Culex quinquefasciatus and Cx. pipiens molestus are sibling species within Cx. pipiens complex. Even though they are hard to distinguish morphologically, they have different physiological behaviors. However, the molecular mechanisms underlying these differences remain poorly understood.Both Transcriptome sequencing was conducted on antennae of two sibling species. The identification of the differentially expressed genes (DEGs) was performed by the software DESeq2. Database for Annotation, Visualization and Integrated Discovery was used to perform GO pathway enrichment analysis. The protein–protein interaction (PPI) network was constructed with Cytoscape software. The hub genes were screened by the CytoHubba plugin and Degree algorithms. The identified genes were verified by quantitative real-time PCR.OBP10 was expressed 17.17 fold higher in Cx. pipiens molestus than Cx. quinquefasciatus. Eighteen resistance-related DEGs were identified, including 15 from CYP gene family and three from acetylcholinesterase, in which CYP4d1 was 86.59 fold more highly expressed in C. quinquefasciatus. Three reproductive DEGs were indentified with the expression from 5.01 to 6.55 fold. Among eight vision-related DEGs, retinoic acid receptor RXR-gamma in Cx. pipiens molestus group was more expressed with 214.08 fold. Among the 30 hub genes, there are 10 olfactory-related DEGs, 16 resistance-related DEGs, and four vision-related DEGs, with the highest score hub genes being OBP lush (6041148), CYP4C21 (6044704), and Rdh12 (6043932). The RT-qPCR results were consistent with the transcriptomic data with the correlation coefficient R = 0.78.Most annotated transcripts  were expressed in both sibling species. Among 15 identified odorant-related DEGs, OBP lush, CYP4C21, and Rdh12 may be key hints to the potential molecular mechanisms behind the two sibling species' biological differences.The study provided clues that antennae might play special roles in reproduction, drug resistance, and vision, not only the traditional olfactory function. The online version contains supplementary material available at 10.1186/s13071-022-05482-6. Culex mosquitos are vectors of West Nile virus, Japanese encephalitis virus, and lymphatic filariasis [Mosquitoes are not only annoying but can also transmit pathogens, resulting in billions of potential infections and approximately 700,000 deaths worldwide each year. Different species of mosquitoes carry and transmit different pathogens , 2. For lariasis .Culex quinquefasciatus  and Cx. pipiens molestus  are sibling species within Cx. pipiens complex whose morphologies are difficult to distinguish [Cx. p. molestus and Cx. quinquefasciatus have very different physiological behaviors. For example, Cx. p. molestus mainly mate in confined spaces. Their ovaries can develop normally, and they lay eggs without blood-feeding in their first life cycle. On the other hand, Cx. quinquefasciatus mate in open areas. There are also differences in their preference for mammalian and bird blood sources [tinguish . However sources . AlthougAnopheles sinensis, and their expression levels are significantly regulated after the blood meal [Culex pipiens complex [Aedes aegypti [Antenna is the main chemosensory organ of mosquito and plays an important role in smell , hearingood meal , quite s complex  and Aede aegypti –15. Besi aegypti .Cx. p. molestus and Cx. quinquefasciatus and then explored the differences in the expression and interaction of transcripts. Besides olfaction, antennae could also have special roles in reproduction, drug resistance, and visual function. The study provides hints about the potential molecular mechanisms behind the two sibling species' biological differences.We conducted transcriptome sequencing on antennae of Cx. p. molestus and Cx. quinquefasciatus were obtained from long-term laboratory-reared strains, which had been characterized by male genitalia and/or cytochrome oxidase subunit I (COI) barcoding in advance [Both  advance , 18. TheCx. quinquefasciatus (Cqui) and antennae of Cx. p. molestus (Cmls). Biological samples containing 50 antennae were set in four replicates for each group. RNA was extracted with TRIzol (Takara.9108). Libraries were constructed using the NEB Next Ultra RNA Library Prep Kit and finally sent to Beijing MacroMicro-test Biotechnology Co., Ltd., for transcriptome sequencing.Two groups of samples were designed in this study: antennae of After the raw data were filtered, clean reads were compared to the reference genome using HISAT2 software . Referenpadj  < 0.05 and |log2(foldchange)|> 1. Functional annotation and GO enrichment analyses were carried out using the R and clusterProfiler [A differential analysis was performed using DESeq2 software . The scrProfiler , 29 to cProtein-protein interaction (PPI) network analysis obtained the interaction network file through the STRING database . The fil18S) to verify the accuracy of the transcriptome results. The primers were designed with Oligo Primer Analysis software version 4.0  in total. The Q20 values were all > 95%, suggesting a high degree of quality ; two other DEGs (OR67d and OR7a) were odorant receptors (OR) and one (GR22) a gustatory receptor (GR). Of these, OBP10 in cmls groups were specially more highly expressed at 17.17 fold times compared with the Cqui group  were odorant-binding proteins (/15 were CYP) gene family and three from acetylcholinesterase (AchE). Eight CYP family DEGs were more expressed in Cqui; for example, CYP4D1 was 86.59 fold more highly expressed. Three AchE genes were differentially expressed with fold change ranging from 0.39–4.59  and one location of vulva defective 1 (lov-1) DEGs were more expressed in the Cmls, with expression from 5.01 to 6.55 fold. These three genes were all related to insect mating  RXR-gamma in Cmls group was more expressed at 214.08 fold. Vertebrate ancient (VA) opsin in Cqui group was more expressed with 12.43 fold change  and AchE family and vision-related genes (RBP and Rdh). The highest scores for specific functional hub genes were CYP4C21 (6044704), OBP lush (6041148), and Rdh12 (6043932), respectively. Hub genes were closely related internally in each specific function. The hub gene score and function are presented in Additional file Hub genes were the nodes with higher degree, i.e. nodes with more connections in related pathways. With the cytoHubba plug-in and the Degree algorithm, we calculated the hub gene and drew the PPI Networks, PPI Fig. A. PPI waRdh12 604932, respOBP, Vg, CYP, and so on. All of these genes were significantly different between Cqui and Cmls group on expression of RNA-seq and RT-qPCR  were highly expressed in Cx. p. molestus, which may be related to the autogenous habit [Cx. p. molestus developed normally, which might have led to higher expression of the Vg gene.The two us habit . In the RBP1, RXP Rax, eye-specific DGK, and VA opsin were more highly expressed in the Cqui group. Retinal oxide-binding protein was abundantly expressed in locust antenna and associated with olfactory-related behaviors in solitary locusts [DGK was essential for the photoreceptor function of the Drosophila retina [Rdh12 functioned as part of the visual cycle, which was a series of enzymatic reactions required for the regeneration of the visual pigment and detoxification of lipid peroxidation products [RAR was involved in the retinoic acid signaling pathway, which was crucial for the control of embryonic development [Cx. p. molestus mated mainly in enclosed spaces, while Cx. quinquefasciatus mated mainly in open areas) might interact with the differential expression of their vision-related genes.Of the eight differentially expressed vision-related genes,  locusts . Eye-spea retina , 52. Vera retina . Rdh12 fproducts , 55. Retproducts , 57. RARelopment . The difCx. quinquefasciatus were mainly enriched in cell communication, G protein-coupled receptor signaling pathway, ion channel complex, cell junction, signaling receptor activity, and other pathways. Previous studies have found that G protein-coupled receptor signaling pathway was closely related to insect feeding behavior [Cx. quinquefasciatus by regulating P450-mediated detoxification [GO enrichment results showed that genes with higher expression in behavior , which afication .Cx. p. molestus were mainly enriched in microtubule-based movement, innate immune response, integral and intrinsic component of endoplasmic reticulum membrane synthesis, and cell division-related pathways. Due to the autogenous habit of Cx. p. molestus, the body of Cx. p. molestus was richer in carbohydrates, lipids, proteins, and other nutrients [Genes with higher expression in utrients , 61, 62,OBP lush (6041148) had the highest score for olfactory-related hub gene and consisted of a large family of low-molecular-weight, highly divergent proteins expressed exclusively in the chemosensory sensilla of insects. It was required for normal olfactory behavior in Drosophila [OBP lush mutant flies were abnormally attracted to high concentrations of ethanol, propanol, and butanol but had normal chemosensory responses to other odorants [The screened high scored hub genes would be the key to explore mechanisms behind the two sibling species biological differences. osophila . OBP lusodorants .CYP4C21 (6044704) had the highest score, which was 5.4 times higher in the resistant strain compared with the wild Ae. aegypti strain in Vietnam [Rdh12 (6043932), an NADPH-dependent retinal reductase, which is expressed in the inner segments of the photoreceptors [Rdh12 could enzymatically reduce toxic lipid 4-hydroxynonenal in vitro [The resistance-related hub gene  Vietnam . The hubeceptors . Rdh12 cin vitro , protectin vitro .Cx. quinquefasciatus and Cx. p. molestus, not only focused on olfactory-related genes in common view, but also expanded the orientation of resistance-, reproduction-, and vision-related genes of two sibling species of mosquitoes. Even though the RT-qPCR results confirmed the RNA-Seq prediction to some extent, further molecular and behavioral investigations are needed. This study provides hints about the potential molecular mechanisms behind the two sibling species' biological differences, like blood-feeding, detoxication, mating, host-locating, and other physiological behaviors, which could facilitate the design and development of more targeted repellents or insecticides. By interfering with or silencing the specific genes with RNAi or CRISPR techniques, mosquito host localization ability and resistance to insecticides were affected, leading to the decrease of vector competence. The screened key genes can help to control mosquito-borne diseases effectively and efficiently.Our transcriptome sequencing on antennae of Additional file 1: Table S1. The qPCR primers designed to verify RNA-seq results. Table S2. Olfactory-related differentially expressed genes. Table S3. Resistance-related differentially expressed genes. Table S4. Reproduction-related differentially expressed genes. Table S5. Visual-related differentially expressed genes. Table S6. The hub gene score and function.


Bacillus cereus group species; however, misinterpretation of WGS results may have severe consequences. We report 3 cases, 1 of which was an outbreak, in which misinterpretation of B. cereus group WGS results hindered communication within public health and industrial laboratories.Whole-genome sequencing (WGS) is being applied increasingly to  Bacillus cereus group of bacteria is a complex of closely related species with varying pathogenic potential  is used increasingly in clinical and industrial microbiology laboratories to characterize B. cereus group species assignment misinterpretations in 3 continents: Europe, North America, and Africa  sequencing . Each B. cereus group strain was assigned to a species by comparing its genome to the genomes of all B. cereus group species type strains and identifying the most similar species type strain by average nucleotide identity (B. anthracis using this approach (B. anthracis label). However, subsequent investigation by M.W. and L.M.C. revealed that, although the strains most closely resembled B. anthracis, neither strain belonged to the historical, clonal B. anthracis lineage typically associated with anthrax toxin production   and some group III B. cereus strains capable of causing diarrheal foodborne illness  . The inq illness , panel AB. cereus group strains during routine surveillance of meat products  represented false-positive scenarios, in which group III B. cereus strains incapable of causing anthrax were incorrectly assumed to be anthrax-causing agents  was assigned to an established pathogen (group III B. cereus) and directly hindered an outbreak investigation  represented a worst-case, false-negative scenario, in which a WGS-assigned species label with limited clinical interpretability or previous associations to foodborne illness (tigation . We anti in 2017  (7).We e in 2017 . OverallBacillus cereus group species, 2018–2022Additional information about misidentifications resulting from taxonomic changes to


M. bovis Bacille Calmette-Guerin (BCG) has directed researchers to explore progressive approaches to halt the eternal TB pandemic. Mycobacterium tuberculosis (M.tb) was first identified as the causative agent of TB in 1882 by Dr. Robert Koch. However, TB has plagued living beings since ancient times and continues to endure as an eternal scourge ravaging even with existing chemoprophylaxis and preventive therapy. We have scientifically come a long way since then, but despite accessibility to the standard antimycobacterial antibiotics and prophylactic vaccine, almost one-fourth of humankind is infected latently with M.tb. Existing therapeutics fail to control TB, due to the upsurge of drug-resistant strains and increasing incidents of co-infections in immune-compromised individuals. Unresponsiveness to established antibiotics leaves patients with no therapeutic possibilities. Hence the search for an efficacious TB immunization strategy is a global health priority. Researchers are paving the course for efficient vaccination strategies with the radically advanced operation of core principles of protective immune responses against M.tb. In this review; we have reassessed the progression of the TB vaccination program comprising BCG immunization in children and potential stratagems to reinforce BCG-induced protection in adults.The pursuit to improve the TB control program comprising one approved vaccine,  Mycobacterium tuberculosis (M.tb). Since the 1800s, TB was the leading cause of health menace worldwide. Despite being declared a global health emergency in 1993 by World Health Organization (WHO), TB continues to be the leading cause of morbidity and mortality amongst bacterial infections  is caused by the facultative intracellular pathogen orldwide . The hosM.tb was originally identified in 1882 by Dr. Robert Koch as the causative agent of TB however, it has lurked among living beings since ancient times. 139 years post-discovery of this pathogen, it still endures as an eternal scourge ravaging globally. Current strategies fail to control TB, due to the upsurge of multidrug-resistant strains, increasing incidents of co-infections in immune-compromised individuals, and the emergence of TB-IRIS (Immune Responsive Inflammatory Syndrome). Globally in 2020, approximately 10 million people were disease-ridden with TB and an aggregate of 1.3 million lives were lost (together with 208000 people with HIV). Furthermore, almost one-fourth of humankind is infected asymptomatically (latently) with M.tb, with a 5-15% risk of progressing into clinical manifestations  which has existed for 100 years fails to impart long-term immunity and has limited efficacy against adult pulmonary TB . BCG immatent TB . Furtheratent TB . The effatent TB . Escalatatent TB . It is catent TB . Neverthatent TB . This pratent TB . The emeatent TB . Advanceatent TB . Hence, atent TB . To imprvia oral route after which it was mass vaccinated to protect infants from disseminated forms of TB. To justify the escalating demand for vaccine strain worldwide, several laboratories around the world began sub-culturing BCG owing to which individuals around the world are vaccinated with characteristically distinct BCG strains  receptors on macrophages  function as a nexus between innate and adaptive immune responses by presenting processed antigens to T lymphocytes post BCG immunization  pathway . BCG imm pathway . Nonethetiveness . Nonetheotection . Apart fotection . Howevervia epigenetic reprograming enhances host protective immune responses  knockout mice  deficient mice incapable of restricting M.tb infection  via alteration of immune metabolic pathways which consequently reduces the protective efficacy against TB. Depletion of Tregs can result in enrichment of Th1, cytotoxic T cell responses with enhanced bacterial extermination upon infection . We have demonstrated that intranasal immunization with PTL along with BCG drastically condensed the bacterial burden, enhanced host protective T cell responses with expansion of polyfunctional T cells as well as memory T cell subsets. Furthermore, host protective immune responses were M.tb specific owing to which spectrum of host protective signaling pathways critical to control TB were activated in response to PTL BCG co-immunization  along with successive effector and memory T cell populations  resulted in improved clinical outcomes and a reduction in bacterial burden in C3HeB/FeB mice , sodium (Na+), and chloride (Cl-) ions within immune cell populations (2+) levels play a vital role in the regulation of antimycobacterial mechanisms such as autophagy, maturation of phagosome, and induction of apoptosis  impede induction of autophagy (2+ currents through P2X purinoceptor 7 (P2X7) receptor heighten autophagy induction and intracellular extermination of M. bovis BCG in macrophages. Calcimycin, an ionophore binds to P2X7 receptor which leads to rise in intracellular Ca2+ levels and exerts antimycobacterial activity against M. bovis BCG  blocker – verapamil, an FDA approved drug is utilized to treat abnormalities in heart rhythms, angina (2+ discharge from the endoplasmic reticulum (ER) leading to inhibition of macrophage activation. So as to bypass host immune responses M.tb upregulates the expression of VGCCs in APCs. Verapamil administration inhibits LTCC currents thereby escalating Ca2+ concentration in the cytosol which upregulates autophagy and bacterial clearance in M.tb  is a conventional first-line drug used for leprosy treatment along with RIF and dapsone  enhances the efficacy of BCG vaccination in a murine model of TB by specifically promoting the expansion of the T central memory cell population (TCM). Furthermore, CFZ enriches stem cell memory T cell responses upon BCG revaccination  are invug  is known to inhibit the mammalian target of rapamycin (mTOR) which negatively regulates autophagy. It is majorly employed in organ transplantation owing to the immunosuppressive nature of the drug  is another prospective autophagy inducer. It is an established antitumor agent as well as in mice it triggers, a stimulator of IFN genes (STING) dependent autophagy mechanism  release is directed via HIF-1-dependent glycolytic pathways  an inhibitor of hexokinase enzyme, can limit the IL1-β generation in LPS-activated macrophages and result in succinate accumulation , a protease inhibitor widely used as antiretroviral medication to treat HIV infections -4-propylnaphthalene-1-carboxylic acid]) is known for induction of oxidative stress and reduction in tumour advancement  stimulates anti-microbial responses by activating autophagic pathways in macrophages. AMPK activation by AICAR further controls the biogenesis of mitochondria and metabolic state in macrophages by inducing peroxisome proliferator-activated receptor gamma coactivator-1 (PPARGC1) associated pathways (C75 and cerulenin are prospective targets for the augmentation of efficacious immune responses. Inhibition of lipid-derived droplets by C75 can lead to polarization of macrophages from the M1 to M2 subset causing enhancement of ROS and NO production (GW9662 is known to regulate vital processes such as metabolism, inflammation, and disease progression (M. bovis BCG (M.tb infected THP-1 cells. SIRT-1 downregulates RelA/p65 unit of NF-κB so as to modulate inflammation (M.tb infection (M.tb infection in mice model of TB (M.tb infection, SIRT2 expression increases along with translocation to nucleus to induce immune dampening epigenetic modifications. However, chemical inhibition of SIRT2 using established inhibitor, AGK2 dramatically augmented bacterial clearance and host protective immune responses. Since, existing literature is available regarding SIRT2 induced metabolic programming (feration . Foamy md as HDT . Metabold as HDT . An apprd for TB . Metformory gene . In M.tb and ETH . Metform and ETH . This HDpathways . Componeoduction . C75 andoduction . PPARγ agression  in macroovis BCG . Link beovis BCG . This inovis BCG . SIRTs aovis BCG . It has ammation . SIRT-6 nfection . Furtherel of TB . It was gramming  and signgramming . FurtherM.tb infection (M.tb infection (M.tb temper host immune responses for survival in an antimicrobial milieu. Diverse immune mechanisms such as phagolysosome maturation in APCs, cytokine stimulation by immune cell populations, and antigen processing and presentation are dynamically manipulated by M.tb. These cellular processes are strictly regulated by an assortment of miRNAs in the host (M.tb is additionally known to alter miRNA expression associated with key biological responses to escape host immune responses (miRNAs are non-coding RNAs that are involved at post-transcriptional levels to regulate array of genes decisive of immune responses . Over 20nfection . Differenfection . Furthernfection . Owing tnfection . Severalthe host . M.tb isesponses . AdditioM.tb infection miR-125b inhibits TNF biosynthesis in human alveolar macrophages (M.tb infection results in differential expression of miRNAs which determines the fate of immune responses (M.tb induced autophagy (M.tb infection, diverse cell populations respond varyingly in conjunction with variations in miRNA expression. Upregulation of miR-155 has been observed in bone marrow-derived macrophages of mice infected with M.tb (M.tb infection (M.tb H37Rv and avirulent strain M. bovis BCG. Downregulation of miRNA involved in the regulation of inflammation and lipid metabolism was observed. Furthermore, miR-145 known for induction of apoptosis has been reported to be downregulated upon infection with a virulent M.tb strain, which results in overexpression of targets which inhibit apoptosis (M.tb H37Rv and M.tb H37Ra infected THP-1 cells. These differentially expressed miRNAs such as miR-30a, miR-30e, miR-155, miR-1275, miR-3665, miR3178, miR-4484, miR-4668-5p, and miR-4497 contribute in diverse physiological aspects. miR-155 interacts with negative regulators involved in TNF- α generation (M. bovis BCG infection significant increase in miR-155 expression is observed in macrophages, which modulates diverse innate immune responses including ROS generation (M. bovis BCG inoculation which modulates cellular physiology and immune responses (M.tb infected human alveolar macrophages, miR-125b inhibits TNF generation (M.tb infection (M.tb infection (M.tb, further assessment is necessitated to establish the prominence of miRNA as therapeutic. We required more studies to comprehend the contribution of miRNA in host-pathogen interactions and a progressive strategy for augmenting conventional therapies.miRNAs involved in immune pathways are extensively studied for mycobacterial infections . It is wrophages . Severalesponses . Howeverutophagy  and apopith M.tb . In contnfection . Mycobacpoptosis . Based oneration . Evaluatneration . Elevatineration , TLR signeration . Subsequneration . It addiesponses . In M.tbneration . miR-29 nfection . miR-223nfection . It has nfection . miR-27anfection . Anothernfection . As revenfection , antisennfection  to manipvia vaccination. Hence, progressive approaches were pursued with the utilization of known immune correlates of protection. To surpass the previously failed attempts, it is vital to redirect focus on immunological pathways that can be augmented for better clinical implications (M.tb transmission, advances to tackle the inadequacies of BCG to impart long-lasting immunological memory should be highlighted as a better immunization stratagem. With extraordinary scientific efforts to counteract the most fatal pathogens known to humankind, we have progressed to a situation wherein we can harness establish knowledge and immunological concepts to deal with the shortcomings of existing approaches and improvise for robust clinical trajectories. Since the most of individuals worldwide are already vaccinated with BCG, it is judicious to keep BCG in reflection while developing new vaccination strategies. Scientific communities are attempting stratagems to prevent millions of deaths from escalating infectious diseases by investing on immunotherapeutic approaches to augment immunological responses.Despite incessant debates on the variable protective efficiency of BCG, it prevails as the only vaccine for TB prevention. Owing to considerable protection in children against disseminated forms of TB, it remains a key component of TB control programs in various countries . Throughications . Since aKN, VD wrote the manuscript. AB edited the manuscript. VD conceived the hypothesis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Refractory apical periodontitis (RAP) is an oral infectious disease characterised by persistent inflammation, progressive alveolar bone destruction, and delayed bone healing. RAP has received increasing attention, because it cannot be cured after repeated root canal therapies. The aetiology of RAP is related to the complex interplay between the pathogen and its host. However, the exact pathogenesis of RAP remains unclarified and includes several factors, such as microorganism immunogenicity, host immunity and inflammation, and tissue destruction and repair. Enterococcus faecalis is the dominant pathogen involved in RAP, and has evolved multiple strategies to ensure survival, which cause persistent intraradicular and extraradicular infections.Objective: To review the crucial role of E. faecalis in the pathogenesis of RAP, and open new avenues for prevention and treatment of RAP.Methods: The PubMed and Web of Science databases were searched for pertinent publications, employing the search terms “Enterococcus faecalis”, “refractory apical periodontitis”, “persistent periapical periodontitis”, “pathogenicity”, “virulence”, “biofilm formation”, “dentine tubule”, “immune cell”, “macrophage”, and “osteoblast”.Results and Conclusion: Besides its high pathogenicity due to various virulence mechanisms, E. faecalis modulates the macrophage and osteoblast responses, including regulated cell death, cell polarisation, cell differentiation, and inflammatory response. An in-depth understanding of the multifaceted host cell responses modulated by E. faecalis will help to design potential future therapeutic strategies and overcome the challenges of sustained infection and delayed tissue healing in RAP. Apical periodontitis (AP) is an inflammatory disease characterised by periapical tissue injury of the affected teeth caused by microbial infection, which often requires root canal therapy (RCT) to control infection and eliminate inflammation. A meta-analysis investigating the global prevalence of AP showed that half of the adult population worldwide has AP, while the frequency of AP in root-filled teeth is as high as 39% . FailureEnterococcus faecalis, a gram-positive facultative anaerobic bacterium, is an opportunistic pathogen that is commonly found in the human oral cavity and gastrointestinal tract. Evidence has shown that E. faecalis is one of the most common pathogenic microorganisms in root canals with RAP, as it is frequently isolated from root canals with endodontic failure [E. faecalis survives for a long time in root canals, because it can tolerate a highly alkaline and oligotrophic environment to form biofilms [E. faecalis can also be detected in the periradicular lesions and extraradicular biofilms in patients with RAP [E. faecalis LTA participates in biofilm formation and adhesion to eukaryotic cells due to its alanylation that modulates the net surface charge of the bacteria [E. faecalis and its virulence factor, LTA, in the pathogenesis of RAP is pivotal. failure . E. faecbiofilms ,12, invabiofilms , and easbiofilms . Moreovewith RAP . Lipoteiwith RAP . E. faecbacteria . Signifibacteria ,6. ColleE. faecalis and these two cell types. Integrating and summarising the evidence can improve our understanding of the pathogenic mechanisms of E. faecalis in RAP and further reveal the complex causes of persistent periapical inflammation and delayed bone healing, thus helping to optimise clinical treatment strategies and improve the success rate of treatment of periapical diseases.Macrophages function as crucial responsive cells in the host’s innate immune system. They recognise and phagocytose pathogens, present antigens, secrete a variety of cytokines or chemokines to regulate the body’s immune and inflammatory responses, and polarise or differentiate into different subtypes of cells to regulate tissue homeostasis . InfectiE. faecalis and its regulatory mechanisms in host macrophages and osteoblasts. We focused on the underlying mechanisms of E. faecalis-modulated responses in these two cell types, including regulated cell death (RCD), cell differentiation, cell polarisation, and inflammatory response, and their roles in the progression of RAP.In this review, we summarise the pathogenic properties of E. faecalis in the treated complex root canal system. Accumulating evidence has indicated different causative factors associated with E. faecalis pathogenicity. Since biofilm formation facilitates survival and virulence of microorganisms, herein, we discuss the ability of E. faecalis to invade dentinal tubules and form biofilms in adverse environments after RCT.RAP is due, in part, to the long-term survival of E. faecalis contains many virulence factors, such as cytolysin activator (cylA), gelatinase (gelE), extracellular surface protein (esp), aggregation substance (asa), adhesion of collagen (ace), E. faecalis regulator B (fsrB), and endocarditis antigen A (efaA), which are involved in bacterial adhesion, biofilm formation, resistance to killing, and tissue damage [E. faecalis to alkaline root canal medications [e damage . The gene damage ,31–33. Ae damage . Under sications ,36.E. faecalis maintains its biofilm-forming capacity in many ways under stress. In starvation and alkaline environments, the cell-surface hydrophobicity of E. faecalis increased, which is conducive to bacteria adhesion and aggregation [E. faecalis defence against environmental stress [E. faecalis and biofilm formation in the root canals [et al., E. faecalis strain that produced higher levels of gelE and three serine proteases outcompeted Lactobacillus salivarius and Streptococcus gordonii in multispecies biofilms [et al. found that E. faecalis can lower the environmental pH by exporting lactic acid to antagonize P. aeruginosa growth within biofilms under iron-restricted conditions [regation ,36. Undel stress . Moreovel stress . After al stress . Furthert canals . Accordibiofilms . Restricnditions .E. faecalis leads to low-grade chronic inflammation, which may be associated with and RAP. Mathew et al. found that biofilm-derived E. faecalis cells exhibited higher intracellular survival potential and produced lower levels of the pro-inflammatory mediators IL-6 and TNF-α, compared with planktonic cells [In addition to facilitating survival in root canals or periapical tissues, biofilm growth of ic cells . Similaric cells .E. faecalis under harsh root canal conditions, including expression of various virulence genes, alterations in intracellular and extracellular biochemical compositions, and interactions between microorganisms. These findings will inform further research on how to control infections by inhibiting bacterial biofilm formation. Biofilm inhibitors will be the focus of research into RAP preventative approaches. Novel targeted molecule inhibitors have been investigated for endodontic treatment. For example, antisense walR RNA, which interferes with the expression of the two-component signal transduction systems of E. faecalis, reduces virulence gene expression, exopolysaccharide synthesis, and biofilm formation [walR RNA increases bacterial susceptibility to root canal disinfectants and inhibits the pathogenicity of E. faecalis in RAP [In general, a variety of factors influence biofilm formation by ormation ,45. Furts in RAP ,47.E. faecalis is the ability to invade dentinal tubules, making it difficult to completely remove the pathogen, thus leading to reinfection [E. faecalis cells were still able to migrate into dentinal tubules, suggesting that invasion of dentinal tubules by E. faecalis may involve an electrokinetic and osmotic process [E. faecalis is able to form biofilms but has a significantly decreased invasive ability to dentinal tubules, which may be associated with enhanced cell-surface hydrophobicity [Another important pathogenic property of nfection . Researc process ,49. Undehobicity .E. faecalis bacteria residing in dentinal tubules [E. faecalis at the time of root filling can lead to subsequent reinfection [E. faecalis is thought to result from its ability to enter the viable but nonculturable state  when exposed to stressful environment after root canal disinfection, thus becoming insensitive to disinfectants, waiting for the opportunity to restore growth and proliferation, and then causing reinfection [E. faecalis to the VBNC state may direct development of therapeutics that target the chronicity of infections by this bacterium.It has been reported that root canal irrigation with different protocols seemed unable to completely eradicate  tubules ,52. Resinfection . The pernfection . Deeper E. faecalis infection in the pathogenesis of RAP and related mechanisms.Periapical lesions can be regarded as the result of the fight and interactions between pathogenic insults and the local defence system of the periapical tissue. In RAP, pathogenic microorganisms and their toxins inside and outside the root canals continuously attack the periapical tissue, perpetuate local lesions, and delay healing. As research has progressed, investigators have recently focused on the role of host cell responses modulated by E. faecalis in RAP have focused on three aspects, namely, regulated cell death (RCD), macrophage polarisation, and macrophage differentiation, all of which involve inflammatory response  followed by cytokine secretion [Studies on the mechanisms of macrophage immune responses modulated by response . E. faececretion ,67.TablE. faecalis-modulated RCD in macrophages is an important mechanism involved in RAP. The Nomenclature Committee on Cell Death updated the cell death classification in 2018, defining more than 10 RCDs from a molecular mechanism perspective [E. faecalis infection, current studies have suggested that E. faecalis infection can modulate macrophage apoptosis, pyroptosis, necroptosis and autophagy-dependent cell death through different signalling pathways  of 10:1 [E. faecalis infection of bone marrow-derived stem cells prior to their differentiation into macrophages, with an MOI of 1:1 for a prolonged time to mimic chronic infection, decreased apoptotic activity in subsequently differentiated macrophages [E. faecalis proliferation reached a high concentration , CASP-3 transcript and protein expression were significantly upregulated, and macrophage apoptosis was induced [E. faecalis may inhibit apoptosis in macrophages to survive for an extended time and promote infection spread. But the relationship between E. faecalis infection and macrophage apoptosis is so complex that further studies are warranted.Apoptosis is a highly conserved process that includes two pathways, intrinsic and extrinsic, aimed at maintaining tissue homeostasis by eliminating senescent and damaged cells . Caspase of 10:1 . This anrophages . In addi induced . These rE. faecalis induces CASP-1- and NLRP3 inflammasome-dependent macrophage pyroptosis [E. faecalis root canal isolates (CA1 and CA2) and OG1RF strain proliferated to high concentrations, they activated apoptosis, pyroptosis, and necroptosis in macrophages to various degrees, possibly associated with PANoptosis [E. faecalis-infected macrophages may target immune-escaping pathogens [E. faecalis to sustain persistent infections, and activation of PANoptosis may help to more effectively prevent pathogens from evading immune clearance. However, our understanding of E. faecalis-induced PANoptosis is limited. Further studies should determine the specific sensor that recognises E. faecalis and initiates assembly of the PANoptosome. The PANoptosome components and their interaction network are still unclear. Furthermore, an important remaining question is whether and how we can reduce the release of excessive pro-inflammatory cytokines when controlling infections via modulation of PANoptosis.Unlike apoptosis, pyroptosis and necroptosis are two pro-inflammatory necrotic RCDs whose key morphological feature is cell membrane rupture . In termroptosis ,58,61. Wroptosis . Notablyroptosis . A studyE. faecalis can activate macrophage autophagy through Beclin1 and inhibition of the PI3K/Akt/mTOR pathway, which attenuates macrophage-mediated killing [E. faecalis-infected macrophages, and some bacteria escaped from the monolayer membrane vesicles [E. faecalis in macrophages and may be a potential new therapy for the treatment of RAP. However, the specific immune mechanisms and key molecules involved in E. faecalis-modulated autophagy should be identified.Autophagy, as a fundamental cellular degradation mechanism in eukaryotes, is equally important in innate immune defence and pathogenic microbe clearance ,87. Bact killing . Howevervesicles ,59. EnhaM1 and M2 macrophages have opposite polarities and the ability to mediate killing and repair responses, respectively. They coexist in various tissues throughout the body to regulate immune responses. Macrophages produce the killing molecule nitric oxide (NO) and the repair-promoting molecule ornithine by metabolising arginine to initiate M1- and M2-type responses, respectively . M1 macrE. faecalis and macrophages utilizing transposon insertion sequencing (TIS) coupled with transcriptome sequencing. TIS was performed using a transposon insertion mutant library of E. faecalis OG1RF, to comprehensively assess metabolism-related genetic determinants of E. faecalis resistance to macrophage-mediated killing [E. faecalis downregulates carbohydrate metabolism to promote its survival in macrophages. Mechanistically, attenuation of carbohydrate metabolism in E. faecalis decreases the polarisation of macrophages to M1 phenotype to reduce NO production, thus facilitating the resistance of E. faecalis to macrophage-mediated killing [Of note, metabolism plays a key role in macrophage activation and polarisation ,94. A re killing . Analysi killing .E. faecalis induces macrophage polarisation into an atypical M1-like phenotype, which expresses an aberrant cytokine mRNA profile with reduced levels of the pro-inflammatory cytokines IL-1β and IL-12 and increased levels of the immunosuppressive cytokine IL-10. This atypical M1-like phenotype was retained even in the presence of IL-4 and IL-13 [E. faecalis infection undergo M2 polarisation [E. faecalis infection. These studies provide new insights into the mechanisms involved in intracellular survival of E. faecalis.Studies have suggested that M1 macrophages induce production of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-12, and TNF-α, while IL-4, IL-10, and IL-13 polarise macrophages toward the M2 phenotype . Intrigund IL-13 . Furtherrisation . Macrophrisation . AtypicaE. faecalis (HKEF) stimulation downregulated IL-12 and upregulated IL-10, TNF-α, and NO production in the presence of recombinant interferon-γ, without affecting MCP-1, IL-1α, and IL-6 production [E. faecalis, increased expression of TNF-α and NO suggested that E. faecalis virulence factors may contribute to M1 polarisation, triggering pro-inflammatory and antimicrobial responses during the initial stages of infection. Downregulation of IL-12 and upregulation of IL-10 are consistent with the findings by Mohamed et al [E. faecalis.In addition, heat-killed oduction . Since Hed et al . And mayE. faecalis affects macrophage polarisation  signalling pathway [E. faecalis may attenuate the ability of macrophages to differentiate into osteoclasts, thus maintaining their ability to phagocytose pathogens and induce inflammation [E. faecalis may inhibit RANKL-induced osteoclast formation via the transcription factor RBP-J [RAP is essentially a disease of inflammatory bone destruction, which is widely considered as an imbalance of the bone remodelling process regulated by osteoclasts and osteoblasts. Macrophages can differentiate into osteoclasts as osteoclast precursor cells, affecting bone healing in periapical tissues . Osteoclpathways . E. faec pathway . In a ma pathway . Conversammation ,71. LTA or RBP-J ,74.E. faecalis exerts a positive or negative regulatory effect on osteoclastogenesis of macrophages is unclear, according to current studies. These discrepant results may be attributed to the fact that no other immune cells are tested in in-vitro experiments, which cannot mimic the complex environment of the human oral cavity. Immunomodulators, such as IL-6, IL-1, and TNF-α, play a crucial role in the regulation of osteoclastogenesis and bone resorption, and dysregulated or prolonged immune responses often affect bone metabolism [E. faecalis infection is mediated mainly by host cellular immune responses [E. faecalis does not directly promote macrophage differentiation into osteoclasts, but rather that secreted cytokines mediate the bone destruction process. Thus, there is a need to develop cell co-culture models that better mimic the local microenvironment of human periapical tissues, and more advanced assay techniques to explore the underlying mechanisms.Whether tabolism . Based oesponses . It is pE. faecalis on osteoblast RCD and osteoblastic differentiation has also attracted the attention of researchers  via different signalling pathways.Studies on RCD in osteoblasts have indicated that E. faecalis affects osteoblast apoptosis, researchers initially focused on the virulence factor LTA and found that it inhibits proliferation of human osteoblast-like MG63 cells and induces apoptosis [E. faecalis induces apoptosis in MC3T3 osteoblasts by upregulating expression of Bax and CASP-3, and downregulating expression of Bcl-2 [E. faecalis induces and accelerates osteoblast apoptosis in an MOI-dependent and time-dependent manner, activating not only the CASP-8-dependent extrinsic apoptotic pathway, but also the CASP-9-dependent intrinsic apoptotic pathway. Specifically, the intrinsic pathway is time-dependent, whereas the extrinsic pathway is more sensitive to the MOI [E. faecalis promoted osteoblast pyroptosis and apoptosis via the NLRP3 inflammasome, which was inhibited after downregulation of the NLRP3 gene with small interfering RNAs [et al., indicating that E. faecalis may not induce pyroptosis. The results of these two studies are contradictory and require further investigation. New evidence showed that E. faecalis infection induces necroptosis in MG63 cells via the RIPK3/MLKL signalling pathway. Silencing MLKL using inhibitors or short hairpin RNA significantly reduced osteoblast death [E. faecalis may induce PANoptosis in osteoblasts.To explore how poptosis . Later, of Bcl-2 . Further the MOI . In addiing RNAs . Howeverst death . Taken tE. faecalis-infected human calvarial osteoblasts revealed that Bcl-2 family members acted as regulators of osteoblast apoptosis. Therefore, Bcl-2 family members may be potential therapeutic targets for RAP [E. faecalis-induced RAP since it reduces pro-inflammatory cytokine release rather than just inhibits cell death. However, mechanistic studies remain an unmet need, as we are not quite aware of feasible interventions that target the transcription and expression processes of PANoptosis-related genes in osteoblasts. In addition, more in vivo studies should be performed to determine whether inhibition of PANoptosis (or just apoptosis or pyroptosis or necroptosis) contributes to the reduction of osteoblast death and resolution of RAP.Recently, a polymerase chain reaction (PCR) array analysis of 84 apoptosis-related genes in  for RAP . NotablyE. faecalis inhibits the osteogenic differentiation of ovine osteoblast-like cells [E. faecalis on osteogenic differentiation of pre-osteoblasts MC3T3-E1 is mainly dependent on the p38 pathway and partially on the ERK1/2 pathway [E. faecalis infection inhibits osteoblast differentiation. Nevertheless, the understanding of the molecular mechanisms is insufficient, and more in-depth studies may contribute to exploring new therapeutic modalities by integrating the effects of E. faecalis on osteogenesis and the immune response.Bone remodelling is a sequential and orderly process that includes three stages: initiation, reversal, and termination. Osteoblasts play a key role in the reversal phase, where, in addition to inhibiting bone resorption, recruitment and differentiation of osteoblasts ultimately promote bone formation ,112. A ske cells . HKEF doke cells . Further pathway . These fE. faecalis is a pathogen widely used in experimental infection models for research on the underlying pathogenesis of RAP. Interference with E. faecalis-modulated host cell responses may help to treat RAP. In vivo experiments demonstrated that inflammation and bone destruction were alleviated in RIPK3-deficient mice with E. faecalis-induced RAP [E. faecalis or the host should be adopted as adjuncts and how to deliver these agents to the periapical zone with benefits to the patients. In future therapeutic strategies at the molecular level of RAP, further investigation of E. faecalis-modulated host cell responses and relevant signalling pathways will be of paramount importance, as it will lead to clinical translation to achieve the ideal effect.uced RAP . Moreoveuced RAP . The chauced RAP , suggestE. faecalis has been widely recognised as the dominant species associated with RAP; however, the pathogenic mechanism of E. faecalis in RAP remains unclear. E. faecalis resists adverse conditions after RCT by altering the expression of relevant virulence genes and the synthesis and activity of biochemical components to maintain its biofilm formation capacity [E. faecalis, but contributed to our understanding of host cell responses modulated by E. faecalis. To date, efforts have been made to elucidate the modulatory effects of E. faecalis on macrophages and osteoblasts. However, there is very little research on its regulatory effects and mechanisms on other immune cells, such as dendritic cells, lymphocytes, and neutrophils. As immune response is a complicated process that appears as a series of tissue reactions, different types of immune cells should be considered when exploring the pathogenic mechanisms of E. faecalis infection.More recently, capacity . With adE. faecalis resists autophagy to promote intracellular survival in macrophages and inhibits macrophage apoptosis at low bacterial concentrations [E. faecalis induces macrophage PANoptosis and promotes an inflammatory response [E. faecalis may induce PANoptosis in osteoblasts [E. faecalis-modulated RCD in key cells should focus on the following: (i) Given the crucial role PANoptosis plays during infection, further investigation should shed some light on ways to modulate it for the promotion of pathogen removal or reduction of osteoblast death with minimised inflammatory pathology. Therefore, it is necessary to reveal specific mechanisms of PANoptosis activation in immune cells and osteoblasts, as well as the crosstalk with other pathways. (ii) Autophagy is a form of cellular self-degradation that rarely induces inflammation. It is awaited to investigate potential therapeutic targets to clear E. faecalis from the perspective of the molecular mechanisms of autophagy. (iii) Are there other types of RCD modulated by E. faecalis that play a dominant role?Previous studies have revealed that trations ,60. Whenresponse . Additioeoblasts ,104. FutE. faecalis infection are polarised to an M2 or atypical M1 phenotype, causing persistent bacterial infection and chronic RAP [E. faecalis regulates macrophage polarisation will help to tune macrophage polarisation states, thereby removing pathogens early during infection.Macrophages that remain viable after onic RAP ,65. M1 ponic RAP . Hence, E. faecalis infection, as well as the progression of inflammation and tissue injury. However, it is not yet clear which virulence factors/genes are involved in the induction of host cell responses by E. faecalis. Conceivably, there are uncharacterised virulence factors/genes related to host-pathogen dynamics [E. faecalis interactions.The expression of virulence genes can lead to a spectrum of events favouring the development or maintenance of dynamics . ProspecE. faecalis and its host are never-ending. E. faecalis can develop new strategies to resist host defence mechanisms. A more in-depth study of the pathogenicity of microorganisms and the host responses they modulate is pivotal for detecting and completely removing pathogens from root canals, identifying potential therapeutic targets, controlling infection, and preventing the occurrence of RAP.Microbial factors, immune regulation, and bone homeostasis interact with each other and their complex associations make it challenging to precisely unravel the pathogenetic mechanisms involved in RAP ,118–120.


A common bioinformatics task in single-cell data analysis is to purify a cell type or cell population of interest from heterogeneous datasets. Here, we present scGate, an algorithm that automatizes marker-based purification of specific cell populations, without requiring training data or reference gene expression profiles. scGate purifies a cell population of interest using a set of markers organized in a hierarchical structure, akin to gating strategies employed in flow cytometry. scGate outperforms state-of-the-art single-cell classifiers and it can be applied to multiple modalities of single-cell data . scGate is implemented as an R package and integrated with the Seurat framework, providing an intuitive tool to isolate cell populations of interest from heterogeneous single-cell datasets.https://github.com/carmonalab/scGate (https://doi.org/10.5281/zenodo.6202614). Several reproducible workflows describing the main functions and usage of the package on different single-cell modalities, as well as the code to reproduce the benchmark, can be found at https://github.com/carmonalab/scGate.demo (https://doi.org/10.5281/zenodo.6202585) and https://github.com/carmonalab/scGate.benchmark. Test data are available at https://doi.org/10.6084/m9.figshare.16826071.scGate is available as an R package at Bioinformatics online. Single-cell RNA sequencing (scRNA-seq) is becoming increasingly popular, enabling high-throughput exploration of cell types and states from complex tissues. Cell types are generally defined based on biological function and the markers used to physically isolate them, but these can change depending on the source tissue. In scRNA-seq data analysis, knowledge of cell type-defining marker genes is typically used to manually identify relevant cell populations within custom bioinformatics workflows, requiring several steps and parameters.Alternatively, when high-quality transcriptomic profiles are available for the cell type of interest, training multinomial machine learning classifiers to predict cell type identity has been shown to be a powerful approach  alone allows purifying B cells with 99.7% precision and 99.7% recall  cells with 100% precision and 95% recall  a gene expression matrix or Seurat object and (ii) a ‘gating model’ (GM), consisting of a set of marker genes that define the cell population of interest. The GM can be as simple as a single marker gene, or a combination of positive and negative markers. For example, the marker % recall . More co% recall  by defind FCGR1A . Our algd FCGR1A , to annoFoxi+ Cftr+) was sufficient to accurately isolate rare pulmonary ionocytes )scGate can also be applied to single-cell modalities other than RNA-seq. On a multi-omics scATAC + scRNA-seq dataset of human peripheral blood mononuclear cells (PBMCs), scGate was able to successfully isolate CD14+ monocytes, T cells, NK cells and B cells from DNA accessibility data  and higher Matthews correlation coefficient (MCC) than SingleR .ity data . In a coity data . scGate ity data . When coMultiple predefined scGate models are provided in a public repository as version-controlled tab-separated text, allowing scGate to automatically synchronize its internal database of GMs. Users can manually edit these models and easily write their own. scGate also provides functions to evaluate the performance of custom GMs, either user-provided or those that accompany the package, on a set of pre-annotated testing datasets. Overall, scGate provides an accurate, scalable and intuitive tool to isolate cell populations of interest that can be seamlessly incorporated into Seurat pipelines for single-cell data analysis.This work was supported by the Swiss National Science Foundation (SNF) Ambizione [180010 to S.J.C.]. A.J.B. was supported by the National Scientific and Technical Research Council of Argentina (CONICET).Conflict of Interest: none declared.btac141_Supplementary_DataClick here for additional data file.


KRAS, HRAS and NRAS oncogenes belong toa family of 40 highly homologous genes, which in turn are a subset of asuperfamily of >160 genes encoding small GTPases. RAS oncoproteinsconsist of a globular G-domain (aa1-166) and a 22-23aa unstructuredhypervariable region (HVR) that mediates membrane targeting. The evolutionarilyorigins of the RAS isoforms, their HVRs and alternative splicing of theKRAS locus has not been explored. We found thatKRAS is basal to the oncogene family and its duplicationgenerated HRAS in the common ancestor of vertebrates. In asecond round of duplication HRAS generated NRAS andKRAS generated an additional RAS gene we havedesignated KRASBL, absent in mammals and birds. KRAS4A arosethrough a duplication and insertion of the 4th exon ofNRAS into the 3rd intron ofKRAS. We found evolutionarily conservation of a shortpolybasic region (PBR1) in HRAS, NRAS and KRAS4A, a second polybasic region(PBR2) in KRAS4A, two neutralized basic residues (NB) and a serine in KRAS4B andKRASBL, and a modification of the CaaX motif in vertebrates with farnesyl ratherthan geranylgeranyl polyisoprene lipids, suggesting that a less hydrophobicmembrane anchor is critical to RAS oncoprotein function. The persistence of fourRAS isoforms through >400 MY of evolution argues strongly fordifferential function. RAS oncoproteins, HRAS, NRAS, KRAS4B and KRAS4A have been extensively studieddue to their frequent activation in human tumors , 2. OncoAt the sequence level, RAS oncoproteins consist of two functionally distinctregions: (i) catalytic GTP/GDP binding domains (G-domain) comprising residues 1-166,which are very similar between isoforms  and fold into a well-characterized protein domains that actsas binary molecular switches, and (ii) an intrinsically disordered C-terminalhypervariable region (HVR) of 22-23 residues that anchors the GTPases to cellularmembranes , 5. G-doDespite these high degrees of homology, the functions of the isoforms do notentirely overlap . WhereasRAS family of smallGTPases, which in humans comprise 40 members . Un. UnKRAS RAS oncogene diversification,prompted us to study the specific evolution of the RAS oncoproteins, in particularthe origins of the HVRs and of the KRAS splice variants. We findthat KRAS is primordial and gave rise to HRASthrough a gene duplication >600 MYA, that NRAS is aduplication of HRAS and that KRAS4A arose 475 MYA with theevolution in the common ancestor of jawed vertebrates (cartilaginous fish and bonyvertebrates) not by gene duplication but rather by capture into the third intron ofthe KRAS locus of exon 4 of NRAS. Despite highlyoverlapping regulation and function, the persistence of four RAS isoforms withunique membrane-targeting sequences for >400 MY of evolution suggestsimportant and distinct functions.These findings and the availability of newly sequenced genomes of basalvertebrates instrumental in tracking RAS oncogene evolution we took advantage ofsequenced genomes of lower vertebrate species near evolutionary branchingpoints, such as the jawless fishes lampreys  and hagfish (E. burgeri), which arebasal to vertebrates, elephant shark (C. milii) basal tocartilaginous fishes, and reedfish  basal tobony fishes (D. discoideum) to mammals (H.sapiens). We did not find orthologous RAS oncoprotein sequences inthe closest relatives of vertebrates, urochordates/tunicates  and in bikonts/diphoda [To study y fishes  21-24].-24.RAS oeolates). For seqSequences related to each one of the human oncoproteins HRAS, NRAS, andsplicing variants KRAS4B and KRAS4A were found in all vertebrates except mostbasal jawless fishes (lampreys and hagfish) that only have HRAS and KRAS4B. In addiBranchiostoma)genes we have designated lancelet1 and lancelet2 in cephalochordates and theyeast S. cerevisiae genes RAS1 and RAS2 in fungi (S.cerevisiae . On. On26]. eQYM) and in single celled choanoflagellatesS. rosseta (AGQEdYSAMRDQYM) , appear oncoprotein-specific sincethey are 100% identical in all representative sequences gathered for this study,as well as in all additional sequences we could retrieve by BLAST searching NCBIprotein databases . LikewisAMRDQYM)  . In comgAMReQYM.Thus, tThe primary association of RAS isoforms with cellular membranes as wellas trafficking between membrane compartments and steady-state subcellularlocalization is specified by post-translational modifications of cysteine andserine residues and by the presence of polybasic regions in the HVR  4, 5]. , 5. 4, 5Caenorhabditis genus . A singlhras and kras) in jawless fishes the mostbasal extant vertebrates, and four  in jawed vertebrates , and kras andhras evolve distinct HVR vertebrate characteristics: thelocation of a phosphorylation acceptor residue (T/S) tree amino acids upstreamof the CaaX motif in KRAS and, the formation of PBR1 instead of the NB motif,and the acquisition of two additional cysteines instead of the PBD motif in HRAS while hras genes are paralogous(evolved by duplication) , lrrc56 disappeared from the nrasgenomic area while ampd1 was maintained after the duplication evolved by speciation to formtebrates .During  in HRAS. The facication) . Moreoveequences . Interesmic area. Howeverdatabase , suggestlication further hrasgenerated nras, and kras generatedkrasBL , dkrasBL . Inagreand nras; (ii) phequences ; and , the second round of WGD took place by a genomeduplication following interspecific hybridization  fromtwo now extinct progenitor species that fuse their genomes in the commonancestor of jawed vertebrates . Inthisuncitons .KRAS by mutually exclusivealternative splicing, a tightly regulated process  three residues upstream of the CaaX motif, suggestingthat modulation of the negative charge by phosphorylation is functionally important.Importantly, we found that, with the exception of HRAS in cartilaginous fish andKRASBL, all RAS oncogene orthologs have a CaaX motif that signals for farnesylationrather than geranylgeranylation. In contrast, the majority of RAS superfamily smallGTPases as well as orthologs of RAS oncoproteins prior to jawless fishes have CaaXmotifs that signal for geranylgeranylation. Thus, it appears that diminishing thehydrophobicity of the prenyl lipid that modifies RAS oncogenes was essential fortheir function. This may explain why KRAS4BL was lost from mammals and birds. Thissuggests that the modulation of the membrane affinity of farnesylated RASoncoproteins by palmitoylation/depalmitoylation or phosphorylation/dephosphorylationwas an important adaptation for vertebrate oncoproteins.Although evolution of the RAS superfamily of small GTPases has been thesubject of several analyses, the focus has been on the G domains shedding littlelight on the origins of the multiple RAS oncogene products themselves. Rasoncoproteins are composed of two distinct domains, a guanine-nucleotide bindingdomain (G domain) with a highly conserved structure and an unstructured HVR. Theexpanded availability of sequenced genomes of basal vertebrates has allowed us toanalyze the evolution of the HVRs and obtain insight into the alternative splicingof the KRAS is primordial and gave riseto HRAS by gene duplication 615 MYA, which in turn generatedNRAS by another duplication event. In contrast, KRAS4A evolvednot by gene duplication but rather by incorporation of exon 4 ofNRAS into the 3rd intron of the KRAS locus 475MYA. RAS oncogenes have been the focus of intense investigation by cancer biologistsfor more than 40 years because of the prevalence of oncogenic RAS mutations in humancancer. Through most of those decades RAS proteins were thought to be functionallyinterchangeable. The persistence of the four RAS isoforms through >400 MY ofevolution argues strongly against this view and suggests unique functions driven bythe HVRs, including the HVRs of the splice variants of the KRASlocus. This revised view is consistent with recent analysis of tumors driven byoncogenic RAS mutations where differences are observed not only among the isoformsbut also among different mutations of the same isoform [Our analysis suggests that  isoform , 49. Tarhttps://www.ncbi.nlm.nih.gov). Hagfish and pacific lampreysequences were retrieved from Ensembl (http://www.ensembl.org/) and SIMRBASE (https://genomes.stowers.org) databases,respectively. Whole sequences, organisms’ scientific names,identification numbers, and individual sequence details are provided in This paper analyzes existing, publicly available data. We collected theprotein and DNA sequences used in this study from online genomic, proteomic andnucleotide NCBI databases  [http://smart.embl-heidelberg.de/) [http://www.uniprot.org/help/uniprotkb) [Alignments of protein and DNA sequences were performed using the MAFFTserver (server/) . The SMAerg.de/)  was usederg.de/)  with theerg.de/) . Alternaiprotkb) .https://itol.embl.de/) [https://www.phylogeny.fr/) [All amino acid and nucleotide sequences used in the phylogeneticanalyses are provided in mbl.de/) . The proeny.fr/) .Suppl. File 1Suppl. File 1. List of all sequences, accessionnumbers, and species scientific names used in this study. Exons arerepresented with alternate black and blue colors. Amino acids in red lettersmeans they are split between two exons. G-domains are underlined.Suppl. Fig 1Suppl. Fig. 1. Lancelet2 has distinct gene structure and HVRcharacteristics. Clustalw MAFFT alignment of lancelet1 andlancelet2 cephalochordate sequences. Exons are depicted in alternate blackand blue letters. Whereas lancelet1 has homolog introns a-c like vertebrateoncogenes and intron d like other invertebrates, lancelet2 has a singleintron (indicated by a red arrow). The dicysteine motif that includes theCaaX cysteine of Lancelet2 (highlighted in orange) is also different fromother oncogenes. Amino acid identity: identical (*); strongly similar (:);weakly similar (.).Suppl. Fig 2Suppl. Fig. 2. Eukaryotic G-domain sequences are highlysimilar. The alignment of the full-length G-domain oncoproteinsequences from amoebas to humans was generated by the MAFFT server withdefault parameters. Upper panel effector lobe (residues 1-86) and lowerpanel allosteric lobe (residues 87-166). Switch-regions I and II (greenboxes) and GTP/GDP binding elements (dashed pink boxes). Arrows andrectangles above the sequences α-helices and β-sheets,respectively. Amino acid identity: identical (*); strongly similar (:);weakly similar (.).Suppl. Fig 3Suppl. Fig. 3. Eukaryotic G-domains pairwise amino acidsidentity. Heat map representing amino acids Blastp pairwiseidentities in comparison with human oncoproteins. Numerical values aredepicted inside the cells, which are colored from green (low similarity) tored (high similarity).Suppl. Fig 4Suppl. Fig. 4. Evolutionary relationships between eukaryoticG-domain sequences. (A) DNA sequences. (B) Protein sequences.Evolutionary relationships between DNA and protein sequences correspondingexclusively to G-domains, where analyzed using the tools on the Phylogeny.frweb site [52. Bootstrap values are indicated below tree brancheswith value at group branch points boxed in red. G-domain sequences fromRRAS, MRAS and TC21 which are the closest members to classical oncoproteinsin the RAS family, where used as outgroup [web site , MUSCLE web site , 63, 65.outgroup .Suppl. Fig 5Suppl. Fig. 5. Oncoprotein specific signature conservation ineukaryotes. Protein Blast searches in NCBI protein eukaryoticdatabases were performed in Blastp with default parameters using as a baitthe oncoprotein specific signature(EYDPTIEDSYRKxxxxxxxxxxxxxxxxAGQEEYSAMRDQYM) corresponding to the effectordomain. Single non identical amino acids in one choanoflagellate species(S. rosseta) and in all Fungi are indicated with blackrectangles.Suppl. Fig 6Suppl. Fig. 6. The CaaX motifs of oncoproteins. Whereasthe sequences predict geranylgeranylation signal in KRASBL, HRAS from cartilaginous fishes, and in most non-vertebrate groupsthey predict farnesylation for all vertebrate oncoproteins since jawed fishother than KRASBL that is not found in birds and mammals. CaaX terminalresidues from BLASTP searches are shown as multiple alignment outputs.Suppl. Fig 7Suppl. Fig. 7. Conservation of oncoproteins genomicarchitecture (synteny) from humans to lancelets. The figuredepicts Ras oncogene genomic regions from cephalochordatesto mammals. Genes are depicted as arrows pointing to the direction oftranscription. Evolutionarily conserved solitary genes (hashed), duplicatedgenes (solid), and non-conserved (outline) are shown. Genomic coordinatesare indicated below each species name. GenBank assembly accession numbersare human, GCF_000001405.39; lizard, GCF_009819535.1; frog, GCF_000004195.4;coelacanth, GCF_000225785.1; reedfish, GCF_900747795.1; skate,GCF_010909765.1; elephant shark, GCF_018977255.1; hagfish, GCA_900186335.2;lamprey, GCF_010993605.1; and lancelet, GCF_000003815.2.Suppl. Fig 8KRAS Exon 4A invertebrates.Suppl. Fig. 8. Appearance of  Genomic regions of shark, hagfish and lampreyshowing the locations of exons 4B (blue letters) and 4A (green letters).Stop codons are highlighted in red. Genomic regions: shark NW_006890288.1(547536..553042); hagfish, Eburgeri 3.2: FYBX02009586.1: 2433624:2434677:1;lamprey Chr. 65: NC_046133.1, 5454894 to 5455895. GenBank assembly accessionnumbers: shark, GCF_000165045.1; hagfish GCA_900186335.2; lamprey,GCF_010993605.1.Suppl. Fig 9Suppl. Fig. 9. Alternative carboxyl terminal splicing variantsin the Ras superfamily. NCBI sequences with exons in alternatecolors, and screen captures of the genomic gene structures with the splicedexons indicated by arrows. Time of appearance of isoforms was deduced fromNCBI protein BLAST searches. Isoforms as described in the Uniprot serverthat are produced by alternative splicing affecting the carboxyl terminalregion are shown in the box.


Burn-out is quite common in hospitals especially among young doctors. It results from a mismatch between expectation and professional reality.To determine the prevalence of sever burnout and to identify its associated socio-demographic and psychological factors among young residents.Analytical and descriptive cross-sectional study conducted among residents and interns working at the Hedi Chaker and Hbib Bourguiba University hospital in Sfax, Tunisia, during the month of July 2019. The characteristics of the participants were collected using a questionnaire. Burnout was evaluated using the Maslach Burnout Inventory(MBI) differentiating 3 components: emotional exhaustion, depersonalization and luck of personal achievement.Out of 85 questionnaires disturbed,60 were selected corresponding to a response rate of 72.94%. The sex ratio (M/F) was 0.87. The middle age was 28.22. Forty three percent of the participants were married. More than half consumed tobacco and 45% of them consumed alcohol. The majority of doctors were residents(81.7%). The average working time was 55 hours per week. Burn-out was severe in 30% of our population. Furthermore, doctors who suffered from physical aggression(p=0.001) were more likely to develop severe burn-out. The dissatisfaction with the internship (p=0.01) and the feeling of do not satisfy seniors(p=0.02) were statistically associated with severe burnout. Severe burn-out was associated with anxiety (p=0.0073), conflictual partnership (p=0.0001),conflicts with colleagues(p=0.001) and the paramedical framework (p=0.0001)The risk of burn-out is quite high among young doctors. Some factors seem to be associated with this phenomenon. This could affect not only the quality of life, but also the quality of care provided.burnout-young doctors-prevalence-associated factors


Terminalia myriocarpa Vaniot Huerck et Muell.-Arg is a tropical rainforest indicator species in Southern China. The chloroplast genome of T. myriocarpa was analyzed by high-throughput sequence technology, and its genetic relationship to related species was discussed. The chloroplast genome is 159,854 bp in length, with a total GC content of 37%. It has a typical chloroplast tetrad structure, including 88,015 bp of large single copy (LSC), 18,814 bp of small single copy (SSC), and 26,319 bp of inverted repeats (IR). A total of 130 genes were annotated, including 85 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. Phylogenetic analysis indicated T. myriocarpa was closely related to Terminalia phillyreifolia. Terminalia myriocarpa Vaniot Huerck et Muell.-Arg(1870) is an evergreen or semi-evergreen tree belonging to the genus Terminalia of Combretaceae family. It is mainly distributed in the Southern Guangxi Zhuang Autonomous Region, South Central in Yunnan Province, and Southern Tibet Autonomous Region of China  Red List of threatened species . The collection of plant materials was in accordance with local regulations and obtained the permission of local authorities. A voucher specimen (SWFU20210721MFY) was deposited in the Herbarium of Southwest Forestry University, China . Total genomic DNA was extracted from silica gel dried leaf tissues using a modified CTAB method . The complete plastid genome of T. myriocarpa was submitted to GenBank with accession number OM202511.Fresh leaves of T. myriocarpa is 159,467 bp in length with a typical double-stranded circular tetrad structure, containing a large single-copy (LSC) region of 88,015 bp, a small single-copy (SSC) region of 18,814 bp, and a pair of inverted repeat (IR) regions of 26,319 bp each. The overall GC content of the whole genome is 37%. GC content in the IR region (43.02%) was higher than that in the LSC region (34.80%) and SSC region (30.65%). In total, 130 genes were annotated in the plastome, including 85 protein-coding genes (PCGs), 8 ribosomal RNA genes (rRNAs), and 37 transfer RNA genes (tRNAs). A total of 101 SSRs were discovered by the online software MISA-web . The phylogenetic analysis revealed that all species of Combretaceae formed one clade. The phylogenetic tree showed that Terminalia phillyreifolia was sister to T. myriocarpa with strongly supported under current sampling (T. myriocarpa will be used for population genomics research, phylogenetic analysis, and genetic engineering research, which will contribute to the better development and utilization of this species.The phylogenetic tree was reconstructed based on chloroplast genome sequences of sampling . The comL. L. D. conceived the study; X. Y. collected the molecular materials and drafted the manuscript; Z. N. Z. and H. L. P. analyzed the experimental data; L. L. D. revised the manuscript. All authors provided comments and final approval.


Pseudoalteromonas agarivorans Hao 2018 (P. agarivorans Hao 2018) were investigated through pan-genomic and transcriptomic analyses, and key enzyme genes that may encode the process involved in its extracellular polysaccharide synthesis were screened. The pan-genome of the P. agarivorans strains consists of a core-genome containing 2331 genes, an accessory-genome containing 956 genes, and a unique-genome containing 1519 genes. Clusters of Orthologous Groups analyses showed that P. agarivorans harbors strain-specifically diverse metabolisms, probably representing high evolutionary genome changes. The Kyoto Encyclopedia of Genes and Genomes and reconstructed carbohydrate metabolic pathways displayed that P. agarivorans strains can utilize a variety of carbohydrates, such as d-glucose, d-fructose, and d-lactose. Analyses of differentially expressed genes showed that compared with the stationary phase (24 h), strain P. agarivorans Hao 2018 had upregulated expression of genes related to the synthesis of extracellular polysaccharides in the logarithmic growth phase (2 h), and that the expression of these genes affected extracellular polysaccharide transport, nucleotide sugar synthesis, and glycosyltransferase synthesis. This is the first investigation of the genomic and metabolic features of P. agarivorans through pan-genomic and transcriptomic analyses, and these intriguing discoveries provide the possibility to produce novel marine drug lead compounds with high biological activity.The genomic and carbohydrate metabolic features of  Pseudoalteromonas agarivorans is a gram-negative bacterium, obligate marine microorganism, and agar degrader that is motile, flagellated, and able to utilize organic matter as an energy source. The name Pseudoalteromonas was first proposed in 1995, following a reclassification from Alteromonas based on taxonomic analysis [P. agarivorans from seawater samples around the globe [P. agarivorans was first proposed in 2003 according to 16S ribosomal DNA sequencing analysis, DNA-DNA hybridization (DDH), and phylogenetic identification [T (DSM 14585T).he globe ,3. The nP. agarivorans are currently available, due to the specificity of the marine environment, they can secrete a variety of extracellular active substances, such as exopolysaccharides (EPS), antibiotics, and extracellular enzymes, which helps P. agarivorans to absorb nutrients and compete for survival [P. agarivorans established its own complex marine niche using secreted EPS for antioxidant production [O-specific polysaccharides of P. agarivorans KMM232 (R type) [T [O-specific lipopolysaccharides, which had different configurations compared to the extracellular polysaccharides (EPSs) secreted by the R-type and S-type terrestrial bacteria, and this variability reflects the adaptability of P. agarivorans to environmental changes. In the previous study, it was shown that the P. agarivorans Hao 2018 strain was a high-yield producer of EPS and had high biological activity in terms of anti-oxidation properties, moisture absorption and moisturization, adsorption of heavy metal ions, and in vitro flocculation [P. agarivorans Hao 2018 bacterium were methylated at low temperatures, which was beneficial for the expression of the glycosyltransferase gene in this bacterium [P. agarivorans Hao 2018, ultimately leading to changes in the biological activity of the EPS.Although few reports that characterize survival ,5. Also,oduction , protectoduction , flocculoduction , and cheoduction . These soduction . Komandr(R type)  and KMM2type) [T  in 2010 culation ,14. Furtacterium ,16. In aPseudoalteromonas haloplanktis TAC125, and when the solubility of oxygen increases at low temperatures, it responds by multiplying dioxygen scavenging while deleting the whole pathways that produce reactive oxygen species, thereby adapting to the low-temperature environment. Choudhury et al. [P. agarivorans NW 4327 strain using 16S ribosomal RNA (rRNA), gyrase B gene sequences, DDH, and in silico genome comparisons. To the best of our knowledge, the genomic and carbohydrate metabolic features of P. agarivorans strains have been rarely explored, although they are widespread in the marine environment. Furthermore, John et al. [Marinomonas strain isolated from the Antarctic psychrophilic ciliate Euplotes focardii that could be an alternative to conventional antibiotics. In this study, metabolic features were analyzed by phylogenetic and pan- genome analyses of P. agarivorans. The genomic profiles used were closely related to the P. agarivorans strains and were obtained from GenBank. We constructed the carbohydrate metabolic pathway of the bacteria by transcriptome analysis. In addition, we analyzed EPS synthesis at different nodes of time (pre-logarithmic growth phase and early stationary phase). In conclusion, this study helps to understand the phylogenetic diversity of P. agarivorans Hao 2018 and the regulation of its carbohydrate metabolic pathways.Using only one genome as a template for analyses does not provide complete genetic information about a species at the genetic level, especially when studying different subspecies or variants of the same species, in which the differences in unique genomes are typically more important than differences in core genomes. Therefore, the pan-genome approach is particularly significant. The pan-genome approach was proposed in 2005 by Tettelin et al. and has been applied in several fields because of its ability to interpret the genomic features of bacterial lineages as well as to analyze genome evolution. Pan-genome analysis with transcriptome analysis has helped to efficiently study the regulation of metabolic pathways and their applications ,18,19,20y et al.  identifin et al.  also useT, strain Hao 2018, and the closely related strains were used to build a phylogenetic tree . At present, the NCBI database has complete genome sequences for two strains (P. agarivorans DSM 14585T and P. agarivorans Hao 2018). The genome of P. agarivorans DSM 14585T consisted of 2 chromosomes, whereas the P. agarivorans Hao 2018 genome consisted of a 0.12 Mb plasmid in addition to the 2 chromosomes [16S rRNA gene sequences of strain DSM 14585tic tree . The criomosomes .P. agarivorans DSM 14585T, P. agarivorans Hao 2018, P. agarivorans NW 4327, P. agarivorans S816, P. atlantica ECSMB14104, P. sp. P1–11, P. sp. P1–30, P. sp. TB51, P. espejiana DSM 9414T, and P. atlantica T6c were compared. The list of their general features is summarized in Pseudoalteromonas genomes. According to the ANI analysis results  of all other strains were higher than the recommended ANI cutoff values (95–96%), indicating that they belonged to the same species [T and T6c strains were significantly lower compared to the in silico DDH values of all other strains (82–85.1%) as well as lower than the 70% DDH cutoff values. DDH values above the 70% threshold determined that the above two strains belonged to different species, while the remaining eight strains can be classified as P. agarivorans with the type strain DSM14585.The genome sequences of  results , ANI val species ,26,27. SP. atlantica ECSMB14104 was the smallest (3.66 Mb) while the genome of P. sp. TB51 was the largest (4.63 Mb). The G + C content of these strains ranged from 40.8 to 41.0%. However, the genome of P. agarivorans NW 4327 and P. sp. TB51 contained numerous pseudogenes with frame shifts, most likely due to incomplete genes by numerous contigs or high rates of sequencing error, and was therefore excluded from the next pan-genome analysis of Pseudoalteromonas strains.CheckM software was used to evaluate genome sequencing quality. The results are shown in Pseudoalteromonas species increased with an α value of 0.20591, suggesting that may be an open pan-genome that has experienced frequent evolutionary changes and has been subject to effective environmental adaptation through gene gains and losses or lateral gene transfer. As shown in Pseudoalteromonas strains contained a total of 5625 genes, including 2331 genes in the core-genome, 819 genes in the soft core-genome, 956 genes in the accessory genome, and 1519 genes in the unique genome. Unique genes that differ among Pseudoalteromonas strains, possibly reflect the different ecological niches (as residents of different geographic locations) and needs for the survival of Pseudoalteromonas strains [A pan-genome analysis of the eight strains listed in  strains .P. agarivorans is a new type of marine bacterium, so a comparison with similar species could help predict its possible life activities. In order to study the metabolic and functional characteristics of P. agarivorans, we analyzed the COG distributions of the pan-genome and core-genome in this species  to stable growth (24 h)) of P. agarivorans Hao 2018, using the genome as a reference background for the differential expression analysis of genes, we found that the expression of the gene encoding glucokinase was higher in the logarithmic growth phase (2 h) than in the stable phase (24 h). This led to a higher production of glucose-6-phosphate in cells, which could be used to synthesize EPS. Fructokinase gene expression was downregulated, indicating that the intracellular glucose and fructose conversion rates had decreased. At the same time, D-mannitol dehydrogenase gene expression was downregulated, and the conversion rate from fructose to mannose was reduced, which was consistent with the low mannose content of the EPS. The pyruvate pathway is linked to EPS production, and pyruvate carboxylase in the anaplerotic reaction provided intermediates for EPS synthesis [In this study, the pan-genomic analysis shows higher gene abundance of unique genes than core and accessory genes encoding cell wall/membrane/envelope biogenesis in pathways . As showynthesis ,32. Our P. agarivorans Hao 2018, and several of them showed differences in expression values between the 2 h and 24 h cultures , the expression of genes encoding glucokinase and phosphoglucomutase showed an upregulated trend, which may provide more glucose-1-phosphate for the synthesis of extracellular polysaccharides. Interestingly, the expression of the gene encoding rhamnosyl transferase was higher during the logarithmic growth period (2 h) than during the stable period (24 h), and whether this causes differences in the monosaccharide composition of extracellular polysaccharides between the two periods requires further verification. The results provide a new theoretical basis for further studying the metabolic regulation mechanism in response to changes in environmental factors. At the same time, we screened out some of the differentially expressed genes involved in the synthesis of EPS by analyzing the metabolic pathways of the bacterium, which provided inspiration for the targeted production of extracellular polysaccharides with good biological activity.In addition, the analysis of differentially expressed genes showed that a total of 2277 genes were differentially expressed, of which 1189 genes were upregulated and 1088 genes were downregulated. When the strain Strain Hao 2018 was isolated from the surface of abalone seedlings in Shandong Province , China . GenomicP. agarivorans, the genomes of all strains with more than 98.0% 16S rRNA gene sequence similarities to strain DSM 14585T were retrieved from GenBank. More specifically, the ten genomic sequences of strains P. agarivorans DSM 14585T, P. agarivorans NW 4327, P. agarivorans S816, P. atlantica ECSMB14104, P. sp. P1–11, P. sp. P1–30, P. sp. TB51, P. espejiana DSM 9414T, and P. atlantica T6c used in this study were obtained from GenBank.To collect all publicly available genomes putatively belonging to P. agarivorans, the ANI and in silico DDH values as well as the genome sequence-based phylogenetic relationship comparisons were calculated for strains presumed to belong to P. agarivorans and strains belonging to the other Pseudoalteromonas species using the stand-alone program [P. agarivorans DSM 14585T, P. agarivorans Hao 2018, P. agarivorans NW 4327, P. agarivorans S816, P. atlantica ECSMB14104, P. sp. P1–11, P. sp. P1–30, P. sp. TB51, P. espejiana DSM 9414T, and P. atlantica T6c were investigated primarily using the National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Automatic Annotation Pipeline for auto-annotation  and the protein-encoding genes of n Server . After tn Server .P. agarivorans Hao 2018 strain cultured in a medium containing 45 g/L glucose, 2.5 g/L (NH4)2SO4, and 45 g/L bay salt (pH 7.6–7.8). Fermented cells were harvested by centrifugation at 10,000× g for 5 min at 4 °C. Total RNA from the cells was extracted using a TransZol Kit according to the manufacturer’s instructions . RNA quality was evaluated using NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer. The rRNA was removed by Ribo-Zero rRNA Magnetic Kit, and the mRNA was processed using Truseq TM RNA sample prep Kit. Random primers and SuperScript III reverse transcriptase were used to make the first strand of cDNA, and dUTP instead of dTTP was used to synthesize the second strand. The second strand of cDNA with adaptor junction was digested by uracil-N-glycosylase, and then the library fragments were enriched by PCR and sequenced by Agilent 2100 Bioanalyzer on an Illumina HiSeq 2500 sequencing platform. Mapping of the mRNA sequencing reads against all coding sequences (CDS) of strain Hao 2018 was performed using Burrows-Wheeler Aligner software [Following the general rules of biosynthesis, transcriptome sequencing was carried out by selecting the 2 h time node of the exponential phase and the 24 h time node of the stationary phase for the software  based onhttp://bowtie-bio.sourceforge.net/index.shtml, accessed on 5 May 2018) to analyze the distribution of coverage of sequenced reads on the genes. Read count values on each gene were counted by HTSeq 0.6.1p2 [https://htseq.readthedocs.io/en/master/, accessed on 5 May 2018) as raw expression. RPKM (Reads Per Kilobase per Million mapped reads) was used to normalize the expression volume, making the gene expression levels comparable between samples. Expression pattern correlations of genes between samples were assessed and principal component analysis (PCA) was performed using the DESeq2 package [p-value < 0.05, and the screening results were visualized using the graphical visualization package ggplot2. The calculation of p-values for GO terms with significant enrichment (p < 0.05) of differential expressed genes for each mapping to the Gene Ontology (http://geneontology.org/ were accessed on 5 May 2018) database using the hypergeometric distribution method in the context of the whole genome of P. agarivorans Hao 2018 [The Cutadapt (Version 1.2.1) software was used to filter the raw data for reads containing joints and low quality, allowing 20% base mismatches while removing reads with average quality scores below Q20 . The fil 0.6.1p2  (https:/ package  based on package  with theHao 2018 .P. agarivorans. The pan- and core-genomic analyses found that it had an open genome that evolved to adapt to various environmental changes. The COG, KEGG, and BLAST analysis results show that P. agarivorans strains have promising carbohydrate fermentation characteristics. In addition, we screened genes encoding key enzymes that may be related to extracellular polysaccharide synthesis by differentially expressed genes analysis. In summary, our study provided information regarding the phylogenetic characteristics and metabolic fermentation pathways of the P. agarivorans strains. Further analysis of the genomic, transcriptomic and metabolic characteristics of P. agarivorans Hao 2018 might reveal its carbohydrate fermentation properties and its EPS synthesis pathway, providing a reference for the development of extracellular polysaccharide products with promising applications.In this study, we analyzed the phylogenetic and genomic diversity of