Understanding the native role of CRISPR-Cas systems in providing adaptive immunity can lead to new CRISPR-based technologies. Here, we develop CRISPRminer, a knowledge base and web server to comprehensively collect and investigate the knowledge of CRISPR-Cas systems and generate instructive annotations, including CRISPR arrays and Cas protein annotation, CRISPR-Cas system classification, self-targeting events detection, microbe–phage interaction inference, and anti-CRISPR annotation. CRISPRminer is user-friendly and freely available at  Fan Zhang et al. present CRISPRminer, a comprehensive database for exploring CRISPR-Cas systems in more than 3500 microbial species and a web-server for in-depth data mining. CRISPRminer allows researchers to predict CRISPR-Cas systems and investigate self-targeting CRISPRs, microbe-phage interaction networks, and anti-CRISPR features. CRISPR -Cas (CRISPR-associated proteins) systems are prokaryotic adaptive immune systems that are known to protect bacteria and archaea from phage infection. Understanding the native role of CRISPR-Cas systems and their programmable nature is very inspiring for adaptive immune system studies and can lead to the development of new CRISPR-based technologies.1. Bacteria utilize CRISPR-Cas adaptive immune systems for protection against phage infection2. In turn, some phages have been found capable to evade CRISPR-Cas immunity using anti-CRISPR proteins4. Several databases or web servers have been developed to gather information and provide useful applications to study CRISPR-Cas systems, such as CRISPRs web server , CRISPRCasFinder8, CRISPRone9, CRISPI10, and anti-CRISPRdb database11. CRISPRFinder5 and its updated version CRISPRCasFinder8 are tools to detect CRISPR arrays and Cas gene clusters. CRISPRdb6 focus on displaying CRISPR arrays and generating dictionaries of spacers and repeats. CRISPRcompar7 is used to compare CRISPRs. In general, the CRISPRs web server, CRISPRCasFinder8, CRISPI10 and CRISPRone9, focuses on the annotation and characterization of CRISPR arrays of repeat-spacer units, and/or the Cas locus, while the anti-CRISPRdb11 is a database of manually collected anti-CRISPR proteins and their homologs from publications. However, each of these databases only focuses on a specific field of CRISPR biology and provides limited information concerning CRISPR-Cas systems or anti-CRISPRs, without building a connection between the microbe and phage based on CRISPR-Cas immunity. Currently, with the expansion of the diversity and classification of CRISPR-Cas systems, and novel findings on microbe–phage coevolution, the study of CRISPR-Cas systems requires all-inclusive data from multiple aspects/dimensions combined with thorough mining, yet such a comprehensive online resource or web server is still lacking.Bacteria and phages are engaged in an evolutionary arms raceHere, we develop the CRISPRminer database and web server to fill this gap by comprehensively collecting and mining the knowledge underlying these systems from published observations and generating instructive annotations, and developing pipelines for CRISPR-Cas system prediction, classification, and annotation based on python scripts and a series of public tools.The current CRISPRminer database has integrated five categories of information that are experimentally or computationally identified to be important for CRISPR studies Fig. , includiftp://ftp.ncbi.nlm.nih.gov/genomes/all/), we identified 91,685 CRISPR arrays in 62,176 bacterial and 167 archaeal organisms (distributed in 3588 species of 1279 genera) using PILER-CR12, among which 43,140 organisms (including 2467 complete genomes and 262 plasmids) are detected with both the CRISPR arrays and the corresponding Cas proteins. The process for CRISPR-Cas system detection is demonstrated in Fig. 12 program is used with default parameters to identify CRISPR arrays, and then sequences including 15 kbp upstream and downstream of the CRISPR arrays are extracted. Open Reading Frame (ORF) annotation is performed using FragGeneScan13. All ORFs are further annotated using RPS-BLAST against 395 profiles that represent 93 distinct Cas protein families. The CRISPR arrays lacking nearby Cas genes or Cas clusters lacking nearby CRISPRs are defined as isolated ones. The type of each CRISPR-Cas locus is determined according to the signature cas genes, such as major effector module genes including cas7, cas5, cas8, cas10, csf1, cas9, and cpf114. At the genus level, Mycobacterium, Escherichia, Clostridioides, and Salmonella contribute to approximately 50% of the detected CRISPR systems. At the species level, Mycobacterium tuberculosis, Escherichia coli, Clostridioides difficile, and Salmonella enterica are the dominant species. However, the enriched genus and species may be due to the largest number of strains sequenced for health-related issues, but not the actual distribution of these CRISPR-Cas systems in the microbial world.By investigating completely or partially sequenced archaeal and bacterial genomes downloaded from the NCBI FTP site  and the Cas proteins  with detailed information for the CRISPR array  and Cas genes . In addition, using the “compare” tool, users can compare the CRISPR systems among the organisms belonging to the same species or genus.In the CRISPR-Cas module, users can browse the precalculated CRISPR-Cas systems through the taxonomy tree, or fuzzy search of the bacterial and archaeal description or using RefSeq accession numbers. Both global and local views are provided to visualize the detailed information. The global view provides the locations of the CRISPR regions on the genome coupled with the predicted genomic islands (GIs) using IslandViewer19 , as well as two putative new types V  and VI 21. Users can conveniently browse the bacterial organisms belonging to each (sub) type with detailed information, such as bacterial description, RefSeq ID, Genbank ID, and the Cas operon composition.The Classification module provides (sub) types annotation of the CRISPR-Cas systems based on the signature genes and distinctive gene architectures according to the most recent classification studies19 Fig. . The cla22, and has been used as a genomic marker to screen CRISPR-Cas inhibitor genes in Listeria monocytogenes23. The Self-Targeting module includes 22,110 self-targeting events collected from publications24 and 6260 putative self-targeting events detected from 4136 CRISPR containing organisms using a similar methodology to previous reports13  against an integrated database of 2281 phage and archaeal virus genomes from the NCBI viral Genome Resource, the EMBL EBI phage genomes and the phagedb databases  genes with the HTH (helix-turn-helix) domain usually located downstream of the anti-CRISPRs  of all validated anti-CRISPR proteins, extracted the surrounding genomic content , and determined their functions using rpsblast with an e-value cutoff of 10e−2 against the Conserved Domain Database (CDD)The Prediction module is used to annotate CRISPR-related knowledge  for an existing or a newly sequenced genome by directly uploading or pasting the nucleotide sequence in FASTA format. The prediction procedure underlying CRISPRminer is implemented using python scripts and a series of public tools including blastn/blastp, rpsblast, HmmScan, FragGeneScan, PILER-CR, and CRISPRCasFinder Fig. . At the CRISPRminer is implemented using the ThinkPHP framework and the front-end design is created using the Bootstrap framework, which is a well-established tool for intuitive design. Besides, the front-end design is also created using the cross-platform JavaScript library jQuery, which is designed to simplify the client-side scripting of HTML. In addition, CRISPRminer also uses DataTable, the plugin for jQuery, to conveniently display data. CRISPRminer back-end runs on top of an Apache server, and the communication with the server is made through a Common Gateway Interface. The server works by redirecting or sending the user input to the python script for handling and displaying the results in numerical, textual, and visual output.In summary, we comprehensively collected information about CRISPR-Cas systems and anti-CRISPRs to construct the CRISPRminer web server, seeking to accelerate our understanding of the co-evolutionary relationship between microbe and phages. As a specifically designed CRISPR knowledge database, CRISPRminer not only integrates the dispersed data but also provides a convenient way for in-depth data mining. First, we provide a Prediction module to investigate all five categories of CRISPR-related information for an existing or a newly sequenced genome by uploading or pasting the sequence. Second, we provide several techniques for browsing and comparing the detected CRISPR-Cas systems in the CRISPR-Cas module. Users can look up desired bacterial and archaeal genomes through the taxonomy tree, or by a fuzzy search of the microbial description or use of the RefSeq accession number. The CRISPR systems could also be compared at different taxonomy levels (species or genus) and coupled to the annotation of genomic islands  and prophage regions. Third, by studying self-targeting spacers from all detected CRISPR arrays in currently sequenced bacterial genomes, users could further explore their biological meanings in CRISPR-based autoimmunity. Fourth, by blasting 918,168 spacers against an integrated database of 2281 phage genomes, a large-scale microbe–phage infection network is inferred and could support further characterization of the interaction patterns and underlying processes. Finally, we incorporated experimentally validated anti-CRISPR proteins and their homologs, and annotated their surrounding gene content to facilitate anti-CRISPR discovery. This comprehensive and user-friendly CRISPRminer offers a large collection of information that could be useful to the CRISPR or microbiology community and is a much-needed tool that will facilitate research on CRISPR biology and complement other available tools for CRISPR-based applications such as genome editing. Our database will be updated regularly with newly released bacterial and archaeal genomes and provide all five categories of CRISPR-related information for these genomes automatically by utilizing the underlying prediction procedure.https://blast.ncbi.nlm.nih.gov/Blast.cgi), HmmScan (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan), PILER-CR (https://www.drive5.com/pilercr/), FragGeneScan (http://omics.informatics.indiana.edu/FragGeneScan/), and CRISPRCasFinder (https://crisprcas.i2bc.paris-saclay.fr/Home/Download). CRISPRminer user manual is available at http://www.microbiome-bigdata.com/CRISPRminer/index.php/Home/Index/help. The source code for CRISPRminer is available in the GitHub repository The public tools used in this study can be downloaded from their official website, such as blastn/blastp and rpsblastp (


Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials. Intracellular pathogens are considered to be among the major bacterial public health threats . The outDespite the ability of certain antibiotics to cross cell membrane, their intracellular efficacy can be poor due to: (I) intracellular concentrations below the minimum inhibitory value; (II) intracellular environment  that may affect the antibiotic activity; (III) antibiotic accumulation in subcellular compartments that are different from those in which pathogens reside.hyaloid (vitreous) and uronic acid. Further work by Karl Meyer and his associates led to the resolution of the chemical structure of HA by the 1950s . Erophages . CD44 istrophils , possiblal cells  where it contact . This IpLeishmania in primary and RAW 264.7 macrophages [Leishmania replicates; once internalised, HA provides Leishmania with essential nutrients for growth and virulence. With the aim to investigate this aspect the strategy adopted by the Nader group was generate N-acetylglucosamine (GlcNAc) acetyltransferase (GNAT) deficient Leishmania (Δgnat). This mutant was unable to grow or survive even when macrophages were cultivated in the presence of exogenous GlcNAc, suggesting intracellular HA provides Leishmania with essential carbon sources [Once internalised, the ability of the microorganism to proliferate intracellularly is another advantage to survival. HA also seems play a role in this stage of infection. Evidence suggests that HA is important for the growth of the parasite M. tuberculosis and Mycobacterium bovis bacillus Calmette-Guerin  have been shown to be able to utilise HA as a carbon source for proliferation [3H-labeled HA was added to an infection cell culture model where it was found to be incorporated into the live BCG, demonstrating HA uptake by the pathogen. Further work using l-Ascorbic acid 6-hexadecanoate , which is an inhibitor of HAase, suppressed the enhancing effect of HA on the growth of Mycobacteriae, suggesting: (I) short HA chains are preferred as a carbon source; (II) Mycobacteriae utilise the exogenous HA [Other microorganisms have also been shown to utilise HA as a nutrient source for intracellular groth; for example strains of feration . In ordeenous HA .M. tuberculosis grew when co-cultured with HA-synthase1 (HAS1) and HAS3  but not HAS2 (which synthesises HA with molecular weights higher than 2 × 106), confirming shorter HA chains are preferential for growth. Specifically, HAS1 appeared to be the major HA synthase in Mycobacteriae-infected mouse lungs [ase inhibitors, such as Vcpal [Mycobacteriae in mouse lungs, evidencing that HAase or a potential transporter of short size HA fragments could be potential targets for therapies against such pathogens.Further experiments by the Matusmoto group also demonstrated that both BCG and se lungs  Treatmense lungs ) could bse lungs . Among tse lungs . The appas Vcpal , apigenias Vcpal  have beease that are produced by several pathogens, such as Staphylococcus spp. and Streptococcus spp. [ase.Taking together these data, it is reasonable to assume HA may be a suitable biomaterial for building nano-carriers to target intracellular pathogens, acting as ‘Trojan Horse’, as: (I) a number of host cells  highly express CD44 and internalise HA; (II) HA can enter cells through CD44 receptor that is also used by such pathogens for the cell invasion; (III) like other nanoparticles, HA nano-carriers can be engineered in order to target sub-cellular compartments  where the microorganism grows and replicates; IV) HA nano-carriers may be cleaved by HAcus spp. , facilitcus spp. , and at N-acetyl group (following deamidation). In the last decades, several kinds of HA-based nano-carriers have been developed, including self-assembling nanohydrogels (NHs) [In recent years, HA has received enormous attention as a biomaterial for building nano-carriers, thanks to its biocompatibility, low-toxicity, biodegradability, hydrophilicity, ability to protect the entrapped drug and to enhance the solubility of hydrophobic molecules. Furthermore, HA chains can be easily functionalised, in order to develop materials suitable for drug delivery. Chemical modifications of HA have been extensively reviewed ,70 and tls (NHs) ,72,73, cls (NHs) ,75 or phls (NHs) ,77 or inls (NHs) ,79 and bls (NHs) ,81, and ls (NHs) ,83. A nuls (NHs) ,85.l-lysine), in order to achieve self-assembled HA NHs, capable to physically encapsulate and deliver genetic material [For example, HA-based nanoparticles are produced by using a number of strategies and are classified by the type of cross-linking from which they are formed: the most common nanoparticles are made by hydrophobic associations , chemicamaterial .Chemically cross-linked nanoparticles are usually more stable than the physically cross-linked analogues. However, the methods used for forming cross-linked nanoparticles, such as the micro-emulsion method , generalThe ionotropic gelation process represents another useful technique to prepare physically cross-linked HA nano-carriers. An example is represented by HA/chitosan nanoparticles : the strAmong the advantages that HA-based nano-carriers offer, the binding of HA to CD44, is one of the most relevant; this property ensures both an active targeting to cells that over-express CD44 and the intracellular delivery of therapeutics. Among the cells, certain cancers, such as breast cancer, over-express CD44 and, for this reason, HA-based nano-carriers have been especially studied for cancer therapy and theranostics purposes. The synthesis and application of HA-based nano-carriers for targeting tumours have been extensively reviewed in a number of works ,94, and The ability of HA to cross the cell membrane is another important advantage; this attribute represents an extremely useful strategy to deliver certain drugs intracellularly. Small molecules ,88, polyMycobacteriae can survive and replicate within macrophages by resisting lysosomal delivery by residing in early phase endosomal compartments [Salmonellae [Brucellae [Shigella spp. [Listeria spp. [Rickettsiae [S. aureus [Keratinocytes, activated macrophages and fibroblasts are cells that express CD44 and highly take up extracellular HA, representing another interesting target for HA-based nano-carriers. Recently, the cellular uptake of HA/chitosan nanoparticles by activated macrophages, for a targeted therapy and CD44-mediated nucleic acid delivery, has been investigated in depth ,99. All artments , whilst monellae  and Brucrucellae  survive lla spp. , Listeriria spp.  and Rickkettsiae  are able. aureus ,107 and . aureus  appear t. aureus ,109; typ. aureus  and macr. aureus  predomin. aureus , whereas. aureus  have beeTherefore, among all the drug delivery systems for antimicrobial activity, the HA-based nano-carriers may represent a novel paradigm for targeting to cells where pathogens persist, enhancing the intracellular drug concentration in the specific sub-cellular compartments. Indeed, such nano-carriers can be customised in order to target lysosomes  or to esw = 2.2 × 105, CH degree of functionalisation = 15% , mean diameter~180 nm) quickly accumulate (within 1 h) in acidic endosomal and lysosomal compartments of human keratinocytes (HaCaT cells), reaching the highest co-localisation with those vesicles in 4 h [Even if a number of works have shown HA-based nano-carriers, are internalised by cells especially by CD44 receptors ,116,117,s in 4 h . Intraces in 4 h .Self-assembling oleic acid-ethylendiamine nanoparticles, coated with HA (mean diameter~150 nm) showed co-localisation with lysosomes of colon cancer cells (HCT-116) over 6 h. Moreover, it was found that this nano-system is taken up by cells through both CD44 and clathrin-dependent endocytosis routes .w (from 5.0 × 103 to 1.6 × 106) and degree of grafting density  have been employed with the aim to investigate the impact of these parameters on the liposome uptake (the mean diameter ranged from 120 to 180 nm). Results showed HA-liposomes bind to CD44; this binding increases by increasing either HA Mw or grafting density . Moreover, CD44-mediated uptake of HA-liposomes happened through lipid raft-mediated endocytosis (that is a cholesterol-dependent route) and it was independent from clathrin-coated vesicles or the caveolae or macropinocytosis pathways. HA-liposomes were found to be predominantly localised in acidic endosomal and lysosomal compartments [The intracellular pathway of polycarbonate: polyethylene: cholesterol (65:5:30) liposomes grafted with HA has been investigated on human lung carcinoma epithelial cells (A549) and human breast cancer cell lines (MB-231 and MCF7) . A549 anartments .S. aureus, Leishmania and, possibly, M. tuberculosis.Taking together these results, it is reasonable to assume that negatively charged and amphiphilic HA-based nano-carriers  may enter cells especially through CD44 and predominantly accumulate into lysosomes, by following the endosomal-lysosomal pathway. Consequently, these nano-systems may be particularly suitable for targeting to pathogens that accumulate and replicate into these vesicles, such as l-lysine, chitosan) or cationic lipids, which have been especially employed for gene therapy [l-histidine) micelles [Shigella spp. and Listeria spp.The nanoparticle uptake through endosomal-lysosomal pathway may show some drawbacks: (I) in lysosomes, the low pH (~5) and the presence of an array of hydrolytic enzymes may lead to the destruction of such therapeutic molecules; (II) drugs do not reach the desired site of action, showing low effectiveness ,120. The therapy . HA/chit therapy . Their i therapy  and huma therapy , showing therapy  and selfmicelles . In ordemicelles  by simulThe endosomal escape can be also achieved by using fusion proteins able to catalyse the membrane fusion between the particle and endosomes or to generate a pore on the membrane or the lysis of the membrane in order to empty their ‘cargo’ in the cytoplasm . Also thase that are produced by several pathogens, such as Staphylococcus spp. and Streptococcus spp. [ase. In this scenario, HA-based nano-carriers have been started to be developed and studied for the intracellular delivery of antimicrobials, both in vitro and in vivo.With increasing interest in the potential application of HA-based nano-carriers for the intracellular delivery of antimicrobials, there have been a growing number of investigations looking at their in vitro and in vivo efficacy. The use of HA-based nano-carriers for antimicrobial purposes could show several advantages in comparison to those of nano-carriers made of other polymers/materials: (I) a number of host cells  express CD44 and highly internalise HA, providing an active targeting; (II) HA can enter cells through CD44, the receptor also used by pathogens for the cell invasion; (III) HA-based nano-carriers may be cleaved by HAcus spp. , thus facus spp. , and at M. tuberculosis or Mycobacterium avium showed that treatment with an antimicrobial peptide (LLKKK18) entrapped into self-assembling HA-based NHs provided cellular/sub-cellular targeting and prevented the degradation of LLKKK18 by proteases [mycobacteriae was enhanced using HA NHs even though the exact kind of vesicles in which the co-localisation occurred was not identified; (IV) experiments carried out with LLKKK18-loaded NHs showed the new system was more effective against both M. avium and M. tuberculosis than the free LLKKK18 in vitro and in vivo; (V) un-loaded HA NHs reduced the infection in mice.A study by using bone-marrow derived macrophages infected with roteases . SpecifiS. aureus or Listeria monocytogenes in phagocytic cells (RAW macrophages) or non-phagocytic cells (VERO) [It has also been reported that HA-streptomycin bio-conjugate showed the ability to enhance the antimicrobial activity of free streptomycin against s (VERO) . Authorss (VERO) . HoweverP. aeruginosa and S. aureus infected HeLa cells (a model cell line) were incubated with levofloxacin (LVF), a broad spectrum and highly active and cytosolic antibiotic [S. aureus [S. aureus, but they did not improve the antibacterial activity of GM, which showed a significant effect without the employment of NHs. As it has been demonstrated that NHs co-localise with lysosomes of HaCaT cells and it is known that free LVF predominantly accumulates in the cytoplasm, these results suggested NHs may be able to change the intracellular fate of LVF from cytoplasm to lysosome, thereby targeting intracellular S. aureus, illustrating the importance of a targeted antibiotic treatment, and the the opportunity to enhance the intracellular activity of such antibiotics by using HA-based nano-carriers. It should be noted that, in both works, extracellular pathogens were previously eliminated, and the LVF-loaded NHs were tested only against the intracellular microorganisms. Though it is still not clear where the sub-cellular sites in which the loaded antibiotic was acting, the experiment was designed to remove any extracellular pathogen, so only intracellular microorganisms were counted after treatment. Moreover, as the free NHs was not effective against both intracellular and extracellular pathogens, it can be stated the antibacterial activity was only due to the intracellular LVF [Investigations have also begun to focus on microorganisms that have not traditionally been thought as intracellular bacteria as there in increasing evidence suggesting that this mode of growth may facilitate persistent and chronic infections, and may be a cause of treatment failures. For example, axenic tibiotic , loaded . aureus ; infecteular LVF ,133.P. aeruginosa, S. aureus and L. monocytogenes; HA did not improve the MIC of amikacin on planktonic pathogens, but did show a high capability to enhance the antimicrobial activity against the three pathogens within RAW 264.7 macrophages. The bio-conjugate was then tested in vivo, on mice infected intraperitoneally with L. monocytogenes, which received subcutaneous injection of HA-amikacin or its controls. Like the in vitro results suggested, the bio-conjugate showed an improvement of the antimicrobial activity of amikacin, evidencing the HA capability to enhance the efficacy of the antimicrobial against such intracellular pathogen. To the best of our knowledge, these are the only examples which describe the development and use of HA-based nano-carriers for targeting to intracellular pathogens. None of these works has shown a significant increase in toxicity of the loaded antimicrobials, both in vitro and in vivo. In contrast, a significant toxicity has been noted for chitosan-based nanoparticles [Recently, HA-amikacin was synthesised by ‘click’ reaction between HA-propargyl amide and amikacin-azide . The obtarticles , HoweverS. aureus) [uptake and catabolism of HA into cells that express LYVE-1 or HARE . This aspect reduces the efficacy of the targeted therapy, representing an important disadvantage for HA-based injectable formulations. Several strategies have however been found for overcoming these drawbacks; for example, stealth HA nanoparticles have been shown to reduce the uptake by liver and kidney, improving the targeting to the desired site of action [The relationship between HA, CD44 and the invasion and survival of a number of pathogens within the cells is becoming increasingly evident. Several microorganisms utilise CD44 and/or HA to enter and replicate within the cellular micro-environment. Furthermore, a number of host cells highly express CD44 and internalise HA, ensuring an intracellular uptake of antimicrobials loaded into HA-based nano-carriers. This scenario makes HA a possible candidate for the development of ‘Trojan Horse’ systems to target intracellular microorganisms, thus overcoming the ineffectiveness of many antibiotics intracellularly. Previous works showed the ability of HA-based nano-carriers to enhance the intracellular activity of certain antimicrobials; however, little has been carried out in biology, microbiology and drug delivery fields, therefore further studies are necessary. HA is already extensively used for topical administration in cosmetics or ophthalmology. The incorporation of antimicrobials within HA-based nanogels may represent an interesting approach for enhancing the intracellular delivery of some drugs, thus targeting to pathogens that are a common cause of chronic topical infections  . Moreovef action . ToxicitAntibiotic resistance is one of the major public health threats, and the novel approach based on HA nano-carries may represent an interesting strategy for overcoming some antibiotic failures in the treatment of intracellular infections.


Prostate cancer (PCa) is the main cause of cancer-related mortality in males and the diagnosis, treatment, and care of these patients places a great burden on healthcare systems globally. Clinically, PCa is highly heterogeneous, ranging from indolent tumors to highly aggressive disease. In many cases treatment—generally either radiotherapy (RT) or surgery—can be curative. Several key genetic and demographic factors such as age, family history, genetic susceptibility, and race are associated with a high incidence of PCa. While our understanding of PCa, which is mainly based on the tools of molecular biology—has improved dramatically in recent years, efforts to better understand this complex disease have led to the identification of a new type of PCa–oligometastatic PCa. Oligometastatic disease should be considered an individual, heterogeneous entity with distinct metastatic phenotypes and, consequently, wide prognostic variability. In general, patients with oligometastatic disease typically present less biologically aggressive tumors whose metastatic potential is more limited and which are slow-growing. These patients are good candidates for more aggressive treatment approaches. The main aim of the presented review was to evaluate the utility of liquid biopsy for diagnostic purposes in PCa and for use in monitoring disease progression and treatment response, particularly in patients with oligometastatic PCa. Liquid biopsies offer a rapid, non-invasive approach whose use t is expected to play an important role in routine clinical practice to benefit patients. However, more research is needed to resolve the many existing discrepancies with regard to the definition and isolation method for specific biomarkers, as well as the need to determine the most appropriate markers. Consequently, the current priority in this field is to standardize liquid biopsy-based techniques. This review will help to improve understanding of the biology of PCa, particularly the recently defined condition known as “oligometastatic PCa”. The presented review of the body of evidence suggests that additional research in molecular biology may help to establish novel treatments for oligometastatic PCa. In the near future, the treatment of PCa will require an interdisciplinary approach involving active cooperation among clinicians, physicians, and biologists. Prostate cancer (PCa) is the main cause of cancer-related mortality in males and the diagnosis, treatment, and care of these patients places a major burden on healthcare systems globally. In countries with a large proportion of elderly men, the morbidity rate associated with PCa remains high . ClinicaBRCA2, ATM, BRCA1, PALB2, FANCA, CHEK2, and CDK12. The available data show that homologous recombination deficiency-associated mutations correlate with a worse prognosis. These genetic aberrations are inherited in ~10–12% of men with lethal PCa  or as a pattern of restrained recurrence, also known as oligo-recurrence  process, ultimately leading to dissemination of the PCa cells from the primary tumor and metastases in epithelium-derived carcinoma , 8. The thal PCa . Furtherthal PCa –15. In gthal PCa . In mostthal PCa . Howeverthal PCa . Oligomecurrence . An oligcurrence . There icurrence , 22. Thecurrence . Most mecurrence . Availabcurrence . Also gecurrence .For these reasons, there is need to identify biomarkers that could potentially improve the treatment of advanced PCa, which often involves difficult to detect bone metastases  27). Id. Id27). Circulating nucleic acids, both RNA and DNA, are extracellular nucleic acids found in cell-free serum, plasma, and other body fluids from healthy subjects or patients . SeveralUnderstanding the biology of oligometastases should lead to a detailed acknowledgment of molecular events responsible for the shift from an organ-confined to a disseminated metastatic disease, with future clinical implications and the possibility to identify specific biomarkers able to predict cancer evolution in patients diagnosed with a localized disease. Although specific data supporting this assumption are still lacking, different strategies are likely to be adopted to approach the molecular genetics of the oligometastatic status .In this context, the primary objective of the present study is to discuss the most characteristic features of liquid biopsy as a potential tool for the diagnosis and treatment of oligometastatic PCa.99mTechnetium-methylene diphosphonate (99mTc-MDP) bone scan, computed tomography (CT), or magnetic resonance imaging (MRI) (11C-choline positron-emission tomography (PET)/CT; PET/MRI; 18fluorodihydrotestosterone PET; 68Ga-labeled prostate-specific membrane antigen (PSMA); combined ultra-small superparamagnetic particles of iron oxide-enhanced and diffusion-weighted MRI (USPIO-enhanced MRI); and ferumoxytol-enhanced MRI  . Howeverng (MRI) . For thinced MRI .18F-sodium fluoride (Na18F), 18F-fluoromethylcholine (18F-FCH), and 68gallium- and prostate-specific membrane antigen-labeled PET/CT (68Ga-PSMA-PET-CT)  and volumetric-modulated arc therapy (VMAT) can be used to optimize the dose distribution to more precisely target the tumor while reducing the dose to surrounding healthy tissue. The combination of high-dose RT and systemic treatment reduces the number of PCa cells in the prostate and pelvic lymph nodes . Active -PET-CT) –37. PET/ failure –40. In t99mTc-MDP bone scan, CT, and MRI—are commonly used to determine the extent of disease spread in men with a high-risk primary, recurrent, or metastatic PCa. However, as mentioned above, the main limitation of these conventional techniques is their low sensitivity to detect small volume metastatic lesions  PET combined with CT (NaF-PET/CT) has a higher sensitivity and specificity , a metabolically active radiotracer, is used to image different tumor subtypes. However, its role in metastatic PCa is limited due to the low glycolytic activity of hormone-naive PCa cells, which restrains 18F-FDG uptake , which is these tracers are promising tools for the diagnosis of oligometastatic PCa  to treat patients with oligometastatic PCa (≤3 synchronous active lesions). Those authors found that the use of repeated salvage 18F-choline PET/CT-guided SBRT was well-tolerated and could delay the implementation of systemic therapy in selected patients with oligometastatic PCa  without clinically-relevant side effects  for all metastatic PCa (mPCa) subtypes . Howeverp < 0.0001] but did not improve OS. However, in the subgroup of patients with low metastatic burden (according to the CHAARTED definition) (p = 0.007) and FFS . In another trial (HORRAD)  , 68 EBRT(HORRAD)  ADT + EB(HORRAD) , 71. ThaAnother important question under investigation is the optimal approach to definitive local treatment, with two ongoing trials currently underway: (1) the phase II MD Anderson Cancer Center trial (NCT01751438), with initial results expected in 2019)  and (2) The results of the trials described above suggest that an aggressive, multimodal, multitargeted approach may be curative in well-selected patients. Such an approach would involve radical treatment of the primary tumor together with metastasis-directed therapy. In this context, two trials are currently underway—the Memorial Sloan Kettering METACURE trial (NCT03436654)  and the A range of salvage approaches can be used to manage loco-regionally recurrent PCa, including radiotherapy (particularly brachytherapy and SBRT) and prostatectomy. The treatment of choice for distant metastases is ADT due to its well-documented capacity to improve survival, despite the sometimes significant adverse effects . HoweverThe SABR-COMET trial  includedThe typical progression pattern in all of the treatment arms in the aforementioned trials was polymetastatic progression, a pattern that suggests that there was a subgroup of patients whose “oligometastatic” disease was actually only “oligo-visible” lesions (present from the start of treatment), and thus these patients should not have been enrolled in those trials because they did not actually met the enrolment criteria. This observation is relevant because the precise definition of oligometastatic disease depends on the quality of the imaging technique. Nevertheless, based on the current evidence, it seems reasonable to escalate MDT (using either SBRT or surgery) to all detectable metastatic lesions in patients with low-volume disease and good performance status.One approach to identifying the most appropriate treatment scheme for a particular candidate could be liquid biopsy to assess certain biomarker. Despite the undeniable efficacy of the new anti-androgens, the primary resistance  it was aIn the ongoing Dutch CABA-V7 study NCT03050866) (6 (86), tGenerally, accurate diagnosis of oligometastatic disease constitutes a problematic procedure because all radiological modalities are strongly limited by certain thresholds for the detection of size-limited metastases. Nieder et al.  performein vitro for drug testing or other purposes , such as circulating tumor DNA (ctDNA); circulating cell-free RNA (ccfRNA) or extracellular vesicles (exosomes) as well as a cargo of exosomes (nucleic acids and proteins) . CTCs capurposes . For thepurposes . The maipurposes . Additiopurposes , 94. Despurposes . For thi6-108 white blood cells. Due to their small size and relatively low prevalence, it is challenging to detect, quantify, and particularly isolate single cells. These tasks are further complicated by the EMT process that CTCs undergo, which means that the expression profile of their markers evolves over time , 966-108-targeting to impair PCa metastasis prior to it can start . H. H100). Most CTCs are single cells. However, they can also form clusters known as microemboli, particularly in the circulating blood of patients with advanced disease. CTC clusters play an essential role in the formation of metastases . However−EpCAM+ population of DTCs has an erythroid-like rather than the expected prostate gene expression profile. Moreover, two distinct populations of DTCs may occur: (1) CK+, which presents features of EMT such as high expression of Twist, TGF-β, Slug, and Zeb1 or (2) CK−/low, AR−, neuroendocrine, and/or CSCs phenotypes (Disseminated tumor cells (DTCs), which are CTCs that can home to the bone marrow, represent another interesting potential target. DTCs can be detected in peripheral blood years before clinically-detectable metastases are identified . Bone maenotypes .+/CD24−/low–and in the case of unique tumorigenic properties—also ALDH+ , which consist of the subpopulation of the CTC fraction. As a consequence of the EMT process, CTCs lose EpCAM and CK markers, leading to differentiation from the CSC subgroup. CSCs are largely resistant to radio- and chemotherapy-based treatment, and thus responsible for most metastases. CSCs remain CD44so ALDH+ –115.An interesting trial was conducted by Mandel et al. . They de+ or CK19 and CD45−. The major disadvantage of this method is that not all CTCs present the EpCAM marker on the cell membrane. Additionally, EpCAM expression may be barely detectable, and thus part of the CTC population will be lost during manipulation . Because PCR-based methods measure the whole amount of DNA, it is not possible to use this technique to differentiate between DNA-derived apoptotic and viable cells. However, RT-PCR enables the detection of mRNA derived only from viable cells, and thus identification of nucleic acid from cells responsible for the metastatic process. RT-PCR may be more useful to characterize CTCs rather to detect them. Consequently, a multi-marker approach is required to identify CTCs , 123. ThCurrently, label-free methods based on cell size and morphology, which is why as three-dimensional (3D) microfilters and bilayers seem to be desirable approaches. Their efficiency depends on pore size, rigidity, and blood flow rate. The blood flow rate may cause both “squeezing” of CTCs through pores as well as leukocyte accumulation and blood clotting if inappropriately chosen. Microfluidic devices (CTC- or herringbone-chip) isolate CTC clusters based on difference in physical features and specific antibody-binding features , 129. AnIn general, morphology-based enrichment techniques are not recommended for routine clinical procedures because a relatively large amount of cells are lost during these procedures .Cell-free DNA (cfDNA), also known as cell-free-tumor DNA (ctDNA), originates from apoptotic and necrotic cells, and is considered a biomarker of advanced solid tumors. These DNA fragments may be released by apoptotic cells in the form of nucleosomes . Tumor dCfDNA, which consist of ~160 base pairs in length, are routinely isolated from different body fluids, mainly blood. In most studies, cfDNA is used to detect point mutations and copy number variations, and can also detect larger chromosomal aberrations. Screening of epigenetic changes in cfDNA is a growing focus of research , 134. InIn healthy individuals, the normal cfDNA concentration level is <5 ng/ml, mainly derived from hematopoietic cells. Elevated levels of cfDNA in plasma are characteristic in patients with PCa, in whom the plasma contains both circulating tumor DNA (average 30%) and DNA from healthy tissue. In patients with advanced cancer (including but not limited to PCa), the average concentration of cfDNA is 17 ng/ml, with the highest concentration in patients with mCRPC (53 ng/ml) .NKX3.1, point mutations in FOXA1, TP53, and SPOP, as well as PTEN-deletion. That study found that those genomic lesions are involved in early carcinogenetic processes, suggesting that multiple distinct tumor clones give rise to metastatic disease  release DNA continuously and automatically, which may explain why it is possible to detect cfDNA in early-stage cancers. In addition, the amount of cfDNA increases as the tumor grows, which also supports the hypothesis that cfDNA is derived from living tumor cells. The hypothesis that cfDNA is released from CTCs is supported by three main factors: first, both cfDNA and CTCs are characterized by identical genetic mutations; second, CTCs can escape macrophages and easily enter the bloodstream; third, blood that contains CTCs also contains cfDNA. These facts strongly suggest that CTCs may be an alternative source of cfDNA . In one  disease . The rel disease .As an emerging biomarker for disease biology, RNA possesses several advantages over DNA, including the following: (i) RNA expression is a dynamic process that changes according to the internal needs of cancer cells; (ii) the expression profile of specific RNA molecules or fragments such as lncRNA is highly tissue- or disease state-specific; (iii) this RNA-based approach enables investigation of non-coding RNAs, fusion transcripts, splice variants, and RNA editing events , 145. CuMicro RNAs are small single-stranded non-coding RNA molecules (18–22 nucleotides in lenght) which control transduction of mRNA. Altered microRNA is related to cancer development  and metastasis formation. Therefore, microRNAs have a great potential to serve as biomarkers. The tumor tissue releases those molecules to the biological fluids such as blood, urine, saliva) inside exosomes. Therefore, they are ideal candidates used in non-invasive biopsies, known as liquid biopsy . OligomeCancer-associated RNAs can be detected in the peripheral blood of patients with PCa and for this reason they are good prognostic or predictive biomarkers. Endogenous small RNAs include miRNAs, transfer RNAs, ribosomal RNA, and RNA fragments . In patiA comprehensive overview of miRNAs and their role in EMT process in PCa was provided by Sekhon et al. . We are ZEB1 expression and cell migration. Overexpression of miR-200 members represses EMT by directly targeting and downregulating ZEB1 and ZEB2 via miR-200-binding sites located within their 3′ UTRs, resulting in enhanced E-cadherin expression and inhibition of tumor cell migration and cancer cell motility  . The assSeveral lncRNAs—not only prostate cancer antigen 3 (PCA3) but also prostate cancer gene expression marker 1 (PCGEM1), and prostate cancer associated ncRNA transcript 1 (PCAT1)—are highly prostate-specific and thus attractive candidates for use as biomarkers . Based oGAS5 expression levels are believed to promote basal apoptosis and enhance the response to apoptotic stimuli. By contrast, a low level of GAS5 expression diminishes the intensity of programmed cell death in response to physical and chemical stimuli (Tumor suppressor growth arrest-specific 5 (GAS5)-encoded small nucleolar RNAs (snoRNAs) are upregulated during PCa progression. High  stimuli .Localized treatment of the primary tumor leading to a radiation-induced immunological response is responsible for regression of metastasis disease. This phenomenon, known as the abscopal effect, contributes to the induction of antitumor immunity, thereby resulting in broader systemic effects , 165. Thvia an IR-mediated global stress response  receptor family members, are involved in the propagation of additional activating signals. The release of mixed proinflammatory mediators  plays an essential role in generating a specific immunologic response that can disrupt non-targeted tumor growth response . Immunogresponse , 170. Neresponse , 172.The application of immunotherapy, particularly immune checkpoint inhibitors, can enhance the systemic anti-tumor response to RT. For this reason, the combination of RT and immunotherapy is considered a highly promising approach. Patients with mCRPC benefit from the combination of anti-CTLA-4 (ipilimumab) immunotherapy with RT (single fraction 8 Gy) . PD-1 exMoreau et al.  describeAs noted above, the abscopal effect probably has a significant impact on reducing metastases in PCa. For this reason, we firmly believe that liquid biopsy is likely to be a highly valuable tool to assess the abscopal effect in oligometastatic PCa.The abscopal effect, which was first described in 1953, refers to the regression of metastatic lesions distant from the irradiated site , an effep = 0.053)—which was the primary endpoint of the study—the authors did observe, in an exploratory subgroup analysis, a strong benefit in terms of OS for patients with favorable prognostic features , who may meet criteria for oligometastatic disease. Another option is to use “disseminated radiation” from radiopharmaceuticals to stimulate the immune system. Preclinical data on radium-223 suggest it has an immunomodulatory effect (Data from clinical and preclinical studies have suggested that RT can activate the immune system, thereby promoting immune cell infiltration into the tumor , 186. Thy effect . Ongoingy effect  and sipuy effect . In addiy effect  has descUnfortunately, at present, no specific biomarkers have yet become available to guide prostate cancer treatment. The availability of specific biomarkers would be highly valuable in order to personalize treatment for each individual patient, but also to determine important questions such as the optimal timing of immunotherapy administration, switching between methods, and the possibility of withdrawing therapy. Studies evaluating the role of soluble markers in blood samples in PCa patients are currently ongoing .The main aim of the present review was to evaluate the utility of liquid biopsy for diagnostic purposes in PCa and for use in monitoring disease progression and treatment response, particularly in patients with oligometastatic PCa. This review should help to improve our understanding of the biology of PCa, particularly the recently defined condition known as “oligometastatic PCa.” In this paper, we have discussed the processes and mechanisms that are most likely to underlie the development of this distinct clinical entity. We have discussed several different possible biomarkers or gene therapy targets that could be used in future clinical practice. However, more research is needed to better elucidate this poorly-understood area. The present review of the body of evidence suggests that additional research in molecular biology may help to establish novel treatments for oligometastatic PCa. In the near future, the treatment of PCa will require an interdisciplinary approach involving active cooperation among clinicians, physicians, and biologists. The most promising novel areas of research pertain to CTCs, cell-free DNA, and RNA. Liquid biopsies offer a rapid, non-invasive approach whose use is expected to play an important role in routine clinical practice to benefit patients. However, more research is needed to resolve the many existing discrepancies with regard to the definition and isolation method for specific biomarkers, as well as the need to determine the most appropriate markers. Consequently, the current priority in this field is to standardize liquid biopsy-based techniques.ES wrote the parts concerning biological aspects. MK wrote the parts concerning clinical issues. AG designed figures. WS gave a main concept, supervised work, and she is a grant recipient. All authors took part in preparation and modification of figures and manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Naltrexone attenuates craving, and the subjective effects of methamphetamine and extended-release naltrexone (XR-NTX) reduces functional connectivity between regions of the striatum and limbic cortex. Naltrexone modulates neural activity at dopaminergic synapses; however, it is unclear whether naltrexone has an effect on large-scale brain networks. Functional networks interact to coordinate behavior, and as substance-use disorders are associated with an imbalance between reward and cognitive control networks, treatment approaches that target interactive brain systems underlying addiction may be a useful adjunct for behavioral therapies. The objective of this study was to examine the effect of XR-NTX on large-scale brain networks and to determine whether changes in network relationships attenuate drug use, craving, and addiction severity. Thirty-nine participants in or seeking treatment for methamphetamine-use disorder were enrolled in a clinical trial of XR-NTX between May 2013 and March 2015 . Functional magnetic resonance imaging (fMRI) and questionnaires were conducted before and after double-blinded randomization to a 4-week injection of XR-NTX or placebo. In the XR-NTX group, methamphetamine use was reduced along with a decrease in the coupling between executive control (ECN) and default mode (DMN) networks. As decoupling of ECN and DMN networks was associated with change in the severity of dependence, the results suggest that XR-NTX may modulate and enhance ECN attentional resources and suppress DMN self-referential and emotional processing. This study identifies the effect of naltrexone on changes in the intrinsic functional coupling of large-scale brain networks and provides a more systematic understanding of how large-scale networks interact to promote behavioral change in methamphetamine-use disorder. Although methamphetamine (MA) is a highly addictive psychostimulant causing severe physical, neurological, and emotional disruptions , there aNaltrexone, a competitive mu-opioid receptor antagonist, attenuates craving and subjective effects of MA in humans , 7. AlthAdvances in understanding the functional organization of brain systems suggest that a collection of interconnected brain areas work together to form functional networks that interact to coordinate behavior. Core networks include the Default Mode Network (DMN), which is comprised of the posterior cingulate cortex, temporal and medial prefrontal cortices and is associated with self-monitoring function and internal attention; the Executive Control Network (ECN), which includes the dorsolateral prefrontal cortex and posterior parietal cortices and is important for cognitive control; and the Salience Network (SN), which is comprised of the insula, anterior cingulate cortex, amygdala, ventral striatum, dorsomedial thalamus, hypothalamus, and substantia nigra/ventral tegmental area and is responsible for processing motivational stimuli and reward saliency . The goaThirty-nine participants were enrolled in a randomized, double-blind, placebo-controlled clinical trial of XR-NTX . Participants were recruited from community-based treatment programs and primary care clinics in Portland, Oregon, USA, between May 2013 and March 2015 and were included in a previous study examining seed-based resting-state connectivity . InclusiThe study was approved by the Oregon Health and Science University and Veterans Affairs Portland Health Care System Joint Institutional Review Board, and all participants provided written informed consent. At baseline, participants underwent resting-state functional MRI and completed survey assessments (Visit 1). Using a double-blind design, participants were randomized to XR-NTX (n = 19) or placebo (n = 20) groups based on the output from a computerized random number generator. To reduce issues with study drug adherence challenges associated with daily-dosed oral naltrexone, both the XR-NTX and placebo conditions involved a single 4-week injection, which was donated by the manufacturer. Survey and brain imaging assessments were repeated 4 weeks after baseline scans (Visit 2).A Mini International Neuropsychiatric Interview (MINI)  was cond2*-weighted image was acquired using an echo planar imaging scheme , while subjects stared at a white cross on a black screen for 6 min. One high-resolution T1-weighted magnetically prepared rapid acquisition gradient echo  was acquired for co-registration with functional images and statistical overlay.Imaging was performed on a 3 Tesla Siemens TIM Trio MRI scanner. A localizer scan was acquired in order to guide slice alignment during anatomical and functional scans. A Twww.fmrib.ox.ac.uk/fsl). Images were skull-stripped, spatially smoothed [5-mm full width at half maximum (FWHM) Gaussian kernel], and realigned to compensate for motion . Three eligible participants declined randomization. Of those randomized, 39 completed baseline and follow-up assessments that were available for analysis. Reasons for exclusion from analysis of those randomized included scheduling conflicts/no-shows and MRI confounds.At baseline, groups were well-matched on demographic variables Table 1.On average, the number of days in the past 30 days of self-reported MA use decreased from 5.05 to 1.56 in the XR-NTX group but increased in the Placebo group from 3.55 to 3.87 in the Placebo group. The repeated measures ANOVA resulted in a significant time by treatment interaction (p = 0.03), with the XR-NTX group showing greater reductions in MA use compared to the Placebo group. Mean craving scores decreased from 32.83 to 20.07 in the XR-NTX group and from 23.84 to 18.63 in the Placebo group. There were no significant time by treatment interactions on craving and (p = 0.52) or on SDSS (p = 0.13).Correlations between networks for each subject and scan are depicted in As a group by time interaction on Left ECN and DMN correlations was detected, we explored how changes in Left ECN–DMN coupling affect MA use, craving, and substance dependence severity. When examining the relationship between change in MA use and change in network coupling, we found a significant group interaction , National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and NIH Roadmap for Medical Research [1 UL1 RR024140 01 Oregon Clinical and Translational Research Institute (OCTRI)], and Department of Veterans Affairs Clinical Sciences Research and Development Merit Review Program, I0CX001558 (WH) and Development Career Development Award IK2CX001790 (MK), Oregon Health and Science University Collins Medical Trust Award APSYC0249, and Medical Research Foundation of Oregon APSYC0250. The manufacturer, Alkermes, donated extended-release naltrexone and placebo injections for use in this trial.The contents of this paper do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.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.


Objective: To analyze the factors associated with recent suicide attempts including socio-demographic and clinical characteristics in major depressive disorder (MDD) patients in China.Methods: The data were from a nationwide sample from 13 major psychiatric hospitals or the psychiatric units of general hospitals in China, from September 1, 2010 to February 28, 2011. Melancholic features and suicide attempts in the past month were defined according to the melancholic feature module and the suicide module of the Mini International Neuropsychiatric Interview (MINI). Socio-demographic and clinical characteristics were compared between MDD patients with and without recent suicide attempts. Further analyses regarding the factors associated with recent suicide attempts in MDD patients were performed via multivariate logistic regression analysis.Results: Among 1,172 MDD patients, 57 (4.9%) were reported to have made a suicide attempt in the past month. Compared to the MDD patients without recent suicide attempt, significantly higher percentage of patients in the recent suicide attempters group had previous suicide attempts  and depressive episodes with melancholic features . Logistic regression analysis indicated that previous suicide attempts  and depressive episodes with melancholic features  were independently associated with recent suicide attempts in MDD patients.Limitations: Cross-sectional design, retrospective recall of suicide attempt data.Conclusion: Recent suicide attempts are associated with melancholic features and previous suicide attempts in MDD patients in China. These data may help clinicians to identify MDD patients at high risk of suicide attempt behavior. Suicide is a serious public health problem that has received increasing attention worldwide , 2. AccoA national case-control psychological autopsy study conducted in China showed that mental disorders are present in up to 63% of people who died by suicide . The mosPrevious studies in Western countries reported risk factors for suicide attempts in MDD patients. They include family history of psychiatric disorders , adverseThere has been limited study of the risk factors for attempted suicide in MDD patients in China. A case-control study of 215 suicide attempters with MDD showed that hopelessness, negative life-events and family history of suicide were risk factors for attempted suicide . The smaThe Diagnostic Assessment Service for People with Bipolar Disorder in China (DASP) is the first nationwide, multicenter and large sample study conducted in patients with major affective disorders in China. A previous study using this data analyzed factors associated with suicide risk in MDD patients including social-demographic and clinical features . The folThe data were obtained from DASP –23, whicPatients with MDD who were receiving treatment in the participating hospitals/units were consecutively referred by their treating psychiatrists to the research team to be screened for eligibility. Patients fulfilling the study entry criteria were invited to participate in the study . The patPrior to the study, all 13 raters  of research team were trained in the use of the Chinese version of the Mini International Neuropsychiatric Interview (MINI), Version 5.0 , 26 in 2Recent suicide attempts were assessed by one of the questions in the suicidal module of MINI . “Did yoMelancholic features were defined using the melancholic features module of MINI, which is in accordance with the DSM-IV criteria . The crit-tests or Mann-Whitney U-tests for continuous data and chi-square tests for categorical data. Multiple logistic regression analysis was performed to identify the factors associated with recent suicide attempt in MDD patients. The variables with a univariate p-value < 0.05 and possible clinical variables related to suicide such as age, gender, depressive episodes with psychotic symptoms, and depressive episodes with anxiety symptoms or disorder were included in the multiple logistic regression analysis. The level of significance was set at 0.05 (two-tailed).The data were analyzed using SPSS, version 20.0 . Group differences between the recent suicide attempters and a non-suicide attempters control group were measured using independent-sample The total number of screened patients was 1,757. Of these patients, 270 patients refused or failed to complete the interview, 306 patients were excluded because of meeting DSM-IV criteria for bipolar disorder based on the MINI, and 9 patients were lacking information on the suicidal module of the MINI. Ultimately, 1,172 patients were included in the present study, and 57 (4.9%) of the subjects had a suicide attempt during the past month.2 = 171.861, p < 0.001) and depressive episodes with melancholic features .Table p-value < 0.05 on univariate analysis and clinical relevant variables including age, gender, depressive episodes with psychotic symptoms, depressive episodes with anxiety symptoms, or disorder were entered into multivariable model. Multiple logistic regression analysis with the forward stepwise method also showed that previous suicide attempts  and depressive episodes with melancholic features  were the risk factors for recent suicide attempts in MDD patients  study also found that subjects with prior suicide attempts had greater current severity of medical illness than non-attempters in MDD patients (There are several limitations in the present study. First, the DASP was a cross-sectional survey in which most of the data were retrospectively collected. Therefore, recall bias might have affected the accuracy of the data. Second, the cross-sectional nature of the study could not establish a causal relationship between the demographic and clinical features and the suicide attempts. A prospective study should be carried to evaluate risk factors of suicide in patients diagnosed with MDD. Third, the severity of depression in patients with MDD was not evaluated. Perroud et al. found that MDD patients with suicide attempts were more severely ill than subjects who had not attempted suicide . The Seqpatients . Howeverpatients . Fourth,patients  and co-mpatients , 44, 45 In conclusion, two clinical features were found to be related to recent suicide attempt in MDD patients in our study. Focusing on these clinical characteristics may help clinicians to identify the patients with high-risk of suicidal behavior and implement effective preventive interventions to reduce suicide.Y-AS and T-MS designed research. F-DY, GW, Y-RF, ZL, H-CY, JH, Z-YC, YH, JS, X-PW, H-CL, and J-BZ performed research and collected data. L-MX and LC analyzed data. L-MX, Y-AS, and T-MS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fundamental data on the distributions, diversity, and threat status of terrestrial snakes and lizards (hereafter squamates) is limited. This is due to the cryptic nature of species in this faunal group, and to limitations in the effectiveness of the survey methods used to detect these species. Camera-traps are a useful tool for detecting numerous vertebrate species, yet their use for detecting squamates has been limited. Here, we apply recent methodological advancements in camera-trapping and assessed the utility of camera-traps for inventorying a squamate assemblage by comparing camera-trapping survey results with two widely used labour-intensive methods: artificial refuges and pitfall traps.We conducted a 74-day survey using camera-traps and, concurrently, four by four-day surveys using labour-intensive methods. Given the duration and three detection methods, we compared seven variants of survey protocol, including using each method alone or all methods simultaneously. We compared both the effectiveness and cost-effectiveness of each survey protocol by estimating the number of species detected at the transect level, and by calculating the costs of conducting those surveys.We found the camera-trapping survey was most cost-effective, costing 687 AUD (CI 534–912) per squamate species detected, compared with the 2975 AUD (CI 2103–4486) per squamate species detected with the labour-intensive methods. Using all methods together was less cost-effective than using camera-traps alone. Additionally, there was a 99% probability that camera-traps would detect more species per transect than the labour-intensive methods examined.By focusing the analysis at the level of the survey, rather than the level of the device, camera-traps are both a more effective and cost-effective technique for surveying terrestrial squamates. Where circumstances are appropriate, those wildlife researchers and managers currently using camera-traps for non-squamate surveys, can adopt the methods presented to incorporate squamate surveys with little upfront cost. Additionally, researchers currently using traditional techniques can be confident that switching to camera-traps will likely yield improved results. Still, camera-traps are not a panacea and careful consideration into the benefits and usefulness of these techniques in individual circumstances is required. Snakes and lizards (hereafter squamates) are one of the most speciose groups of terrestrial vertebrates . Yet, coe.g. Varanus komodoensis; [et al. [Camera-traps have become an increasingly useful tool for detecting various vertebrates and answering a range of research questions . Althougdoensis; ), with ldoensis;  demonstrdoensis; . Comparidoensis; . Adams e [et al.  used timAs squamates are morphologically diverse and exploit various niches, numerous survey methods have been variously devised for detecting them . Using mTo examine the cost-effectiveness of camera-traps for detecting a squamate assemblage, we compared outcomes and costs of conducting squamate surveys with camera-traps and two often used complementary labour-intensive methods, artificial refuges and pitfall traps. We determined the probability that one survey protocol would detect more squamate species, at the transect level, than another to evaluate the effectiveness of each technique. By determining survey costs, we evaluated cost-effectiveness of the different approaches. Although surveys were conducted in a temperate heathland on the south-east coast of Australia, the results are nevertheless broadly applicable to similarly structured environments.We conducted the study on Beecroft Weapons Range (BWR), ~135 km south of Sydney, Australia. Beecroft Weapons Range has a temperate climate and is dominated by coastal heath from less than a metre to above two metres in height. The camera-trap and labour-intensive methods surveys were conducted concurrently between 23 November 2014 and 4 February 2015. Forty transects (~2 x ~100 m) that had vegetation cleared to understory level had been established across BWR perpendicular to vehicle tracks, starting ~2 m from track edges. However, only 10 transects could be surveyed due to site access and equipment constraints.We deployed four Reconyx HC600 camera-traps to each 100 metre transect, positioning camera-trap stations at approximately the 20, 40, 60, and 80 m intervals . Each stReconyx HC600 camera-traps can be triggered by both PIR and time-lapse triggers . As suchi.e., ~30 x 600 cm total with the bucket centrally located), were used at each pitfall trap. Vegetation, a small bait ball of peanut butter and oats, and a wet sponge were placed in the bottom of each pitfall trap to provide shelter and reduce trap mortality [The labour-intensive methods survey consisted of four sub-surveys between 23–26 November 2014, 9–12 December 2014, 6–9 January 2015, and, 1–4 February 2015. We conducted each sub-survey for four consecutive days following Fisher and Rochester [Pitfall traps were installed in late October 2014 to allow the habitat to recover from trap installation . Artificλij) for the ith survey protocol at the jth transect using the following Bayesian hierarchical model:Given the three detection methods, and total survey length, we compared seven variants of survey protocol . We detes) was Poisson distributed, and α and β were sampled from vague normal distributions with mean = 0 and variance = 1E12. The transect was incorporated as a hierarchical (random) effect from the following distributions:We assumed the number of squamate species detected , the entire period was considered a single survey and costs calculated as such. Thus, the total cost (Ti) of m such surveys with the ith survey protocol, and the marginal cost (Mi) of the ith survey protocol were calculated from:E = equipment cost; V = vehicle cost; C = cost of consumables; L = hours of labour required for the survey; l = hours of labour required to deploy artificial refuges and pitfall traps; v = vehicle cost to deploy artificial refuges and pitfall traps; r = rate of replacement (RoR) of equipment where 0 < r < 1; and, h = hourly rate in dollars.Since survey effectiveness estimates were determined for the entire survey period  [R package CODA was used to assess chain convergence [Analyses were conducted using on 3.4.2 . The pacOpenBUGS  was usedn 3.2.3) . Bayesiavergence . Convergvergence . IteratiAlthough the total camera-trap deployment was 74 days, five days were removed from the final camera-trap analysis due to maintenance. Additionally, although 40 camera-traps were deployed during the survey, three camera-traps, each at different transects, failed shortly after deployment and continued to fail after each maintenance event. Nevertheless, these transects were not removed from the analysis. Overall, camera-traps resulted in 2492 camera-trap days. The labour-intensive methods resulted in 960 trap days and 1920 trap inspections, consisting of 640 pitfall trap days and 320 artificial refuge ‘trap’ days. Across all protocols, 10 squamate species were detected, all of which were detected by camera-traps. In contrast, seven species were detected by artificial refuges and six squamate species were detected by pitfall traps .i.e., labour-intensive methods, LI; i.e., All-74) would increase species detections per transect, yet the overall number of squamates detected was equivalent (Cyclodomorphus michaeli) and the marsh snake  detected the 10 squamate species, the 74-day camera-trapping survey detected more squamate species per transect ~85% of the time. Pitfall trapping alone was the least effective technique tested; however, detections with pitfall traps complemented detections with artificial refuges (There was a 99% probability that the 74-day camera-trapping survey (CT-74) detected more squamate species per transect than artificial refuges and pitfall traps, either alone or together  was less costly than using both protocols individually, as vehicle and labour costs were not duplicated. This was not the case for including camera-trapping with the labour-intensive methods , as most costs were additive.Initial equipment outlay was the most expensive component of the camera-trapping surveys, whereas implementation expenses were the most expensive component of the labour-intensive methods . The equMCT-74 = 6014, SD = 2469) was less than the means of all other survey protocols  and hourly labour was very low , or where RoR of the camera-trapping protocol was much higher than the RoR of the labour-intensive methods protocol. Still, under a realistic scenario where RoR is < 10% and cost of hourly labour is > $10 camera-trapping is less costly than labour-intensive methods  is less cost-effective than camera-trapping alone . For those researchers using traditional methods to survey squamates, this study supports a growing body of literature demonstrating that camera-traps present a new tool for answering existing questions and asking new questions [The most important finding is that the 74-day camera-trapping protocol (CT-74) is effective and more cost-effective than the labour-intensive methods tested for inventorying a squamate assemblage. While using camera-traps and labour-intensive methods in combination may increase the number of species detected at the transect level (All-74), it may not improve overall detections and has considerable financial and ethical (see below) costs associated. Consequently, those wildlife researchers and managers currently using camera-traps to survey non-squamate fauna can incorporate squamate surveys into their fauna monitoring with few additional equipment requirements (uestions –10, 31. Although camera-traps are more effective, they are initially more expensive and require a number of surveys to be carried out before becoming less costly overall. The specific number of surveys will depend upon the research context; for example, if field researchers are volunteers or paid technicians. Additionally, in some situations camera-traps can be more expensive than labour-intensive methods. Due to the high equipment cost of camera-traps, high rates of theft can result in overall higher costs compared with other methods. We did not experience theft of any equipment, but other researchers have reported numerous camera-traps stolen or damaged during surveys . AttemptThat said, these results understate the financial advantage of using camera-traps, in several ways. First, we calculated survey costs assuming field researchers would not be required to stay in the field. This assumption greatly reduced the cost of the labour-intensive protocols relative to camera-traps. Labour-intensive methods typically require field researchers to remain in the field for protracted periods, often with associated financial costs for incidentals and accommodation, whereas camera-traps can be deployed, maintained, and retrieved in shorter timeframes. Second, detections and costs associated with camera-trapping surveys are less susceptible to adverse weather in the following sense. During one of the labour-intensive methods sub-surveys, heavy rain prohibited pitfall traps from being opened. Surveying during that occasion was abandoned and conducted the following week. The two days that field staff spent at the site were not included in cost calculations in this study since we aimed to compare camera-traps to the best possible case of using traditional methods. Yet, ordinarily these costs would be part of the overall survey liability. Additionally, detections of squamates can be impacted by weather . Read anC. michaeli and H. signatai at more transects than did camera-traps. This is most likely related to the behavioural ecology of these two species. Although C. michaeli will bask during the day, it is normally crepuscular [H. signata is active both day and night, but it is often nocturnal during warm periods [While camera-traps were more effective overall, the current methodology is limited. The labour-intensive methods detected puscular . Similar periods , 40, suc periods , who fou periods  this is Further improving effectiveness with the methods tested can be achieved in two ways: modifying the detection device; and/or, increasing survey effort. While the equipment setup in the current experiment was apt for the comparison, the setup of both camera-traps and pitfall traps was likely less than optimal than if these methods were used in isolation. Drift fence lengths and array designs have been the focus of considerable research . As cameL. delicata, which is apparently abundant and among the smallest squamate at this location [2, and the tile occupies ~900 cm2 where most detections occur. The pitfall trap opening is ~71% smaller than the tile, covering ~638 cm2. Thus, a component of the difference in detection of L. delicata between these methods was likely due to the spatial extent being sampled, but given the stark disparity, much of the difference may result from trap-avoidance, which has been observed in studies of other species (e.g., Komodo monitors, Varanus komodoensis, [Pitfall traps of the size used here are more effective at capturing small lizards than large lizards or snakes . Therefolocation , pitfallIncreasing survey effort can be achieved by increasing the number of sampling stations at a sampling location and/or by increasing the number of days of sampling. In this respect camera-traps have a considerable advantage over labour-intensive methods if the number of days of sampling are to be extended. Once deployed, there is little extra cost associated with leaving camera-traps in the field for longer periods. Still, it is important to note that extending the number of sampling days cannot be done indefinitely as assumptions of population closure will become an issue and invalidate certain modelling approaches , 46. IncOne final point goes to the issue of animal welfare. The camera-trapping approach presented here appears less impactful than pitfall trapping. Mortality of fauna in pitfall traps, due to exposure or predation, is widely discussed in the literature –52. Our We compared the cost-effectiveness of using camera-traps with methods commonly used to survey a squamate assemblage and found camera-traps were more effective and clearly more cost-effective. These results demonstrate that, under the given circumstances, camera-traps provide wildlife researchers and managers with a versatile tool with which to inventory squamates. Still, camera-traps are not a panacea. Researchers need to closely assess their requirements before investing in camera-trapping equipment or adopting camera-traps for surveying squamates. Important considerations are the species assemblage, the environment in which sampling is to be conducted, and the resource at the researcher’s disposal. Continued research is required to overcome the identified limitations of surveying squamates with camera-traps, particularly issues relating to detection of crepuscular and mostly nocturnal species. By further developing camera-trapping methods, and perhaps adopting lessons learnt from pitfall trapping, camera-traps may help fill some of the fundamental missing data on squamates.


Agrobacterium-mediated transformation has been a desirable tool to manipulate single or multiple genes of existing genotypes of woody fruit crops, for which conventional breeding is a difficult and lengthy process due to heterozygosity, sexual incompatibility, juvenility, or a lack of natural sources. To date, successful transformation has been reported for many fruit crops. We review the major progress in genetic transformation of these fruit crops made in the past 5 years, emphasizing reproducible transformation protocols as well as the strategies that have been tested in fruit crops. While direct transformation of scion cultivars was mostly used for fruit quality improvement, biotic and abiotic tolerance, and functional gene analysis, transgrafting on genetically modified (GM) rootstocks showed a potential to produce non-GM fruit products. More recently, genome editing technology has demonstrated a potential for gene(s) manipulation of several fruit crops. However, substantial efforts are still needed to produce plants from gene-edited cells, for which tremendous challenge remains in the context of either cell’s recalcitrance to regeneration or inefficient gene-editing due to their polyploidy. We propose that effective transient transformation and efficient regeneration are the key for future utilization of genome editing technologies for improvement of fruit crops.Genetic engineering based on  There are 494 culinary fruits (excluding fruit vegetables such as watermelons and tomatoes) and 14 nuts listed as of submission in Wikipedia. The increasing consumer awareness regarding health benefits and growing population worldwide are boosting a market for more and higher-quality fruits and nuts. For example, the total global production of fresh fruit increased from 13.6 million metric tons in 1996 to 33.3 million metric tons in 2016 (FAO). On the other hand, many adverse impacts, including emerging diseases , abiotic stress  due to global warming, and natural resource depletion  due to the growing population, have threatened fruit yield and quality of F&N crops . Thus, tormation , and carormation .Agrobacterium-mediated transformation. The transgenic plum is on the horizon to be commercially released and the non-browning apples are in the stores. Agrobacterium-mediated transformation protocols for stable and transient expression remain a major platform for gene editing technologies in F&N species  or gene blocks . Except  species . In thisAgrobacterium tumefaciens-mediated stable transformation of tobacco plants ] have been demonstrated to be effective in producing selectable marker gene (SMG)-free apple (RNA interference (RNAi) technologies. The efforts to develop/improve transformation protocols for more F&N crops or cultivars continued and as a result blueberry  and souree apple , apricotee apple , and citee apple . The mosee apple , 2013; aee apple .Agrobacterium to produce stable transgenic plants expressing either editing reagents or small RNAs inducers. Ideally, transient expression of editing reagents leading to stable editing of a GOI or a regulatory DNA sequence, similar to those demonstrated in annual crops  are still recalcitrant for transformation, and most of the transgenic F&N crops were produced using ormation . The scaormation . For exaeen made .Efficient protocols for transformation rely on effective gene delivery, easy but accurate selection, and prolific regeneration from treated explants .Agrobacterium-mediated gene transfer is a powerful tool for delivery of transgenes although optimal conditions for efficient gene delivery vary for different species, genotypes and strains of Agrobacterium   . A. tumen plants , althougapproach –3.neomycin phosphotransferase II (nptII) gene that confers kanamycin resistance, the hygromycin-B-phosphotransferase gene (hph) conferring hygromycin B resistance, and the bar gene conferring resistance to phosphinothricin, a glutamine synthetase inhibitor produced by plants from the herbicide bialophos  species, regeneration can be achieved mostly on the basis of the use of seed explants , the cultivated F&N crops, for example, blueberries  and cherexplants . In term of plum . More reormation ; and theormation .Genetic transformation of F&N crops might be necessary when a breeding goal is not easily achievable through traditional breeding approaches . Of manyDsRED) from transgenic rootstock to non-transgenic scion of walnut (Juglans regia) (nptII gene) was not detected in non-transgenic sweet cherry scions grafted on transgenic rootstock through RT-PCR  is a widely used agricultural practice with over 3000-year history for horticultural crops, especially for F&N crops . Traditis regia) ; in conth RT-PCR . More reh RT-PCR . In facth RT-PCR ; this ish RT-PCR . Using tc scions . Thus, tc scions .FLOWERING LOCUS T, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, LEAFY, and APETALA1) or repression of flower repressing genes  is an effective approach to enable a fast introduction of genes of interest from wild germplasm through the early flowering seedlings from both crosses and backcrosses  or transgenic plants from which the SMG was removed . While t/LoxP and Flp-FRT (flippase recognition target)] are effective in generating SMG-free apple  for crop improvement . RemarkaFokI) which disrupts DNA adjacent to the recognition zones. Both ZF- and TALE-nucleases, require two effectors (left and right) in order to define the nuclease cutting site (Programmable DNA binding proteins such zinc finger (ZF) and transcription activator-like effector (TALE) emerged as the first generation engineered nucleases to create targeted mutagenesis, which is an alternative to classical protocols for random mutagenesis. These tools have a recognition capability of specific target DNA sequences based on customized arrangements of one (TALE) or three (ZF) nucleotides, and in such way bringing to these places a nuclease  or gene regulatory sequence(s). The progress made in genetic engineering for fruit crops prior to 2013 has been well-documented .A. tumefaciens-mediated transformation of nine F&N crops using 24 genes for improving seven traits , progress has been made for n traits . This prent rate . For insg et al.  describeg et al. .Agrobacterium-mediated transformation, such as transgrafting, fast-track breeding, intragenesis, and genome editing will be employed more frequently for solving problems facing tree fruit industry worldwide.Low success rate and recalcitrance of some species to transformation remain as major challenges for adoption of the new breeding techniques to fruit crops. As those challenges are overcome, several new technologies based on All datasets generated for this study are included in the manuscript and/or the supplementary files.HP wrote the gene editing part. G-qS and VO drafted the rest of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Peptidoglycan (PG) is an essential molecule for the survival of bacteria, and thus, its biosynthesis and remodeling have always been in the spotlight when it comes to the development of antibiotics. The peptidoglycan polymer provides a protective function in bacteria, but at the same time is continuously subjected to editing activities that in some cases lead to the release of peptidoglycan fragments  to the environment. Several soluble muropeptides have been reported to work as signaling molecules. In this review, we summarize the mechanisms involved in muropeptide release (PG breakdown and PG recycling) and describe the known PG-receptor proteins responsible for PG sensing. Furthermore, we overview the role of muropeptides as signaling molecules, focusing on the microbial responses and their functions in the host beyond their immunostimulatory activity. Most bacteria surround themselves with a protective cell wall to repel environmental challenges. These tough cell walls are primarily composed of a peptidoglycan (PG) exoskeleton, also called the murein sacculus . PG is aN-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) covalently attached to a peptide chain containing 2–5 amino acid residues. The archetypical peptide stem structure is L-alanine, D-glutamic acid, a dibasic amino acid [typically meso-diaminopimelic acid (mDAP) or L-lysine], D-alanine, and D-alanine  in a process termed PG turnover. In ell wall . Around ell wall .In recent years, PG has been of much interest not only because it is one of the major antibiotic targets  but alsoIt is well documented that the PG sacculus is remodeled during bacterial growth and that this process causes the release of muropeptides. The discharge of PG fragments can occur as a consequence of the disruption of PG during growth or by the complete lysis of cells.Cleavage of PG is required for fundamental physiological processes in bacteria such as enlargement of the PG sacculus during bacterial growth and cell separation during cell division ; incorpoN-Acetyl-β-glucosaminidases (N-acetylglucosaminidases), while muramidases  cleave the bond between sequential NAM and NAG residues , which can be divided into soluble periplasmic LTs (named Slts) or membrane-attached LTs (named Mlts), and can perform the cleavage at the end of the glycan strands (exolytic) and/or in the middle of the PG chains (endolytic)  . Althougdolytic) , a uniqudolytic) . Particureatment .E. coli, the gate of entry for the internalization of soluble anhydromuropeptide monomers (NAG-anhNAM-peptides) into the cytoplasm is the AmpG permease, an inner transmembrane protein that specifically takes up anhydromuropeptides or free anhydrodisaccharides (N-acetylglucosaminidase) and AmpD  on molecules presenting anhNAM structure yield NAG, anhNAM, and free peptides in the cytoplasm  or released to the environment by a currently unknown mechanism . In E. ccharides . Deletiocharides  revealincharides . The speytoplasm . Resultiytoplasm  into triytoplasm  or attacytoplasm . Ligatedynthesis , 2006.The internalization of anhydromuropeptides and subsequent breakdown in order to supply a demanding nutrient or energy sources seems unlikely under favorable growth conditions , and eveN-acetylglucosaminidases and amidases, have been described to act at the cell wall compartment in Gram-positive bacteria  through an AmpG-like transporter is vital for PG-synthesis  , some ofFurthermore, many Gram-negative bacteria can interact with other microbes and the host by releasing outer membrane vesicles (OMVs) to the environment . FormatiSpecific roles for a variety of soluble PG fragments as messenger molecules have been known for decades  and haveDespite the abundance of PG-containing microbiota and the numerous studies implicating PG as an immunostimulatory signal , little In the last two decades, multiple structural motifs and proteins have been described to bind PG. Interestingly, not a single class of microorganism is sensed by only one type of receptor, hence ensuring a rapid and potent response while allowing for some specificity during, for example, infection. In this review, we summarize those receptors that recognize and bind PG.E. coli lytic tranglycosylase MltD  is considered a general PG-binding domain that binds specifically to molecules containing repetitions of NAG such as chitin, peptidoglycan, and short oligosaccharides . The Lysase MltD , Enterocase AtlA , B. subtase CwlS , or Lactase AcmA ], LysM iase AcmA . The stuase AcmA . In planase AcmA . Furtherase AcmA .Penicillin-binding and Ser/Thr kinase-associated (PASTA) proteins are essential tools for bacteria to sense and respond to the host environment and antibiotic stress as they play a central role in virulence and β-lactam resistance via their ability to regulate metabolism, cell division, and cell wall homeostasis through the recognition of muropeptides (mDAP in the third position in the peptide stem) has also been described   are intracellular regulatory proteins that respond to a variety of signaling molecules including PG-derived fragments . NLRs sheptides) , which alantarum , while Nlantarum . PG fraglantarum ] or are via OMVs , where tvia OMVs .Drosophila has 13 PGRPs (Peptidoglycan recognition proteins (PGRPs) are evolutionarily conserved innate immunity molecules homologous to bacteriophage type 2 amidases found in animals and humans that present bactericidal activity . All PGR13 PGRPs ] and mam13 PGRPs ] that re13 PGRPs . PGRP-PG13 PGRPs , 2017. S13 PGRPs .C-type lectin-like receptors (CTLRs) are a major class of PRR that present an extracellular carbohydrate recognition domain that putatively binds sugar moieties within the glycan backbone of bacterial PG or the fungal glucan mannan, in a calcium-dependent manner . Upon livia disruption of the glycolytic pathway and the mitochondrial function  and (2) modifications of the stem peptides to evade immune recognition . Thus, these modifications constitute a tactic that provides resistance to cell lysis and helps bacteria to evade the host immune system. For specificities about PG modifications, we refer to excellent reviews on the topic  released by actively growing cells (S. pneumoniae (StkP), S. mutans (PknB), M. tuberculosis (PknB), C. difficile (PrkC), and S. aureus (PknB)  . Interespeptides , suggestMicrococcus and Mycobacterium through the resuscitation-promoting factor (Rpf), a muralytic enzyme that cleaves the β-1,4 glycosidic bond in the glycan backbone of PG  (The predatory bacterium o spores . One keyo spores . This beubtilis) .via the detection of cell wall fragments in the environment, which seems to be a widely used strategy in the microbial world  and also in prokaryote-prokaryote encounters. For example, zosphere . PG isol unknown .C. albicans, muramyl dipeptides exhibit a potent hypha-inducing activity by directly binding to adenylyl cyclase Cyr1p LRR domain that stimulates cAMP production and subsequent hyphal growth , a phenomenon that is tightly linked to PG recycling -infected patients has caused the emergence of β-lactam-induced vancomycin-resistant MRSA (BIVR-MRSA). The presence of β-lactams inhibits PG biosynthesis, producing the accumulation of large amounts of PG precursors  with free D-Ala-D-Ala terminals that bind with vancomycin, depleting its concentration. Even if it is well characterized that the muropeptide NAG-NAM-L-Ala-D-Gln-L-Lys-(ɛ-amino-4Gly)-D-Ala-2Gly triggers vancomycin resistance , which in synergy with LPS derivatives triggers the normal morphogenesis of the light organ. Morphogenesis involves the loss of ciliated epithelium, the shortening and eventual loss of appendages  by NOD proteins can stimulate the innate immune response too  of released anhydromuropeptides and other PG fragments; however, few studies have focused on this kind of protein . A recenThe development of highly sensitive analytical methods and the use of synthetic muropeptides could help to elucidate the specific receptors that are able to bind PG as much as to determine the agonist PG structures and their role as signaling molecules. Overall, PG sensing seems to be a global mechanism that leads to different responses, so it is conceivable the existence of multiple PG-sensing pathways. More research is needed to clarify the different sensing mechanisms, to determine the interplay level of the different receptors (or to identify new ones), or understand the responses generated by muropeptides during bacteria-bacteria or bacteria-host interactions.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.