMTs) has increased greatly . In the gastric carcinoma tissues, there were also two TP53 alterations (NM_000546: exon4: c.375G>A: p.T125T and NM_000546: exon7: c.715A>G: p.N239D). In the right-sided colon carcinoma tissues, there was one TP53 mutation (NM_000546: exon8: c.821T>G: p.V274G)  using a pan-cancer 599-gene panel  on July 20, 2020. NGS demonstrated the TMB value was 10.11 Muts/Mb, 11.37 Muts/Mb, 6.32 Muts/Mb in the tumor tissues of esophagus, stomach and colon, respectively, which ranked 29.58% (moderate), 6.45% (high) and 62.73% (low) in patients with corresponding malignancy. p.V274G) .ATRX (NM_000489: exon21: c.5424T>G: p.Y1808*), CHEK1 (NM_001114121: exon7: c.676dup: p.T226fs), copy number variations (CNVs) of CCND1 and FGF3/4/19. In the gastric carcinoma tissues, the same CHEK1 variation was also detected. In the colon carcinoma tissues, there were two APC variations    targeted against TP53 variations is ongoing.Study of Dranka-Bojarowska and Lewinski  revealedATRX variation (NM_000489: exon21: c.5424T>G: p.Y1808*) was also detected in the esophagus carcinoma tissues. According to the annotations of OncoKB, the ATRX mutation may be pathogenic. Previous studies have shown that loss of ATRX is associated with a variety of tumors such as sarcomas, hepatic angiosarcomas, gliomas and so on  in the tissues of esophagus carcinoma and gastric carcinoma  of the patients with esophageal carcinoma showed positive expression of CCND1 through immunohistochemistry .The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital Zhejiang University School of Medicine. Written informed consent for participation in the study, publication of clinical details and images was obtained from the patient.YW: conceptualization. XZ: writing-original draft. SC and EM: conducting bioinformatics analysis. LH and KS: writing-review and editing. All authors contributed to the article and approved the submitted version.SC and EM are employees at ChosenMed Technology. The remaining 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.


Coxiella burnetii. This disease typically manifests as a self-limiting, febrile illness known as acute Q fever. Due to the aerosol transmissibility, environmental persistence, and infectivity of C. burnetii, this pathogen is a notable bioterrorism threat. Despite extensive efforts to develop next-generation human Q fever vaccines, only one vaccine, Q-Vax®, is commercially available. Q-Vax® is a phase I whole-cell vaccine, and its licensed use is limited to Australia, presumably due to the potential for a post-vaccination hypersensitivity response. Pre-clinical Q fever vaccine development is a major area of interest, and diverse approaches have been undertaken to develop an improved Q fever vaccine. Following a brief history of Q fever vaccine development, current approaches will be discussed along with future considerations for an improved Q fever vaccine. Q fever is a zoonotic disease caused by the intracellular pathogen  Coxiella burnetii and is typically acquired by inhalation of infectious aerosols generated from livestock. Although C. burnetii can cause disease in various organisms, this review focuses on Q fever vaccine development in the context of human disease. C. burnetii is endemic nearly worldwide [C. burnetii is classified as a Select Agent by the United States Centers for Disease Control and Prevention’s Division of Select Agents and Toxins (DSAT) and is a noted bioterrorism pathogen of interest [C. burnetii in Australia and the United States in the late 1930s. Early Q fever vaccine development efforts were led by NIH scientists at Rocky Mountain Laboratories (RML) [C. burnetii with 10% egg yolk sac [C. burnetii strains. Initial injections were marked by severe adverse reactions, yet following a series of dilutions, the vaccine was generally well tolerated. This vaccine and related iterations likely reduced the risk of C. burnetii laboratory-acquired infections since their inception [C. burnetii whole-cell vaccine (WCV) efficacy in humans was directly tested, demonstrating protection against high-dose aerosol challenge in US Army volunteers [Q fever is a zoonotic disease that typically presents as an acute, febrile, self-limiting disease but can also lead to more chronic manifestations such as endocarditis or vasculitis . Q feverorldwide  and is cinterest . Accordies (RML) . After yes (RML) . The vacyolk sac . These vnception . Additiolunteers .Early egg derived WCVs displayed remarkable efficacy but were also associated with post-vaccination hypersensitivity (PVH) responses characterized by localized induration, sterile abscesses, and granulomas at the site of injection ,9 along C. burnetii LPS phase variation was first described in 1956 by Stoker and Fiset [C. burnetii virulence [O-antigen is a crucial component of C. burnetii virulence and vaccine-induced protection [Full-length or phase I lipopolysaccharide (LPS) was identified as an important protective factor in vaccine-induced immunity ,19,20. Tnd Fiset . This phirulence ,23 and Wirulence . Historiirulence . After tirulence . In respirulence . Althougirulence  and protirulence . Recent otection , yet the® has demonstrated nearly 100% efficacy in human trials [® requires pre-vaccination screening including serology, patient history, and a skin test. The potential for hypersensitivity reactions following vaccination has likely precluded licensing of Q-Vax® beyond Australia. The RML-derived Q58-A WCV was used as an investigational new drug (US BB-IND-26)  followinn trials  and was n trials . Importa® has demonstrated durable protective efficacy, but its widespread use has been hampered by the potential for severe adverse reactions and the cumbersome pre-screening process associated with vaccine distribution. As the only licensed vaccine available for human Q fever pre-exposure prophylaxis, Q-Vax® was deployed in the 2011 Q fever outbreak in the Netherlands [®. With a better understanding of C. burnetii–host interaction, including identification of immunogenic antigens and correlates of protection, Q fever vaccine development will be greatly enhanced.Overall, Q-Vaxherlands . This prherlands ,35. Modeherlands , the resherlands , and insC. burnetii vaccination, investigating and defining correlates of protection following both primary infection and secondary immunity are worthwhile pursuits. Indeed, these results may vary depending on factors such as the vaccine type, vaccine or challenge dose, and host organism, but a general understanding of WCV-induced immunity and corresponding protective factors are crucial for the design of an improved Q fever vaccine.For effective vaccine design and implementation, identification of protective correlates of immunity is integral. Typically, these correlates involve protective immunologic components of the vaccine response that are required, responsible for, or closely related to protection . HistoriC. burnetii, the adaptive immune response appears to be imperative for protective responses following both vaccination and natural infection. In murine models, an early role for T and B cells was established in primary clearance following infection, with T cells demonstrating importance in pathogen control and elimination [C. burnetii challenge further emphasizes the potential protective capability of B cells following C. burnetii vaccination [C. burnetii challenge, something that transfer of neither sera nor B cells could do. Contrastingly, both cellular and humoral components were able to reduce clinical disease, as reflected by a lack of significant weight loss in SCID mice following transfer and challenge. Ultimately, this study demonstrated the overall importance of cell-mediated immunity for host clearance of C. burnetii in a murine model contrasted by the dispensability of humoral immunity for this function. Similarly, Read et al. demonstrated that SCID mice reconstituted with CD4+ or CD8+ T cells were able to control pulmonary infection better than their non-reconstituted counterparts or those reconstituted with B cells [Generally, adaptive immune responses are associated with vaccine-mediated protection, with antibody titers and cellular readouts representing primary correlates of protection for many vaccines. For mination ,40. The cination ,42. Adopcination , further B cells . Indeed,C. burnetii infection clearance [+ T-cell-dependent and -independent mechanisms in vaccine-induced protective responses [C. burnetii infection model, with Tbet−/− mice displaying reduced ability to control bacterial spread, experiencing persistent infection and increased granuloma formation [+ T cells from secondary lymphoid organs in mice vaccinated with phase I WCV compared to that of phase II WCV vaccinated mice [C. burnetii infection. Tbet has demonstrated significance beyond the orchestration of CD4+ Th1 responses [C. burnetii phase I WCV vaccine–challenge model [Cd4−/− and Tbet−/− mice demonstrated variable protection following vaccination and challenge, with Tbet−/− mice appearing to experience more severe disease and less efficient bacterial clearance. These data suggest that Tbet and Th1-mediated responses are involved in both primary and WCV-induced C. burnetii clearance in murine models, although more investigation is warranted to elucidate specific underlying mechanisms. The apparent importance of T-cell-independent responses involving Tbet and MHC-II illustrate the complexity of protective responses. In a guinea pig model, draining lymph node cellularity and T-cell metrics were distinct two weeks following intraperitoneal infection in naive and phase I WCV vaccinated animals, indicating heterogeneity in primary and secondary immune responses [C. burnetii remain to be fully investigated, particularly in relation to T-cell subset behavior and T-cell-independent processes. Together these studies indicate that T-cell responses are critical for bacterial clearance following infectious challenge. Building on these findings, murine models revealed roles for MHC-I and MHC-II molecules in primary learance  and MHC-esponses . Furtherormation . These formation . Despiteted mice . Similarted mice . This stesponses , and Th1ge model . Accordiesponses . OverallC. burnetii infection and have been studied extensively in this context. Antigen-presenting cells, such as macrophages and dendritic cells, are known to contribute to vaccine-mediated immunity to various intracellular pathogens, primarily due to the fact of their role in bridging innate and adaptive immune responses and initiating cell-mediated immune responses. Indeed, cell-mediated immunity in response to C. burnetii phase I WCV has been associated with T-cell-dependent activation of macrophages in a guinea pig model [C. burnetii protein antigens  were able to confer slight protective effects  following vaccination [C. burnetii. In a separate study, BMDCs from C57Bl/6 mice were simulated with phase I or II WCV ex vivo and assayed for maturation markers and migratory ability, with phase I WCV stimulated cells displaying increased maturation and migratory ability [Ccr7−/− mice are able to control C. burnetii infection following phase I WCV vaccination, although this protection may be mediated by anti-phase I LPS IgG production in Ccr7−/− mice [C. burnetii, and antigen-presenting cells may play an important part in the orchestration of these responses. Macrophages are a major target cell for ig model ,52. Muricination . Althoug ability ; these f−/− mice . In muriC. burnetii-specific antibody titers and T-cell specific responses, but is this also evidenced in humans? Indeed, B and T cells appear to be involved in various stages of natural and vaccine-induced immune responses in humans, with long-lasting C. burnetii-specific T cell reactivity [C. burnetii or phase I WCV. Acute Q fever patient sera has revealed an early C. burnetii phase II-specific humoral response followed by mounting Phase I-specific IgA, IgG, and IgM antibody responses [C. burnetii Phase I-specific responses. In line with infection responses, human seronegative volunteers receiving WCV have exhibited phase I-specific IgM responses with a minor phase II-specific IgM and IgG components, while seropositive volunteers receiving WCV displayed Phase I-specific IgA, IgG, and IgM responses along with phase II-specific IgG and complement fixing responses, mirroring the humoral profile of those experiencing a positive skin test [C. burnetii growth in vivo.In mice, WCV-induced protective responses are associated with increased activity ,55 and aactivity ,57 persiesponses . Patientkin test ,59. SeruC. burnetii-specific humoral responses. Following the 2007–2011 Q fever outbreak in the Netherlands, C. burnetii-specific IgG half-life averaged 318 days, exemplifying the potential for long-lasting humoral responses in humans following natural infection [C. burnetii exposure may impact these data but does not appear to account for most persistent antibody responses. Although humoral responsiveness has been routinely described in human subjects and patients, definite correlation of antibody titer/function and protection has not been established. In fact, antibody seroconversion rates generally appear to be lower and less durable than cell-mediated markers following phase I WCV vaccination [In a south Australian clinical trial with more than 1600 abattoir workers, vaccination with phase I WCV resulted in seroconversion of 30–60% of vaccines [nfection . In anotnfection , approxicination . Given t®). In this trial, vaccine-induced protection was measured by the absence of Q fever cases in vaccinated subjects . Short-term cellular and humoral responses were quantified and appeared to correlate with vaccine-induced protection [C. burnetii-specific antibodies were detected in 80–82% of vaccinees shortly following vaccination  characterized by phase I and II IgM, followed by largely phase I IgG responses 20–60 months after vaccination, with an overall seropositivity rate of 64% among vaccinees at these later time points. Despite waning antibody titers, nearly two years following WCV vaccination, cellular immune responses were maintained in approximately 95% of vaccinated individuals five years following vaccination [C. burnetii, which reside in divergent genomic groups, displaying the potential for heterologous protection by WCV, which has since been demonstrated in animal models [C. burnetii exposure [Human-derived humoral data suggest a role for immunologic components beyond antibodies in long-lived protective responses following both infection and WCV vaccination. Substantial information can be gleaned from reports of the 1981–1988 clinical trial of a phase I WCV  epitopes were identified as potential MHC-I antigens [+ IFN-γ recall responses and induce some level of protection when added to a live Listeria monocytogenes vaccine vector in a murine vaccine challenge model. Recently, computational epitope identification was employed in mice expressing human MHC alleles [+ T Cells (both CD4+ and CD8+CD73+ subpopulations) by day 10 post-vaccination , increased CD44 expression on CD4+ T cells by day 35 post-vaccination (possibly representing TCM cells), reduced circulating innate myeloid cells, and a potential circulating immune signature  associated with bacterial clearance in WCV vaccinated animals. Ly6C expression and its association with T cell activation, CD8+ memory cells, and a CD4+ Th1 phenotype is certainly reasonable in the context of existing knowledge of murine C. burnetii infection and vaccine responses. A Ly6C homologue has not been identified in humans [Accordingly, more data are available regarding T-cell antigenic identification in the context of analyses . These eantigens . Several alleles . Followin humans , but theC. burnetii proteins were able to induce durable T cell IFN-γ release responses in convalescent human patients from the 2007–2010 Netherlands Q fever outbreak, nearly four years following the resolution of the outbreak and presumed C. burnetii exposure [T-cell-specific antigen identification has been carried out in humans as well, with recent studies revealing epitopes potentially related to adaptive immune responses to natural infection. Bioinformatically predicted T cell epitopes (HLA class II) from exposure . The samexposure . In bothC. burnetii have been revealed through bacterial fractionation and extraction methods. Kazar et al. tested the protective efficacy of phase I WCV and phase I trichloroacetic acid (TCA) extract in a murine model of homologous and heterologous intraperitoneal challenge [C. burnetii proteins as antigenic stimuli, these proteins alone do not appear to provide sufficient immunostimulation to confer protection comparable to phase WCV [Additional insight into the protective components of hallenge . Protecthase WCV ,82,83,84C. burnetii undergoes LPS phase variation in vitro which results in truncation of phase I LPS to a phase II counterpart [C. burnetii components such as proteins [Phase I LPS appears to be an integral protective component of WCV-induced protective responses. nterpart . Recentlnterpart . Accordiproteins . The reqproteins . Zhang eproteins . By compC. burnetii prior to infection. This suggested a direct role for phase I LPS epitopes in protective responses. Additionally, a LPS mimic peptide-keyhole limpet hemocyanin (KLH) conjugate vaccine candidate demonstrated immunogenicity and protective potential against C. burnetii challenge, although not to the same degree as that of phase I WCV. These data paired with human phase I-specific adaptive responses following vaccination and challenge suggest that phase I LPS (O-antigen) epitopes are major players in protective C. burnetii responses. Based on the observation that phase I LPS plays a major role in WCV-induced protective responses, Peng et al. developed an LPS peptide mimic vaccine based on protective monoclonal antibody (mAb) reactivity . This stFurther investment is warranted in antigen discovery for Q fever vaccine development to aid in the rational design of modern vaccines. Additionally, a better understanding of the role of phase I LPS and relevant protective epitopes in vaccine-induced protection is needed. Although significant advancements have been made in both areas, much remains unknown, and this information is likely integral for an improved Q fever vaccine that retains potent efficacy and durability.® and other phase I WCVs as theoretical stepping stones will likely allow for the rational establishment of these features. Phase I WCVs display many advantages including potent immunogenicity, stimulation of durable immunity, and a single-dose immunization regimen. The downsides of phase I WCVs include the potential for a PVH response and the accompanying cumbersome pre-vaccination screening process along with production difficulties  vaccines for Q fever [C. burnetii-specific IgM responses, 20–40% mounting C. burnetii phase II-specific IgG responses, and 30–40% displaying PBMC T cell proliferation responses to C. burnetii [C. burnetii challenge [Modification of  Q fever . Broadly Q fever ; multipl Q fever ,90,91,92burnetii . Next, tburnetii , and thehallenge . This sthallenge . AdditioC. burnetii. Accordingly, a recent study demonstrated protective efficacy of genetically modified phase I WCV in a guinea pig vaccine–intraperitoneal challenge model [C. burnetii dot/icm locus, which encodes the bacterium’s Type IV Secretion System (T4SS), the resultant strain retained phase I LPS expression but was avirulent in a high-dose (106) guinea pig intraperitoneal challenge model. As this avirulence is due to a non-revertible, large genetic deletion, these data demonstrate potential for DSAT select agent exclusion, a clear advantage for vaccine production efforts. This genetically modified WCV also demonstrated protection comparable to phase I WCV but did not completely mitigate reactogenicity, suggesting that the T4SS is dispensable for vaccine-induced protection but not solely responsible for reactogenicity. While efforts to modify WCVs capitalize on the potent protective efficacy of C. burnetii and its seemingly multifactorial antigen repertoire, issues with immunogenicity and reactogenicity remain. Notably, the development of an axenic media for C. burnetiid [The development of modified WCVs has evolved along with the development of genetic tools for ge model . By geneurnetiid  has imprC. burnetii WCVs. As previously noted, subunit vaccines have been developed for C. burnetii but have not demonstrated comparable protection as that of phase I WCV. Early subunit vaccine efforts were conducted by Williams et al. and involved the creation of multivalent subunit vaccines [C. burnetii burden compared to mock-vaccinated mice, although phase I WCV was not included in these experiments as a positive control. Subsequent efforts to identify and evaluate proteins suitable for subunit vaccine use yielded an outer membrane protein composed of 17 amino acids and devoid of LPS, which induced immunogenicity  and protective efficacy in rodent models [C. burnetii components, such as LPS, may play a role in the protective efficacy demonstrated by some isolated proteins. Beyond isolation of bacterial proteins for subunit vaccines, recombinant-based approaches have been employed. Zhang et al. demonstrated a lack of protective efficacy of individual recombinant proteins Mip (CBU0630), P1 (CBU0311), and P28 (CBU0952) in a BALB/c murine vaccine–challenge model [C. burnetii loads appeared to be higher in P1-HspB-vaccinated animals following challenge. Despite the addition of adjuvant and booster vaccinations, this multivalent recombinant subunit vaccine was unable to completely match the protection afforded by a single administration of phase I WCV, demonstrating the powerful protective effect of this formulation. Subunit vaccines are typically composed of antigenic components   with supvaccines . This stt models . The potge model . Improvege model ; both vaC. burnetii antigens into this adjuvant system, immunogenicity and protective efficacy of resultant vaccines were evaluated in murine and guinea pig models. Various vaccine formulations demonstrated promising protective efficacy in a guinea pig vaccine–intratracheal challenge model following a booster vaccination and 49 day prime-challenge window. Body weight change, lung histopathology scores, and lung weight were comparable between phase I WCV and subunit vaccinated animals following challenge, and splenomegaly appeared to be consistently lower in phase I WCV vaccinated animals. The use of various adjuvant formulations yielded insight into innate immune mechanisms associated with vaccine-induced protective immunity. Specifically, TLR4, 7, and 9 appeared to be imperative for optimal subunit vaccine-induced protective responses, presumably (at least in part) due to the fact of their role in the induction of Th1 effector responses. Although these subunit vaccines require a multi-dose regimen, this demonstration of protective efficacy is promising. Post-vaccination hypersensitivity modeling revealed a range of dermal histopathologic reactions among the subunit vaccine candidates, some comparable to phase I WCV. Notably, C. burnetii antigens in the absence of adjuvant did not induce reactogenicity, indicating that C. burnetii antigens combined with the innate stimuli of adjuvants may be required for induction of the hypersensitivity response observed following phase I WCV vaccination. Subunit vaccine development for C. burnetii has been hindered due to the lack of understanding of immunogenic antigens and epitopes and the apparent requirement for additional stimuli beyond single proteinaceous antigens for strong protective responses. Novel antigen and adjuvant formulations will likely be introduced for C. burnetii subunit vaccines in the future, and these candidates will need to address the issues of sufficient immunogenicity, the implications of multi-dose immunization regimens, and reactogenicity.The apparent requirement of additional stimuli beyond proteinaceous subunit components and single adjuvants has been appreciated, as evidenced by several recent vaccine design strategies. In accordance with the importance of phase I LPS in vaccine-induced protective responses, an LPS peptide mimic-KLH conjugate vaccine was developed by Peng et al. based on mAb screening, demonstrating some protective efficacy, albeit not to the level of phase I WCV . HarnessC. burnetii, Coxiellosis, are a subject of current study and past developments. Coxiellosis is marked by abortion and infertility and can be rapidly spread among livestock, causing significant economic losses, livestock morbidity, and initiating human Q fever outbreaks. Accordingly, livestock vaccination has been implemented as a preventative and control measure. Inactivated WCVs have been primarily utilized for this purpose. Adverse effects, such as injection site reactions and increased body temperature, can occur in livestock following WCV vaccination [C. burnetii control in livestock, as they have demonstrated reduced reactogenicity in both livestock and humans [C. burnetii vaccination of livestock has yielded variable results in terms of protective efficacy and population control [C. burnetii vaccines, Coxevac®  and Chlamyvax FQ® , are available for veterinary use in many parts of the world. These vaccines are inactivated WCVs, with Chlamyvax FQ® also containing inactivated Chlamydophilia abortus, a bacterial pathogen also known to cause adverse fertility effects. Coxevac® is a phase I vaccine, while Chlamyvax FQ® is a phase II vaccine. The LPS composition of these WCVs appears to affect protective efficacy as demonstrated by a goat vaccination study showing reduced bacterial shedding and abortions in phase I vaccinated animals compared to that of the phase II vaccinated animals which experienced no clinical benefit [C. burnetii vaccination has contributed to limited vaccine usage in this context. Although this article focuses on Q fever vaccines for human use, the use of Coxiellosis vaccines for pathogen control at the reservoir level appears to be a promising strategy to prevent future naturally occurring outbreaks of C. burnetii associated with livestock that may be transmitted to humans [Vaccines for the livestock disease caused by cination , althougd humans . C. burn control ,99. Gene benefit . The queo humans ,102. AccC. burnetii is a unique pathogen due to the fact of its intracellular lifestyle, natural history, and ability to induce complex immune responses that are not yet fully understood. Currently, most studies have utilized phase I WCV to probe C. burnetii vaccine immune responses and identify protective bacterial components in both animal models and humans. Although phase I WCV was first introduced for Q fever prophylaxis shortly after its discovery in the 1930s, the only licensed Q fever vaccine of modern times, Q-Vax®, is a modern iteration of the original version. Years of progress have unfolded in the study of C. burnetii and Q fever, resulting in enhanced understanding of this pathogen and its interactions with host organisms. Accordingly, novel vaccine development strategies, such as modified WCVs, subunit vaccines, and LPS mimic vaccines, have been employed and evaluated in both animal models and humans. Additionally, basic scientific and clinical studies have contributed to our understanding of C. burnetii virulence determinants, protective responses, protective antigens, and more. C. burnetii and protective correlates of Q fever vaccination. Potent immunity induced by phase I WCVs appears to require a complex milieu of bacterial components, likely including phase I LPS, proteins, and bacterial structures serving as adjuvants. Additionally, the stimulatory effects of phase I WCV may be related to the potential slow elimination of WCV and persistent local immune stimulation, although this remains to be investigated. Vaccine design strategies ignoring this complex interplay are likely to encounter challenges. As such, an understanding of the bacterial determinants of immunity and resultant host immune responses will be extremely valuable for continued vaccine development strategies. Despite this progress, a substantial barrier in the development of improved Q fever vaccines appears to be a lack of understanding of the antigenic determinants of Additional considerations for modern Q fever vaccine development include the (1) importance of heterologous protection, particularly against epidemic strains; (2) route of vaccination; (3) continued development and refinement of animal models.C. burnetii strain composition, with eight genomic groups established by diverse genomic analysis methods [C. burnetii strains [C. burnetii, offering good news for future Q fever vaccine development efforts. Regardless, with novel C. burnetii strains emerging worldwide, the potential for strain-specific antigenic escape is a relevant consideration, particularly considering more reductionist vaccine approaches such as subunit formulations; (1) Genomic typing efforts have revealed diverse  methods ,104,105. methods ,63 and h strains . Histori strains , and mon strains . ObservaC. burnetii phase I WCV to humans and animals. While this has not appeared to alter protective efficacy, as this vaccine appears to be capable of inducing potent immune responses, consideration of alternative routes of vaccination is intriguing for several reasons. First, the route of immunization may be important for non-WCV vaccines that are not as immunologically potent. Stimulation of mucosal immunity, as demonstrated by intratracheal administration of CMR in mice [C. burnetii, aiding in modern vaccine design efforts. Secondly, the route of exposure may have a mitigating effect on post-vaccination hypersensitivity. Lastly, with general advancements in non-parenteral vaccine delivery [(2) Historically, the subcutaneous route has been employed to administer  in mice , may enhdelivery  and renedelivery , similarC. burnetii–host interactions. Recent advancements, such as the validation of intratracheal C. burnetii infection models from rodents to non-human primates [®  for Anthrax post-exposure prophylaxis [(3) Animal modeling remains an integral tool in our understanding of primates , are impphylaxis . FurtherC. burnetii (C. burnetii and the host immune system. Indeed, many novel vaccine technologies spark interest but require knowledge of antigenic targets and immunologic responses that the C. burnetii field has not yet ascertained. As we continue to unravel the mechanisms of C. burnetii–host interactions we will move closer to a next-generation Q fever vaccine that will benefit people around the world.The path to a non-reactive Q fever vaccine that is simple to produce and administer and that induces potent and durable protection against burnetii c will be


Synanceia) species, presented a table of clinical case reports and provided up-to-date information about the development of polyspecific stonefish antivenom. We have also presented an overview of recent advancements in the biomolecular composition of stonefish venom, including the analysis of transcriptomic and proteomic data from Synanceia horrida venom gland. Moreover, this review highlights the need for further research on the composition and properties of stonefish venom, which may reveal novel molecules for drug discovery, development or other novel physiological uses.Stonefish are regarded as one of the most venomous fish in the world. Research on stonefish venom has chiefly focused on the in vitro and in vivo neurological, cardiovascular, cytotoxic and nociceptive effects of the venom. The last literature review on stonefish venom was published over a decade ago, and much has changed in the field since. In this review, we have generated a global map of the current distribution of all stonefish ( Synanceia: Synanceia horrida (previously referred to as S. trachynis), S. verrucosa, S. alula, S. nana and S. platyrhyncha. Synanceia spp. can grow 35–50 cm in length and have evolved grey and mottled skin to camouflage themselves amongst encrusted rocks and coral for predation and defense . Ea. EaS. vesa venom . Like SNsa venom   and scorpions (53%) [S. horrida appears to be unique when compared to other venomous species, containing several proteins that have not been previously recognized in proteomic studies of other venoms [roteases ,69. Analroteases . The perns (53%) . Additions (53%) . The lowns (53%) . Overallr venoms .S. horrida venom also contains small molecules such as norepinephrine, dopamine and tryptophan, which are known regulators of cardiac physiology [Synanceia venom thus far, even though these symptoms are consistent with stonefish envenomation [Crude ysiology  and may ysiology . In addiysiology . Investiysiology . Interesnomation ,79.S. verrucosa; however, a 59 kDa hyaluronidase  acts in synergy with nitric oxide (NO) [+ channels, ultimately leading to the SNTX-induced muscle relaxation observed [In vitro studies using purified SNTX on rat aortic rings have demonstrated potent vasorelaxant activity . This vaide (NO)  to bind observed . Furtherobserved ,90. ThisSynanceia envenomation may also contribute to the lethal activity of stonefish venom [S. horrida envenomation could ensue from direct skeletal muscle paralysis, as neuromuscular conduction through the phrenic nerve–diaphragm junction is not inhibited even when respiration has stopped in anesthetized rabbits [S. verrucosa venom, irregular or weakened respiration is observed, often followed by cessation of respiration when lethal doses of crude venom or VTX are administered in anesthetized rats [S. verrucosa crude venom [S. verrucosa. One of the victims required CPR and continued to show dyspnea and bilateral rattles, where acute pulmonary edema was hypothesized, although not confirmed [The respiratory failure observed in experimental sh venom . Respira rabbits . In the zed rats . Furtherde venom . Apart fonfirmed . In two onfirmed .S. verrucosa venom appears to act on several GPCRs. For example, when examining venom effects in guinea pig ventricular myocytes, VTX appeared to modulate ion channels by stimulating the ß-adrenoceptor–cyclic adenosine monophosphate–protein kinase A (cAMP-PKA) pathway [2+ channels, which promotes an inotropic effect in cardiomyocytes [G-protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins . This fa pathway . When stmyocytes .S. horrida venom also appears to act on bradykinin receptors. A ß2-receptor antagonist, acting as a noncompetitive inhibitor of bradykinin, was shown to inhibit the relaxation response induced in pig coronary arteries by reconstituted crude S. horrida venom or bradykinin [S. horrida venom also seems to act on muscarinic receptors by stimulating endogenous acetylcholine production or mimicking its effect on the cardiac membrane [Synanceia venom has some action at muscarinic receptors [Synanceia envenomation. VTX can also stimulate muscarinic receptors and activate Ca2+ currents, leading to arrhythmia and hypoxia, preventing the activation of cardioprotective KATP currents and promoting cardiovascular collapse [ATP current through activation of adenosine receptors or α1-adrenoceptors. Instead, it was strongly suggested that VTX activates the M3 receptor–protein kinase C (PKC) pathway [Reconstituted crude adykinin ,94. Bradadykinin , such asadykinin . Therefoadykinin . In addimembrane ,69,97. Weceptors . Researccollapse ,92. VTX  pathway .S. horrida stimulates the release of endogenous tachykinins. S. horrida venom-induced bronchoconstriction responses were reduced by an NK1 receptor antagonist in anesthetized guinea pigs in vitro [1 receptors possibly through the release of substance P (SP) [There is evidence that venom from in vitro , indicate P (SP) . SP is ae P (SP) . The vene P (SP) .S. horrida and S. verrucosa venoms produce potent inflammatory responses in in vivo studies and in patients, where stonefish victims show signs of tendon inflammation, pulmonary edema and other cardiac complications [S. horrida venom causes edema when administered intradermally into rat hind paws and mouse footpads [Synanceia envenomation does not appear to be triggered by histamine release because diphenhydramine, an antihistamine, does not seem to have an effect on the edema-inducing properties of SNTX [Crude ications ,27,99. Rfootpads ,69,100.  of SNTX . Instead of SNTX .S. verrucosa and S. horrida, as well as VTX and SNTX, exhibit strong lytic actions against diluted blood and washed erythrocytes of a variety of mammal species [S. horrida does not cause hemorrhage or have any measurable dermonecrotic effects in mice but seems to possess some anticoagulant activity in rabbit blood [S. horrida venom appears to prevent clotting of human fibrinogen when Ca2+ is present, and this anticoagulant activity seems to be concentration-dependent [S. horrida venom appears to cause cell lysis in cultured murine cortical neurons [Crude venoms from  species ,56,58,69ependent . Notably neurons . This laS. verrucosa venom on recalcified human plasma [Apart from these, thromboelastography assays were performed using fresh and lyophilized n plasma . Althougn plasma . Fibrinon plasma . Furthern plasma  suggesten plasma .Synanceia venom in some animals has been linked to the formation of pores in cell membranes [S. verrucosa venom resulted in elevated levels of the oxidative stress marker malonaldehyde caused by lipid peroxidation, which indicates that venom-induced damage to the cell membrane may be caused by the generation and action of free radicals [S. horrida venom, in particular, is one of the largest naturally occurring toxins isolated to date that possess pore-forming activity [N-terminal domain is homologous to the membrane attack complex–perforin/cholesterol-dependent cytolysin (MACPF/CDC) pore-forming domains [S. horrida that is homologous to perforin-1-like proteins found in several teleost fish species [The observed hemolytic activity produced by embranes ,103. Expradicals . Moreoveactivity . SNTX suactivity . Each SN domains . Protein domains . Interes domains . Additio species . This pr species , which c species , and may species .Synanceia venoms and the functional structure of their isolated fractions, inactivation studies have been performed with SNTX and neoVTX. The hemolytic activity from both neoVTX and SNTX is inhibited by modified anionic lipids [To understand the mechanism behind the toxicity of c lipids ,102. Furc lipids ,109,110.c lipids ,109. Intc lipids .S. horrida venom have demonstrated that the venom may also block neurotransmitter synthesis, ultimately leading to a depletion in neurotransmitter stores [S. horrida venom can cause irreversible neuromuscular blockages, which may lead to fatal respiratory paralysis [Synanceia venom is myotoxic and likely to induce the permanent damage observed by electron microscopy at the neuromuscular apparatus in skeletal muscle [Experiments to determine the neuromuscular effects of crude r stores . When aparalysis . Interesaralysis . This inaralysis . SNTX haaralysis . This isl muscle ,114.S. verrucosa investigated the activity of the venom on mimotopes of nicotinic acetylcholine receptors (nAChRs) from four different taxa [S. verrucosa venom might act on postsynaptic nAChRs, agreeing with previous research on chick biventer cervicis nerve preparations using S. horrida venom [Recent research on crude fresh and lyophilized venom from ent taxa . The freent taxa . This bida venom . Interesda venom .V1.2 channel mimotopes of domains I–IV (DI–DIV) using biolayer interferometry (BLI) showed that both lyophilized and fresh venoms bound only to DIV, indicating that the toxins responsible for this particular activity might be small peptides or an amine-type molecule, which are more stable than proteinaceous or enzymatic molecules [2+ channels by Synanceia venom [2+ channel activity of the venom acts as an antagonist or agonist [Further testing on mammalian L-type Caolecules . These ria venom ,65. Unfo agonist , and so,2+-dependent exocytosis of large dense-core vesicles in the presence of extracellular Ca2+ in bovine chromaffin cells [N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent exocytosis was shown to be independent of Ca2+ influx through voltage-activated Ca2+ channels, as blockade of the L-, N- and P/Q-type channels resulted in a minimal change in catecholamine secretion [2+, as depletion of intracellular Ca2+ with caffeine, thapsigargin or ryanodine resulted in a reduced SNTX-induced catecholamine response [In addition, SNTX promotes the secretion of catecholamine from neuroendocrine cells through a Cain cells . This soecretion . Intraceresponse .Synanceia venom have a time- and dose-dependent relationship. S. horrida venom, for example, causes permanent depolarization of muscle fibers at high concentrations, whereas at low concentrations it causes massive release and depletion of neurotransmitters in frog nerve–muscle preparations [Synanceia venom also blocks the recycling of synaptic vesicles [Synanceia venom to other animal toxins with similar neuromuscular blocking activity via the release and depletion of neurotransmitters from the nerve terminal, such as α-latrotoxin from the spider genus Latrodectus [Synanceia venom is differentiated by also causing muscle depolarization and microscopic muscle damage. An exception to this is the pardaxins, toxins found in the dorsal and anal fin gland secretions of the Pacific sole (Pardachirus pavoninus) and Red Sea Moses sole (P. marmoratus). Synanceia venom mimics the action of the pardaxins, acting as a presynaptic neurotoxin at low concentrations and severely damaging skeletal muscle fibers at high concentrations [Synanceia venom and the pardaxins lyse dog erythrocytes, only the pardaxins lyse human erythrocytes [Many of the effects of arations . Additiovesicles . When corodectus , Synancetrations ,118. Inthrocytes ,119.V) [V1.8 [V1.9 [Stonefish venom-elicited release of neurotransmitters has been reported not to involve voltage-gated sodium channels (NaV) . This waV) [V1.8  or NaV1..8 [V1.9  sodium c.8 [V1.9  and is cSynanceia are widely distributed, and while stonefish cause serious envenomations worldwide, the extent of involvement of other venomous fish species in serious envenomations around the globe is not fully understood.Stings caused by venomous fish species occur worldwide. While the causative agent of many of these stings is considered to be “stonefish”, it should be noted that the identity of the offending animal cannot be confirmed in most cases. As a result, reports of stonefish stings should be viewed with caution when the animal has not been captured. Notably, individuals belonging to the genus Synanceia venom at the cellular, tissue and organ levels have significantly advanced since the 1990s. Of note, all published work on Synanceia venom has been performed only on two species thus far: S. horrida and S. verrucosa. Characterizing the venom of additional species for future research is likely to increase understanding of the pathophysiological mechanisms and effects of stonefish venom.Studies investigating the pathophysiological mechanisms and effects of Synanceia venom, many of which appear to be novel. Indeed, only one of these enzymes, hyaluronidase, has been characterized on a molecular level, and yet its role in the envenomation effects remains poorly understood. Although the rich bioprospecting potential of Synanceia venoms has yet to be realized, the results of recent transcriptomic- and proteomic-based approaches to venom characterization highlight the need for more research into these medically important fish species. The study of Synanceia venom variability, composition and immunological cross-reactivity may also have a direct impact on human health by contributing to the development of improved antivenoms, thereby reducing the burden of venomous fish species on human health worldwide.Of note, while the cytolytic activity of stonefish venom appears to be the result of pore-forming action on cell membranes, the precise mode of action of the venom and its individual components still remains largely unknown. It has become clearer that the biological symptoms and pathologies observed in sting victims cannot solely be attributed to the presence of a single venom protein, such as SNTX or VTX, as initially thought. Instead, these effects are likely a result of the combined activity of the many enzymes and proteins that have now been discovered in


Mus musculus musculus-derived BLG2/Ms, NJL/Ms, CHD/Ms, SWN/Ms, and KJR/Ms; M. m. domesticus-derived PGN2/Ms and BFM/Ms; M. m. castaneus-derived HMI/Ms; and JF1/Ms and MSM/Ms, which were derived from a hybrid between M. m. musculus and M. m. castaneus. These strains were established by Prof. Moriwaki in the 1980s and are collectively named the “Mishima Battery”. These strains show large phenotypic variations in body size and in many physiological traits. We resequenced the genomes of the Mishima Battery strains and performed a comparative genomic analysis with dbSNP data. More than 81 million nucleotide coordinates were identified as variant sites due to the large genetic distances among the mouse subspecies; 8,062,070 new SNP sites were detected in this study, and these may underlie the large phenotypic diversity observed in the Mishima Battery. The new information was collected in a reconstructed genome database, termed MoG+ that includes new application software and viewers. MoG+ intuitively visualizes nucleotide variants in genes and intergenic regions, and amino acid substitutions across the three mouse subspecies. We report statistical data from the resequencing and comparative genomic analyses and newly collected phenotype data of the Mishima Battery, and provide a brief description of the functions of MoG+, which provides a searchable and unique data resource of the numerous genomic variations across the three mouse subspecies. The data in MoG+ will be invaluable for research into phenotype-genotype links in diverse mouse strains.Laboratory mouse strains have mosaic genomes derived from at least three major subspecies that are distributed in Eurasia. Here, we describe genomic variations in ten inbred strains: The online version contains supplementary material available at 10.1007/s00335-021-09933-w. Mus musculus domesticus, M. m. musculus, and M. m. castaneus  and JF1/Ms (JF1) that are members of the Mishima Battery. A comparative genomic analysis of the strains and a reference strain C57BL/6 J (B6) suggested that the ancestor of the JF1 strain provided the M. m. molossinus genome in classical laboratory strains and largely contributed to its intersubspecific genome diversity , NJL/Ms (NJL), CHD/Ms (CHD), SWN/Ms (SWN) and KJR/Ms (KJR); M. m. domesticus-derived PGN2/Ms (PGN2) and BFM/Ms (BFM); M. m. castaneus-derived HMI/Ms (HMI); and JF1 and MSM) and conducted a comprehensive comparative genomic analysis across these mouse strains. A simple presentation of the short-reads from the sequenced Mishima Battery strains with mapping to the reference sequence was first made available in 2018, and the data was released as an updated database (NIG_MoG2).We have now extended genome resequencing to all strains of the Mishima Battery (https://molossinus.brc.riken.jp/mogplus/). In this study, we performed a new comparative genomics analysis with dbSNP data . Our analyses identified 8,062,070 new SNP sites in the 81,866,820 nucleotide coordinates at variant sites following the addition of the Mishima Battery data to the dbSNP data. The MoG+ database allows visualization of the genome-wide pattern of intersubspecific nucleotide variants across both the Mishima Battery and some commonly used laboratory mouse strains. The web browser can be used to retrieve information on nucleotide variants from genomic regions of interest and in genomic segments from specific subspecies in the classical laboratory strains. This feature enables exploration of genes involved in disease susceptibility and phenotypic variation in genetic studies using intersubspecific crosses. MoG+ stores a wide range of data on genomic variations that cannot be captured by comparison among the standard laboratory strains. Since different human ethnic populations contain considerable genomic diversity, which has an influence on individual phenotypes, the mouse intersubspecific nucleotide variants will be a useful source for identifying novel genomic variations responsible for differences in phenotypes and disease susceptibilities. We have therefore added links from MoG+ to other public databases related to human diseases and phenotypes. In addition, we have added a catalog of experimental mouse strains that are available from RIKEN BRC.In 2019, operation of this database was transferred to the RIKEN BioResource Research Center (BRC) . Mice  were used for the data collection, which is summarized in Fig. To expand the value of MoG+ for biomedical research, we systematically phenotyped the Mishima Battery strains as well as B6 . The phenotypic measurements mainly focused on body size and physiological traits. The raw measurement data is downloadable from the RIKEN BRC website . As described above, genomic information on the Mishima Battery strains is extremely useful because they have a wide variety of phenotypes, some of which might be related to human disease-related phenotypes such as fat accumulation and blood chemistry values. In addition, the phenotype data from the Mishima Battery strains may also provide insights into genomic variations that might underlie phenotypic variations in mouse inbred strains with a M. m. molossinus-derived mosaic genome.The Mishima Battery strains show a wide range of phenotypes with regard to morphological and physiological traits. In cross comparisons of the Mishima Battery strains with B6, the rate of each pair that indicates statistically significant values was 61.9% with https://molossinus.brc.riken.jp/pub/Wild10_2020/) to summarize the relationships between the strains in the Mishima Battery . Low-quality regions around indels were excluded in this analysis.The resequencing of the genome of the Mishima Battery MSM strain updated the data on nucleotide variants by the addition of 78.9-fold short-reads to the previously reported data set based on 2.4-fold capillary sequencing and 16.5-fold short-reads . Low-quality regions around indels were included in this analysis in order to store the maximum amount of genome variant information in MoG+. The variant classification is depicted in Fig. ftp://ftp-mouse.sanger.ac.uk/). The compilation of the MoG+ data and the Mouse dbSNP 142 showed that a total of 81,866,820 sites, corresponding to about 3% of the mouse genome (mm10), was polymorphic (Table S6). The inclusion of the MoG+ data in the comparison increased the number of SNPs by 9.8%. The subspecies-specific SNPs are also listed in Table S6. We observed large differences in the number of variants across strains even within the same subspecies. For example, the comparison of the castaneus-derived strains HMI and CAST/EiJ identified 14,427,289 SNPs, a frequency equivalent to that found in comparisons between subspecies (Table S3).Nucleotide variant datasets include SNPs and short indels for each strain of the Mishima Battery, named pgn2v1 (PGN2), bfmv1 (BFM), blg2v1 (BLG2), njlv1 (NJL), chdv1 (CHD), kjrv1 (KJR), swnv1 (SWN), hmiv1 (HMI), msmv4 (MSM), and jf1v3 (JF1), respectively, were annotated using SnpEff , an online catalog of human genes and genetic disorders; and ‘TogoVar’ (https://togovar.biosciencedbc.jp), a publicly accessible and comprehensive database of genetic variants in the Japanese population. These various links enable users to connect data on mouse genomic variations with those of human diseases and phenotypes. On the top page of MoG+ , a search by gene symbol retrieves information on human diseases, which are caused by a defect in the gene of interest . The function to search for gene annotations which includes links to the human diseases information has also been improved . Data retrieval using VariantTable , which integrates multiple datasets from human variant databases such as GEM Japan Whole-Genome Aggregation (GEM-J WGA) Panel (https://www.amed.go.jp/en/aboutus/collaboration/ga4gh_gem_japan.html), JGA-NGS, JGA-SNP (https://humandbs.biosciencedbc.jp/en/), Exome Aggregation Consortium (ExAC: https://gnomad.broadinstitute.org), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), Human Genetic Variation Database (HGVD: https://www.hgvd.genome.med.kyoto-u.ac.jp/download.html), and ToMMo 4.7KJPN Allele Frequency Panel (4.7KJPN: https://jmorp.megabank.tohoku.ac.jp/202001/downloads/#variant). Since basic gene functions are evolutionarily conserved and defects in orthologues may cause similar phenotypes in different organisms, MoG+ will be modified to integrate genetic variation data from other mammals and other vertebrates. The expansion of the database to include genome data in different taxa will be of value for exploring gene function using information of synteny in different organisms. Integration of data on genomic variation in different species will necessitate the development and use of common vocabularies such as Sequence Ontology (http://www.sequenceontology.org) and Variation Ontology (http://variationontology.org/instructions.shtml). Currently we are working on the collaboration to develop a common data format for genetic variation databases including MoG+ and TogoVar based on the resource description framework (RDF), a framework to provide a formal description of knowledge and common application programming interface (API) . At present, “Mishima Battery” mouse strains are distributed to the research community via RIKEN BRC (https://web.brc.riken.jp/en/) and NIG (https://shigen.nig.ac.jp/mouse/nig/). C57BL6/JJcl was purchased from CLEA Japan  and bred at the NIG. For measurement of all traits, mice 9–11 weeks of age (67–79 days after birth) were fasted from 6 to 10 am (± 30 min) prior to blood sampling. In total, 613 animals  were used for body size and physiological measurements (Table S1). Environment and housing, and body size and physiological measurements were performed as described in our previous report  and has been described by Koide et al.  and Furuhttp://www.repeatmasker.org), was obtained from the Project website (http://hgdownload.cse.ucsc.edu/goldenpath/mm10/chromosomes/) and used as the mapping reference. Filtered reads were aligned to the mouse reference genome by our in-house program, which was set at > 95% sequence identity (indels were treated as mismatches) with the reference sequence. Multi-mapping, where it is difficult to map a sequencing read to a particular position, is often the cause of incorrect genotyping. A read was considered as being multi-hit if the difference between the first and second highest sequence identities between the read and the reference genome was 2% or less. All other reads were regarded as uniquely mapped reads. Uniquely mapped pair read alignments were analyzed further. The alignments were converted from sequence alignment/map (SAM) format to sorted, indexed binary alignment/map (BAM) files  was used to remove duplicate reads and the output BAM files were realigned using GATK IndelRealigner (https://gatk.broadinstitute.org/).We resequenced genomic DNAs from the Mishima Battery mouse strains BLG2, HMI, NJL, PGN2, KJR, CHD, BFM, SWN, and MSM. Sequence data from JF1 (DRA000323) was obtained previously  using the allele sharing distance (ASD) criterion. The dendrogram was explored using the general UPGMA methods. The analysis for acquisition of genomic data and the data analysis for the genomic variation detection and the dendrogram construction were performed at NIG and ROIS-DS.The BAM files produced above were used for calling SNPs and short indels; SAMtools mpileup . The filtering conditions for the nucleotide variant detection were the same as those described above. New vcf files are available in the download page (https://molossinus.brc.riken.jp/pub/Wild10_2020/).In 2020, the BAM files produced above were used for calling SNPs and short indels using HaplotypeCaller implemented in GATK software tools (ftp://ftp-mouse.sanger.ac.uk/). The homozygous variants were used in this comparison . The count of variants was applied up to the third candidates. The results are downloadable from https://molossinus.brc.riken.jp/pub/Wild10_2020/.Nucleotide variant datasets of the Mishima Battery, consisting of pgn2v1, bfmv1, blg2v1, njlv1, chdv1, kjrv1, swnv1, hmiv1, msmv4, and jf1v3, respectively, were used to compare with dbSNPs  were donated to Dr. Kazuo Moriwaki of NIG by Dr. P. Michael. BFM is an inbred mouse strain of the subspecies M. m. domesticus. This strain was established from a few wild mice trapped by Dr. F. Bonhomme on the University Campus of Montpellier, France in 1976. In 1980, founder mice  were donated to Dr. Kazuo Moriwaki of NIG by Dr. F. Bonhomme. BLG2 is an inbred mouse strain of the subspecies M. m. musculus. This strain was established from wild mice trapped near General Toshevo in Bulgaria in 1980 by Dr. F. Bonhomme; the mice were brother-sister mated at the Institut Pasteur. In 1981, founder mice  were donated to Dr. Kazuo Moriwaki of NIG by Dr. F. Bonhomme. NJL is an inbred mouse strain of the subspecies M. m. musculus. This strain was established from wild mice trapped by Dr. J.P. Hjorth 15km west of Aarhus in 1980. In 1980, founder mice  were donated to Dr. Kazuo Moriwaki of NIG from by Dr. J.P. Hjorth. CHD is an inbred mouse strain of the subspecies M. m. musculus. This strain was established from wild mice trapped in Chengdu, China. In 1981, founder mice  were transferred to Dr. Kazuo Moriwaki of NIG.KJR is an inbred mouse strain of the subspecies M. m. musculus. This strain was established from wild mice trapped on Kojuri Island, Republic of Korea. In 1984, founder mice  were transferred to Dr. Kazuo Moriwaki of NIG. SWN is an inbred mouse strain of the subspecies M. m. musculus. This strain was established from wild mice trapped on the campus of Agricultural College, Suweon, Republic of Korea. In 1984, founder mice  were transferred to Dr. Kazuo Moriwaki of NIG by Dr. Cho. HMI is an inbred mouse strain of the subspecies M. m. castaneus. HMI is descended from wild mice trapped in He-mei, Taiwan by Dr. Sakaizumi in 1986. Founder mice  were given to Dr. Kazuo Moriwaki of NIG by Dr. Sakaizumi in the same year. MSM is an inbred mouse strain of the subspecies M. m. molossinus, collected in 1978 in Mishima, Shizuoka-ken, Japan . The divergence time between M. m. molossinus and the west European subspecies M. m. domesticus, from which most classical laboratory strains are derived, is estimated to be roughly one million years . JF1 is an inbred mouse strain of the subspecies M. m. molossinus. The ancestors of JF1 were found at a pet market in Denmark and transferred to NIG in 1987, where they were established as an inbred strain in 1993. Figure S2. Box-plots of body size, tissue weights, and physiological characteristics of Mishima Battery strains. (a) BW: Body weight, (b) TOTL: Total body length, (c) BL: Body length, (d) TL: Tail length, (e) BMI: Body mass index, (f) TES: Testis, (g) SPL: Spleen, (h) LIV: Liver, (i) KID: Kidneys, (j) HEA: Heart, (k) VFAT: Visceral fat tissues, (l) SFAT: Subcutaneous fat tissues, (m) DBAT: Dorsal brown adipose tissues, (n) AI: Adiposity index. Blood concentrations of selected physiological characteristics: (o) IP: Inorganic phosphate, (p) HDL: High-density lipoprotein cholesterol, (q) TCH: Total cholesterol, (r) TG: Triglyceride, (s) ALB: Albumin, (t) ALP: Alkaline phosphatase, and (u) AMY: Amylase. Figure S3. Trait-strain matrices of the Mishima Battery strains. Significance levels between strains in the Mishima Battery were confirmed using a Bonferroni correction to account for multiple-hypothesis testing. A measured value was considered significant if the corrected t-test P value was less than 0.05  and highly significant if the P-value was less than 0.001 (red). (a) BW: Body weight, (b) TOTL: Total body length, (c) BL: Body length, (d) TL: Tail lengths, (e) BMI: Body mass index, (f) TES: Testis, (g) SPL: Spleen, (h) LIV: Liver, (i) KID: Kidneys, (j) HEA: Heart, (k) VFAT: Visceral fat tissues, (l) SFAT: Subcutaneous fat tissues, (m) DBAT: Dorsal brown adipose tissues, (n) AI: Adiposity index, (o) IP: Inorganic phosphate, (p) HDL: High-density lipoprotein cholesterol, (q) TCH: Total cholesterol, (r) TG: Triglyceride, (s) ALB: Albumin, (t) ALP: Alkaline phosphatase, and (u) AMY: Amylase. Figure S4. Dendrogram of the Mishima Battery constructed using the whole set of SNP variants. Low-quality regions around indels were excluded in this analysis. Supplementary file1 (PDF 6397 KB)Figure S1. The Mishima Battery of wild-derived inbred strains. The figure illustrates strains belonging to Below is the link to the electronic supplementary material.


Propionibacterium acnes, newly reclassified as Cutibacterium acnes, is an anaerobic Gram-positive bacterium causing acne, found mainly on the skin. In addition, P. acnes is responsible for inflammation of the gums (gingivitis) and blood vessels, consequently leading to various diseases in the human body. In recent years, the evolution of microorganisms, such as P. acnes, that have become resistant to many commercial antibiotics due to the widespread use of antimicrobial drugs in the treatment of infectious diseases has emerged as a major clinical problem. We here analyzed the potential use of 37 medicinal plant extracts as plausible candidates for treating P. acnes, in terms of total phenolic and flavonoid contents, antioxidants scavenging and antimicrobial activity. Consequently, methanol extracts from 14 medicinal plants showed promising antimicrobial activities against P. acnes. In particular, as the extracts from Chrysosplenium flagelliferum F. and Thuja orientalis L. exhibited distinct antimicrobial activities in both the broth dilution and disc diffusion assay, they could be effectively used as active ingredients for preventing or treating inflammatory periodontal diseases, such as periodontitis. Propionibacterium acnes (newly reclassified as Cutibacterium acnes) is a non-spore-forming anaerobic Gram-positive bacterium frequently found in the sebaceous glands of the human body. It is also a causative organism closely linked to various inflammatory diseases by infecting other organs, including the skin, airways, heart valves, eyes, gums, and others -4H-1-benzopyran-4-one) and Kuraridin ((E)-1--3-prop-2-en-1-one) were the principal active antimicrobial substances isolated from the A. continentalis K. extract. Choi et al. [Terminalia chebula R. and Angelica reflexa L., have antimicrobial activity against P. acnes and that the degree of antimicrobial activity varies depending on the medicinal plant species and type of extraction solvent. The highest amount of active antimicrobial substance can be obtained when ethanol is used as an extraction solvent. In their study, methanol extracts were used for comparison, and the antimicrobial activity differed between ethanol and methanol extracts.As shown in n et al.  reportedP. acnes. The antimicrobial activity of propolis is closely linked to its phenolic compounds, which are similar to those from typical plant extracts. However, the antimicrobial effect of propolis varies depending on its native habitat and extraction method. In the study, they used 70% ethanol extract from propolis for an antimicrobial assay, as a relatively high flavonoid content was extracted. Cho et al. [Laurus nobilis L.) exhibited antimicrobial activity against P. acnes, and used deionized water and 100% methanol as controls for activity validation.Kim et al.  reportedo et al.  also repP. acnes not only depends on the extraction solvent, but also on various factors, such as the plant part, extraction time, and temperature. These characteristics provide different results when conducting comparative analyses using reported data.Water ,21, ethaP. acnes is a common species throughout the human body that causes inflammation- related diseases, such as mild, moderate, and severe acne; chronic endophthalmitis; and inflammation caused by the implantation of artificial aids [P. acnes. We verified that methanol extracts from 14 plant extracts, including I. integra T., C. japonica L., C. flagelliferum F., P. sagittatum var. sieboldii L., S. stoloniferum B., P. vulgaris var. lilacina L., L. glauca S. and Z., R. pseudo-accacia L., C. siderosticta H., G. sibiricum L., T. orientalis L., H. rhombea S. and Z., A. continentalis K., and R. rugosa T., had promising antimicrobial activities. While many studies have been conducted on P. acnes for preventing or treating skin diseases, studies on other diseases are rarely conducted. In particular, the mechanism by which P. acnes causes oral disease has not received much attention. P. acnes is a causative pathogen of edema and bleeding in the gums and increases the bioadhesiveness of biofilms formed around prostheses [Streptococcus mutans. Although this study suggests the potential use of methanol extracts from 14 medicinal plants as suppressive agents for P. acnes, we did not further identify or isolate active antimicrobial ingredients in plant extracts. However, along with their antimicrobial activity, the free radical scavenging activity and total phenolic and flavonoid contents of the 14 plant extracts could provide insight into how they inhibit the cell growth of P. acnes.ial aids ,28,29,30ial aids ,32,33,34ostheses . HoweverP. acnes. The data provide evidence that extracts from some medicinal plants could be used as basic materials for identifying substances that can prevent or treat skin and oral diseases, such as tooth decay and stomatitis, and be used as oral hygiene products, such as mouthwashes, toothpaste, varnishes, or the like. In particular, as the extracts from C. flagelliferum F. and T. orientalis L. exhibited excellent antimicrobial activities in both the broth dilution and disc diffusion assay, they could be effectively used as active ingredients for preventing or treating inflammatory periodontal diseases, such as periodontitis.In conclusion, the results of this study indicate that methanol extracts from 14 medicinal plants have promising antimicrobial activity against Platycladus orientalis, Camellia japonica, and Sparganium erectum (600 g of each sample) were purchased from the Gyeong-dong (Korea) oriental medicine market, while freeze-dried methanol extracts of other plants (approximately 20 mg of each sample) were obtained from the Korea Plant Extract Bank. The medicinal plants used in this study are listed in Medicinal plants in Korea were selected based on their antibiotic activity and their traditionally known medical uses. Dried whole-plant materials of P. acnes 5527, a typical skin and gingivitis pathogen, was selected as a candidate to evaluate the antimicrobial activity of the plant extracts and was purchased from the Korean Collection for Type Cultures (KCTC) in the Korea Research Institute of Bioscience and Biotechnology . P. acnes was grown at 37 °C for 72 h in YPD  broth using an anaerobic culture kit consisting of an anaerobic jar and Anaerocult® A . The presence or absence of oxygen was confirmed using an anaerotest .T. orientalis L., C. japonica L., and S. stoloniferum B. (50 g) were extracted three times with 1.3 L of 95% methanol for 3 d at room temperature (25 °C). Thereafter, the extracts were centrifuged at 8000× g for 30 min. The combined supernatants were evaporated using a rotary vacuum evaporator . The freeze-dried solids (approximately 20 mg) were dissolved in 1 mL of methanol to assess the antimicrobial activities.The dried ground raw materials of P. acnes KCTC 5527, prepared to determine the antimicrobial activity of plant extracts, were typically cultured in a YPD solid medium, selected as a single colony, and then inoculated in 10 mL of liquid medium. The antimicrobial activity of the plant extracts was primarily determined following the broth dilution method according to a previous report [The cells of s report . To this6 cells/mL) were inoculated onto YPD solid medium following the pour plate method, then dried for 15 min at room temperature. The 6 mm filter paper discs were impregnated with 10 μL of varying concentrations of a plant extract  and then placed on the inoculated agar plates. After cultivation for an appropriate time (24–48 h), the total diameter of the growth inhibition zone, including the paper disc, was measured for each extract concentration. The MIC for each plant extract was defined as the lowest concentration of each tested methanol extract that prevented cell growth (diameter > 8 mm). All experiments were conducted in triplicate to confirm reproducibility.Considering the ecological niches of the human body , the antimicrobial activities of plant extracts were further determined following the diffusion method using disc paper. Precultured fresh cells (5 × 103. The resulting mixture was incubated in a water bath at 25 °C for 20 min. To remove precipitates, the reaction mixture was centrifuged at 3000 rpm for 20 min. After centrifugation, the absorbance of the supernatant was measured at 735 nm. The total phenolic content was calculated using a gallic acid calibration curve and expressed in gallic acid equivalents.The total phenolic content was determined following the method reported by Folin-Denis et al. . BrieflyThe total flavonoid content was determined following the Nieva Moreno method . BrieflyThe antioxidant activity was measured following the method reported by Abe  and YamaAll tests presented in


Spirea japonica var. acuminata Franch. (Rosaceae) is a Chinese herbal medicine distributed in southwest and east China. The first complete chloroplast genome of Spirea japonica var. acuminata Franch. was assembled and reported in this study. The genome is 153,822 bp in length and contained 125 encoded genes in total, including 80 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The phylogenomic analysis showed that Spirea japonica var. acuminata Franch. was closely related to Spirea blumei, Spirea trilobata, Spirea mongolica and Spirea insularis according to the current sampling extent. Spirea japonica L. f. (1782) is a perennial shrubby species with pink flowers occurring in clusters at the tips of branches. It is widely dissimilar in 14 intraspecific varieties. Spirea japonica var. acuminata Franch. is one variant of Spirea japonica L. f. which is a Chinese herbal medicine classified in the Rosaceae. It is mainly distributed in southwest and east China . The specimen and extracted DNA was deposited at the College of Life Sciences and Medicine, Zhejiang Sci-Tech University  under the voucher number ZSTULSM0001. The total genomic DNA was extracted from its fresh leaves using the CTAB method in accordance with the manufacturer’s instructions. The plastome sequences were generated using the Illumina HiSeq 2500 platform . In total, ca. 20.6 million high-quality clean reads (150 bp PE read length) were generated with adaptors trimmed. These clean data were de novo assembled to complete chloroplast genome using GetOrganelle  as a reference.The leaves of Spirea japonica var. acuminata Franch. chloroplast sequence (GenBank Accession No. MZ981784) is 153,822 bp, consisting of a large single copy region , a small single copy region , and two inverted repeat regions . The overall GC content of Spirea japonica var. acuminata Franch. chloroplast genome was 36.6% and the GC content of the LSC, SSC, and IR regions are 34.3%, 30.3%, and 42.5%. A total of 125 genes were contained in the genome . Sixteen genes had two copies, which were comprised of fine PCG genes , seven tRNA genes , and all four rRNA species . In the genome, nine protein-coding genes  had one intron, rps12 and ycf3 gene contained two introns.The full length of Spirea japonica var. acuminata Franch., we obtained 11 published complete chloroplast genomes of Rosaceae from NCBI. Hippophae neurocarpa was used as outgroup for constructing the phylogenetic tree. The 13 complete cp sequences were aligned using MAFFT v7.3 (Katoh and Standley Spirea japonica var. acuminata Franch. was closely related to Spirea blumei, Spirea trilobata, Spirea mongolica and Spirea insularis according to the current sampling extent (To confirm the phylogenetic position of g extent .


Studies on ticks infesting equids are lacking in various parts of the world, including Khyber Pakhtunkhwa (KP), Pakistan. The aim of this study was to investigate the diversity of ticks infesting equids, associated risk factors and rickettsial detection in ticks from equids in KP.gltA and ompA genes were used for Rickettsia detection in ticks.Inspection of 404 equid hosts from November 2018 to October 2019 resulted in the collection of 550 ticks. Data on tick-associated risk factors were collected from equid owners by means of a questionnaire. After morphological identification, partial DNA sequences of the tick mitochondrial 16S rRNA gene were used for taxonomic confirmation of species. Partial sequences of the Rhipicephalus microplus , Rh. haemaphysaloides , Rh. turanicus , Rh. sanguineus (s.l.)  and Hyalomma anatolicum . The most prevalent tick life stage was adult females  followed by adult males  and nymphs . Higher tick infestations were observed on male equids  and adult equids . Ticks were frequently attached to the axial region of horses , sternum of donkeys  and belly of mules  (P < 0.04). Temporal patterns of tick infestation in association with temperature and humidity were highly significant (P < 0.05). Risk factors, such as animal housing (P < 0.0003), living management (P < 0.006), grazing type (P < 0.01) and location in hilly areas (P < 0.02), significantly enhanced the chances for tick infestation. Tick species analyzed in this study were phylogenetically related to species from Afghanistan, China, South Africa and Taiwan. Partial sequences of the gltA and ompA genes obtained from Rh. microplus and Rh. haemaphysaloides were 100% identical to the spotted fever group pathogen Rickettsia massiliae.A total of 550 tick specimens were collected on 324 (80.2%) of the equids inspected, of which 161 were horses (50%), 145 (45%) were donkeys and 18 were mules (5%). The ticks were identified as belonging to the following five species: R. massiliae.Equids exposed to significant risk factors were infected by one or more of at least five tick species in KP, Pakistan, and some of the ticks harbored the human pathogen  Rickettsia, and several protozoans and viruses that cause disease and are a threat to human and veterinary health ) ) . PositivpA) gene , 24 (seewww.ncbi.nlm.nih.gov/blast) to infer closest identities with the organisms available in GenBank [ompA rickettsial gene, we constructed a phylogenetic tree in MrBayes using Bayesian statistics [The obtained sequences were assembled and trimmed in SeqMan v 5.00  and subjected to BLASTn analyses  was used for differences, and relative risk (RR) was analyzed using GraphPad Prism v. 5.00 , with the 95% confidence interval (CI). Significance was set at P < 0.05.The recorded observations were assembled and arranged in the spreadsheets of Microsoft Excel V 2016. The chi-square test (χP < 0.002).A total of 404 animals were sampled, including 200 horses, 171 donkeys and 33 mules, of which 324 (80.2%) were found to be infested with ticks . Of the equids inspected, horses were the most infested  followed by donkeys  and mules . The highest tick burdens were found in equids inspected in Peshawar , Mardan , Nowshera , Swabi  and Charsadda ; the lowest burdens were observed in Upper Dir , Malakand , Bajaur  and Buner   and the lowest tick infestation during the winter between November  and January  (P = 0.1907)  and temperature .The trend line of tick infestation increased from May  to August  of 2019, with the highest tick infestation found to be throughout the summer  and Hyalomma . The most numerous tick species was Rhipicephalus microplus  followed by Rh. haemaphysaloides , Rh. turanicus , Rh. sanguineus (s.l.)  and Hyalomma anatolicum .A total of 550 collected ticks were morphologically identified into two genera and five species as follows: Rh. microplus (70% of the ticks from horses), 44 were Rh. haemaphysaloides (17%), 18 were Rh. turanicus (0.06%), ten were Rh. sanguineus (s.l.) (0.03%) and six were H. anatolicum (0.03%). From donkeys, 206 ticks  were collected, of which 110 were Rh. microplus (53% of the ticks from donkeys), 62 were Rh. haemaphysaloides (30%), 13 were Rh. turanicus (06%), 18 were Rh. sanguineus (s.l.) (8%) and three were H. anatolicum (1%). Mules were infested by 81 tick specimens , of which 46 were Rh. microplus (57% of the ticks from mules), 20 were Rh. haemaphysaloides (24%), eight were Rh. turanicus (10%), five were Rh. sanguineus (s.l.) (6%) and two were H. anatolicum (2%)  were collected from horses, of which 185 were χ2 = 18.66, 10; P < 0.045) , the sternum of donkeys  and the belly of mules . The lowest tick-infested body parts were the dewlap in horses , the axial region in donkeys  and the scrotum of mules . Statistical analysis of infestation of the body regions of the equid hosts revealed significant differences , Rh. turanicus (15 specimens), Rh. sanguineus (s.l.) (2 specimens), Rh. haemaphysaloides (12 specimens) and H. anatolicum (5 specimens). BLAST of obtained 16S rRNA sequences for Rh. microplus, Rh. haemaphysaloides and H. anatolicum resulted in the screening of homologous sequences from Pakistan, India and China that showed 98–99% identity. The BLAST of Rh. turanicus showed closest identity (95–97%) with conspecific sequences reported from India, Afghanistan, Iran, South Africa and China, and the Rh. sanguineus (s.l.) sequences showed highest identities (96–98%) with conspecific sequences from Taiwan, USA and Singapore. The resultant consensus sequences after trimming were deposited in the GenBank for each species; Rh. microplus (accession MW071171), Rh. turanicus (MW074299), Rh. sanguineus (s.l.) (MW113239), Rh. haemaphysaloides (MW113238) and H. anatolicum (MW172215). The phylogenetic analysis of the collected five species revealed an evolutionary relatedness with homologous species reported in Pakistan’s neighboring countries. In a phylogenetic tree, the obtained sequences of prevalent tick species clustered with the same species reported in the bordering countries  MW11239, Rh. Rh. microplus, Rh. turanicus, Rh. haemaphysaloides and H. anatolicum). Among these, ten speciments showed high Cq values (> 35) and were excluded from further analyses. The remaining 13 samples were found to be positive for Rickettsia through conventional PCR targeting the rickettsial gltA and ompA genes. These Rickettsia-positive ticks included Rh. microplus-infected ticks  from all sampled districts, and Rh. haemaphysaloides-infected ticks  from Bajaur, Mardan and Charsadda districts. No Rickettsia spp. were detected in Rh. turanicus, Rh. sanguineus (s.l.) and H. anatolicum through PCR. A consensus partial sequence (333 bp) of the highly conserved Rickettsia gltA gene, detected in Rh. microplus and Rh. haemaphysaloides, was 100% identical to several sequences of R. massiliae from GenBank . From these same ticks, a consensus partial sequence (592 bp) of the highly polymorphic Rickettsia ompA gene was 100% identical to a sequence of R. massiliae from GenBank (CP003319). The obtained sequences of gltA and ompA were uploaded to GenBank with accessions numbers MW922872 and MW928497 respectively. The obtained sequence of the ompA gene for Rickettsia spp. clustered with sequences of R. massiliae in a phylogenetic tree  and H. anatolicum. These ticks were found to be infesting equid hosts  throughout the year in extreme cool to extreme hot weather conditions. The bacterium Rickettsia massiliae was detected in all Rh. microplus ticks collected in all nine study districts and in Rh. haemaphysaloides collected in Bajaur, Mardan and Charsadda districts. Ecological conditions and biotopes drive the geographic distribution and the risks associated with the possible harmful effects of tick species. The favorable environmental conditions in the region extend tick adaptation and also result in infestation of novel hosts [Ecological changes and an increasingly evident increase in global temperature have widely impacted the distribution of ticks and tick-borne diseases, ultimately increasing their threat to public and veterinary health , 10. Basel hosts , 16, 17.Risk factors, such as age, altitude, housing type, grazing, living management and food conditions, of equid hosts with respect to tick infestation were statistically significant, with the exception of food conditions. A strong correlation was observed between the intensity of tick infestation and temperature and relative humidity, with the highest infestation recorded in the summer (June–August) and lowest infestation recorded in the winter (December–January); these results agree with previous reports in the region , 17, 32.The younger age group (1–years) of equid hosts was found to have lower tick infestations, possibly correlated with a strong immune response, shorter exposure to questing ticks, shorter time in free grazing conditions and frequent grooming, all of which protect younger equids from tick infestation –36. HighRh. microplus, which has been reported to the dominant tick species in the region [Haemaphysalis and Hyalomma species other than H. anatolicum are also abundant tick species in Pakistan [Ticks collected across the different study regions belonged to two genera and five species. The most prevalent tick was e region , 16, 17.Pakistan , 16, 17,Pakistan –44, 51. Pakistan –46. The Rickettsia spp. have been collected from several Rhipicephalus ticks infesting different hosts in various regions of the world [Rickettsia infection. In this study, Rickettsia massiliae was detected in Rh. microplus and Rh. haemaphysaloides using gltA and ompA rickettsial markers. There have been several reports of spotted fever due to R. massiliae in Europe [R. massiliae associated with equid ticks in Pakistan. Our findings highlight that human illness due to this spotted fever group of pathogens could be underreported in Pakistan, where to date tick-borne rickettsioses have not been reported.Tick-borne he world , 47–51. n Europe . NoteworR. massiliae, a tick-borne pathogenic spotted fever group Rickettsia species, was detected in Rh. microplus and Rh. haemaphysaloides. These results demonstrate that it is important to investigate the vector potential of ticks for other pathogens infesting equids in different regions of Pakistan to avoid the emergence of zoonotic infections. They will also provide valuable data for use in developing integrated control management strategies against ticks and tick-borne diseases in Pakistan.To our knowledge this is the first report of equid ticks from selected regions of KP. The risk factor analyses showed that temperature, humidity, age, keeping and grazing systems, sex and host species considerably affected the intensity of tick infestations. The phylogenetic tree of the collected ticks based on the tick mitochondrial 16S rRNA gene (2 genera and 5 species) revealed a close resemblance to tick species reported from Afghanistan, China, South Africa and Taiwan. The human pathogen