Many prokaryotes encode CRISPR-Cas systems as immune protection against mobile genetic elements (MGEs), yet a number of MGEs also harbor CRISPR-Cas components. With a few exceptions, CRISPR-Cas loci encoded on MGEs are uncharted and a comprehensive analysis of their distribution, prevalence, diversity, and function is lacking. Here, we systematically investigated CRISPR-Cas loci across the largest curated collection of natural bacterial and archaeal plasmids. CRISPR-Cas loci are widely but heterogeneously distributed across plasmids and, in comparison to host chromosomes, their mean prevalence per Mbp is higher and their distribution is distinct. Furthermore, the spacer content of plasmid CRISPRs exhibits a strong targeting bias towards other plasmids, while chromosomal arrays are enriched with virus-targeting spacers. These contrasting targeting preferences highlight the genetic independence of plasmids and suggest a major role for mediating plasmid-plasmid conflicts. Altogether, CRISPR-Cas are frequent accessory components of many plasmids, which is an overlooked phenomenon that possibly facilitates their dissemination across microbiomes. These sference) . BrieflyCRISPR-Cas systems are broadly distributed across the genomes of about 42% of bacteria and 85% of archaea . DespitePlasmids are extrachromosomal, self-replicating MGEs that are ubiquitous across microbiomes on Earth. They are known to shape the ecology and evolution of microbial communities by, for example, promoting horizontal gene transfer (HGT) between taxa ,18. AlthEven though some plasmids have been reported to encode CRISPR-Cas loci ,29–36 thhttps://github.com/Russel88/CRISPRCas_on_Plasmids. Analyses were made with a combination of shell, python 3, and R 3.6.3 scripting. Plots were made with ggplot2, heatmaps with pheatmap, phylogenetic trees with iTOL   , togethee/plsdb) . A totale/plsdb)  to re-ane/plsdb)  with thee/plsdb)  as seconcas gene or matched (95% coverage and 95% identity) with any previously defined high confidence CRISPR repeat: CRISPRCasFinder evidence level 4 or archived in CRISPRCasdb   were considered associated with a cas operon if the direct repeat sequence was at least 85% identical to the direct repeat sequence of an array adjacent to that cas operon (The prediction and classification (at the subtype or variant level) of asTyper) . CRISPR s operon . Furthers operon . When pon = 9999) and a Bonferroni adjusted P-value threshold of 0.05 was used.Enrichment of certain CRISPR-Cas subtypes on either plasmids or host chromosomes was investigated with an indicator species analysis, using the indicspecies R package. For the comparison between all plasmids and chromosomes the IndVal.g statistic was used, which controls for difference in group sizes. For the direct comparison between plasmids and hosts chromosomes, where both carry CRISPR-Cas, the IndVal statistic was used. Statistical significance was determined by permutation (mob_typer function (The conjugative transfer functions and incompatibility (Inc) typing of all plasmids in PLSDB was predicted with MOB-suite v3.0.1 using function  using dehttps://typer.crispr.dk, model version 2021_03  were masked in order to avoid false positive matches to spacers in arrays. Furthermore, for matches to plasmids only matches to high confidence ORFs were included, also to rule out any matches to possibly undetected CRISPR arrays. Spacers from orphan arrays whose consensus repeat could not be typed by repeatTyper from CRISPRCasTyper ( 2021_03 ) were exe-value cutoff of 0.05. To reduce redundancy bias, spacers were only counted once, no matter the absolute number of matches.Viral genomes were obtained from the IMG/VR v3  only inNetworks were visualized in gephi with layout generated by a combination of OpenOrd and Noverlap algorithms. For calculating taxonomic confinement of spacer-protospacer matches between plasmids, each pair of plasmids connected by at least one spacer-protospacer match was counted as one matching pair. Cross-targeting plasmids were included as two separate plasmid pairs. Confinement was calculated as the number of matches found exclusively within a specific taxonomic rank, such that each plasmid-plasmid pair was only counted once. For estimating confinement of random spacer-protospacer matching, the taxonomic annotations were permuted among the plasmid-plasmid pairs with observed spacer-protospacer matches. This was repeated 100 times and the median number of matches was used as an estimate of confinement for hypothetically random matches. For estimating targeting bias towards conjugative versus non-conjugative plasmids each unique spacer was counted with a weight of 1 with the targeting bias proportional to the number of matches to conjugative and non-conjugative plasmids, respectively. For example, a spacer matching four conjugative plasmids and one non-conjugative plasmids is counted as 0.8 for conjugative matches and 0.2 for non-conjugative matches. The spacer-protospacer matches identified for plasmid and associated host chromosome-derived CRISPR array contents are found in cas loci. To reduce the confounding effect of sequencing biases, we removed identical or highly similar plasmids from further analyses. This resulted in a non-redundant dataset of 17 608 bacterial and 220 archaeal plasmid sequences, spanning 30 phyla and 771 genera. For a total of 13 265 non-redundant plasmids, we were able to collect the corresponding set of host chromosome sequences (n = 6979). Overall, our survey identified a total of 338 complete and 313 putatively incomplete loci (207 orphan CRISPR arrays and 106 orphan cas), indicating that ∼3% of sequenced plasmids naturally carry one or more CRISPR and/or cas loci  and archaeal (253) plasmid genomes in search of CRISPR and i Figure , top. Thi Figure . Strikini Figure , bottom,cas operons were also commonly identified. These putatively incomplete systems are more frequent on plasmids than chromosomes  for array expansion and maintenance. Furthermore, we found a less frequent association of plasmid-encoded systems with adaptation modules in plasmids compared to chromosomes  that is thought to be compensated via in trans use of chromosomally-encoded adaptation machinery  CRISPR arrays and s Figure . Intriguachinery ,7,10,13.achinery . TogetheWe then sought to investigate the diversity of CRISPR-Cas systems across plasmid genomes. Our analysis revealed a broad range of plasmid-encoded subtypes and marked differences in their abundances Figure . Except cas loci on plasmids   . Among tng loci) ,57.Rhodothermia, Deinococci and Clostridia lies between 19 and 27%, in other taxa their incidence is very low or even zero. Strikingly, the prevalence and diversity of CRISPR-Cas subtypes on plasmids correlates poorly with their abundance across the chromosomes of plasmid-host taxa . These results support the notion that conjugative plasmids facilitate HGT of CRISPR-Cas systems in the environment and, given the remarkably broad transfer ranges of some proteobacterial plasmids   , possiblnd IncN) ,60. For nd IncN) ,66 and hnd IncN) , mobiliznd IncN) , transdund IncN) ,70, and nd IncN) . TherefoWe then sought to examine other biological characteristics of the plasmids and searched especially for common or distinctive patterns shared by CRISPR-Cas-encoding plasmids. We focused on exploring the link between plasmid genome size and the presence of CRISPR-Cas modules. In contrast to the collection of non-CRISPR-Cas-encoding plasmids—which displayed the previously reported bimodal size distribution ,60—plasmGiven the relatively large sizes of CRISPR-Cas systems, a bias towards larger genomes is unsurprising and possibly stems from size-related constraints associated with certain plasmid life history strategies. Larger plasmids allocate considerable portions of their genomes to transfer, stabilization and accessory modules that enhance their persistence . This isNext, we examined whether CRISPR-Cas systems in plasmids have short-lived associations or whether we could identify signs of retention by specific plasmid lineages. To this end, a common plasmid classification scheme types plasmids into incompatibility (Inc) groups and is deeply rooted in plasmid eco-evolutionary dynamics, i.e. based on the observation that plasmids sharing replication or partitioning components cannot stably propagate within a given cell host lineage . We therOverall, we found that only a reduced number of Inc types (15/50) include plasmids carrying CRISPR-Cas Figure . Most CRWe then focused on understanding the possible function(s) of plasmid-encoded CRISPR-Cas systems. CRISPR arrays are uniquely suited to provide ecological and biological insights; the origins of many spacer sequences can be backtracked, providing valuable clues about the functions of CRISPR-Cas and their selective benefits ,74–76. In = 11 080) were extracted from the bacterial and archaeal plasmid-encoded CRISPR arrays and searched against comprehensive virus and plasmid sequence datasets . For comparison, analogous searches were performed with the collection of spacers originating from: (i) the host chromosomes associated with the plasmids in this study  and (ii) plasmid-host chromosome pairs where both the plasmid and associated host chromosome carry at least one CRISPR array . Only a limited fraction of spacers yielded significant matches to protospacer sequences , consistent with previous studies , while a substantially smaller fraction matched viruses (27%) Figure . In contThe abundance of plasmid spacers targeting other plasmids raised the question of whether the reported plasmid-targeting preference of type IV CRISPR-Cas systems  could beThe identification of extensive plasmid-plasmid targeting provides a practical framework for investigating plasmid eco-evolutionary dynamics and offers a unique opportunity to gain insights into HGT routes. This prompted us to build a global network of plasmid-plasmid interactions based on the linkage information provided by the CRISPR-targeting data Figure . The corNotably, the clustering analysis demonstrates a pronounced inverse relationship between the number of plasmid connections and the phylogenetic hierarchy of the cognate bacterial hosts. Whereas targeting between plasmids within a single species, genus and family account for the bulk of all predictions , matches confined to higher taxa comprise less than 2% Figure . Indeed,Given the self-transmissible properties of conjugative plasmids, we wondered whether their effective spread through bacterial populations could render them common targets for CRISPR-Cas compared to non-conjugative plasmids. In support of this, we observed an over-representation of plasmid spacers predicted to target conjugative plasmids Figure . This maThe study of CRISPR-Cas biology has primarily focused on chromosomally-encoded systems and their adaptive antiviral functions in bacteria and archaea. While recent work has started to uncover the common association of CRISPR-Cas systems with diverse MGEs and the importance of this phenomenon for CRISPR-Cas ecology and evolution , their rcas loci may be remnants of decaying CRISPR-Cas systems , suggest an active contribution of plasmids to the conspicuous dissemination of CRISPR-Cas systems across microbiomes. These results are in agreement with the proposed bacterial pan-immune concept, where defense systems are continually lost and (re)gained by bacteria through HGT mechanisms , and furPseudomonas aeruginosa (Notably, we found that plasmid-encoded CRISPR arrays tend to carry a larger fraction of spacers predicted to target other plasmids, while plasmid-host chromosome-encoded systems show the commonly observed targeting bias towards viruses. This contrasting targeting preference was consistently observed across taxa and the different CRISPR-Cas subtypes, indicating that plasmids may primarily exploit CRISPR-Cas systems to target other plasmids, and thus likely play a less dominant role in host protection against viral predators Figure . These oruginosa .Vibrio cholerae phage encodes a I-F CRISPR-Cas system to restrict the phage satellite PLE, a MGE that parasitizes ICP1 (Together, our results are consistent with previous reports of the co-option of CRISPR-Cas systems, or components thereof, by different MGEs for waging inter-MGE conflicts. For example, the ICP1 zes ICP1 ,16. Addizes ICP1 ,9,10,93.zes ICP1 , wherebyMore broadly, the implications of our findings have practical applications beyond CRISPR-Cas biology. Plasmid sequences may hide an uncharted diversity of CRISPR-Cas systems with promising biotechnological applications, e.g. in genome engineering. Furthermore, plasmid-derived CRISPRs can be exploited to determine information about a plasmid's direct relationships with other elements across evolutionary timescales. When available, spacer-protospacer match prediction data could comprise an added layer of information during retrospective plasmid host-range inference analyses, similar to how chromosomal CRISPR contents are leveraged for bioinformatic deconvolution of virus-host associations ,95–98. FOverall, CRISPR-Cas systems constitute powerful barriers against MGE-mediated HGT in microbial communities. While the investigation of CRISPR-Cas biology has focused on chromosomally-encoded systems, our work uncovers their pervasive association with plasmids across a broad phylogenetic breadth, where they appear to play a major role in mediating plasmid-plasmid conflicts. We anticipate that MGE–MGE warfare likely constitutes an important, yet largely overlooked, factor influencing the dynamics of gene flow across microbiomes.https://github.com/Russel88/CRISPRCas_on_Plasmids.Scripts for downloading data and reproducing all analyses are available at gkab859_Supplemental_FilesClick here for additional data file.


Tertiary dentin results from the interplay between the host defense and dental injury or infection. Modern endodontics aiming vital pulp treatment take the tertiary dentin formation as the interim step, with the final goal of a physiological pulp-dentin like tissue regeneration. Dental pulp stem cells have been nominated for contributing to differentiating into odontoblast-like cells who are responsible for reparative dentin formation. Understanding the original dentin formation mechanism provides us a blueprint while exploring the reparative dentin formation mechanism builds bridge to bonafide pulp-dentin tissue regeneration. Among all the regulators, growth factors have long been revealed under the spotlight. The insulin-like growth factor (IGF) family has been implicated in critical events of inducing dentin formation, which is essential for pulp treatment. The expression of IGF family members including IGF1, IGF1R, IGF2, and IGF2R has been well characterized in dental papilla cells, dental pulp stem cells, and periodontal ligament cells. Recent studies indicated IGF binding to the receptors activated pathways, including MAPK pathway, and AKT pathway, orchestrated proliferation, and differentiation, and finally, contributed to dentin formation. This review summarizes the role of IGF family in dentin formation during tooth development and tertiary dentin formation during dentin-pulp repair and sheds light on key parts of research for future treatment improvements. Both trauma and caries can lead to dentin-pulp injuries. Pulp diseases or injuries are not fatal diseases, however, markedly affect the quality of life. Over the last decades, improvements in the performance of capping materials have substantially decreased the pulp loss associated with caries and dental trauma. Although dental pulp diseases are common, and capping materials are more advanced, our ability to preserve dental pulps and recover their functions remains limited and challenged.When the dentin-pulp is impaired, the interaction between direct tissue injury, infection, inflammation, and the host defense responses determines the final outcomes . While tGrowth factors (GFs) are a kind of proteinic bioactive agents who are essential to tissue growth and regulate cell fate . GFs areTo achieve the injury repairing and promote pulp healing, multiple GFs reveal their essential roles in the formation of odontoblasts and odontoblast-like cells, subsequently leading to enhancement of dentin formation and tertiary dentin formation. Among these factors, the IGF family stands out in the spotlight and shows great potential for future bioactive pulp treatment.The original dentin formation during tooth development exhibits us the biological process which we expect to work during dentin regeneration. Tooth development is initiated by the interaction between epithelium and mesenchyme . Dentin Upon injury, would healing process in dentin-pulp initiates. It is a pathological event reflecting the extent and severity the dentin-pulp complex has suffered, and alongside the tertiary dentin formation, reactionary, and reparative dentinogenesis included. When the injury is mild enough for primary odontoblasts to survive, the following formed dentin can contain a completely regular tubular structure, which is defined as reactionary dentin. However, more intense injury strikes the dentin-pulp complex causing local death of the primary odontoblasts at the injury site and triggering a cascade of progenitors, most possibly dental pulp stem cells, recruited to the specific site, and ultimately forming reparative dentin with atubular morphology.Zooming in to the cellular level, following tooth injury, cell death and cell replacement take place. In recreationary dentinogenesis, primary odontoblasts survive and, upon mild stimuli, keep secreting regular tubular dentin. While in reparative dentinogenesis, primary odontoblast death is followed by new generation of odontoblast-like cells, lining the pulp interface with dentin and secreting atubular dentin-like matrix. However, lacking unique molecular or morphological markers and unclear morphological evidence makes the identification of odontoblast-like cells and its origination difficult . SeveralRegenerative endodontics including vital pulp therapy, pulp revitalization, and cell homing treatments aims at healing the pulp injury with natural manners . BonafidDentin formation is a complex process involving sequential and ordered deposition of an extracellular matrix, followed by its mineralization . The forβ/BMP signaling were proved to be vital for odontoblast maturation and dentin formation in both temporal and spatial manner [BMP family regulates various biological processes, such as cell proliferation, differentiation, migration, and extracellular matrix remodeling . BMP-2 al manner , 16. BMPβs has long been proven indispensable in dentin formation [β1 increased the mineralization and ALP activity of dental pulp cells which marked the enhancement of odontogenic differentiation by regulating transcription of two critical noncollagenous proteins, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP1), in odontoblasts [β2 strongly upregulated when pulp cells differentiated into odontoblasts in vitro [β2 signaling in dentin resulted in sex-related enamel defects [β3 enhanced odontogenic differentiation and the formation of ectopic dentin [Among TGF family, TGF-ormation . TGF-β1 toblasts . TGF-β2 in vitro . In addi defects . Upregulc dentin . TGF famIGFs have also been investigated and have shown great potential in regulating dentin formation.IGF family composes of IGF-1 and IGF-2 participant in massive physiological activity, including development, growth, organogenesis, metabolism, cell proliferation, and cell differentiation . IGF-1 aαCT domains and the low affinity-binding site composed of the Fn-III-1 domain. The type 2 IGF receptor (IGF-2R) is a single-chain type I intramembrane protein, which entraps the extracellular IGF-2 exclusively.IGF receptors have different affinities for different ligands. Both IGF-1 and IGF-2 can act via the type 1 IGF receptor (IGF-1R), which is a dimeric transmembrane glycoprotein expressed ubiquitously both pre- and postnatally. IGF-1R contains two separate IGF-contacting sites, the high affinity-binding site made up with L1 and IGF-binding proteins (IGFBPs) are a group of proteins who regulate IGF ligand function. IGFBP1-6 share 50% homology and have high affinities for IGF-1 and IGF-2. 80% of the total IGF-1 are bound by IGFBP-3, who is the most important subtypes of IGFBPs for IGF-1, while IGFBP-6 is essentially IGF-2 specific. Most IGFBPs compete for activity of IGFs at the receptor level and antagonize IGF function, while some  appear to amplify IGF signaling. IGFBP7-10 also play a role in IGF axis. They share conserved N-terminal domain of the conventional IGFBPs. But unlike IGFBP1-6, IGFBP7-10 show low affinity for IGFs. IGFBP7 is proved to be at least 5-fold to 25-fold lower than conventional IGFBP1-6 in binding IGFs with its IGF-dependent and IGF-independent cell growth regulating function . All theOnce binding to the cognate receptor, their inner kinase domain starts to autophosphorylate, leading to activation of two main downstream pathways, phosphatidylinositol 3-kinase (PI3K)-AKT/mammalian target of rapamycin (mTOR) pathway, and Ras-mitogen-activated protein kinase (RAS-MAPK) pathway. PI3K-AKT/mTOR pathway activated by insulin receptor substrate (IRS) predominantly leads to metabolic outcomes. The SHC-initiated RAS-MAPK pathway controls mitogenic outcomes, both play vital roles in the host defense of dental tissue injury, wound healing, and pulp regeneration , 29.IGFs have been revealed to drive cell proliferation and differentiation during the tooth development. Recent studies showed that IGF-1 had an essential role in cellular migration and cell proliferation in many dental tissues . ImmunohDental papilla cells turning into odontoblasts through cell fate determination is a pivotal step for dentinogenesis, where multiple signaling pathways and factors are involved, and IGF axis has a place. Similarly, the odontogenic differentiation of stem cells originated from dental tissues is also governed by IGF axis. In stem cells from dental papilla (SCAPs), IGF-1 were investigated to bind to IGF-1R, activating phosphorylation of MAPK pathways, leading to enhance the differentiation and mineralization of SCAPs by increasing key markers' expression, including Alp, Runx2, Osx, Ocn, Col-I, Dspp, and Dmp-1. This effect could be enhanced by microRNA hsa-let-7c depression whilst reversed by the overexpression . EvidencWhen pulp injury happens, pathological events emerge , 48. IGFWith the roles of growth factors in dentin formation and regeneration getting more and more defined, proper carriers facilitating clinic application are gaining more and more attention. Exosomes, small double-lipid layer particles containing proteins, mRNA, and miRNA, can convey a multiple of bioinformation and stimuli. Exosomes secreted by MSCs showed strong regenerative potential and immunomodulation . Cai et The underneath mechanism of dental wound healing and repair sheds light on advanced healing strategies inducing pulp regeneration processes. IGFs orchestrating crucial cell events in dentin formation makes it one of the potential candidates for dentin regeneration. Yet, there are still gaps between our current knowledge about dentin formation and what is really going to there. Previous research proved spatio-temporal expression of IGF axis in dentin development, arousing interests in probing into the role of IGFs in tooth development. Further research revealed IGF axis' involvement in dentin and dentin-like tissue formation. Additionally, Ras/Raf-1/MAPK and PI3K/AKT/mTOR pathways were found activated by IGF-axis during this biological process. Yet, the adequate release and spatio-temporal expression of IGF-axis in an active form and the manual control release of IGF-axis imitating bioprocess have not yet been investigated. The gaps mentioned above also reminds us of potential barriers within the IGFs' application in the future. Moving forward, it will be critical to further investigate growth factors both at the genomics level in biologically relevant cell types and in the potential therapeutic application .


Splice-disrupt genomic variants are one of the causes of cancer-causing errors in gene expression. Little is known about splice-disrupt genomic variants.HLA-A in prostate cancer, MSR1 in familial prostate cancer, and EGFR in both castration-resistant prostate cancer and metastatic castration-resistant had the highest allele frequencies of splice-disrupt variations. Some splice-disrupt variants, located on coding sequences of NCOR2, PTPRC, and CRP, were solely present in the advanced metastatic castration-resistant prostate cancer. High-risk splice-disrupt variants were identified based on computationally calculated Polymorphism Phenotyping (PolyPhen), Sorting Intolerant From Tolerant (SIFT), and Genomic Evolutionary Rate Profiling (GERP) + + scores as well as the recorded clinical significance in dbSNP database of NCBI. Functional annotation of damaging splice-disrupt variants highlighted important cancer-associated functions, including endocrine resistance, lipid metabolic process, steroid metabolic process, regulation of mitotic cell cycle, and regulation of metabolic process. This is the first study that profiles the splice-disrupt genomic variants and their target genes in prostate cancer. Literature mining based variant analysis highlighted the importance of rs1800716 variant, located on the CYP2D6 gene, involved in a range of important functions, such as RNA spicing, drug interaction, death, and urotoxicity.Here, pattern of splice-disrupt variants was investigated using 21,842,764 genomic variants in different types of prostate cancer. A particular attention was paid to genomic locations of splice-disrupt variants on target genes. This is the first study that profiles the splice-disrupt genomic variants and their target genes in different types of prostate cancer. Unravelling alternative splicing opens a new avenue towards the establishment of new diagnostic and prognostic markers for prostate cancer progression and metastasis.The online version contains supplementary material available at 10.1007/s11033-022-07257-9. Alternative RNA splicing is an emerging topic in molecular and clinical oncology , 2. AlteDysregulation of post-transcriptional regulation can result in defective proteins or transcripts without causing genetic diseases . AlternaPrecise pre-mRNA splicing is vital for correct protein translation. Precise pre-mRNA splicing is related to the presence of consensus “cis” sequences, identifying exon-intron boundaries and regulatory sequences rby splicing machinery . Point mNBAS, SLC16A1, RHD, PNPLA2 have been accounted as genetic basis of recurrent liver failure, ketoacidosis, variant D phenotype, progressive severe myopathy, respectively [CHEK2 genes in many cancers has been documented in individuals carrying a single pathogenic splice-disrupt variant [Within different types of cancer-associated genomic variants, splice-disrupt genomic variants are the less studied, particularly in prostate cancer. There is an ample evidence of the discovered point mutations that prevent appropriate splicing by disruption of exonic splicing enhancers . Point mectively . Pathoge variant .Our knowledge about abnormal splice-disrupt genomic variants in prostate cancer, and their prospective contribution to cancer progression (castration-resistant prostate cancer and metastatic castration-resistant prostate cancer) is limited. Furthermore, their relative abundance and their potential to use splice-disrupt in cancer diagnosis and treatment has been largely neglected .Whole genome sequencing projects, particularly 1000 Genomes Project, have resulted in bulk identification of human variations, including splice-disrupt variants . The ideFor the first time, we mined a large dataset of genomic variants in different types of prostate cancer, identifing the splice-disrupt variants and their target genes. Furthermore, we investigated the association between splice-disrupt variants and advanced types of prostate cancer. Finally, computational systems biology was applied to investigate the functional consequences of the discovered splice-disrupt variants.The National Center for Biotechnology Information (NCBI) database of genetic variation (dbSNP database)  was usedTranslational impact of variants including missense, splice-disrupt, coding sequences insertion or deletion (CDs indel), nonsense, misstart, and non-stop were retrieved and variants were filtered for splice-disrupt variants using SQL Table of Pathway Studio tool. As a result, splice-disrupt variants were identified for different types of prostate cancer .Allele (variant) frequency stands for a gene variant  in a specific locus in a population, commonly presented as percentage/fraction. Minor allele frequency explains the frequency where the second most common allele occurs in a population; for example, allele frequency of 1% mean that the variant happens in 1% of population. We used 1000 Genomes Project  to extraGenomic locations of variants were identified based on dbSNP, and variants were assigned to the following locations: CDs (coding sequences), 3′UTR (untranslated region), 5′UTR, intergenic, and intronic variants.High-risk splice-disrupt variants were identified using dbNSFP –25, a daSIFT algorithm is developed to predict the effect of coding variants on protein function based on sequence homology and the physical properties of amino acids. SIFT is considered as a standard tool for characterizing missense variation , 27. SIFPolyPhen algorithm, like SIFT, use sequence homology of related proteins to evaluate whether an amino acid substitution can be deleterious to protein function based on the degree of conservation of the affected base throughout evolution. SIFT relies solely on sequence homology  while PoGERP is an evolutionary conservation score which have a good correspondence with clinical significance and pathogenicity level. GERP++ demonstrates the constrained elements in multiple alignments by quantifying substitution deficits. These deficits identify substitutions that would have happened if the element were neutral DNA, but did not happen as the element has been experienced functional constraint . Low valPathogenic status of splice-disrupt variants was obtained from dbSNP database. dbSNP is the main global reference and repository of single nucleotide variations , 33, 34.Genes targeted by splice-disrupt variants in each type of prostate cancer  were used as input for Gene ontology (GO) enrichment analysis in three terms of biological process (BP), molecular function (MF), and cellular component (CC). To this end, the following tools were employed: (1) Comparative GO , 36, andLiterature mining more was used as a validation of in silico discovered splice-disrupt variants, to to investigate which of the computationally selected splice-disrupt variants have evidence of involvement in different types of prostate cancer.Literature mining was performed using Pathway Studio Mammal database (Elsevier) , Versionhttps://www.minitab.com). Graphs were visualized using GraphPad Prisim 7 (https://www.graphpad.com/).Analysis of Variance (ANOVA) followed by Mean comparison using Tukey test was performed to compare the allele frequency of GERP++ conservation score between different types of prostate cancer. Two-sample proportion test was used to compare the clinical significance (occurrence of pathogenic status) between types of prostate cancer. Leven’s test was used to compare variance of allele frequency, and GERP++ between types of prostate cancer. Analysis was performed in MINITAB 18  (Supplementary 2–5). In more advanced type of prostate cancer (MCRPC), splice-disrupt variants, located on CDs of BRCA1 in all types of prostate cancer was the target of pathogenic splice-disrupt variants. Interestingly, CYP2D6 genes harbors splice-disrupt variants with high allele frequency.Highly pathogenic splice-disrupt genomic variants and their corresponding genes in different types of prostate cancer  is presented in Fig. Variance analysis of CDs-located splice-disrupt variants showed that advanced type of prostate cancer, MCRPC, has remarkable higher variation in GERP++ score Fig. . High diSupplementary 6 presents functional annotation of high-risk splice-disrupt variants in different types of prostate cancer by enrichment analysis using GO database as well as KEGG pathways. According to KEGG pathway analysis, damaging splice-disrupt variants are significantly (p < 0.01) involved in Endocrine resistance and different types of cancer.ESR1 are NCOR1 are involved in this function that can explain the link between UTR damaging variants and microRNA regulation.Lipid metabolic process, steroid metabolic process, regulation of mitotic cell cycle, negative regulation of metabolic process, negative regulation of signal transduction, and response to lipid are the key Biological Processes enriched by damaging splice-disrupt variants. One of the significant functions were negative regulation of production of miRNAs involved in gene silencing which variants on CHEK2 kinase is an important genetic change involved in FPC , highlighting the importance of rs1800716 splice-disrupt variants, located on Noticeably, no cutoff was used for minor allele frequency of splice-disrupt variants in this study. This allowed us to investigate the contribution of less frequent  and rare variants  to different types of prostate cancer. While GWAS commonly the cutoff of 5% for minor allele frequency, recent publications suggest the potential contributions of less variants to the risk of different diseases have been discussed , 43. It ESR1 are NCOR1 are involved in this function.Apoptosis and angiogenesis are the main cancer-associated processes, where alternative splicing plays a crucial role in their regulation . FunctioPTPRC is involved in cell adhesion, facilitating the tumour proliferation and lymph node metastasis in cervical cancer patients [PTPRC reduced the rates of tumour growth and metastasis in vivo [PTPRC has also been noticed in colon cancer metastasis [PTPRC, as an adhesion molecule, is involved in the spread of the tumour and immortalisation of the tumour cells during malignancy [CTSF is involved in the growth and apoptosis where CTSF knockdown promotes proliferation by inhibiting apoptosis [CTSF gene may function as a tumour suppressor with high potential therapeutic value [PTPRC and CTSF genes and their involvement in prostate cancer. We found that splice-disrupt variants on CTSF and PTPRC is specific to MCRPC and may contribute to prostate cancer progression.Metastasis is the cause of more than 90% of cancer-related deaths and is the most complex function of cancer cells . Metastapatients . In anot in vivo . The roltastasis . It has lignancy . In gastpoptosis . It has ic value . To bestThis is the first study that profiles the splice-disrupt genomic variants and their target genes in prostate cancer. Noticeably, we found an association between specific splice-disrupt variants with advanced prostate cancer. The major limitation of this study was the remarkable difference between number of splice-disrupt genomic in different types of prostate cancer. The variants in FPC, CRPC, and MCRPC are more detrimental and harder to detect compared to early stage of PC. Alternative splicing contributes to a range of phenotypic traits of tumours as they progress and undergo metastasis and is a potential target for gene therapy , 11. UnrBelow is the link to the electronic supplementary material.Supplementary file 1 (DOCX 98 KB) List of splice-disrupt variantsin different types of prostate cancer. Prostate cancer (PC), castration-resistant prostate cancer (CRPC), and metastatic castration-resistant prostate cancer (MCRPC)Supplementary file 2 (DOCX 25 KB) High-risk splice-disrupt variants in prostate cancer, based on PolyPhen, SIFT, GERP++ scores and reported clinical significance in dbSNPSupplementary file 3 (DOCX 23 KB) High-risk splice-disrupt variants in familial prostate cancer (FPC) based on PolyPhen, SIFT, and GERP++ scores as well as reported clinical significanceSupplementary file 4 (DOCX 23 KB) High-risk splice-disrupt variants in castration-resistant prostate cancer (CRPC) based on PolyPhen, SIFT, and GERP++ scores as well as reported clinical significanceSupplementary file 5 (DOCX 23 KB) High-risk splice-disrupt variants in metastatic castration-resistant prostate cancer (MCRPC) based on PolyPhen, SIFT, and GERP++ scores as well as reported clinical significanceSupplementary file 6 (DOCX 24 KB) Functional annotation of high-risk splice-disrupt variants by enrichment analysisSupplementary file 7 (XLSX 53 KB) Mined references underpinning literature-mining network of splice-disrupt variants, their annotated genes, and interactions with different types of prostate cancer. The network of relationships/interaction is visualised in Figure 6)Supplementary file 8 (XLSX 11 KB)Supplementary file 9 (DOCX 13 KB)


Polymer-drug conjugation  was firsPolymer-drug conjugates have since gradually increased in their clinical application, now with more than 29 marketed products that vary widely in polymer architecture (linear and branched), molecular weight (0.3–60 kDa per polymer), and degree of conjugation (1–69-82 per drug) and nearly all of which employ the synthetic polymer, PEG, a polyether typically produced by the ring-opening polymerization of ethylene glycol . In addiin vivo fluorescence imaging, photodynamic therapy, and sonodynamic therapy  food ingredient by the FDA in 1973 and has a long history of safe use in humans. It is the most widely used stealth polymer in drug delivery and is typically regarded as a non-immunogenic polymer. Early studies by Richter and Akerblom in 1984 found that 0.2% of treatment-naïve individuals , had antibodies specific to PEG in their plasma . Since tIn addition to treatment-naïve immunity, anti-PEG antibodies are also drug-inducible and associated with systemic administration of PEGylated proteins , nucleicHypersensitivity reactions, including anaphylaxis has been reported in association with many PEG-containing formulations including PEG-protein conjugates  of PEGylated compounds was identified in mice in 1999, and in patients treated with pegaspargase in 2007  and is ce.g. PEG-IFNα) may be susceptible to polymer-specific ABC whereas the estimated threshold concentration of anti-PEG antibodies needed to accelerate the clearance of nucleic acid drug carriers (e.g. Patrisan) overwhelmingly exceed those observed in patient blood  following local administration are therefore warranted.In addition to formulation-dependent susceptibility to polymer immunogenicity, mode of administration can also modulate the impact of antibody recognition. Most clinically approved polymer-drug conjugates are intravenously administered and thus their interaction with plasma IgG and IgM is higher than may be expected following intramuscular or intratumoral injection, as is common among many mRNA indications including both BNT162b2/Comirnaty and mRNA-1273/Spikevax . Thus, tHaving established that PEG immunogenicity can limit the clinical utility of PEG-drug conjugates and that nanoparticle-based formulations may be less vulnerable to some of these effects relative to polymer-protein drug conjugates, how can one minimize the impact and risk of immunogenicity-diminished efficacy from future polymer-conjugated drugs and vaccines? As discussed above, PEG immunogenicity can arise through a variety of mechanisms  and inclin vivo. While all clinical PEG-drug conjugates are chain-terminated by methoxy groups, recent preclinical studies suggest that hydroxy-terminal PEG conjugates generate lower amounts of backbone-specific anti-PEG IgM , these -PEG IgM , ethoxy,-PEG IgM  moietiesin vivo  PEG, has also been shown to reduce liposome-induced anti-PEG antibodies in preclinical studies  the therapeutic impact of PEG on drug immunogenicity, 2) the possible increasing prevalence of pre-existing and drug-induced PEG immunity, 3) the growing public need for safe and effective mRNA vaccines, and 4) our prevailing reliance on PEG for use in clinically approved nucleic acid and polymer-drug conjugate therapies , it is cGiven the challenging risk-reward of advancing non-PEG-based polymer-drug conjugates towards clinical translation, what can governments and funding agencies do to facilitate continued innovation in polymer-drug conjugate development and ensure the capacity for safe and effective vaccination at-scale? 1) Biosimilar-like regulatory guidelines for conjugable polymers (i.e. polysimilars) may be one approach to formalize and streamline the approval of new polymer-drug conjugates, albeit one likely requiring increased rigor given the wide structural diversity and potential health hazards of various polymer subunits relative to proteins. 2) Funding or federal lab support to perform large-scale longitudinal studies of immunogenicity towards polymers and other drug conjugates/excipients  would elucidate current  risks of polymer immunogenicity to human health, drug conjugate efficacy, and the strategic national need for mRNA vaccine-stabilizing polymers. 3) Federally subsidized R&D to offset the risks taken-on by companies exploring PEG- and other polymer-conjugates would greatly incentivize further innovation in this space. 4) Funding to improve our poor mechanistic understanding of polymer-induced immunogenicity and associated short- and long-term health risks would accelerate the discovery of new PEG derivatives and alternatives or propel historically utilized polymers through clinical translation. 5) Federal partnerships to ensure the financial viability of domestically manufactured, pharmaceutical-grade PEG and other polymers, as a matter of national interest, would ensure our readiness for future pandemics  surely yet-to-come. In closing, while it is tempting to suggest a singular direction for polymer-drug conjugate development in the future, we also acknowledge that the ideal properties for a conjugation partner vary substantially with drug class, mode of administration, dosing frequency, and disease indication as discussed above; thus, with proper incentives, funding, and tools we anticipate that future conjugates will not only increase in diversity but also diverge based upon drug type and/or indication.


Low-quality evidence suggests that COVID-19 may trigger reactive arthritis one to four weeks after the infection. Post COVID-19 reactive arthritis resolves within a few days, and no additional treatment is required. Established diagnostic or classification criteria for reactive arthritis are missing, and a deeper understanding of the immune mechanism related to COVID-19 prompt us to further investigate the immunopathogenic mechanisms capable of promoting or contrasting the development of specific rheumatic diseases. Caution should be exerted when managing post-infectious COVID-19 patient with arthralgia. SARS-CoV-2 Infection (COVID-19) may trigger reactive arthritis at its post-infectious stage through immuno-mediated sequelae, including vasculitis, antiphospholipid antibody syndrome, myositis, and lupus . COVID-1Recently, several case reports originating from India, Pakistan, Italy and the United Kingdom have been published on reactive arthritis following COVID-19 , 36–40. Given the intrinsic nature of case reports, it is unclear how generalisable these findings are. High variability in patient characteristics, diagnosis, examinations, and treatment were evident. Established diagnostic or classification criteria for reactive arthritis are missing, and a deeper understanding of the immune mechanism related to COVID-19 may offer a useful opportunity to further investigate the immunopathogenetic mechanisms capable of promoting or contrasting the development of specific rheumatic diseases.Concluding, low quality evidence suggests that COVID-19 can target the musculoskeletal system causing reactive arthritis at its post-infectious stage. Caution should be used while managing post-infectious COVID-19 patient with arthralgia. It is unclear whether this condition can be prevented, whether previous vaccinations exert a preventive effect, and what the long-term sequelae of COVID-19-related reactive arthritis are.


Obesity and overnutrition induce inflammation, leptin-, and insulin resistance in the hypothalamus. The mediobasal hypothalamus responds to exercise enabling critical adaptions at molecular and cellular level that positively impact local inflammation. This review discusses the positive effect of exercise on obesity-induced hypothalamic dysfunction, highlighting the mechanistic aspects related to the anti-inflammatory effects of exercise. In HFD-fed animals, both acute and chronic moderate-intensity exercise mitigate microgliosis and lower inflammation in the arcuate nucleus (ARC). Notably, this associates with restored leptin sensitivity and lower food intake. Exercise-induced cytokines IL-6 and IL-10 mediate part of these positive effect on the ARC in obese animals. The reduction of obesity-associated pro-inflammatory mediators , and the improvement in the gut–brain axis represent alternative paths through which regular exercise can mitigate hypothalamic inflammation. These findings suggest that the regular practice of exercise can restore a proper functionality in the hypothalamus in obesity. Further analysis investigating the crosstalk muscle–hypothalamus would help toward a deeper comprehension of the subject. Obesity increases the susceptibility to cardio-metabolic and neurodegenerative diseases, representing a major public health concern and an economic burden . An arraThe hypothalamus is key to integrating hormonal, environmental, and neural signals in the effort to provide adequate outputs to properly modulate feeding behavior and energy expenditure . ObesityExercise represents an important challenge for organs regulating whole-body homeostasis , inducinThis review discusses animal and human evidence to highlight the positive effect of regular exercise on obesity-induced hypothalamic inflammation/dysfunction. This remodulation is hypothesized to activate a systemic anti-inflammatory network, favorable for the hypothalamus.WAT stores the energy surplus in the form of triglycerides (TG) . PhysicaSkeletal muscle plays a pivotal role in the regulation of systemic inflammation and metabolic health , and varThe MBH regulates BAT and WAT metabolism and governs energy balance ,33,34. WObesity and overnutrition induce chronic low-grade inflammation in the MBH, primarily in the ARC nucleus . High faWAT-released pro-inflammatory molecules can mediate the inflammatory effect on the hypothalamus ,36,39. IThe development of chronic inflammation in the hypothalamus has detrimental effects on energy expenditure and body weight  6]. Hyp. Hyp6]. Exercise-induced adaptive response in the hypothalamus leads to the improvement of insulin and leptin signaling in the ARC and positively modulates the activity of POMC and AgRP neurons, resulting in decreased food intake and body weight control .Findings from animal models suggest that exercise ameliorates obesity-associated hypothalamic inflammation and dysfunction . In obesIn accordance with animal evidence, early findings in humans show changes of circulating levels of NPY/α-MSH in trained overweight and obese individuals ,63, suggDifferent mechanisms can be addressed to explain the positive effects of exercise on the hypothalamus in obesity, although the exact pathophysiology in still poorly explored. Exercise-produced cytokines and mediator are able to cross the brain–blood barrier (BBB), regulating vascularization, neuronal plasticity, and inflammation in diverse areas of the brain . ChangesSkeletal muscle is a secretory organ that responds to either exercise or inactivity by releasing myokines mediating the communication of the skeletal muscle with central and peripheral organs .Interleukin (IL)-6 is a cytokine presenting a bimodal activity, being implicated in both pro-inflammatory and anti-inflammatory functions, depending on the differential signaling pathways activated in targeted tissues ,66. IL-6Recent studies suggest a role of muscle derived IL-6 in the mitigation of inflammation and metabolic dysfunction in the hypothalamus  55], wh, wh55], 2+ and calcineurin via a p38 mitogen-activated protein kinase (p38MAPK)-mediated mechanism ) [Resistin was demonstrated to decrease in humans undergoing both resistance and endurance training . In over2max])  and 12 m2max])  signific2max]) .2max) produced a significant decline in CCL-2 and resistin levels [In randomized-controlled trials in obese-dysmetabolic subjects, the regular practice of endurance exercise was shown to significantly lower CCL-2 plasma levels ,123. In n levels . A 10% dn levels ,121,122,Overnutrition and obesity have detrimental effects on the gut. Obesity is associated with a pro-inflammatory switch of microbiota , lower sFaecalibacterium prausnitzii and Akkermansia muciniphila [While there is no current experimental evidence showing that the positive effect of exercise is mediated by the gut functional improvement, available findings suggest that exercise-induced gut-microbiota/gut permeability improvement can positively influence hypothalamic inflammation/functionality . The reginiphila , whose piniphila . Interesiniphila , suggestiniphila ,144,145.iniphila . SCFAs piniphila . One of iniphila ,149. Impiniphila .2max) lowered endotoxemia in overweight-to-obese diabetic subjects.The restoration of a proper gut permeability contributes to lower the translocation of bacterial antigens and LPS into circulation , with poFuture studies examining in-depth the three-way relationship “exercise-gut-hypothalamus” are warranted to support this pathophysiological model.Obesity and overnutrition induce inflammation and dysfunction in key hypothalamic nuclei governing energy homeostasis and metabolic health. The findings discussed in the present review suggest that moderate exercise is capable of improving hypothalamic inflammation in obesity, restoring leptin and insulin sensitivity, and exerting positive effects on food intake and body weight . Importa


The non-canonical functions of cGAS-STING signaling have recently gained extensive attention. These functions occur in various cellular processes, including senescence, autophagy, and cap-dependent mRNA translation2.Cellular communication is necessary for organizing multicellular organisms, and is critical in tumorigenesis and progression. Immune and tumor cells use the cGAS-STING mechanism to sense and respond to genomic instability, DNA damage, and mitochondrial dysfunction induced by extra- and intracellular stresses. As an essential component of innate nucleic acid sensing, cGAS-STING signaling is critical in various pathological processes, including microbial infection, autoimmunity, inflammation, organ degeneration, and malignancy. The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) senses aberrant or displaced dsDNA in the cytosol and synthesizes 2′3′-cyclic GMP-AMP (cGAMP). Endoplasmic reticulum (ER)-resident adaptor stimulator of interferon genes (STING), mobilized by this second messenger, is trafficked among the ER, ER-Golgi intermediate compartment, and Golgi apparatus, and forms distinct STING signalosomes, which in turn trigger various molecular programs controlling mRNA translation, autophagy, organelle reorganization, and interferon (IFN) productionFigure 1) that has recently been established in several seminal discoveries. The effective intercellular transfer of cGAS-STING signaling enables the sharing of critical information arising from infected, injured, or cancerous cells and the activation of multiple bystander cells in innate immune responses. In cancers, intercellular communication of cGAS-STING signaling connects the tumor microenvironment and pathogenic, immune, and stromal cells, thus promoting or compromising various immune responses and substantially altering disease progression. Understanding this intercellular interplay would greatly aid in developing therapeutics against malignancy. In this perspective, we discuss current knowledge, effects on cancer biology, and implications of in cancer progression and therapeutics.Notably, the complexity of cGAS-STING signaling is further enhanced by an intriguing feature of intercellular transmission .Unlike normal cells, free cytoplasmic dsDNA commonly arises in tumor cells because of chromatin instability and altered metabolism. These dsDNAs are derived from multiple sources, including genomic, mitochondrial, apoptotic, and transposable elements, all of which activate cGAS-STING signaling and subsequently induce innate immune responses4.dsDNA transfer between cancer cells and immune cells typically occurs through phagocytosis in the tumor microenvironment. Tumor cell-derived DNA accumulates in the cytoplasm of phagocytes, such as dendritic cells and macrophages, and triggers the activation of cGAS-STING signaling, thus facilitating antigen presentation and promoting acquired immune responses5.High levels of dsDNA are released into extracellular space after cell death. This DNA is typically non-immunogenic because of its rapid degradation. However, some antimicrobial peptides, such as LL37, bind extracellular dsDNA and facilitate its internalization into monocytes, which produce type I IFNs6. Tumor cell-derived EVs with dsDNA cargo regulate the tumor microenvironment, thereby promoting tumor progression and metastasis. In Crohn’s disease, EVs carrying dsDNA are shed from sites of inflammation and subsequently activate cGAS-STING signaling and enhance innate immune responses in macrophages, thus aggravating the disease7. Upregulation of the mitochondrial DNA-binding protein Lon also induces the secretion of EVs with mtDNA and PD-L1, thereby further inducing the production of IFN and IL-6, and attenuating T-cell immunity. Because dsDNA is found in many stages of cancer and has the potential to strengthen cancer immunity and mediate metastasis, further exploration of its generation and transmission is expected to yield exciting findings in cancer biology.dsDNA associated with small EVs (<200 nm in diameter) has been found to mediate intercellular communication in scenarios including infection, inflammation, and malignancy. EVs are lipid bilayer-bound vesicles in biological fluids and are released and captured by cells. EV-mediated delivery of foreign DNA to bystander cells is frequently detected in pathogen infections8.cGAMP is secreted by cancer cells into the extracellular space and subsequently activates immune responses in receptor cells through various paracrine pathways. Transfer of cGAS-synthesized cGAMP to bystander cells is a primary mechanism underlying cytokine-independent activation of innate and adaptive immune responses. The extracellular hydrolase ENPP1 degrades extracellular cGAMP, thereby controlling the levels of cGAMP in the extracellular environment9. This macrophage transactivation amplifies positive-feedback loops of antitumor immune responses. However, cGAMP transfer also promotes tumor growth and metastasis. Brain metastatic cancer cells use gap junctions to transfer cGAMP into astrocytes in a process mediated by protocadherin 7 and connexin 43, thus resulting in the propagation of cytokines that support tumor growth, such as interferon-α (IFN-α) and tumor necrosis factor (TNF)10. In this scenario, the modulators of gap junctions that break this paracrine loop may serve as targets to treat established brain metastasis.Cells transfer cGAMP to neighboring immune cells and activate the immune response through gap junctions, intercellular structures consisting of arrays of intercellular channels in the plasma membrane that allow for the direct transfer of ions and small molecules between cells. Connexins form gap junctions in vertebrates, and transmission of cGAMP relies on the expression of connexins. For instance, connexins 43 and 45 enable the transfer of cGAMP between epithelial and macrophages11. Other cGAMP transporters, including solute carrier family 19 member 1 (SLC19A1) and SLC46A2, have been found to have roles in cells such as THP-1, Nu-DUL-1, U937, and CD14+ monocytes, according to two recent studies13. ATP-gated P2X purinergic receptor 7 (P2X7R) is another protein that may potentially transport cGAMP14. Opening the ATP-gated P2X7R channel requires a high concentration of ATP, which may be present during cell damage or death, thus allowing nanometer-sized molecules such as cGAMP to pass through. Therefore, receptor-based transport of cGAMP requires various yet-to-be-identified transporters and functions in a context-dependent manner.Unlike gap junctions, receptor-based transport facilitates the transmission of cGAMP to distant cells. The volume-regulated anion channel (VRAC) mediates cGAMP import and export depending on the cGAMP chemical gradient. Knockdown of leucine-rich repeat containing 8 (LRRC8A) has been found to eliminate VRAC activity and to inhibit 50%–70% of cGAMP uptake in various primary and cultured cells+ macrophages. The intercellular transfer of cGAMP through fusion sites augments type I IFN responses in macrophages15. HIV-1 also rapidly transfers cGAMP to surrounding cells through viral particles, as validated by mass spectrometry analysis. These viral particles containing cGAMP stimulate robust STING-mediated immune responses in cGAS-deleted dendritic cells17. However, the role of cGAMP transmission by viral particles and membrane fusion is less understood in cancer biology.Expressing HIV-1 Env in donor T cells leads to a membrane fusion reaction with uninfected CD418 has recently revealed that activated STING proteins are packaged into RAB22A-induced EVs and transported intercellularly through non-canonical autophagy. EVs containing activated STING induce downstream effects of cGAS-STING signaling in recipient cells. Host-derived STING proteins have been detected in exosomes isolated from HSV-1 infected cells, although no secretion signal peptide in STING has been identified. Activated STING proteins are packaged into Rafeesomes, the organelle formed by the fusion of ER-derived and RAB22A-mediated non-canonical autophagosomes with RAB22A-positive early endosomes18. During the process, RAB22A inactivates RAB7, thereby suppressing the fusion of the Rafeesome with the lysosome, and enabling the secretion of these inner vesicles containing activated STING as EVs. These STING-containing EVs induce IFN-β release from recipient cells into the tumor microenvironment, thus promoting antitumor immunity. These intriguing observations provide a new perspective regarding the spread of STING-armed immunity and alternative forms of intercellular transmission of functional membrane proteins.STING is an ER-resident transmembrane protein that traffics to the Golgi after activation, and is degraded through autophagy and lysosomes. The lifespan of STING is associated with the membrane system. Notably, the Kang group19. Type I and III IFNs are crucial secretory proteins downstream of Toll-like receptors, RIG-I-like receptors, and cGAS that activate the JAK-STAT pathway in a paracrine manner by binding receptors of bystander cells such as IFNAR1 and IFNAR2. This ligand-receptor-based intercellular communication of IFNs promotes the generation of hundreds of interferon stimulated genes, thus affecting and modulating IFN signaling19. Intercellular transmission of IFN signals in cancer commonly occurs and is crucial for modulating tumorigenesis and cancer progression, such as through directly prolonging the cell cycle and inducing apoptosis. IFNs restrain tumor metastasis by affecting vascular endothelial cells and increasing antitumor immunity in immune cells3, such as through activating dendritic cells and cytotoxic CD8+ T cells. Consequently, IFN-β is a potential adjuvant for cancer therapy20. However, the effects of IFNs on cancer biology are complex, and the protumorigenic effects of IFNs are also well accepted.IFNs have various functions in immunity and diseases, such as adaptive immune regulation, pathogen defense, antitumor immunity, and autoimmune responsesTable 1). For example, the physiological concentration of cGAMP is controlled by the cGAMP hydrolase ENPP1, which serves as a potential target for leveraging cGAS-STING transmission. A potent membrane-impermeable ENPP1 inhibitor, compound 32, has been found to enhance plasma cGAMP concentrations and alleviate tumor burden21 significantly and has been studied in a clinical trial22. Furthermore, DNA-containing EVs secreted from topotecan-treated cancer cells have been found to activate dendritic cells and to facilitate antitumor immunity via cGAS-STING signaling. In contrast, pharmacological inhibition of gap junctions hinders the transfer of cGAMP from cancer cells to astrocytes and restricts brain metastasis10. The delivery of activated STING via EVs adds another layer for leveraging cGAS-STING signaling by inducing the production of IFN-β and facilitating antitumor immunity18. However, activators and inhibitors of cGAMP transporters, such as LRRC8A, remain to be developed and examined.In most scenarios, the activation of cGAS-STING signaling facilitates antitumor immunity; consequently, leveraging the intercellular transmission of cGAS-STING signaling might serve as a promising target for treating cancers. The intercellular transmission of cGAS-STING signaling might be targeted, probably context-dependent, to benefit disease therapeutics (The beneficial roles of cGAS-STING signaling in cancer progression have recently gained considerable attention. Other critical questions, such as how multiple innate DNA sensors activate the context-dependent response programs and crosstalk with each other and how the cGAS-STING pathway communicates with cancer-specific metabolism and nutrients, are intriguing questions remaining to be answered.


Idiopathic inflammatory myopathies (IIMs) are a group of chronic autoimmune disorders characterized by proximal skeletal muscle weakness. One subtype of the IIMs is immune-mediated necrotizing myopathy (IMNM). IMNM can be further classified according to its autoantibody presence, including anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), anti-signal recognition particle (SRP), and seronegative. Here, we describe the case of a 61-year-old Caucasian female with a prior history of distant lung cancer and current statin use presenting with a subacute onset of bilateral proximal lower extremity muscle weakness and markedly elevated creatinine kinase (CK) and amino transaminases. In the acute inpatient setting, she underwent successful treatment with corticosteroids that were eventually discontinued and replaced with azathioprine three months after hospital admission. At that point, she had attained a 60% increase in muscle strength. Immune-mediated necrotizing myopathy (IMNM) is an autoimmune-mediated muscle disease that leads to severe proximal muscle weakness, markedly elevated creatinine kinase (CK), and myofiber necrosis with minimal to no inflammatory cell infiltrates on muscle biopsy . The proA 61-year-old Caucasian female presented to the ED with five months of progressively worsening bilateral proximal lower extremity weakness, along with acute onset abdominal pain and non-bloody diarrhea. In the week prior to presentation, her lower extremity weakness rapidly progressed to a point where she was not able to ambulate and could not raise herself from a toilet. This was associated with a significantly decreased oral intake and myalgias. Her past medical history is significant for peripheral artery disease status/post (s/p) femoral-popliteal bypass and stent, obesity, coronary artery disease, hypertension, hyperlipidemia, small fiber peripheral neuropathy, ulcerative colitis without immunosuppression, and a distant history of lung cancer several years ago s/p partial lobe resection. Of note, home medications include rosuvastatin 40 mg. She denied inciting trauma, strenuous exercise, shortness of breath, chest pain, or fever. Physical exam revealed hyperalgesia and altered sensation to light touch in bilateral L3-L4 dermatomes. Muscle strength testing graded by the American Spinal Injury Association (ASIA) impairment scale demonstrated weakness in hip flexion  but was otherwise within normal limits. Upper extremity muscle strength and sensation were grossly intact. Deep tendon reflexes (DTRs) of bilateral upper and lower extremities were +2/4. There was no evidence of ascites, abdominal striae, or jaundice. A thyroid examination was unremarkable. There were no signs of a heliotrope rash on the face, Gottron papules on the hands, or erythema. There was no muscle wasting in the upper or lower extremities. Gait evaluation was deferred due to the patient's safety. The patient was admitted for further workup and evaluation. Initial laboratory evaluation is summarized in Table Radiology findingsBecause this initial clinical presentation was presumably consistent with statin-induced myopathy, we immediately discontinued rosuvastatin and performed a full myopathic workup. Unfortunately, subsequent myositis serology testing demonstrated negative findings, including anti-HMGCR and anti-SRP autoantibodies. However, she did have a mild elevation in anti-proteinase 3 antibody and alpha-1 antitrypsin. Notably, primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) autoantibodies were negative on serology. The primary team immediately started her on IV fluids and methylprednisolone 40 mg twice a day. Response to treatment was assessed with down-trending CK levels over two weeks. She was then transitioned to prednisone 40 mg daily and discharged to a subacute rehabilitation facility. At a two-week follow-up visit in the outpatient rheumatology clinic, she endorsed over a 50% improvement in muscle strength while on prednisone 40 mg daily. She was eventually tapered to 15 mg and then to 10 mg while endorsing over 60% improvement in muscle strength and continuing at-home physical therapy. Three months after hospital discharge, she self-tapered off of prednisone and was started on azathioprine 100 mg daily.IMNM is a rare subtype of autoimmune IIM. It is characterized by acute or subacute proximal muscle weakness, rare extra-muscular involvement, and myofiber necrosis with minimal inflammatory infiltrate on muscle biopsy ,3-5. AltThe hallmark clinical feature of IMNM is proximal muscle weakness, and the presence of skin or lung involvement should prompt consideration of other subtypes of inflammatory myopathy ,3-5. ComAutoantibody testing is a critical method of classifying IMNM patients into its three subtypes. Anti-SRP autoantibodies are screened by techniques such as enzyme-linked immunosorbent assay (ELISA), line blot assay, and anti-HMGCR antibodies usually screened by ELISA . Anti-HMIn contrast to patients with other subtypes of myositis, those with IMNM have the highest CK elevations, which is correlated to disease activity . InteresThere are currently no documented randomized clinical drug trials pointing to an effective treatment for IMNM. However, systemic glucocorticoids are the mainstay of initial treatment for IIMs in general -15. GlucIMNM is a type of IIM characterized by severe proximal muscle weakness. Seronegative IMNM is one subtype that is not associated with either anti-SRP or anti-HMGCR autoantibodies and has a more significant association with malignancy. It is characterized by prominent myofiber necrosis on muscle biopsy, and minimal-to-absent lymphocytic inflammatory infiltrates. Early and prompt recognition is critical to prevent long-term and irreversible damage; however, most patients do not regain full muscle strength even after treatment. While the literature supports an initial regimen of a steroid and steroid-sparing agent for the treatment of IMNM, this case report demonstrates that steroid monotherapy with eventual transition to a steroid-sparing agent, along with physical rehabilitation, maybe a beneficial approach for the long-term management of these patients.


Our study demonstrates the significant effects of both depth and symbiosis on forming genomic characteristics of Thaumarchaeota, and provides novel insights into their niche adaptation in deep-sea sponges.Thaumarchaeota account for a large portion of microbial symbionts in deep-sea sponges and are even dominant in some cases. In this study, we investigated three new sponge-associated Thaumarchaeota from the deep West Pacific Ocean. Thaumarchaeota were found to be the most dominant phylum in this sponge by both prokaryotic 16S rRNA amplicons and metagenomic sequencing. Fifty-seven published Thaumarchaeota genomes from sponges and other habitats were included for genomic comparison. Similar to shallow sponge-associated Thaumarchaeota, those Thaumarchaeota in deep-sea sponges have extended genome sizes and lower coding density compared with their free-living lineages. Thaumarchaeota in deep-sea sponges were specifically enriched in genes related to stress adapting, symbiotic adhesion and stability, host–microbe interaction and protein transportation. The genes involved in defense mechanisms, such as the restriction-modification system,  The deep sea is one of the least explored ecosystems on earth. Despite the nonluminous and oligotrophic conditions with extreme hydrostatic pressure, the deep sea still harbors diverse taxa. Sponges (phylum Porifera), as benthic sessile invertebrates, account for high biodiversity and biomass in the benthic ocean . Within Sponges often form stable and sometimes species-specific relationships with microbial symbionts , whose tPhylogenetic analyses have shown that sponge symbionts often include a substantial amount of Archaea  and are Axinella Mexicana) belonged to Cenarchaeum symbiosum A  were separated from a deep-sea sponge collected at 2407.9 meters. Together with other 21 sponge-associated Thaumarchaeota and 28 free-living Thaumarchaeota  from bot3 pieces, stored in RNAlater, and immediately frozen at −80°C, as described previously  near the Mariana Trench during the diving cruise SY219 on October 22, 2019. The manned submersible “Shen Hai Yong Shi” was used during the mission. The sponge sample SY219HM1 was cut into 1 cmeviously . DNA wasTwo methods were used to test the quality of DNA samples: (i) DNA degradation degree and potential contamination was monitored on 1% agarose gels and (ii) DNA concentration was measured using Qubit® dsDNA Assay Kit in Qubit® 2.0 Flurometer . DNA with OD value between 1.8 and 2.0 and contents above 1 μg was used to construct library.A total amount of 1 μg DNA was used as input material for the DNA sample preparations. Sequencing library was generated using NEBNext® UltraTM DNA Library Prep Kit for Illumina  following manufacturer’s recommendations and index codes were added to attribute sequences to the sample. Briefly, the DNA sample was fragmented by sonication to a size of 350 bp. The DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further PCR amplification. At last, PCR products were purified (AMPure XP system) and the library was analyzed for size distribution by Agilent2100 Bioanalyzer.The clustering of the index-coded sample was performed on a cBot Cluster Generation System according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform and paired-end reads were generated.A flexible pipeline, MetaWRAP version 1.2.1 . Boruta tests if the importance of each individual variable is significantly higher that the importance of a random variable by fitting random forest models iteratively until all predictor variables are classified as “confirmed” or “rejected” at the 0.05 alpha level (In order to identify predicted functions and genes (COG and KEGG) responsible for observed clustering patterns, a “random forest” algorithm was performed using R package Boruta version 7.0.0  based on the data provided in t-test was conducted instead.All significant tests were calculated using GraphPad Prism version 9.1.1 (233).Simple linear regression between genome size and coding density is calculated also using GraphPad Prism version 9.1.1 (233).https://ngdc.cncb.ac.cn. Data for four MAGs and metagenomic assemblies can be accessed through Accession Numbers GWHBGCA00000000, GWHBGCB00000000, GWHBGBY00000000, GWHBGBZ00000000, and GWHBGBV00000000. Raw metagenome reads can be accessed through Accession Number CRA005418.The whole genome sequence data, assemblies, and bins reported in this paper have been deposited in the NGDC , under BA deep-sea sponge was collected at 2407.9 meters depth . After DHertwigia sp. MD-2008 from Lyssacinosida order . Within Archaea, all annotated contigs were identified as Thaumarchaeota  at the phylum level. The high abundances of Thaumarchaeota by metagenomics were consistent with the amplicon sequencing results targeting prokaryotes 16S rRNA , Bacteria , Archaea , and Viruses  genes were annotated in metagenomic assemblies . Within 16S rRNA , which s16S rRNA . EukaryoFour MAGs with >50% completeness and <5% contamination were generated. These four MAGs, named bin1-bin4, were then reassembled to increase the genome quality. After reassembly, the completeness of bin1 and bin4 improved and contamination decreased for all bins , S5. TheThe reassembled bins were compared with reference bacterial or archaeal genomes for taxonomic classification based on Genome Database Taxonomy GTDB; . GTDB clTotally 60 Thaumarchaeota and two Aigarchaeota outgroups were aligned to 122 archaeal marker genes  then buiNitrosopumilus_sp._ESC recovered from deep-sea sponges. Overall, the ANI results were highly consistent with the phylogenetic tree based on 122 archaeal marker genes.The ANIs between all 60 Thaumarchaeota were calculated . All ourN = 13) was significantly greater  than that of shallow-water free-living Thaumarchaeota . However, there was no significant difference  between those of deep-sea sponge-associated Thaumarchaeota  and deep-sea free-living Thaumarchaeota .We analyzed the GC contents of all Thaumarchaeota from sponges and the water column based on . The GC p = 0.0007, two-tailed, unpaired t-test) were found in deep-sea free-living Thaumarchaeota  than in shallow-water free-living Thaumarchaeota . However, there was no significant difference  in genome size between deep-sea sponge-associated Thaumarchaeota  and shallow-water sponge-associated Thaumarchaeota . In a horizontal comparison, i.e., samples from the same depth but different habitat types, the genome size of deep-sea sponge-associated Thaumarchaeota  was significantly larger  than that of deep-sea free-living Thaumarchaeota . Similarly, the genome size of shallow-water sponge-associated Thaumarchaeota  was significantly larger  than that of shallow-water free-living Thaumarchaeota .The genome size, coding density, coding sequences (CDS), non-coding sequence (nonCDS), and GC contents of reference Thaumarchaeota with >95% completeness were shown in p < 0.0001, two-tailed, unpaired t-test), shallow water , and deep sea . We fitted a linear regression between genome size and coding density for all Thaumarchaeota with >95% completeness and detected a negative relationship  was used to identify the annotations that segregated significantly between sponge-associated and free-living Thaumarchaeota, or between deep-sea sponge-associated and deep-sea free-living Thaumarchaeota for their COG functional categories , together with an additional function “Mobilome: prophages, transposons” (X) found only in deep-sea sponges, were further examined through presenting all possible COGs with at least one copy within all 52 Thaumarchaeota that have been annotated under these COG functions . The genAnkyrin repeat (COG0666), CRISPR/Cas system—associated endoribonuclease Cas2 (COG1343) and Prophage maintenance system killer protein (COG3654); while four other sponge-specific COGs were Phosphohistidine phosphatase SixA (COG2062), Preprotein translocase subunit YajC (COG1862), CRISPR/Cas system—associated endonuclease/helicase Cas3 (COG1203) and Restriction endonuclease Mrr (COG1715).COGs that were only found in all sponge-associated Thaumarchaeota, deep-sea sponge-associated Thaumarchaeota and free-living Thaumarchaeota were shown in Boruta “random forest” analysis was used to identify the COGs that were significantly different between sponge-associated and free-living Thaumarchaeota, or between deep-sea sponge-associated and deep-sea free-living Thaumarchaeota . In summChromosome segregation protein Spo0J, contains ParB—like nuclease domain (COG1475) that enriched in both all sponges and deep-sea sponges was detected in KEGG annotation (K03497). The 3′, 5′—cyclic AMP phosphodiesterase CpdA (COG1409) that deprived in both all sponges and deep-sea sponges was detected in KEGG annotation (K03651). Sponge-specific Preprotein translocase subunit YajC (COG1862) was only detected in sponge-associated Thaumarchaeota in KEGG annotation (K03210). However, the sponge-specific Phosphohistidine phosphatase SixA (COG2062) was not found in sponge-associated Thaumarchaeota but only in free-living Thaumarchaeota in KEGG annotation (K08296).Moreover, some sponge-enriched or sponge-specific genes found in COG annotation were also detected through KEGG annotation . ChromosProphage maintenance system killer protein (COG3654) in “Mobilome: prophages, transposons” (X) was exclusively annotated in deep-sea sponges . Deep-sea sponge-associated Thaumarchaeota were also highly enriched in CheY chemotaxis protein or a CheY—like REC (receiver) domain in both COG database (COG0784) and KEGG database . The copies of COG0784  and map02030  in deep-sea sponge-associated Thaumarchaeota were higher than both deep-sea free-living Thaumarchaeota  and shallow-water sponge-associated Thaumarchaeota  was found for these comparisons based on unpaired two-tailed Welch’s t-test, which was possibly caused by the limited sponge-associated Thaumarchaeota genomes with >95% completeness. Those results demonstrated the importance of “Signal transduction mechanisms” (including Chemotaxis) for the deep-sea sponge-associated Thaumarchaeota. The only common result of sponge-enriched and deep-sea sponge-enriched COG was Chromosome segregation protein Spo0J, contains ParB—like nuclease domain .In general, more sponge-specific and sponge-enriched COGs were shown in deep-sea sponges than in all sponges compared to their free-living counterparts . COGs inCRISPR/Cas2 of bin1 was found in COG annotation (COG1343) as well as KEGG annotation (K09951), but the CRISPR/Cas1 was not found in KEGG annotation.Similar to COG annotation, genes annotated based on KEGG also showed relevant enrichments of sponge-associated Thaumarchaeota in some frequently-discussed systems: CRISPR/Cas system, RM system and TA system . In summIn this study, the classification of both MAGs and 16S rRNA-gene amplicons showed the dominance of Thaumarchaeota within our deep-sea sponge . For metDifferences in genome size between symbionts and free-living counterparts in the seawater column have been debated for a long time. A genome reduction was found in symbiotic bacteria and was attributed to adaptation within the host environment . Smallerp < 0.05) in sponge-associated Thaumarchaeota compared to free-living Thaumarchaeota was found both in deep sea and in shallow water. This genome expansion within both deep-sea and shallow-water sponge-associated Thaumarchaeota might indicate that the symbiotic strategies in shallow-water and deep-sea environment are similar. The gene content and organization may be influenced by both nutrient levels and habitat types. For example, Thaumarchaeota species at a neutral soil pH that occupy nutrient-enriched environments tend to have larger genomes and lower coding densities  coding densities than those of free-living Thaumarchaeota, irrespective of depth. The lower coding density in sponge-associated Thaumarchaeota might be explained by the nutrient-rich conditions. Relatively enriched carbon and nitrogen compounds can be directly obtained from sponge hosts . At the gene level (level 3), five deep-sea sponge-enriched COGs (p < 0.05), one deep-sea sponge-specific COG and one sponge-specific COG accounted for the enrichment of the function “Signal transduction mechanisms”. However, all five deep-sea sponge-enriched COGs were not enriched anymore when considering shallow-water Thaumarchaeota genomes (sponge and free water) together. This might be due to the high copies of these COGs in shallow-water free-living Thaumarchaeota that undermine the presence of these COGs detected within shallow-water sponge-associated Thaumarchaeota. Moreover, the difference might suggest the unique characteristics and adapting mechanisms of Thaumarchaeota within deep-sea sponges.In COG functional categories, the function of “Signal transduction mechanisms” (T) was enriched in both all sponges and deep-sea sponges  was found significantly enriched in deep-sea sponge-associated Thaumarchaeota compared to deep-sea free-living Thaumarchaeota. The conserved universal stress proteins (USPs) are found in bacteria, archaea, fungi, plants and even invertebrates animals , CheY chemotaxis protein or a CheY—like REC (receiver) domain (COG0784 and K03413), and Chemotaxis protein CheY—P—specific phosphatase CheC (COG1776). TCST systems play major roles in the process of a variety of biological processes  found in this study is a kind of HK, while CheY (COG0784) and CheC (COG1776) were genes related to RR. Moreover, CheY and related “Bacterial Chemotaxis” pathway (KEGG map02030) were found higher in deep-sea sponge-associated Thaumarchaeota compared to Thaumarchaeota from shallow-water sponges and deep-sea  were specifically enriched in deep-sea sponge-associated Thaumarchaeota compared to deep-sea free-living Thaumarchaeota, which were rocesses . In TCSTrocesses . The Sigdeep-sea . A givendeep-sea  that alldeep-sea , temperadeep-sea , small mdeep-sea  and antideep-sea . In H. pdeletion . TherefoPrevious study has found several genomes of sponge microbiome contained gene families encoding adhesive molecules and type IV pilus structures, which were expected to counteract the strong shear foreces from high seawater flow rate induced by sponge pumping . In our Ankyrin repeat (COG0666) was annotated in Nitrosopumilus sp. ESC from a deep-sea sponge. Ankyrin repeat (ANK) proteins constitute one of the most abundant families of repeat proteins, usually implicated in specific protein–protein interactions  was found as deep-sea sponge-enriched COG. The signal recognition particle (SRP) is a ribonucleoprotein complex involved in the targeting of nascent proteins to translocation sites contained in Eukarya, Bacteria and Archaea  was detected in a shallow-water sponge-associated Thaumarchaeota . YajC complex could transiently interact with the translocon, assisting the processes of posttranslational protein translocation and cotranslational integration of membrane proteins (This speculation could be further supported by our result that the only proteins . Howeverp < 0.05). We found the enrichments of those common-shared genes were involved in CRISPR-Cas system, RM system, and TA system in all sponge-associated Thaumarchaeota, suggesting that these functions have vital and common roles for their adaptation to the hosts.In our study, genes in sponge-associated Thaumarchaeota were significantly enriched for the function of “Defense mechanisms” (V) at COG level 2 compared to genes in free-living Thaumarchaeota (Sponge symbionts are likely to be exposed to numerous kinds of phages due to the high filtration activity of their hosts . Some spCRISPR/Cas1 (COG1518), CRISPR/Cas2  and CRISPR/Cas3 ] were only detected in sponge-associated Thaumarchaeota in this study. Moreover, CRISPR/Cas2 was only detected in bin1 in our analysis, while Cenarchaeum symbiosum A was the only Thaumarchaeota genome that encoded CRISPR/Cas3. CRISPR/Cas protein families function in the immune system in many prokaryotic genomes, providing cell immunity against phage infection and foreign DNA expression (CRISPR and CRISPR-associated (Cas) genes are often more abundant in the genomes of sponge-associated bacterial symbionts than in their free-living counterparts , and sucpression . Sponge pression . TherefoMrr (COG1715). Moreover, the unique Thaumarchaeota bin1 also harbored three copies of Type 1 restriction-modification system (COG0286) and Restriction endonuclease S subunit (COG0732). The high abundance and uniqueness of these genes suggested that RM systems are important for Thaumarchaeota in sponges. RM systems represent a primitive immune system in bacteria. They are broadly found in prokaryotes due to their role in defense against invading DNA and in additional cellular processes, thereby increasing survival rates . Doc belongs to the type II TA system and could kill host cells . More high-quality sponge-associated Thaumarchaeota genomes are needed in the future to detect genes related to phage defense, the regulation of host cell death, and adaptation to the symbiotic community.3′, 5′—cyclic AMP phosphodiesterase CpdA (COG1409) was the only sponge-deprived COG found in both deep-sea sponges and all sponges. The regulation of intracellular concentration of cAMP is dependent not only on the synthesis of cAMP but also on the hydrolysis of cAMP by cAMP phosphodiesterase  was found enriched in deep-sea sponge-associated Thaumarchaeota compared to deep-sea free-living Thaumarchaeota. Class III adenylate cyclases are signaling proteins present in bacteria, archaea, and eukaryotes. This cAMP-producing enzymes were reported to be able to translate diverse intracellular and extracellular stimuli into a uniform intracellular signal  was considered as both sponge-specific and deep-sea sponge-specific COG, showing the importance of this gene in all sponge-associated Thaumarchaeota. Low-copy-number genomes like bacterial chromosomes and certain plasmids have evolved partitioning (Par) mechanisms to ensure that daughter cells receive a full complement of the genetic material, in contrast to high-copy-number plasmids that rely on random partitioning , chemotaxis protein CheY (biofilm formation), archaellum (swimming ability) and ankyrin repeat (host-microbe interation), suggesting the unique genomic characteristics and adapting mechanisms of Thaumarchaeota within deep-sea sponges. Such enrichments might be important for their adaption under various stresses, such as shear forces from host pumping, catabolite repression and invading phages in deep-sea sponges. Genes involved in defense mechanisms and symbiotic relationships, such as genes related to the RM systems (defense invading DNA and symbionts fitting), CRISPR/Cas systems (defense phages), and TA system (defense phages and manipulate host cell death), were highly enriched in both shallow and deep-sea sponge-associated Thaumarchaeota, suggesting the pivotal and common roles of those genes for the adaptations of those Thaumarchaeota to the hosts. The high enrichment of genes involved in defense mechanisms indicated that those sponge-associated Thaumarchaeota species continuously face invading phages within sponges. Moreover, different enriched genes and dispersed clustering of different sponge-associated Thaumarchaeota suggest that taxa are associated with different ecological niches and evolutionary stages within sponges.https://ngdc.cncb.ac.cn/search/?dbId=gwh&q=GWHBGBV00000000.The data presented in the study are deposited in the NGDC repository, accession number GWHBGCA00000000, GWHBGCB00000000, GWHBGBY00000000, GWHBGBZ00000000, GWHBGBV00000000, CRA006398, and CRA005418. Available at NGDC from China National Center for Bioinformation (CNCB): WX and LD designed research. WX, PW, ML, and CZ performed research. WX and PW analyzed data and wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) [SML311019006 and SML311020006], and the Project of China Geological Survey [DD20191002 (WX)].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.


Given that FA oxidation and glucose-derived FA synthesis are two antagonistic mitochondrial processes in the same cell, it has long been questioned how brown adipocytes run FA oxidation and FA synthesis simultaneously. In this review, I summarize mechanisms regulating mitochondrial substrate selection and describe recent findings of two distinct populations of brown adipocyte mitochondria with different substrate preferences. I further discuss how these mechanisms may permit a concurrent increase in glycolysis, FA synthesis, and FA oxidation in brown adipocytes.Brown adipocytes is a specialized fat cell that dissipates nutrient-derived chemical energy in the form of heat, instead of ATP synthesis. This unique feature provides a marked capacity for brown adipocyte mitochondria to oxidize substrates independent of ADP availability. Upon cold exposure, brown adipocytes preferentially oxidize free fatty acids (FFA) liberated from triacylglycerol (TAG) in lipid droplets to support thermogenesis. In addition, brown adipocytes take up large amounts of circulating glucose, concurrently increasing glycolysis and  Brown-leceptors –5. Notabeceptors , 7 and heceptors –14 increde novo fatty acid synthesis (FAS), and fatty acid oxidation (FAO)  –19, whicon (FAO) . In most2 in the mitochondrial matrix. These reduced electron carriers donate their electrons to the electron transport chain (ETC) system, which is composed of four multi-subunit complexes (I-IV) located in the inner mitochondrial membrane (IMM) and two mobile electron carriers coenzyme Q and cytochrome c (+ ions) from the mitochondrial matrix to the intermembrane space, creating an electrochemical proton gradient, also known as the proton motive force (PMF). The PMF is a form of potential energy composed of an electrical charge gradient (ΔΨm) and a chemical gradient (ΔpH) across the IMM  cycle, which generates NADH and FADHchrome c , 22. Sub the IMM . The reshosphate . For thehosphate . Fluctuahosphate .+ transport protein, allows the re-entry of protons into the mitochondrial matrix independent of ATP synthesis  in the IMM , 28 alonynthesis , 30. Thuomplexes –33.3-adrenergic receptor agonists, brown adipocytes liberate free fatty acids (FFA) by lipolysis of TAG stored in lipid droplets as well as take up extracellular nonesterified fatty acids (NEFA) from the circulation . Accordingly, FAO is more efficient in generating NADH and FADH2 and FA has been viewed as the primary substrate for energy-demanding brown adipocyte mitochondria  and UCP1 activity (proton leak) in the IMM. Upon adrenergic stimulation of the cell by cold-dependent activation of the sympathetic nervous system or βculation –38. FFA culation , 39. Thechondria , 40.de novo lipogenesis (DNL) through multiple mechanisms (de novo FA synthesis (FAS); ii) Glucose-derived glycerol-3-phosphate serves as the structural backbone for TAG synthesis; iii) Glycolytic intermediates support the pentose phosphate pathway generating NADPH needed for DNL; and iv) Cytosolic ATP production during glycolysis meets energy requirement for DNL as well as compensates for the loss of mitochondrial ATP synthesis. Therefore, it has been suggested that, while oxidizing FA to fuel thermogenesis, brown adipocytes concurrently increase glycolysis and de novo FAS to replenish intracellular TAG pool in lipid droplets .More interestingly, recent studies have shown that cold-activated BAT in rodents and humans utilizes additional substrates such as branched-chain amino acids (BCAA) , 51, glude novo FAS  that catalyzes the conversion of glucose-derived pyruvate to acetyl-CoA in the mitochondria  and pyruvate kinase (PK)  , 55–57.de novo synthesized FA. Remarkably, malonyl-CoA produced by ACC2 allosterically inhibits CPT1  increases citrate production in the mitochondria. Under high glucose, excess citrate is exported to the cytosol and hydrolyzed back to acetyl-CoA and OAA by ATP-citrate lyase (ACLY). Acetyl-CoA is then carboxylated to malonyl-CoA by two acetyl-CoA carboxylases, ACC1 and ACC2 . Malonylits CPT1 , 60 thatde novo FA synthesis catalyzed by fatty acid synthase (FAS). In contrast, ACC2 is associated with the OMM and regulates FAO through malonyl-CoA-mediated CPT1 inhibition  and peridroplet mitochondria (PDM) –78. PDM chondria Figure 2chondria , FA-deriThe association between mitochondria and lipid droplets has been overserved in other tissue/cell types including skeletal muscle, heart, and adipocytes , 81–83. de novo FAS, and FAO. Upon activation, brown adipocytes increase glycolysis and de novo FAS to replenish intracellular TAG pools that are depleted due to increased lipolysis and FAO. The co-existence of FA-oxidizing and lipogenic mitochondria within the brown adipocyte in addition to heterogeneity of brown adipocytes may in part explain the unique capacity of brown adipocytes to be involved simultaneously in FAO and FAS.Brown adipocytes have two unique features: (1) UCP1-mediated dissipation of the PMF, which provides a mechanism for maximal substrate oxidation in the mitochondria and (2) concurrence of glycolysis, The author confirms being the sole contributor of this work and has approved it for publication.