Vs) are membrane proteins that are involved in the generation and propagation of action potentials in neurons. Recently, the structure of a complex made of a tetrodotoxin-sensitive (TTX-s) NaV subtype with saxitoxin (STX), a shellfish toxin, was determined. STX potently inhibits TTX-s NaV, and is used as a biological tool to investigate the function of NaVs. More than 50 analogs of STX have been isolated from nature. Among them, zetekitoxin AB (ZTX) has a distinctive chemical structure, and is the most potent inhibitor of NaVs, including tetrodotoxin-resistant (TTX-r) NaV. Despite intensive synthetic studies, total synthesis of ZTX has not yet been achieved. Here, we review recent efforts directed toward the total synthesis of ZTX, including syntheses of 11-saxitoxinethanoic acid (SEA), which is considered a useful synthetic model for ZTX, since it contains a key carbon–carbon bond at the C11 position.Voltage-gated sodium channels (Na Vs) are membrane proteins involved in neuronal excitation and transmission . The. TheVs)  are not ,21,22,23reatment ,27,28,29reatment ,31.1) is a guanidine alkaloid with potent and specific inhibitory activity towards NaVs  is algae .,45.1) bif TTX-s) ,47.1), C11 , or C13 (R4) in the common structure shown in 2) is hydroxylated at N1, decarbamoylsaxitoxin  has a hydroxyl group at C13, and gonyautoxins I–III  have a sulfate ester at C11; all of these analogs show similar NaV-inhibitory activity to STX (1). To date, more than 50 kinds of natural analogs of saxitoxin have been reported, of which most are modified at N1 (R1) derivatives, zetekitoxin AB  has an unusual structure . T III (7) C.10, which was previously developed by their group . . 9) . Th. Th42 fr59  by the iodination of 44 with iodine and PPh3, acetylation of the hydroxyl group, and reductive cleavage of the pyran ring with zinc in acetic acid . Al. Al59  has been observed at 156.5 ppm . Af. Af72 as8, ZTX). Although this goal still remains elusive, there have been some significant synthetic advances in the construction of characteristic structures of ZTX, such as (i) the carbon–carbon bond at C11 in the STX structure, (ii) stereoselective construction of the substituted isoxazolidine moiety at C15 and C16, and (iii) the macrocyclic structure from C6 to C11. Since ZTX has potent inhibitory activity, even towards tetrodotoxin-resistant (TTX-r) NaVs, a total synthesis of ZTX and its analogs is expected to provide useful tools for chemical biological studies of NaVs, overcoming the severely restricted availability of natural ZTX.Here, we have reviewed recent progress towards the total synthesis of zetekitoxin AB (


FPR1, FPR2, and FPR3 are members of Formyl Peptides Receptors (FPRs) family belonging to the GPCR superfamily. FPR2 is a low affinity receptor for formyl peptides and it is considered the most promiscuous member of this family. Intracellular signaling cascades triggered by FPRs include the activation of different protein kinases and phosphatase, as well as tyrosine kinase receptors transactivation. Protein kinases and phosphatases act coordinately and any impairment of their activation or regulation represents one of the most common causes of several human diseases. Several phospho-sites has been identified in protein kinases and phosphatases, whose role may be to expand the repertoire of molecular mechanisms of regulation or may be necessary for fine-tuning of switch properties. We previously performed a phospho-proteomic analysis in FPR2-stimulated cells that revealed, among other things, not yet identified phospho-sites on six protein kinases and one protein phosphatase. Herein, we discuss on the selective phosphorylation of Serine/Threonine-protein kinase N2, Serine/Threonine-protein kinase PRP4 homolog, Serine/Threonine-protein kinase MARK2, Serine/Threonine-protein kinase PAK4, Serine/Threonine-protein kinase 10, Dual specificity mitogen-activated protein kinase kinase 2, and Protein phosphatase 1 regulatory subunit 14A, triggered by FPR2 stimulation. We also describe the putative FPR2-dependent signaling cascades upstream to these specific phospho-sites. Post-translational modifications (PTMs) regulate many proteins functions thanks to the addition of a functional chemical group. Phosphorylation is the most important PTMs in eukaryotic cells as it plays a critical role in regulating many eukaryotic cellular processes, including metabolism, cell cycle progression, cellular proliferation, survival, migration and differentiation . PhosphoFormyl Peptides Receptors (FPRs) belong to the GPCR superfamily and include three members  that recognize formyl peptides and/or other ligands with different range of affinity ,13. FPR2FPRs were firstly identified on myeloid cell membrane, but subsequently their expression was also observed in neuronal, endothelial and epithelial cells . Their aphox and p67phox which are required for NADPH oxidase-dependent reactive oxygen species (ROS) generation [Intracellular signaling cascades triggered by FPRs include the activation of different kinases and phosphatases, such as protein kinase C (PKC) isoforms, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mitogen-activated protein kinase (MAPK), p38MAPK, PTEN, PTP-PEST, and DUSP3/VHR ,18,19,20neration ,22. Furtneration ,24,25,26These signaling cascades regulate several cellular functions and their impairment significantly contributes to the development of human diseases. In fact, FPRs are functionally expressed in cells and tissues of central and peripheral nervous system, where they play a key role in the development of neurodegenerative diseases ,27, suchWe previously performed a phospho-proteomic analysis in FPR2-stimulated cells to shed light on intracellular signaling cascades triggered by this receptor. To this aim, CaLu-6 cells were serum-starved for 24 h and then stimulated for 5 min with the WKYMVm peptide, a selective FPR2 agonist . We iden2-terminal GTPase-binding region. The central domain shows a weak homology to the calcium-dependent phospholipid binding C2 domain of PKC (HR2/C2) and contains an auto-inhibitory region [2-terminus by a hinge region. The 3′-end of the COOH-terminal region of PKN1 and PKN2 is also involved in the binding to RhoA [Protein kinase C-related kinases (PKN/PRK) belong to a subfamily of AGC serine/threonine-protein kinases ,40,41. Ty region ,44,45. Ty region ,47 and cy region ,48,49. Ty region ,51. The y region  that in  to RhoA . PKN2 st to RhoA . Two hig to RhoA ,55. The  to RhoA  and may  to RhoA ,55. In P to RhoA ,57.PKN1 and PKN2 kinase activity is regulated by mTORC2-dependent phosphorylation of the turn phosphate motif . The actThe biological responses downstream to the interaction of chemoattractants with FPRs are regulated by Ras and Rho GTPases family, which induce the activation of PI3K, AKT and ERK1/2 ,66,67. PPKNs can be activated by growth factor receptors signaling through different pathways. TKR-induced PI3K activity catalyzes PIP3 synthesis which is required for mTORC2 activation and Thr958 phosphorylation in the turn phosphate motif. Furthermore, phospho-tyrosine residues of TKRs provide docking sites for SH2 and PTB domains. NCK adapter protein binds phosphorylated Epidermal Growth Factor Receptor (EGFR), Platelet-Derived Growth Factor Receptor (PDGFR), Vascular Endothelial Growth Factor Receptor (VEGFR) via its SH2 domain and is a mediator of growth factor-induced signal transduction ,70,71. PPKN2 is one the first identified effector proteins of Rho  and playSchizosaccharomyces pombe, but its expression has been subsequently observed in a variety of human tissues [2-terminus, which is characterized by basic aminoacids repeats, able to act as a NLS, and Arg/Ser (RS) domains, frequently found in pre-mRNA splicing factors whose deletion induces the cytosolic localization of the protein [Cdk, MAPK, GSK, and cdc2-like kinase (Clk) protein families [PRP4 was originally isolated from  tissues . Human P protein . PRP4 be protein ,87 and, families . PRP4 befamilies . MAPK fafamilies . The regfamilies ,91. Thr3families .847PY849 motif in the subdomain VIII of PRP4 is not essential for kinase activity which requires, at least partially, the autophosphorylation on Ser and Tyr residues [PRP4 is involved in pre-mRNA splicing. In fact, it is able to phosphorylate the RS domain of the mammalian splicing factor ASF/SF2 , which bresidues . Neverthresidues . PRP4 isresidues  and playresidues . Previouresidues . We specMAP-microtubule affinity-regulating kinases (MARKs) were discovered for their ability to phosphorylate tau and related microtubule-associated proteins (MAPs) on Ser residues located within the KXGS motifs. Phosphorylation of tau results in the detachment from microtubules and in the impairment of microtubule-based intracellular transport ,99. In C2-terminal header, (ii) a catalytic kinase domain, (iii) a linker, (iv) a UBA domain, (v) a spacer, and (vi) a tail domain [The alpha subunits of AMPK belong to a family of 12 related kinases , namely BRSK1, BRSK2, PAR-1c/MARK1, PAR-1b/MARK2, PAR-1a/MARK3, PAR-1d/MARK4, MELK, NUAK1/ARK5, NUAK2/SNARK, SIK1, SIK2/QIK and SIK3/QSK . PAR-1c/l domain . The roll domain .Interestingly, both PAR-1b/MARK2 and AMPK are regulators of metabolism and show overlapping functions in vivo ,107. In In addition to tau and MAPs, MARKs phosphorylate several substrates in their binding sites, such as mitogen-activated protein kinase scaffolding protein KSR1, tyrosine phosphatase PTPH1, cell cycle-regulating phosphatase Cdc25, class IIa histone deacetylases and plakophilin. Such phosphorylations enhance the binding of these proteins to 14-3-3 modifying their subcellular localization and affecting regulatory pathways ,114.MARK2 is activated by phosphorylation of a conserved Thr208 in the activation loop (T loop) located within the catalytic domain. MARKK/TAO-1 and the tumor suppressor Liver kinase B1 (LKB1) in complex with STRAD and MO25 phosphorylate MARK2 at this site ,116. On MARKK/TAO-1 is regulated by Ras GTPases family ,123 and 2-terminal PBD (p21-GTPase-binding domain), also known as a Cdc42 Rac Interactive Binding (CRIB) domain [2-terminal PBD sequence, there is considerable homology within the PBD between all PAK family members. PAK1-3 bind to Rac and Cdc42, whereas PAK4-6 binds preferentially Cdc42. None of the PAK family members bind to the Rho isoforms RhoA, RhoB, and RhoC [The p21-activated kinases (PAKs) belong to the Ser/Thr kinases family which is composed of six members distributed into a group I  and a group II . The PAK family proteins show a similar structure that can be divided into three main domains: an NH) domain , a centr) domain . Even thand RhoC . The cenand RhoC , and an and RhoC .2-terminal regions of PAK4 and PAK5 can interfere with kinase activity. In fact, removal of the PBD enhances PAK4 and PAK5 kinase activity [2-terminal fragment able to inhibit kinase activity has not been identified in PAK4. The kinase activity of PAK4 can be inhibited by the PI3K inhibitor LY294002 but the mechanism of regulation of PAK4 kinase activity by PI3K is still unclear [2-terminal region, and a nuclear export sequence [The activation mechanisms of groups I and II of PAKs are different and the relative regulatory domains are structurally distinct . The kinactivity ,138, sug unclear ,135. PAKsequence ,132.Therefore, PAK4 is mainly activated by cMet-induced PI3K signaling. Noteworthy, PAK4 and the p85 α-subunit of PI3K specifically interact  and the Previously, we showed that FPR2 stimulation by WKYMVm induces the trans-phosphorylation of Y1313/Y1349/Y1356 residues of c-Met and elicits some of the molecular responses triggered by c-Met/HGF binding, such as STAT3, phospholipase C (PLC)-γ1/PKCα and PI3K/Akt pathways ,143. We 2-terminus, which shows similarity to that of the STE20 family of serine/threonine kinases involved in regulating MAPK cascades, and a long coiled-coil structure at the COOH-terminus. The two domains are separated by a proline-rich region. In yeast STE20 is a component of the MAP kinase cascade required to transmit the pheromone signal from Gβγ subunits to downstream components [In mice, lymphocyte-oriented kinase (LOK) is a member of the STE20 family characterized by serine/threonine kinase activity, whose expression is restricted mostly to lymphoid cells . It consmponents . STE20 pmponents . Severalmponents ,158.STK10 is the human homolog of LOK. It shows high similarity to LOK with a 98% and 93% of identity in the kinase domains and in the coiled-coil regions, respectively . STK10 sSTK10 is a key kinase for tumor progression  and siRNSTK10 phosphorylates ezrin, radixin and moesin (ERM), which mediate linkage of actin cytoskeleton to plasma membrane  and contThe upstream signaling cascades regulating STK10 kinase activity remain to be elucidated, although STK10 catalytic activity seems to depend on dimerization of the kinase domain and, in turn, on STK10 trans-phosphorylation . InteresWe found that Ser438 and Thr952 residues of STK10 are phosphorylated in FPR2-stimulated cells , in humaDual specificity mitogen-activated protein kinase, kinase 2 (MAP2K2) is also known as CFC4, or MAPKK2, or MEK2 , or MKK2 (MAP kinase kinase 2), or PRKMK2. MEK1 and MEK2 are ubiquitously expressed and are encoded by two distinct genes located at different chromosomes . The MAPphox, p47phox and p67phox and their translocation to the membrane, followed by their interaction with cytochrome b558 [phox on Ser and Thr residues [phox phosphorylation [phox phosphorylation and membrane translocation, as well as NADPH-dependent superoxide production [NADPH oxidase activation requires the phosphorylation of the cytosolic components p40ome b558 ,195,196.residues ,198,199 rylation ,201, sugoduction ,22.In a typical signaling pathway triggered by TKRs, the phosphorylated tyrosine residues of the TKRs act as docking sites for several proteins, including Shc, Grb2 and SOS that link the activated TKR to Ras-signaling pathway. The GTP-bound Ras induces the translocation of the Ser/Thr kinase Raf-1 to the plasma membrane, where it is activated by multiple phosphorylation events ,203. Raf2- and COOH-terminal tails and a central PHIN domain that consists of 86 residues [2-terminal tail domain, absent in CPI-17 [PPP1R14A is also known as CPI-17, CPI17 or PPP1NL. Its structure shows intrinsically disordered NHresidues ,209,210.residues . Three Cn CPI-17 .CPI-17 is expressed predominantly in smooth muscles where it plays crucial regulatory functions ,213,214.2+ and an initial rapid MLCP phosphorylation. On the other hand, ROCK stimulates the Ca2+-independent phosphorylation of CPI-17 in the subsequent sustained phase, in parallel with a force generation [2-terminal tail [2+/calmodulin-dependent protein kinase II phosphorylates the Ser130 residue at the COOH-terminal tail of CPI-17 [A major kinase for GPCR-induced CPI-17 phosphorylation is PKC which is activated by the PLCβ-produced signaling messenger diacylglycerol (DAG). It phosphorylates CPI-17 at Thr38 residue that directly docks at the active site of MLCP, thereby inhibiting its activity and promoting an increase of phosphorylation of myosin and of other MLCP substrates ,220,221.neration . CPI-17 neration . CPI-17 neration . Sequencnal tail . In addinal tail ,228,229,f CPI-17 .We observed that FPR2 stimulation induces the phosphorylation of Ser16 and Ser26 residues of CPI-17 . PhosphoTherefore, CPI-17 activity is regulated by PKC and ROCK and several GPCR agonists induce PKC- and ROCK-dependent phosphorylation of CPI-17 at Thr38 residue ,226,230.FPR2 is a versatile transmembrane protein activated by an array of agonists, which include structurally unrelated lipids and peptide ligands, resulting in different intracellular responses. The ability of FPR2 to mediate a multitude of physio-pathological processes can be traced to the different receptor domains used by distinct agonists. FPR2 represents a promising therapeutic target in several human diseases, but further studies are required to fully understand molecular mechanisms responsible for PKs and PPs activation upon stimulation with different agonists.PKs mediate a network of highly complex signals. The understanding of the regulatory functions of kinases may represents a valuable tool to identify more effective therapies against several human diseases. In fact, many drug kinase inhibitors are used in the treatment of different types of cancer and phospho-proteomics represents an important analytical strategy to identify new phospho-sites and phospho-proteins, and in turn to develop new drugs in tumor biology.Many proteins are phosphorylated, even though phosphorylation is only one of several types of reversible covalent PTMs, which also include methylation, acetylation, sumoylation, and ubiquitination. In phospho-proteins, only a limited subset of phosphorylated sites is regulated. Furthermore, the observation that single phospho-sites are differently regulated suggests that proteins function as platforms to integrate multiple incoming stimuli. This integration of signals could act in an independent- or dependent manner. In the first case, the phosphorylation of each site occurs independently from the others, whereas in the second case a “priming site” is necessary for additional phosphorylation events. Inactivating mutations of PKs have been identified only in a small number of human cancers, indicating that modifications in kinase activity by phosphorylation are among the main causes of disease progression, and that phospho-proteomic screens could be useful to monitor cancer development and to identify new potential therapeutic targets.


In recent years, sulfur-containing nanomaterials and their derivatives/composites have attracted much attention because of their important role in the field of biosensor, biolabeling, drug delivery and diagnostic imaging technology, which inspires us to compile this review. To focus on the relationships between advanced biomaterials and biosensors, this review describes the applications of various types of sulfur-containing nanomaterials in biosensors. We bring two types of sulfur-containing nanomaterials including metallic sulfide nanomaterials and sulfur-containing quantum dots, to discuss and summarize the possibility and application as biosensors based on the sulfur-containing nanomaterials. Finally, future perspective and challenges of biosensors based on sulfur-containing nanomaterials are briefly rendered. As a by-product of oil refining and natural-gas purification, sulfur usually exists in the form of sulfide, sulfate or elementary substance in nature and is one of most abundant elements ,2,3. Sin2S quantum dots), exhibit high absorption in near-infrared (NIR) region, which enables their applications in bioimaging, biolabeling, deep tissue imaging, diagnostics and photodynamic therapy [A variety of sulfur-containing nanomaterials have been reported, such as metallic sulfide nanomaterials, sulfur-containing quantum dots, sulfur-containing organosilicon compounds, and lithium sulfide materials ,8,9,10.  therapy . In thisxSn, MxM’ySn, MxM’yM”zSn and MxAiBj…CkSn , respectively. It should be noted that actually metal sulfide nanomaterials also include metal sulfide quantum dots, which will be illustrated in the section of “sulfur-containing quantum dots” below.Metal sulfides contain chemical bonding of one or more sulfur atoms (S) to a metal (M) . They caxSn, e.g., MoS2, NiS, Cu2S, Bi2S3, CuS, SnS, In2S3 and Ag2S) [Binary sulfides. Binary sulfides (Mnd Ag2S) ,16,17,18nd Ag2S) ,20, photnd Ag2S) , antibacnd Ag2S) , ablationd Ag2S) , optoelend Ag2S) , photovond Ag2S) ,26 and mnd Ag2S) .2, WS2 and NiS [Among binary sulfides, transitional metal disulfides, such as ZnS, CuS, CdS, MoS and NiS ,30,31,32 and NiS . SimilarxM’ySn, e.g., Ni3In2S2, Ni3Tl2S2 and NiCo2S4 [Ternary sulfides. Ternary sulfides  cont NiCo2S4 ,36. Thes NiCo2S4 ,38, and xM’yM”zSn where M, M’, M” = Zn, Cd, Mn, Hg, Cu, Ge, Sn, Cd, Fe, Co or Ba [Quaternary sulfides. Quaternary sulfides contain three types of metal and S atoms in their chemical formulas, which have common composition of MCo or Ba ,42,43,443(PS4)2) are an emerging class of non-metallic/metallic hetero-sulfides.Non-metallic/metallic hetero-sulfides. Non-metallic/metallic hetero-sulfides have attracted considerable interests recently, which contain not only metal and S atoms but also other non-metallic atoms in their chemical formulas. For example, phosphor-chalcogenides ,46  have been available in the market, but ternary, quaternary, and non-metallic/metallic hetero-sulfides have not been available in the market.Literature ,50,51,52Metallic sulfide nanomaterials, as important and emerging materials, have arisen quickly in the area of biosensing due to their specific properties, namely, nanometric scale, water-dispersibility, large specific surface area, excellent catalytic activity, conductivity, biosafety, PL quenching abilities, photoactivity, and fascinating optical properties ,48,61.Catalysis: The metallic sulfide nanomaterials had excellent catalytic activity due to their high density of active sites, which can be used as modifiers in fabrication of novel biosensors ,48. CataConductivity: The metallic sulfide nanomaterials had high electronic conductivity due to their low bandgaps, which make them be used as electrode materials in fabricating biosensors via electrochemical or ECL assays. For example, Chen et al.  have conBiosafety: Some metallic sulfide nanomaterials, such as silver, copper and iron sulfides, were non-toxic . These nPL quenching effect: Quencher is one of important component in PL  sensing platforms for detection of biomolecules. In our previous work, we have demonstrated graphene possesses unprecedented PL quenching abilities . Just liPhotoactivity: some metallic sulfide nanomaterials were photoactive materials which can convert light illumination into electrical signals. When excited by light, electrons of metallic sulfide nanomaterials transferred from valence band to conduction band, resulting in the separation of photogenerated electrons and holes ,66.2S quantum dots), emitted tunable fluorescence in near-infrared (NIR) region, which enabled their applications in bioimaging, biolabeling, deep tissue imaging, diagnostics and photodynamic therapy [Fascinating optical properties: Some metallic sulfide nanomaterials, especially 0-D metal sulfide nanomaterials , emitted high fluorescence. In comparison with organic fluorophores or alloy nanoclusters, metallic sulfide nanomaterials are superior as biomarkers due to their water-dispersibility, long lifetimes, resistance to photobleaching and biosafety ,68. More therapy . Even so therapy .Based on these specific properties of metallic sulfides that can be used to prepare biosensors, much research efforts have been devoted to developing biomolecule sensors for understanding physiological or pathological functions of biomolecules in living body or cells. In recent years, metal sulfide nanomaterials mainly have been applied to establish four types of biosensors, including electrochemical, photoelectrochemical (PEC), ECL and PL biosensors, for probing various types of biomolecules.2S4 nanowires due to their novel catalytic properties , a wide linear range  and low detection limit  via amperometric strategy.Metallic sulfide nanomaterials have been applied to establish electrochemical biosensors commonly due to their properties of conductivity, catalysis and biosafety. For example, Guo et al.  have devoperties A. In syn2 nanocrystals. The MoS2 nanocrystals displayed enhanced catalytic activities and fast heterogeneous electron transfer rate because of unsaturated sulfur on the edge sites and stronger quantum confinement. As shown in 2 nanocrystals and Cu submicron-buds on graphene paper (GP) via hydrothermal and electrodeposition method to form GP-MoS2-Cu biosensor. Further coating of lactate oxidase (LOD) on the GP-MoS2-Cu electrode, GP-MoS2-Cu-LOD biosensor for lactate detection was obtained. Due to the electron transport property and high specific surface area of GP, enhanced catalytic activities, fast electron transfer rate and biosafety of MoS2 nanocrystals, the electrochemical biosensor showed excellent sensing performances. For glucose, the electrochemical biosensor had a linear range of 5~1775 μM with a detection limit of 500 nM (S/N = 3). For lactate, the electrochemical biosensor had a linear range of 0.01~18.4 mM with a detection limit of 0.1 μM (S/N = 3).Wang et al.  have als2 nanocrystals, Zhang et al. [2) into MoS2 nanocrystals to obtain binary metal sulfide composites (CoS2-MoS2). Based on CoS2-MoS2, a non-enzymatic electrochemical biosensor for determination of ascorbic acid, dopamine and nitrite have been proposed with linear ranges of 9.9~6582, 0.99~261.7 and 0.5~5160 μM, respectively. In addition, due to its good electrochemical activity caused by synergistic effect between CoS2 and MoS2, low detection limits of the electrochemical biosensor for determining ascorbic acid (3.0 μM), dopamine (0.25 μM) and nitrite (0.20 μM) have obtained, respectively.In order to further enhance the catalytic performances of MoSg et al.  have incDue to their superior photoactivities and conductivity, metallic sulfide nanomaterials have been used in various PEC biosensing systems to be photoactive materials or be one of other components through combining with photoactive materials ,75,76,772 nanowire on Ti mesh (WS2 NW/TM) as photoactive material. Under visible light excitation, photo energy collected by WS2 NW/TM electrode was higher than that of its bandgap. Accordingly, electron was transferred from valence band (VB) to conduction band (CB), and then CB electron was transferred to the surface of Ti mesh, finally the hole in VB was scavenged by H2O2. Based on the electron transfer process, photocurrent was generated. Moreover, to obtain a dual signal PEC amplification strategy, AuNPs modified with glucose oxidase (GOx) and HER2 specific peptide for signal amplification were utilized. The localized surface plasmon resonance (LSPR) of AuNPs generated a collective oscillation of free electrons when excited by visible light. The free electrons can transfer from Au to the CB of the WS2 NW/TM electrode, which enhanced the photoelectric transfer efficiency and then achieved dual signal amplification. GOx modified in AuNPs catalyzed glucose to produce H2O2, which scavenged the hole in VB of the WS2 NW/TM electrode.Guo and Liu et al.  have fab2 NW surface via oxygen containing sulfur species of WS2 NW. The HER2 specific peptides modified on the surface of AuNPs were also utilized to bind with HER2 molecules. When detected HER2 molecules, a sandwich type dual signal PEC amplification biosensor was established with a wide linear range (0.5~10 ng/mL) and low detection limit .For binding with HER2 molecules, HER2 aptamers were modified on the WS2S3 nanorods as the photoactive materials . Manganese based mimic enzymes (MnME) were modified with AuNPs to obtain MnME@AuNPs composites, which could label signal probes (P2). The capture probe on the modified electrode can specifically hybridize with the MnME@ AuNPs-labeled signal probe to form a double-stranded DNA. In the absence of PNK, MnME can catalyze H2O2 with 3,3-diaminobenzidine (DAB) as substrate, and generated MnME catalytic precipitations on the modified ITO electrode. The MnME catalytic precipitations were insulating barriers and blocked the interfacial electron transfer and eventually leaded to a low PEC signal. In the presence of PNK, the double-stranded DNA was phosphorylated and subsequently cleaved by lambda exonuclease to release the MnME@AuNPs from the modified electrode, leading to a high PEC signal. Based on the signal on-off PEC strategy, the PNK biosensor was proposed and exhibited high sensitivity with a detection limit of 1.27 × 10−5 U/mL.Cui et al.  have alsaterials B. The Bi2 sensitized structures as photoactive materials and CuS nanocrystal as electronic extinguisher (2 electrode surface and used to immobilize a double-helix DNA (dsDNA). Then, CuS nanocrystal was efficiently immobilized on the dsDNA via doxorubicin (Dox) inserting into the dsDNA. In absence of PSA, electron donor and radiant light were consumed by CuS nanocrystals, and steric hindrance effect of insulating substances  generated, leading to a low PEC signal. In the presence of PSA, the PSA specifically cleaved the peptide, and DNA/Dox-CuS probes were released from the electrode surface, resulting in a high PEC signal. Take advance of the signal on-off PEC strategy, the PSA biosensor revealed good sensing performance with a linear range from 0.005 to 20 ng/mL and a low detection limit of 0.0015 ng/mL.In addition to being photoactive materials, metallic sulfide nanomaterials also have been one of other components through combining with photoactive materials in PEC biosensing systems. For example, Zhao et al.  have alsnguisher C. For fa2S2O8 as co-reactant, the ECL signal of the MIP-ECL biosensor significantly enhanced with increased concentrations of HIV-1 gene. Taking advantage of both MIP-ECL assays and the ECL properties of EuS nanocrystals, the HIV gene biosensor was sensitive and selective with a wide linear range (3.0 fM~0.3 nM) and low detection limit (3.0 fM).Due to their ECL properties, metallic sulfide nanomaterials have been used to establish ECL biosensors. For example, Babamiri et al.  have pre14S) (14S combined nitrogen doping mesoporous carbons (Au-ZnCd14S/NH2-NMCs) acted as sensing platform and Au-Cu alloy nanocrystals were employed as labels to quench the ECL of Au-ZnCd14S/NH2NMCs. On the basis of the ECL quenching effects between ZnCd14S and Au-Cu alloy nanocrystals, a sensitive ECL immunosensor for insulin detection was successfully constructed with a linear response range from 0.1 pg/mL to 30 ng/mL and detection limit of 0.03 pg/mL (S/N = 3). Although some metallic sulfide nanomaterials did not exhibit ECL properties, they have also been used to construct ECL biosensors via being as electrode materials based on their superior conductivity and large specific surface area. For example, Wei et al. [32+ and the electrode was facilitated. At the present of AML, the ECL intensity of Ru (bpy)32+ increased which induced the development of AML biosensor. Take advance of large specific surface area of rGO-CuS composite and powerful CE separation technique, the ECL biosensor for the detection of AML was successfully fabricated with a linear response range of 0.008 to 5.0 μg/mL and a detection limit of 2.8 ng/mL (S/N = 3).Moreover, Zhu et al.  have als14S) B. Au-ZnCi et al.  have rep2S3 nanocomposite as ECL acceptor and porous α-MoO3-Au structure as ECL donor (2S3 nanocomposite was prepared by hollow In2S3 nanotubes as substrate adsorbing Ru (bpy)32+. For fabricating PCT biosensor, HWRGWVC heptapeptide (H7), which could provide -SH, was immobilized on the surface of nanomaterials through amide bond (with Ru-In2S3 nanocomposite) and Au-S bond (with α-MoO3-Au structures) and used to capture antibody (Ab1 and Ab2). In the presence of PCT, Ru-In2S3 nanocomposite captured Ab1 and α-MoO3-Au structures captured Ab2 connected together, and ECL-RET from Ru-In2S3 to α-MoO3-Au occurred which was further confirmed by testing the overlap between ECL emission of Ru-In2S3 and UV-vis spectra of α-MoO3-Au. Take advantage of huge specific surface area of Ru-In2S3 or α-MoO3-Au and dual-quenching ECL-RET strategy, the ECL biosensor for detecting PCT was obtained with sensitive response, linear range from 0.0001 to 50 ng/mL and low detection limit of 12.49 fg/mL (S/N = 3).Moreover, Xue et al.  have alsCL donor D. SpecifMetal sulfide nanomaterials also have been used to establish PL biosensors due to their fascinating optical properties. However, to our best well know, metal sulfide nanomaterials used to fabricate PL biosensors mainly were 0-D metal sulfide nanomaterials . Thus, PL biosensors based on metal sulfide nanomaterials will be illustrated in the section of “sulfur-containing quantum dots” below.As described above, biosensors based on metal sulfide nanomaterials have been used for detection of various analytes, including glucose, dopamine, proteins, DNA, etc. Moreover, these biosensors displayed good sensing performance toward analytes detection. In addition, these biosensors also showed other outstanding advantages, including simple of preparation, low cost and good selectivity, stability, and great promising practical applications in clinical diagnosis, as shown in Sulfur-containing quantum dots are quantum dots containing central sulfur-containing nanodots and surface functional groups , and possess fascinating photophysical properties, small size , good biocompatibility, and chemical inertness. They can be broken down into three main categories: sulfur quantum dots, sulfide quantum dots and sulfur-doped quantum dots.Sulfur quantum dots. Sulfur quantum dots are pure elemental quantum dots, mainly including S central nanodots and surface functional groups ,82.2O2-assisted “top-down” approach [As a new class of quantum dots, sulfur quantum dots were firstly synthesized by Li’s group through phase interfacial reactions in 2014 . Since tapproach  and oxygapproach . Synthet3 aqueous solution was mixed with CdS quantum dots or ZnS quantum dots solution with a slowly stirring at room temperature. The resulting white mixture was separated by a funnel, and sulfur quantum dots were synthesized as a white suspension in hexane.For phase interfacial reaction, CdS quantum dots or ZnS quantum dots were diluted by n-hexane and then sonicated to form a homogeneous solution. HNOFor “assemble-fission” approach, sulfur quantum dots were synthesized by simply treating sublimated sulfur powders with alkali using polyethylene glycol-400 as passivation agents.2O2-assisted “top-down” approach, sulfur quantum dots were synthesized by dissolved bulk sulfur powder into small particles in an alkaline environment in the presence of polyethylene glycol, followed by the H2O2-assisted etching of polysulfide species.For Hx2−), followed by oxidation of Sx2− to zero-valent sulfur under a pure O2 atmosphere sulfide quantum dots. Sulfide quantum dots commonly included central sulfide nanodots, especially metal sulfide, and surface functional groups. Much research efforts have been devoted to synthesize sulfide quantum dots, such as ZnS, CdS, PbS, Ag2S, SnS2, In2S3 and AgInZnS quantum dots [For oxygen accelerated scalable approach, sulfur quantum dots were synthesized by dissolved bulk sulfur powder into small particles in an alkaline environment in the presence of polyethylene glycol to form polysulfide species , sulfide quantum dots have received extensive attention in the field of biosensing ,126. The2S quantum dots.Due to excellent electrochemical activities of sulfide quantum dots and inexpensive instruments and simple operations of electrochemical methods, electrochemical biosensors based on sulfide quantum dots have attracted increased attention. Zhang et al.  have repPEC sensing, a branch of electrochemistry, is a newly developed technology and has attracted great interest in biosensing fields. For fabricating PEC biosensor, photoactive materials are vital because they can generate photocurrent excited by light. To our best well know, sulfide quantum dots not only have directly been photoactive materials to establish PEC sensors ,131,132,2S released from MCF-7 cells based on heterostructures formed by CdS quantum dots and branched TiO2 nanorods (CdS-B-TiO2). Herein, CdS-B-TiO2 heterostructures in the PEC biosensors were directly as photoactive materials. In addition, due to the formation of CdS-B-TiO2 heterostructures, a significant enhancement in photocurrent was obtain, thus leading to sensitive PEC recording of the H2S level in cellular environments.Wang et al.  have pro2 (ERGO-TiO2) as reduced graphene oxide-TiO2 (ERGO-TiO2) and CdS quantum dots as a PEC signal amplifier. For preparing the PSA biosensor, ERGO-TiO2 was utilized to immobilize capture antibody (Ab1) for PSA detection, and quinone-rich PDA nanospheres (PDANS) loaded with CdS quantum dots were used to load detection antibody (Ab2) for PSA detection. In the presence of PSA, photo-generated electron transferred between PDANS loaded with CdS quantum dots and ERGO-TiO2. Due to the good conductivity of PDANS, ERGO and CdS quantum dots, a PSA biosensor has been proposed with a linear range from 0.02 pg/mL to 200 ng/mL with the detection limit of 6.8 fg/mL.Moreover, Deng et al.  have utiSulfide quantum dots have been widely used as alternative probes for biomolecules  via ECL strategies.Liu et al.  have fab2+, ECL quenching  achieved lie in RET between the CdS: La QDs and AuNPs at a close distance. In the presence of TB, ECL enhance  achieved lie in surface plasmon resonances (SPR) between the CdS: La QDs and AuNPs at a separated distance. The “on-off-on” approach was used to detect TB, and the linear range were 1.00 × 10−16 to 1.00 × 10−6 mol/L with limit of detection (S/N = 3) of 3.00 × 10−17 mol/L.In addition, Wang et al.  have als2S quantum dots (Ag2S: Mn QDs) as ECL materials  obtained by ECL spectrum. Based on a sandwiched ECL immunoassay, the biosensor displayed a wide linear range of 10 pg/mL to 100 ng/mL with a low detection limit of 3.2 pg/mL for LN detection.Moreover, Wu et al.  have preaterials C. The op2O2 which served as coreactant of CdS QDs. Thus, the ECL intensity of CdS QDs was linear correlation with the activity of M.SssI MTase.Furthermore, Zhou et al.  have proSulfide quantum dots, as 0-D metal sulfide nanomaterials, have also attracted great attention to establish PL biosensors due to their fascinating optical properties . Until nPhosphorescence biosensors were proposed based on sulfide quantum dots with phosphorescence emission. For example, Gong and Fan  have pro2 quantum dots, and constructed a glutathione biosensor based on the efficient fluorescence RET from VS2 quantum dots to MnO2 nanosheets and fast redox reaction between MnO2 and glutathione. Adegoke et al. [3+ ion-functionalized fluorescent MoS2 quantum dots for biosensing Guanosine 3′-diphosphate-5′-diphophate (ppGpp)  A. Litera2S quantum dots, displayed fluorescence emission in NIR window, therefore they have been used to establish biosensors. Moreover, Ding et al. [− in living cells based on NIR emitting Ag2S quantum dots. The fluorescence intensity of Ag2S quantum dots enhanced when various rare earth ions were added. In the presence of F−, F− coordinated with rare earth ions leaded to fluorescence quenching of Ag2S quantum dots. Based on the on-off fluorescence findings, a label-free NIR fluorescence biosensor for F− in living has been proposed. Moreover, Shu et al. [2S quantum dots by doping Pb ions to synthesized Pb-doped Ag2S quantum dots. The Pb-doped Ag2S quantum dots emitted fluorescence in NIR-Ⅱ window (950 to 1200 nm). Based on the NIR-Ⅱ emitting Pb-doped Ag2S quantum dots, a biosensor for H2O2 have been proposed  which can achieve higher imaging depth without complications from tissue autofluorescence. Sulfide quantum dots, such as Agg et al.  have fabu et al.  have ameproposed B.− in living cells, etc. These biosensors displayed good sensing performance toward analytes detection. In addition, these biosensors also showed other outstanding advantages, including simple of preparation, low cost and good selectivity, stability, and great promising practical applications in clinical diagnosis, as shown in Carbon or graphene quantum dots have attracted intensive interest and have also been used to determine biomolecules due to their fascinating properties ,147,148.All in all, biosensors based on sulfur-containing nanomaterials have been used for detection of various biomolecules in the last five years, including glucose, dopamine, proteins, DNA, RNA, etc. The biosensors based on sulfur-containing nanomaterials displayed enhanced selectivity, lower sensitivity, faster response time, and low detection limit in comparison to biosensors based on other nanomaterials. Taking glucose as analytes, In summary, this paper provides a brief overview of recent researches on the applications of sulfur-containing nanomaterials, including metallic sulfide nanomaterials and sulfur-containing quantum dots in biosensors. The sulfur-containing nanomaterials have excellent properties, such as nanometric scale, water-dispersibility, excellent catalytic activity, conductivity, biosafety, photoactivity, and fascinating optical properties, and have been proven useful in various biosensing applications via electrochemical, PEC, ECL and PL strategies. Though many achievements have been obtained for biosensors based on sulfur-containing nanomaterials, there are still significant challenges that need to be solved.(1) As an emerging quantum dots, sulfur quantum dots possess excellent optical properties and biocompatibility which make them possible to prepare biosensors. However, researches on biosensing applications of sulfur quantum dots are still inadequate. Therefore, it is urgent to further exploit the biosensing applications of sulfur quantum dots in the next few days. Taking advantage of optical properties of sulfur quantum dots and PL-based technologies , PL probes for detecting various biomolecules based on sulfur quantum dots can be established. (2) Real-time biosensing in vivo or intracellular based on sulfur-containing nanomaterials remains a challenge because typical analytical measurements only capture a single-time-point in samples. Biosensing in vivo or intracellular are a new class of detecting technologies that can be established by means of a number of sophisticated analysis platforms providing an in vivo read-out of the spatial, temporal, and quantitative information of biomolecules. Therefore, the vast majority of analytes detected by biosensors based on sulfur-containing nanomaterials are limited to exist in vitro or extracellular. In order to fabricate vivo or intracellular biosensors based on sulfur-containing nanomaterials, sophisticated analysis platforms providing real-time information of biomolecules should be tried to fabricate by utilizing various of technologies and methods .(3) With the development of materials science and nanotechnology, wide variety of nanomaterials have emerged in our life. Some nanomaterials, such as metal organic frameworks and gold nanoclusters, are easy to synthesize without complicated operations. Considering time cost and experimental safety, more and more researchers have dedicated to exploiting these nanomaterials. In view of the abundant storage and the pressure on the environment of S elements, more and more sulfur-containing nanomaterials should be synthesized and used to construct biosensors. Therefore, green synthetic methods  should be exploited and utilized to synthesize sulfur-containing nanomaterials.


Tff1 represents a gastric tumor suppressor gene in mice. TFFs are also synthesized in minute amounts in the immune and central nervous systems. In mucous epithelia, they support rapid repair by enhancing cell migration (“restitution”) via their weak chemotactic and anti-apoptotic effects. For a long time, as a paradigm, this was considered as their major biological function. Within recent years, the formation of disulfide-linked heterodimers was documented for TFF1 and TFF3, e.g., with gastrokine-2 and IgG Fc binding protein (FCGBP). Furthermore, lectin activities were recognized as enabling binding to a lipopolysaccharide of Helicobacter pylori  or to a carbohydrate moiety of the mucin MUC6 (TFF2). Only recently, gastric TFF1 was demonstrated to occur predominantly in monomeric forms with an unusual free thiol group. Thus, a new picture emerged, pointing to diverse molecular functions for TFFs. Monomeric TFF1 might protect the gastric mucosa as a scavenger for extracellular reactive oxygen/nitrogen species. Whereas, the TFF2/MUC6 complex stabilizes the inner layer of the gastric mucus. In contrast, the TFF3–FCGBP heterodimer  are likely part of the innate immune defense of mucous epithelia, preventing the infiltration of microorganisms.Trefoil factor family peptides  are typically co-secreted together with mucins.  I-V, CysII-IV, and CysIII-VI; th cysteine (CysVII) residues located outside the TFF domain. TFF2 (human: 106 amino acids) contains two TFF domains and the N- and the C-terminals are disulfide-linked via two additional cysteine residues outside the TFF domains  peptides  are characterized by a common structural motif, the TFF domain , which contains six conserved cysteine residues with three intramolecular disulfide bonds .Xenopus laevis, even more TFF peptides exist [In the skin of frogs, such as es exist . Furtheres exist , the froes exist  and “βγ-es exist , and thees exist ,10,11. Ges exist .Mucous epithelia are the predominant expression sites of TFF peptides. Here, the exocrine secretion of TFFs occurs together with different mucins ,6. TFF1 Furthermore, minute amounts of TFF peptides are also secreted in an endocrine manner, e.g., by the central nervous system (CNS), as well as by the immune system . In partTFF peptides, together with epidermal growth factor (EGF), are aberrantly expressed in diverse chronic ulcerative conditions, often in glandular structures termed the “ulcer-associated cell lineage” (UACL) . Thus, TToxoplasma gondii infection [Of special note, TFF1 in particular is ectopically expressed in various chronic inflammatory conditions, even in murine models of encephalitis and asthma, and in the murine spleen after nfection ,34,35,36Further pathological expression of TFF peptides occurs in metaplasias , as wellTff1, Tff2 and Tff3 have been available. The most prominent phenotype is observed in Tff1KO mice. For a long time, mice deficient in Tff1KO mice mainly show a gastric phenotype, i.e., they all develop antropyloric adenomas with ~30% progressing to carcinomas [Tff1KO mice also show strongly reduced Tff2 expression, particularly in the gastric corpus [Cckbr+) and +5 (Mist+) are involved in tumorigenesis in Tff1KO mice [Tff1KO mice, continuous self-renewal is also dysregulated in fundic units, but this does not lead to carcinogenesis [Tff1KO mice show generally increased chemically-induced tumorigenesis [Tff1 represents a gastric tumor suppressor gene in mice [gp130757F mice), showed reduced Tff1 levels and a phenotype  highly similar to Tff1KO mice [rcinomas ,43. Thusrcinomas . Of notec corpus ,45. Antrc corpus ,47. Tumoc corpus . There aogenesis . Furtherigenesis . Taken t in mice ,41. Of n1KO mice ,52.Tff2KO mice do not show a striking phenotype. Other than moderate morphological changes [Helicobacter-induced gastritis and to Yersinia enterocolitica infection, as well as a delayed recovery after dextran sodium sulfate (DSS)-induced colitis [Tff2KO mice show the dysregulated expression of immune response-related genes and their macrophages are hyperresponsive to interleulin-1β [In contrast,  changes , they ha changes . They al colitis ,55,56,57eulin-1β ,54.Tff2KO rats are highly sensitive to oral infection with Escherichia coli K1, leading to bacteremia probably due to the loss of Tff2 in the developing small intestine [Tff2 expression is developmentally regulated in the neonatal rat intestine, reaching a peak at P9 and dropping sharply thereafter [Nine-day-old (P9) ntestine . Of noteereafter .Tff3-deficient mice have strongly increased sensitivity in the DSS colitis model and these animals are particularly sensitive to radiation-induced mucosal injury and chemotherapy [gp130ΔSTAT mice), showed reduced Tff3 levels and a phenotype highly similar to Tff3KO mice [Tff3KO mice show minor morphological differences in the foliation of the cerebellum, as well as motoric deficits [otherapy ,61. Of n3KO mice ,52. Furtdeficits .There is a large body of literature describing the weak motogenic effects of all three TFF peptides for a variety of different epithelial and immune cells in vitro, starting in the year 1994 ,5,63. ThFor all three TFF peptides, an anti-apoptotic effect has also been observed ,76,77,78A special form of rapid wound healing occurs in mucous epithelia after superficial injury, i.e., by the cell migration of neighboring cells. This process is called “restitution” and starts within minutes after damage. Thus, TFFs would be well designed to act as luminal protection peptides, enhancing restitution only after mucosal injury ,82.Lactococcus lactis was effective in preventing and healing DSS-induced colitis [A protective function of TFF peptides for mucous epithelia is in line with numerous in vivo studies using different animal models of disease . Addi colitis .Tlr2KO mice from increased morbidity and mortality during acute colonic injury, probably due to its anti-apoptotic effect [TFF3 is also linked to innate immunity, as its synthesis in intestinal goblet cells is selectively induced after the activation of Toll-like receptor (TLR) 2 by commensal bacteria . This prc effect .−6 to 10−7 M or even higher [−10 M [Generally, the motogenic and anti-apoptotic effects of TFF peptides are rather weak and were observed in a concentration range of about 10n higher ,71. Suchr [−10 M . Thus, ir [−10 M .Deleted in Malignant Brain Tumors 1 (DMBT1) [gp340 in a Ca2+-dependent manner [−7 M [−9 M [−7 M) via the activation of the proteinase-activated receptor PAR4 [−6 M) was claimed to activate PAR2 [However, several transmembrane proteins could be characterized to bind TFF2 with low affinity, such as a porcine β-integrin and a porcine 224k protein (CRP-ductin), with similarity to  (DMBT1) . In addit manner , the latt manner . Later oer [−7 M ,90. Thus M [−9 M . Thus, t M [−9 M , as welltor PAR4 , and TFFate PAR2 .Taken together, TFF peptides cannot be considered as high-affinity ligands for specific transmembrane receptors. As their concentrations in mucous gels are rather high, it seems highly unlikely that their protective function is based solely on their action as low-affinity ligands, e.g., for CXCR4 and CXCR7. It is more realistic to expect additional molecular functions for TFF peptides.VII located outside the conserved TFF domain. Generally, the existence of unpaired cysteine residues is highly unlikely for secretory proteins, as disulfide formation is enforced in the endoplasmic reticulum (ER) [Helicobacter pylori and TFF3 to DMBT1gp340 was reported to depend on dimerization [TFF1-3 are typical secretory peptides. TFF1 and TFF3 are special, as they contain an odd number of cysteine residues, with Cyslum (ER) . Thus, Tlum (ER) ,96,97,98rization ,99.Only later did biochemical studies reveal that TFF1 and TFF3 occur in vivo in different molecular forms and are also capable of forming disulfide-linked heterodimers with at least gastrokine 2 (GKN2) and IgG Fc binding protein (FCGBP). Thus far, the following hetero(di)meric forms have been identified: TFF1-GKN2, TFF1-FCGBP, and TFF3-FCGBP ,102,103.H. pylori lipopolysaccharide in a pH-dependent manner and TFF3 binds to a lesser extent [H. pylori mutants point to N-acetylglucosamine (GlcNAc) being part of the carbohydrate structure recognized by TFF1 [2+- and pH-dependent lectin activity of TFF2 was narrowed down to the unusual O-linked terminal carbohydrate moiety GlcNAcα1→4Galβ1→R of the mucin MUC6 [All three TFF peptides were shown to bind to carbohydrate moieties, but to quite different extents and probably also with different specificities. Dimeric TFF1 interacts with a core oligosaccharide portion of the r extent ,104,105. by TFF1 ,105. TFF by TFF1 ,106; thecin MUC6 ,109,110.Xenopus laevis are capable of forming high-molecular-mass complexes probably via the lectin activities of their intrinsic multiple TFF modules [Furthermore, the frog integumentary mucins FIM-A.1 and FIM-C.1 from  modules . In addi modules . Finallygp340 [Generally, the lectin activities of TFF peptides might explain diverse biological effects, such as their weak motogenic, (anti)-apoptotic, and angiogenic effects . Theoretgp340 ,87. Furtgp340 ,113.X. laevis (ortholog xP1) [Human gastric TFF1 occurs in different molecular forms, i.e., monomeric TFF1, homodimeric TFF1, and heterodimeric forms  ,114. Genlog xP1) . This islog xP1) ,116. Howlog xP1) .Gastric TFF1 is also capable of forming a heterodimer with GKN2, as shown for humans and mice ,103,106.Minute amounts of gastric TFF1 form a disulfide-linked heterodimer with FCGBP, as shown for humans and mice ,103. ThiVII. This has been shown for X. laevis (ortholog xP1) [VII is probably masked by four flanking acidic amino acids (see VII are conserved in X. laevis (…PECVII), mice (…QEEECVIIPF), and humans (…PPEEECVIIEF) [VII is exceptionally reactive (nucleophilic) because of the pronounced steric exposure outside the TFF domain and separation by proline residues [VII, has special intracellular as well as extracellular molecular functions.Systematic biochemical investigations revealed that, in gastric extracts, the major form of TFF1 is a secreted monomer with a free thiol group at Cyslog xP1) , mice [4log xP1) , and humlog xP1) . Normalllog xP1) , but thelog xP1) . Howevercids see , and thuECVIIEF) . It is eresidues . Thus, iTff1KO mice the unfolded protein response (UPR) is activated [Tff1KO mice [Intracellularly, TFF1 is expected to be involved in the folding of cysteine-rich glycoproteins (such as the mucin MUC5AC), as in ctivated . For exa1KO mice ,123. Thi1KO mice , which h1KO mice , and is 1KO mice .2O2 and O2-) is high due to dual oxidases (DUOX) and NADPH oxidases (NOX), respectively. DUOX plays a key role in innate mucosal immunity, preventing gastric colonization with bacteria [3-) from food to nitrite (NO2-) by saliva and microbiota, HO-NO forms in the gastric juice and disproportionates into NO [2− forming peroxynitrite (ONOO−), which is the prototype of a toxic RNS [VII of human TFF1 [Extracellularly, the free thiol group of TFF1 has been proposed to act as a scavenger for reactive oxygen/nitrogen species (ROS/RNS) ,103,115.bacteria ,133. Addbacteria . Further into NO , which i into NO ,137. In oxic RNS . Criticaoxic RNS , as welloxic RNS . Of specman TFF1 . Taken tVII of monomeric TFF1 could not only have intracellular and extracellular protective functions as a gastric tumor suppressor. TFF1 is also expected to play a protective role during inflammatory processes, as there is a fundamental connection to oxidative stress (“oxidative burst”) [T. gondii infection [The reactive Cys burst”) . This as burst”) ,141, as  burst”) , asthma  burst”) , and enc burst”) , and in nfection .X. laevis (ortholog xP4) [2+- and pH-dependent manner [Griffonia simplicifolia [X. laevis gastric mucin [H. pylori infection by inhibiting the cholesterol α-glucosyltransferase of H. pylori [H. pylori co-localizes with MUC5AC in the human stomach [N-acetylglucosaminyltransferase (α4GnT); all mice lacking this enzyme (A4gntKO) spontaneously develop antral adenocarcinomas through an inflammation-associated pathway [In contrast to TFF1, gastric TFF2 is predominantly associated with mucins, as shown for humans , pigs 1, and X. log xP4) . Here, ilog xP4) . Bindingt manner ,109,110.t manner . The unuicifolia ,144. MUCicifolia . This exic mucin . Of spec. pylori ,147. Tha stomach . A key e pathway . Thus, αN,N’-diacetyllactosediamine (LacdiNAc) oligosaccharide [H. pylori adhesin LabA [H. pylori mainly adheres to MUC5AC in the gastric mucus barrier.A characteristic feature of human TFF2 is the N-glycosylation site , which iccharide . There iccharide . The Lacsin LabA . This isX. laevis orthologs of TFF2, i.e., xP4.1 (N-glycosylated) and xP4.2 (non-glycosylated), where glycosylation has no influence on lectin binding to gastric mucin [TFF2 binding to MUC6 differs characteristically between human (mainly N-glycosylated TFF2) and pig (non-glycosylated TFF2). In porcine gastric mucus, the binding of TFF2 is much stronger than in human mucus and is even resistant to boiling in SDS ,109,110.ic mucin .Taken together, the highly specific lectin binding of TFF2 to a conserved O-linked oligosaccharide in MUC6 has implications for understanding the structure of the two-layered gastric mucus–bicarbonate barrier, which is composed of a loosely adherent (outer) layer and a firmly adherent, water-insoluble (inner) layer  108,153153. X. laevis ortholog of TFF2 (xP4) prevents the shrinking of secretory granules of esophageal goblet cells during the processing of the samples for electron microscopy, due to its interaction with mucin [Tff2KO mice show accelerated progression of Helicobaccter-induced gastritis, as the inner mucus barrier is probably no longer very tight [2+-dependent manner [3−) occurs [Gkn2KO mice have increased susceptibility to H. pylori-dependent immunopathology [The outer layer consists mainly of the mucin MUC5AC and only little MUC6 ,153,154,th mucin . Furtherry tight . Thus, Tt manner ,110. Thi) occurs . This ne) occurs . Additioathology ,157. ThiIn contrast to human, pig TFF2 is also expressed in the pancreas. Here, TFF2 is clearly not associated with mucins and rather occurs in an unusual low-molecular homodimeric form, which is resistant to boiling in SDS . This isTff2 deficiency influences the immune system [Finally, it will be a challenging goal for the future to identify the binding partner and to elucidate the molecular function of TFF2 in the lymphoid system, as e system . It coulVII residue  and TFF1 (TFF1–FCGBP) is not known. The repetitive glycoprotein FCGBP is ubiquitous in vertebrates and cephalochordates and it is secreted by most mucous epithelia from a variety of mucin-producing cells (similar as TFF3), and thus appears in the corresponding body fluids ,159. Theimmunity ,88. For  sanguis . The int sanguis . Of note sanguis . TFF3 mais model ,169.Other than playing a role in the innate immune defense of mucous epithelia, FCGBP could also fulfill a structural matrix function, as FCGBP seems to be expressed in the aorta, where it belongs to the most downregulated genes in patients with ruptured abdominal aortic aneurisms .Taken together, within recent years, a body of evidence has been accumulated that TFF peptides fulfill diverse roles in the protection of mucous epithelia, other than acting solely as motogens (old paradigm). Their relatively high abundance in mucous epithelia does not fit with a predominant role as typical high-affinity ligands for receptors in mucous epithelia. However, even as low-affinity ligands, they can synergistically support mucosal repair, e.g., by restitution. In this case, the activation of certain glycosylated receptors, such as CXCR4 and CXCR7, might occur via the lectin activities of TFF peptides and would depend on the glycosylation of these receptors; the latter could vary in different cell types and would thus allow some specificity and diversity.Currently, TFF peptides seem to have rather structural roles as part of the insoluble gastric inner mucus–bicarbonate barrier layer (TFF2/MUC6 complex) or as part of a postulated mucosal clearance/barrier system for different microbes . The latter might also be able to trap viruses with the help of immunoglobulins on mucosal surfaces. Both of these functions would indicate important roles of TFF2 and TFF3/TFF1 for the mucosal innate immune defense against pathogenic bacteria and viruses . This isThe function of TFF peptides in sites other than mucous epithelia is currently less well understood. Particularly, TFF1 might play a protective role during various inflammatory processes, even in the brain. TFF3 could also have a function during immune reactions in the CNS . Furthermore, all three TFF peptides  could also act as neural chemotactic factors, as they show developmental changes, particularly in the cerebellum . In contVII, can prevent Tff1KO mice from developing antral carcinomas.Based on these diverse molecular functions of TFF peptides, interesting clinical applications might arise in the future . For exaIn a further attempt, the region in TFF modules responsible for lectin activities could be defined in detail. In combination with theoretical modeling methods, a mutagenesis program could create variants with new/novel affinities for different carbohydrate moieties. Such a program should be greatly facilitated by the fact that biologically active TFF peptides can be generated, not only by recombinant technologies, but also pure chemical synthesis, where even the correct formation of disulfide bridges is possible ,105. Taken together, TFF peptides appear to share characteristic features with the multifunctional family of galectins . For the


Orthohepevirus C, currently comprising HEV genotypes C1 and C2, by either serological or molecular approach is summarized. The distribution, genomic variability, and evolution of Orthohepevirus C are analyzed. Moreover, the potential risk of cross-species infection and zoonotic transmission of Orthohepevirus C are discussed.Hepatitis E virus (HEV) is an emerging zoonotic pathogen that has received an increasing amount of attention from virologists, clinicians, veterinarians, and epidemiologists over the past decade. The host range and animal reservoirs of HEV are rapidly expanding and a plethora of emerging HEV variants have been recently identified, some of which have the potential for interspecies infection. In this review, the detection of genetically diverse HEV variants, classified into and presumably associated with the species  Hepatitis E Virus (HEV) is the causative viral pathogen of hepatitis E, originally considered a self-limiting disease occurring only in developing countries with poor sanitary conditions . MortaliHEV was first isolated in 1983 and the viral genome was subsequently cloned and sequenced ,7. HEV iHepeviridae, which includes the genera Orthohepevirus and Piscihepevirus. The genus Orthohepevirus is further divided into four species Orthohepevirus A to D. Orthohepevirus A contains HEV variants isolated from human, pig, wild boar, deer, mongoose, rabbit, and camel; Orthohepevirus B from chicken; Orthohepevirus C from rat, greater bandicoot, Asian musk shrew, ferret, and mink; and Orthohepevirus D from bat. The genus Piscihepevirus occurs in cutthroat trout and possibly other fish species [HEV differs from all other human hepatitis viruses in that it is closely related to non-human viruses . HEV is  species . Based o species . NeverthOrthohepevirus C is separated into two genotypes: HEV-C1 and HEV-C2. HEV-C1 contains variants derived from hosts in the orders Rodentia and Soricomorpha, and HEV-C2 from the order Carnivora [Orthohepevirus C has been rapidly extended in the last years [Orthohepevirus C is still under debate, two recent clinical cases describing persistent hepatitis in a liver transplant patient in Hong Kong and severe acute hepatitis in an immunocompetent patient in Canada, respectively, have been attributed to infection with rat HEV [Orthohepevirus C and their potential risk of cross-species infection and zoonotic transmission to humans.The species arnivora ,11. By mst years . Althoug rat HEV ,15. Furt rat HEV . In thisOrthohepevirus C in rodents and suggesting these animals might be the natural reservoirs for this species of HEV [Orthohepevirus C RNA was detected in feces of Norway rats in Germany in 2010 using a nested broad-spectrum reverse transcription polymerase chain reaction (RT-PCR) and the complete genomes were sequenced and found to be highly divergent from human HEV strains [Muridae and Cricetidae of the order Rodentia, respectively, and one species within the family Soricidae of the order Soricomorpha, from eight countries . Additionally, Orthohepevirus C sequences have been detected in 22 countries  and 25 animal species, containing eight species within the family Muridae and 10 species within the family Cricetidae of the order Rodentia, one species within the family Ursidae and two species within the family Mustelidae of the order Carnivore, two species within the family Falconidae of the order Falconiformes, one species within the family Soricidae of the order Soricomorpha, and one species within family Hominidae of the order Primates. Worldwide detection and distribution of Orthohepevirus C is displayed in The first study concerning the prevalence of anti-HEV antibodies in wild rats was conducted in the USA in 1999 . In 2000s of HEV . SubsequRattus norvegicus (Brown rat/Norway rat). In the USA, the seroprevalences of anti-HEV antibodies were 76.9% (83/108 samples tested positive) between 1986 and 1997, 68.5% (135/197) between 1994 and 1998, and 73.5% (144/196) between 2005 and 2006, considerably higher than in other countries [To date, 11 seroprevalence studies have been carried out on ountries ,18,21. Iountries ,23; in Gountries , comparaountries . In Vietountries ,27,28. Iountries .Rattus rattus (Black rat) with results varying considerably. In the USA, one study reported a seroprevalence of 90.2% (102/113) between 1986 and 1997, yet only 38.3% (31/81) between 1994 and 1998 in another [Six studies have been carried out in  another ,18. In I another ,31,32. I another .Rattus tanezumi  of 25.0% (4/16) and 33.3% (2/6) in 2011 and 19.6% (9/46) between 2012 and 2013 [Rattus rattoides losea (Losea rat), Rattus flavipectus (Yellow-breasted rat), Rattus rattus hainanus, and Bandicota indica (Greater bandicoot rat), were 21.5% (26/121), 19.9% (34/171), 11.8% (2/17), and 23.0% (40/174), respectively [Rattus rattus rufescens (Indian black rat), 3.6% (2/55) in 1990 in Rattus rattus andamanesis (Sikkim rat), and 54.5% (12/22) in 1985 in Bandicota bengalensis (Lesser bandicoot rat) [Muridae, Rattus exulans (Polynesian rat) and Mus musculus (House rat), had seroprevalences of 83.3% (15/18) between 1986 and 1997 and 14.3% (2/14) between 1994 and 1998, respectively [Three studies in Vietnam found anti-HEV seroprevalences in and 2013 ,27,28. I19.9% 34/1, 11.8% 83.3% 15/ between Cricetidae. Seroprevalence rates in Clethrionomys gapperi (Southern red-backed vole), Neotoma albigula (White-throated woodrat), Neotoma mexicana (Mexican woodrat), Neotoma micropus (Southern plains woodrat), Oryzomys palustris (Marsh rice rat), Peromyscus boylei (Brush mice), Peromyscus eremicus (Cactus mouse), Peromyscus leucopus (White-footed mouse), Peromyscus maniculatus (North American deermouse), and Sigmodon hispidus (Hispid cotton rat) were 66.7 (4/6), 59.1 (13/22), 57.1 (48/84), 12.5 (1/8), 24.4 (10/41), 8.3 (2/24), 42.9 (3/7), 10.0 (5/50), 11.0 (11/91), and 32.7 (37/113), respectively. Rodent sera were collected during 1994–1998 throughout the USA and assessed by two in-house established serological tests including antigens based on a mosaic protein composed of recombinant HEV ORF2 and ORF3 and a 55-kDa HEV ORF2 protein [Antibodies against HEV were also found in multiple rodent species within the family  protein .Suncus murinus (House shrew) within the family Soricidae of the order Soricomorpha was 10.4% (27/260) for anti-HEV IgG and 4.6% (12/260) for Immunoglobulin M (IgM), respectively, with samples collected between 2011 and 2012 in China [Additionally, seroprevalence in in China . The stuDiffering seroprevalence of anti-HEV antibodies may be due to different serological tests, which have been carried out using commercial anti-HEV enzyme-linked immunosorbent assay (ELISA) kits or conducted with in-house established assays based on diverse antigens (truncated or complete human and rat HEV ORF2 protein) in numerous labs independently. Furthermore, although there is only a single serotype of HEV, the cross-reaction of other known or unknown viral pathogens cannot be excluded and may impact the results. Finally, no HEV seroprevalence studies have been performed on species of the orders Carnivora and Falconiformes.Orthohepevirus C (previously designated rat HEV) RNA from liver, feces, and blood specimens of various animal species. Furthermore, comprehensive sequence and phylogenetic analyses enable genotyping of viral strains. Consequently, an increasing number of novel Orthohepevirus C variants have been discovered around the world whose assignment remain to be determined.Molecular biology methods, including RT-PCR amplification, Sanger sequencing and metagenomic analysis with next-generation sequencing, have facilitated the detection of Orthohepevirus C sequences were initially detected in Germany in the widespread rat species Rattus norvegicus (Brown rat/Norway rat) [Orthohepevirus C genomes have been identified in Norway rats, from countries including the USA, China, Germany, France, Denmark, Lithuania, England, Hungary, Austria, Switzerland, Italy, Spain, Greece, Belgium, the Czech Republic, and Vietnam, with all viral strains belonging to HEV-C1 [Orthohepevirus C genomes have been detected in numerous other members of the family Muridae: HEV-C1 from Rattus rattus (Black Rat) has been detected in Indonesia, China, Kenya, and 12 other European countries comprising Lithuania, Germany, Hungary, Denmark, Austria, Switzerland, France, Italy, Spain, Greece, Belgium, and the Czech Republic [Rattus tanezumi  in Vietnam and China have also been reported to harbor the virus [Rattus losea (Losea rat), Rattus flavipectus (Yellow-breasted rat), and Bandicota indica (Greater bandicoot rat) [Apodemus chevrieri (Chevrier’s field mouse) and Apodemus agrarius (Striped field mouse) were positive for Orthohepevirus C detection; however, these variants are phylogenetically divergent from known HEV-C1 or HEV-C2, and thus, cannot yet be classified [way rat) ,20. To do HEV-C1 ,44,45,46Republic ,41,47,48he virus ,36,44,49oot rat) ,43,44; Aassified .Orthohepevirus C RNA was found in several species within other rodent families, including Cricetidae, containing Eothenomys melanogaster (Père David’s vole), Eothenomys Inez (Inez’s red-backed vole), Myodes rufocanus (Grey red-backed vole), Microtus gregalis (Narrow-headed vole), Cricetulus migratorius (Gray dwarf hamster), and Cricetulus barabensis (Striped dwarf hamster) from China, Microtus arvalis (Common vole) from Hungary and Germany, Myodes glareolus (Bank vole) from Germany, and Necromys Lasiurus (Hairy-tailed bolo mouse) and Calomys tener (Delicate vesper) from Brazil [Consistent with serological studies, m Brazil ,51,52,53Orthohepevirus C sequences have been detected in other animal species of mammals. Within the family Soricidae of the order of Soricomorpha, the species Suncus murinus (House shrew/Asian musk shrew) harbor HEV-C1 in China and Asian musk shrew have been implied as a reservoir for HEV-C1 from wild rats, while the virus in the species Crocidura olivieri (Olivier’s shrew) from Kenya cannot be assigned due to increased genetic divergence [Additionally, vergence ,44,48,50Ursus arctos syriacus, family Ursidae) in a German zoo, most likely the result of a spillover infection from free-roaming Norway rats [Mustela putorius) from the Netherlands [Mustela putorius and Neovison vison belong to the carnivore family Mustelidae. Even though a short (362 nucleotide) partial ORF1 sequence was identified in a red fox , classification into Orthohepevirus C requires comparisons based upon complete genome sequences, as recommended by the International Committee on the Taxonomy of Viruses (ICTV) Hepeviridae Study group [In the order Carnivora, HEV-C1 has been identified in a Syrian brown bear  and red-footed falcon  within the family Falconidae of the order Falconiformes in Hungary, notably the viral strain from kestrel showed higher sequence similarity to members of Orthohepevirus C than to Orthohepevirus B and clustered together with aforementioned rodent HEV variants from China and Brazil; therefore, the kestrel-derived HEV strain is included in this review [Recently, novel HEV variants have been reported from common kestrel  from Apodemus agrarius, which consists of only 6286 nucleotides and has a significant shorter ORF1 with 4334 nucleotides; moreover, RtAa-HEV/JL2014 lacks ORF3. Since it has been recently reported that the ORF3 is essential for the release of membrane-associated rat HEV particles, the absence of ORF3 in Apodemus agrarius needs further verification [Cricetulus migratorius is only 4182 nucleotides in length. However, the short viral genome and ORF1, as well as the absence of ORF3 may be associated with errors in assembling reads to contigs and mapping assembled contigs to references in the metagenomic study [Overall, the genomic length of fication . In addiic study .Rattus norvegicus, Rattus rattus, Rattus tanezumi, and Rattus losea within the family Muridae, two HEV-C1 analogues identified from humans, HEV-C2 strains from the family Mustelidae, and the HEV variants from the species Necromys lasiurus and Microtus gregalis within the family Cricetidae harbor ORF4. In contrast, this putative ORF is not found in other species. A recent study has shown that ORF4 is not necessary for the active replication of rat HEV [Orthohepevirus C, the molecular evolution of ORF4 is as of yet unclear.A unique ORF (tentatively named ORF4), located within the 5′ region of ORF1 has been proposed in certain rat and ferret HEV strains ,55. As s rat HEV . TherefoEothenomys melanogaster, identified from two separate studies, possess ORF2 genes overlapping with ORF1. This is also the case for HEV strains in the species Eothenomys inez, Myodes rufocanus, and Microtus gregalis within the family Cricetidae [Muridae [Orthohepevirus C variants utilize distinct translational frames, as described in our recent study [Typically, HEV ORF2 overlaps partially with ORF3 but not ORF1. However, three HEV variants in the species icetidae ,36,50. A[Muridae . Finallynt study .Orthohepevirus C species have been discovered in diverse wild rodents in China in a study based on metagenomic analysis, each species has only a single viral complete genomic sequence [Although many novel HEV variants within the sequence . The genOrthohepevirus C sequences are partial genomic fragments (<500 bp). In order to determine the genetic variability of HEV variants within the species Orthohepevirus C, we have included 48 currently available complete or nearly complete genomic sequences of Orthohepevirus C from GenBank. The pairwise comparisons of complete genome and deduced ORFs of Orthohepevirus C is illustrated in As mentioned, the majority of described Apodemus chevrieri, Apodemus agrarius, Eothenomys melanogaster, Eothenomys inez, Myodes rufocanus, Microtus gregalis, Microtus arvalis, Necromys lasiurus, Cricetulus migratorius, and Cricetulus barabensis exhibit comparable or even higher divergence than is present between HEV-C1 and HEV-C2, even though HEV-C2 strains have only been found in the carnivore species Mustela putorius. Nucleotide identities in Orthohepevirus C variants from individual rodent species vary between 48.2–67.3%. Higher identities are observed between Eothenomys melanogaster and Eothenomys Inez, ranging from 72.2 to 72.6%, and from 76.0 to 77.3% between Microtus gregalis and Microtus arvalis. Similar results have been observed for the nucleotides and amino acids of the 3 coding regions. Notably, for the comparison of ORF3, most of the HEV variants in their respective group are highly divergent and exhibit low identities, especially at amino acid level (<20%). These data highlight the fact that substantial genetic variability of HEV is present within the order Rodentia, raising a need for increased efforts to characterize the variants circulating in the respective habitats. Only through this will we be able to better assess the risk potential of these viruses to animal and human health.Unexpectedly, the genomic comparisons between HEV-C1 and other newly identified HEV strains derived from the rodent species Microtus arvalis from Hungary, Germany, and the Czech Republic, which is comparable to sequence identities within common vole variants (between 81.5% and 91.9%). Therefore, it is assumed that the detection of HEV variants in birds of prey  may be linked to the consumption of wild rodents, in particular common voles [Due to the high identity (> 75%) to HEV-C1 strains, two HEV variants detected in humans in Hong Kong and Uganda can be considered as HEV-C1 ,15. Furton voles ,53,59.Orthohepevirus C, phylogenetic analysis was conducted using representative complete genomic sequences within the genus Orthohepevirus of the Hepeviridae family  and Père David’s vole  in China and two viral genomes of each animal species were amplified and characterized. Due to the significant divergence from HEV-C1 and HEV-C2, we have proposed these variants be classified as putative HEV-C3 and putative HEV-C4, which has been included and discussed in several recent research articles [Orthohepevirus C variants form a separate branch within the genus Orthohepevirus. Predictably, HEV variants detected in the rodent genus Rattus and the carnivore species Mustela putorius cluster into two independent clades within the species Orthohepevirus C, which have already been defined as HEV-C1 and HEV-C2, respectively. Even though newly identified HEV variants are phylogenetically separate from HEV-C1 and HEV-C2, HEV variants from the rodent genus Apodemus (including species Apodemus agrarius and Apodemus chevrieri), Eothenomys (including species Eothenomys melanogaster and Eothenomys Inez), and Microtus  clustered into three distinct clusters, which are associated with rodent taxonomy and host specificity. Taken together, it is hypothesized that co-evolutionary model, and not the host-switch pattern, might be applied regarding the species Orthohepevirus C [Cricetidae, particularly RtCm-HEV/XJ2016 (GenBank accession no. KY432903) from the species Cricetulus migratorius) and RtCb-HEV/HeB2014 (GenBank accession no. KY432899) from Cricetulus barabensis, demonstrate a high degree of divergence within the Orthohepevirus C clade. With no doubt that the evolutionary history of Orthohepevirus C will be better elucidated when diversified HEV strains are discovered in insectivores and carnivores in the future.In order to investigate the dynamics of the molecular evolution of e family . The disdy group . Recentlarticles ,50,53. Aarticles ,51,53. Cevirus C . HoweverFalco tinnunculus) from Hungary clusters together with the five reported HEV variants from European common voles . Whether Orthohepevirus C variants from common voles indeed can infect common kestrel remains to be determined [The two HEV variants derived from a Chinese and Canadian patient, respectively, cluster within the HEV-C1 branch, providing further evidence of a potential zoonotic origin ,15. Conctermined ,53,59.Muridae and Cricetidae. Considering that the order Rodentia comprises 33 families and 2277 species , therefore forming the largest mammalian order, we believe that these HEV variants are only the tip of the iceberg. Future studies will undoubtably reveal further novel variants within Orthohepevirus C with new Orthohepevirus C genotypes being assigned in the coming years. Although it is evident from our analyses that HEV variants within each genotype of Orthohepevirus C  are highly divergent from one another, subtype differentiation has not yet been proposed by ICTV Hepeviridae study group [Thus far, all HEV variants detected in rodents have been isolated from the families dy group ,31,53.Orthohepevirus A undergoes different patterns between different genotypes (HEV-1 to HEV-8), which is associated with at least two distinct epidemiological profiles of the species [Orthohepevirus C have been identified; to what extent of viral evolutionary discrepancy of the species is largely unclear.It is a well-known fact that evolution of HEV within the species  species . IncreasOrthohepevirus A; domestic swine, wild boar, and sika deer have been confirmed to be the reservoirs. Typically, HEV zoonotic transmission occurs via three routes. Firstly, population groups coming into direct contact with infected animals, such as veterinarians and workers on pig farms or slaughterhouses, who were proven to have significantly higher anti-HEV IgG seroprevalence rates than the general population and other veterinarians in some European countries; secondly, foodborne transmission via consumption of undercooked infectious meat products (mostly pork), such as sausages and liver, which is common in European countries; and thirdly, environmental contamination by animal feces, since the use of pig manure in agriculture may lead to contamination of water sources and agricultural products [Human hepatitis E cases in industrialized countries are mainly caused by zoonotic transmission of HEV-3 and HEV-4 within the species products . Using rproducts ,64. Receproducts . Lastly,products ,67.Rodents are extraordinarily abundant and diversified in multiple continental habitats and live in close proximity to humans, domestic animals, and wild animals. This interface has resulted in the rodent origin of a plethora of zoonotic viruses, such as Lassa virus, Lymphocytic choriomeningitis virus, Sin Nombre virus, Hantaan virus, and Seoul virus, all of which have caused severe diseases in humans in the past decades . IncreasOrthohepevirus C, the potential risk of these viruses concerning cross-species infection and zoonotic transmission to humans remains a controversial topic [Orthohepevirus C variants were understudied and neglected due to their genetic divergence (identities <52%) to the principle human pathogenic HEV strains (HEV-1 to HEV-4). It has been reported that pigs or non-human primates (rhesus monkeys) inoculated with rat HEV showed no evidence of infection, indicating that it is not a source of human infection [Rattus rattus could propagate efficiently in human hepatoma cell lines including PLC/PRF/5, HuH-7, and HepG2 [Orthohepevirus A, rat HEV have caused chronic viral infection in immunosuppressed individuals as well as extrahepatic manifestations [Orthohepevirus C. What is the viral determinant for rat HEV cross-species infection? What is the effectivity of antiviral therapy, especially regarding ribavirin, against rat HEV infection? Will other rat HEV analogues  be capable of infecting humans?Although rodents have been confirmed to be the primary natural reservoirs of HEV variants within the species al topic . In the nfection ,66. In cnfection . In a renfection . Furthernd HepG2 . Intrigund HepG2 ,15. Furtstations . JointlyOrthohepevirus C variants has been characterized in several studies as well. House shrews (Suncus murinus) have been reported as a reservoir of rat HEV worldwide; however, a novel divergent HEV strain has been detected in a Olivier’s shrew (Crocidura olivieri) from Kenya and formed a separate monophyletic branch from HEV-C1, indicating the relationship between the viral species Orthohepevirus C and the animal order Soricomorpha should be more complex than previously considered [Interspecies transmission of nsidered ,43,44,48nsidered .Orthohepevirus C has expanded the known host range of HEV dramatically. This raises critical concerns about the potential risk of cross-species infection and zoonotic transmission to humans, as the biology, ecology, natural history, and pathogenesis of these viruses are largely unknown. After three recent reports identified totally nine human hepatitis E cases linked to rat HEV, the necessity to broaden our knowledge concerning the molecular epidemiology of circulating Orthohepevirus C variants is clear, and the potential risks of these viruses to public health should be reassessed. To elucidate the zoonotic potential of Orthohepevirus C variants, the patterns of transmission, mechanisms of replication, and functional roles of proteins merit further investigation.An increasing number of HEV variants are being detected in diverse animal species, including mammals, avian, and fish, with some of these species having been confirmed as natural reservoirs for HEV and sources of zoonotic infection. With the advancement of techniques such as high-throughput sequencing in metagenomics studies, no doubt more novel HEV variants will be discovered in the near future. The detection of numerous emerging HEV variants within the species


Bradykinin receptor B2 (BDKRB2) has been reported as an oncogene in several malignancies. In glioma, the role of BDKRB2 remains unknown. This study aimed at investigating its clinical significance and biological function in glioma at the transcriptional level. We selected 301 glioma patients with microarray data from CGGA database and 697 with RNAseq data from TCGA database. Transcriptome and clinical data of 998 samples were analyzed. Statistical analysis and figure generating were performed with R language. BDKRB2 expression showed a positive correlation with the WHO grade of glioma. BDKRB2 was increased in IDH wildtype and mesenchymal subtype of glioma. Gene ontology analysis demonstrated that BDKRB2 was profoundly associated with extracellular matrix organization in glioma. GSEA analysis revealed that BDKRB2 was particularly correlated with epithelial-to-mesenchymal transition (EMT). GSVA analysis showed that BDKRB2 was significantly paralleled with several EMT signaling pathways, including PI3K/AKT, hypoxia, and TGF-β. Moreover, BDKRB2 expression was significantly correlated with key biomarkers of EMT, especially with N-cadherin, snail, slug, vimentin, TWIST1, and TWIST2. Finally, higher BDKRB2 indicated significantly shorter survival for glioma patients. In conclusion, BDKRB2 was associated with more aggressive phenotypes of gliomas. Furthermore, BDKRB2 was involved in the EMT process and could serve as an independent prognosticator in glioma. Gliomas are the most common and aggressive primary brain cancers in adult patients . Despite9-BK. However, BDKRB2 is constitutively expressed across different tissues, and it has a high affinity with BK [Bradykinin (BK) is a vasoactive peptide produced from kininogen precursor’s cleavage under kallikrein’s action . BK part with BK . There i with BK .The BK system plays an important role in cancer occurrence and progression . The BK in-vivo and in-vitro experimental studies [Heretofore, some researchers have investigated the potential biological functions of BDKRB2 in glioma based on  studies , 28–30. BDKRB2 expression levels were compared across different WHO grades. The results of both CGGA and TCGA cohorts consistently showed a significant positive correlation between BDKRB2 expression and WHO grade , 1E. In Many other genes have been identified as the mesenchymal subtype’s biomarkers, such as PD1 , PD-L1 , TIM3 33, CD163 p = 1.53E-23), inflammatory response (p = 1.29E-22), leukocyte migration (p = 6.21E-18), immune response (p = 6.30E-17), collagen catabolic process (p = 5.80E-13) and response to lipopolysaccharide (p = 8.58E-12), suggesting that BDKRB2 was tightly associated with EMT process and inflammatory response in glioma. As expected, BDKRB2-negatively-correlated genes were largely associated with normal biological processes, such as cell differentiation (p = 0.001)  .p = 2.12E-12), which was paralleled with the characterization of vascularization in GBM.Given GBM as a distinct subgroup of glioma, we then conducted an independent GO enrichment analysis in this group. In GBM of both datasets, the Venn diagram  exhibiteTo further illustrate the biological process of BDKRB2 in glioma, GSEA analyses were performed in both CGGA and TCGA datasets. We found that BDKRB2 was most correlated with EMT in CGGA  , 3E, whiTo further investigate the relationship between BDKRB2 and EMT, we downloaded seven gene sets from the GSEA website , which wTo further validate the role of BDKRB2 in the EMT signaling pathway, we examined the correlation between BDKRB2 and EMT biomarkers, including E-cadherin, N-cadherin, snail, and slug. Circos plots revealed that BDKRB2 expression was significantly associated with N-cadherin, snail, and slug , 5B. PeaMany other biomarkers have been identified as EMT-related targets in EMT . We addiKaplan-Meier (KM) survival analyses were performed to examine the prognostic role of BDKRB2 in glioma. According to BDKRB2 expression, pan-glioma samples were divided into two groups in each dataset. As shown in Emerging evidence indicates BDKRB2 as a pivotal target in tumorigenesis. BDKRB2 as a frequently amplified molecule has been observed in a range of cancers, including cervical cancer , triple-In the present study, we investigated the transcriptional expression profiles of BDKRB2 in 998 glioma patients and revealed that BDKRB2 expression showed a significantly positive correlation with the WHO grade of glioma. Furthermore, higher BDKRB2 expression was usually accompanied by a more aggressive and malignant phenotype in glioma, including GBM, IDH wildtype, and mesenchymal subtype. Moreover, higher BDKRB2 expression indicated a significantly shorter survival for patients with glioma across different WHO grades. These findings suggested that BDKRB2 played a vital role in the malignant progression of glioma, in line with other malignancies reported previously. Understanding the molecular mechanism of BDKRB2 in glioma may provide a novel therapeutic target to overcome this fatal disease.2+ oscillations and subsequently enhanced glioma cell migration/invasion. In other types of malignancies, BDKRB2 also exhibited the biological function of promoting cell migration, invasion, and metastasis in hepatocellular carcinoma [To elucidate the biological function of BDKRB2 in glioma, we further performed GO and GSEA analysis. GO analysis revealed that BDKRB2 was highly associated with extracellular matrix organization and collagen catabolic process in both pan-glioma and GBM, suggesting that glioma cells through their interactions with BDKRB2 might acquire functions that enhance matrix remodeling, cell migration, invasion, and tumor progression, consistent with the results presented by Montana et al. . They coarcinoma , 13, gasarcinoma , 16, colarcinoma , prostatarcinoma , head anarcinoma  and chonarcinoma . In addiarcinoma –41. ThesTo further validate the role of BDKRB2 in the glioma EMT process, we selected a series of EMT-related signaling pathways and biomarker, which were then analyzed to determine their interaction with BDKRB2. We found that BDKRB2 expression showed a robust correlation with PI3K/AKT, hypoxia, and TGF-β signaling pathway, suggesting that BDKRB2 might promote the EMT process through these pathways. Moreover, most EMT biomarkers, including N-cadherin, snail, slug, vimentin, TWIST1, and TWIST2, were significantly correlated with BDKRB2, which suggested that BDKRB2 might profoundly interact with these key molecules of EMT, further confirming the involvement of BDKRB2 in glioma EMT. Thus, our findings might bring a novel EMT target for potential glioma treatment.in-vitro and in-vivo studies are needed to validate its role in glioma.In conclusion, the present study demonstrated that BDKRB2 expression was associated with more malignant glioma phenotypes and predicted much worse survival for patients. Moreover, BDKRB2 was significantly associated with the EMT process. However, a limitation of the current study was that no experimental validation was performed. Further http://www.cgga.org.cn/) [http://cancergenome.nih.gov/) [Transcriptome and clinical data of glioma patients were available on Chinese Glioma Genome Atlas (CGGA) website (org.cn/) , 43 and ih.gov/) . A totalhttps://david.ncifcrf.gov/) enrichment analysis. The hallmarks.all.v7.1.symbols.gmt gene set was selected for Gene Set Enrichment Analysis . All 301 samples in CGGA and 697 in TCGA were included in pan-glioma GSEA analysis, and 128 GBM samples in CGGA and 167 in TCGA were included in GBM GSEA analysis. The number of permutations was 1000. The enrichment statistic was set as weighted, and the metric for ranking genes was set as Pearson. All statistical tests were two-sided, and a p-value of < 0.05 indicated a statistical significance.For TCGA cohort, RSEM RNAseq data were log2 transformed. For CGGA cohort, microarray data  were directly analyzed. Statistical analysis was performed with R language. Multiple R packages, including ggplot2, pROC, pheatmap, corrgram, circlize, and survival, were used to generate figures. Cox proportional hazard regression analyses were performed with coxph function of survival package. The biological processes of BDKRB2-related genes were annotated using Gene Ontology (GO) (DAVID, Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5


Coronavirus disease 2019 (COVID-19) may cause a cytokine storm, which may lead to respiratory failure or even death, and the mortality rate for critically ill cases reached 60.5% , all 21 As a kind of biological disease-modifying antirheumatic drug, TCZ may weaken the innate immunity in the body, which associates with high risk of serious infections , 8. In a