Two major causes of mortality in the world today are cancer and heart disease. Although these are distinct diseases, their pathomechanisms rely partially on common signaling pathways driven by kinases such as β-adrenoceptor/protein kinase A (PKA) and the mitogen-activated protein (MAP) kinase network. Furthermore, activation of these kinases can lead to both positive and deleterious effects on disease progression. Thus, the goal of therapeutic strategies should be to promote the normalization of these signaling pathways under pathological conditions. Raf kinase inhibitory protein (RKIP) represents a physiological mechanism for achieving this outcome. As a regulator of the cellular kinome, RKIP acts in its unphosphorylated form as a suppressor of metastatic cancer progression by decreasing MAPK signaling. Conversely, in its phosphorylated form, RKIP protects against heart failure by upregulating β-adrenoceptor/PKA signaling. Here we discuss how leveraging RKIP action by using selective targeting strategies has the potential for the cardio-safe treatment of cancer.Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the β-adrenergic receptor (βAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate βAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity. Cancer and heart disease are both progressive diseases that represent the most important unmet clinical needs in medicine today ,2. CanceIn this review, we focus on RKIP and its downstream target, the extracellular signal-regulated kinases 1 and 2 (ERK1/2), two key regulators of cancer and heart disease that can be leveraged as therapeutic targets to treat these diseases while avoiding toxic side effects. Notably, both RKIP and ERK exist in at least two discrete forms, each of which acts upon different targets. In the case of RKIP, the protein assumes two conformational states triggered by a phosphorylation switch that toggles RKIP between repression versus activation of the Raf/MAP kinase and βAR/PKA signaling cascades. For ERK1/2, a specific phosphorylation leads to alternative subcellular localization that results in transcriptional versus cytoplasmic signaling. Here we discuss how utilizing therapeutic strategies to constrain RKIP and ERK signaling states could lead to beneficial MAPK and PKA signaling while minimizing cardiotoxicity and cancer progression.Cellular responses to external and internal stimuli are largely mediated by protein kinase signaling cascades. The importance of the kinome to the integrity of the cellular structure and function necessitates that it is maintained under exquisite regulatory control. One of the proteins that responds to changes in the cellular kinome and resets the relative activity levels of key kinase signaling cascades is RKIP , a member of the highly conserved PEBP family. RKIP regulates both MAP kinases  and βAR/cAMP-dependent protein kinase A (PKA) signaling pathways ,7,8. TheRKIP is a dual function protein whose role depends upon its phosphorylation state. Unphosphorylated RKIP binds to Raf-1 and inhibits its activation. Following the phosphorylation of RKIP at S153 by protein kinase C (PKC), RKIP switches its binding partner from Raf-1 to G protein-coupled receptor kinase 2 (GRK2) ,17,18,19RKIP, which is often absent or downregulated in cancer, is a tumor metastasis suppressor for a variety of cancers including prostate, breast, pancreatic, lung, cervical cancers, and gliomas (reviewed in ). DecreaExperimental studies conducted in a variety of tumor types, including breast and prostate, have demonstrated that exogenous RKIP overexpression in tumor cells blocks metastatic progression without significantly altering primary tumor growth. On a molecular level, RKIP functions by rewiring kinase networks to reprogram tumor cells to a nonmetastatic state . In partAlthough metastatic solid tumors commonly lack normal RKIP expression, the mechanisms by which cancer cells deactivate or eliminate RKIP prior to invasion are not well understood . The genAlthough not as common as the loss of RKIP, significant pS153 RKIP expression has been observed in some tumors. For example, in multiple myeloma where RKIP is often overexpressed, the phosphorylated S153 form is found in about half of the patient cancers . PKA, whThe net consequence is that RKIP can alter tumor cells in three discrete ways: suppressing ERK without activating PKA signaling in the wildtype state; activating ERK and PKA signaling when phosphorylated at S153 by PKC; enabling ERK activation in the absence of robust PKA signaling when depleted or deleted.S153A, can cause RKIP-mediated ERK inhibition in the heart. Thus, the primary function of RKIP in the heart is different from the one in epithelial or cancer cells, and the extent of ERK1/2 inhibition or PKA activation is a function of both cell-type and environmental factors.ERK and PKA signaling are also important signaling pathways in cardiac cells, and RKIP has been shown to be a major coordinator between these signaling cascades in the heart. RKIP exists mainly in its phosphorylated form in the heart and thus inhibits GRK2, which in turn leads to the activation of the most prevalent G protein-coupled receptor (GPCR) in the heart, the βAR. RKIP-mediated βAR activation subsequently leads to increased PKA signaling . As the Phosphorylated RKIP in the heart controls cardiac βAR and PKA signaling that is essential in acute situations. βAR/PKA signaling is responsible for the acceleration of cardiac contraction and relaxation that enables adaptation to hemodynamic stress situations. A pathological stress situation such as heart failure also leads to the activation of βAR and thereby stabilizes the hemodynamic situation of the patient, at least initially. However, a chronic increase in βAR and PKA signaling is detrimental ,28,29. C1AR and β2AR, in a specific manner. RKIP increases cardiac inotropy via β1AR and protects from arrhythmia and remodeling via β2AR. This cardioprotective effect of RKIP on cardiac remodeling, arrhythmia, as well as on survival was shown using mice with cardiac overexpression of RKIP (αMHC-RKIP-tg), RKIP knockout mice (RKIP-KO), and mice treated with an AAV9-RKIP gene therapy in a heart failure model of chronic left ventricular pressure overload [1- or β2AR, respectively [What is the consequence of RKIP expression in this context? Interestingly, cardiac RKIP overexpression in transgenic mice (RKIP-tg) leads to a persistent activation of βAR and PKA, as shown by the increased phosphorylation of targets downstream of βAR and PKA. Consequently, RKIP expression results in the increased speed of cardiac contraction and relaxation. However, in contrast to catecholamines, that are agonists of the βAR, the RKIP-mediated hypercontractility is well-tolerated, at least up to an age of 12–14 months, and RKIP-tg mice are protected from βAR-induced arrhythmia and cardiac remodeling such as interstitial fibrosis and apoptosis ,28,32. Ooverload . The regectively . In sum,These data suggest that RKIP acts as a coordinator of beneficial cell signaling not only in cancer but also in the heart. However, it is not yet known how the cellular environment contributes to the cell-type specific function of RKIP and its overall beneficial signaling outcome. The RKIP switch from inhibitor to activator of PKA may be more pronounced in cardiomyocytes and may even be triggered by RKIP itself, since RKIP coordinates and is coordinated by protein kinase signaling.Protein phosphorylation is an abundant post-translational modification that controls numerous cellular processes. In particular, phosphorylation has been implicated in the formation and dissociation of protein complexes that control protein function and mediate signal propagation within cells. A recent study of RKIP revealed a novel, evolutionarily conserved mechanism for switching protein partners, and thereby protein function, through a phosphorylation-dependent process . As noteHow does S153 phosphorylation or the substitution of two negatively charged residues at these two nearby sites lead to an exchange of RKIP partners and an alteration in RKIP function? The mechanism involves a novel phosphorylation-induced salt bridge theft that utilizes serine or threonine residues located in regions outside the binding interface and thus readily exposed to solvent. Specifically, in its wild type state, the lysine located at residue 157 forms a salt bridge with two negatively charged residues on a neighboring polypeptide chain, D134 and E135 . When RKThere are several advantages to the mechanism by which the phospho-switch in RKIP is triggered. The classic phosphorylation association model only enables facile phosphorylation to promote protein–protein association, as residues that are involved in binding interactions would not be readily accessible to kinases. By contrast, the salt bridge theft model leaves serine and threonine residues that will undergo phosphorylation readily accessible to kinases independent of whether the protein interfaces are free or bound together. Thus, it is more likely that the phosphorylation-induced disruption of the protein interface interactions would occur by the salt bridge theft model rather than by the classic mechanism where the interface is directly phosphorylated. In addition, because there is a high frequency (two-to-three-fold enriched) of salt bridges at the interfaces of hetero-oligomeric complexes, the likelihood that a serine or threonine residue is near a charged residue will be higher at the interface, thus facilitating changes within the interface domain. The salt bridge exchange induced by nearby phosphorylation can lead to local conformational changes within a protein or can serve as a mechanism to establish or uniquely disrupt salt bridge interactions between different proteins. For RKIP specifically, this mechanism causes the disruption of the interacting polypeptide chains within the protein, enabling local conformational changes that facilitate new protein binding interactions. Ultimately this leads to the facile swapping of protein partners following PKC phosphorylation of RKIP.Notably, the presence of S153 as a site of phosphorylation is a relatively new development in the highly conserved PEBP family, but the phospho-theft mechanism probably occurred early in the evolution . A compuEnvironmental signals utilize receptors, generally transmembrane proteins, to activate intracellular signaling cascades and subsequently initiate physiological responses. A central signaling cascade, in which many of those extracellular signals converge, is the MAP kinase cascade, consisting of the kinases Raf, MEK1/2, and ERK1/2. ERK1/2 are activated by various extracellular triggers such as GPCRs, integrins, and receptor tyrosine kinases, and are responsible for the induction of cellular responses such as proliferation, differentiation, and cell survival. The cascade is involved in the development and progression of many diseases including cancer, heart failure, developmental diseases, and autoimmune diseases, but are also vital for many physiological effects such as protection from cell death ,39,40,41T188) has been detected in cancer that does not alter overall ERK1/2 activity but has been shown to promote nuclear ERK signaling and cancer cell proliferation [T188 in cardiomyocytes is discussed in more detail below . The caferation . Interesferation ,27,42. Til below . pERKT18gression .T188 as a potential target to suppress pathological ERK1/2 signaling in the heart.As noted above, the role of the Raf/MEK/ERK1/2 cascade in the heart is a double-edged sword. ERK1/2 signaling is involved in adverse remodeling but is also responsible for cardiomyocyte survival and protection from stress-induced cardiomyocyte death ,35,51,52Post-translational modifications and, in particular, protein phosphorylations are crucial for controlling enzymatic activity, protein conformation, and subsequent protein–protein interactions, as well as the cellular localization of certain proteins. Classically, extracellular signals are transmitted into the cell via receptor tyrosine kinases, integrins, or GPCR, and trigger the recruitment of adaptor proteins such as β-arrestin and the activation of Ras, which in turn facilitate the stepwise activation of the Raf/MEK1/2/ERK1/2 signaling cascade. The phosphorylation of the effector kinases ERK1 and ERK2 at the Thr and Tyr residues within the Thr-X-Tyr motif of their activation loop (Thr183 and Thr185 in mouse ERK2) leads to their activation and the subsequent phosphorylation of a vast array of substrates localized in all cellular compartments. These substrates include protein kinases, cell signaling, receptors, cytoskeletal proteins, and nuclear transcriptional regulators ,57,58. ST188; T208 in ERK1) has been described that modulates nucleocytosolic ERK1/2 signaling. pERKT188 is induced by the activation of GPCR and ERK1/2 that, in turn, can result in the homo- or heterodimerization of ERK1/2. Gβγ subunits released from Gs or Gq proteins can then bind to the ERK dimer and induce the intermolecular autophosphorylation of ERK at threonine 188 [T188, and that this phosphorylation site does not alter ERK activity but instead triggers the nuclear translocation of ERK. Subsequently, pERKT188 has been shown to trigger the activation of nuclear ERK targets such as Elk1, MSK, and c-myc, targets that not only promote cancer but are known to induce cardiomyocyte hypertrophy [T188 is increased in human heart failure and hypertrophic mouse hearts [T188 is only upregulated and involved in the development of pathological, but not in physiological, cardiac hypertrophy, and that it correlates with the speed of disease progression in aortic stenosis patients that suffer cardiac hypertrophy [As mentioned above, an autophosphorylation of ERK1/2 at threonine 188 , although potentially effective against cancer, also leads to the apoptosis of cardiomyocytes, cells with very limited regeneration capacity. In cancer therapy for example, increased Raf/MEK/ERK1/2 signaling is targeted by several small molecule inhibitors of the Raf, MEK or ERK, or upstream kinases or receptors, such as the epidermal growth factor receptor. While the survival of patients with certain cancer types can be extended by these inhibitors, such as in patients with advanced BRAFV600-mutated melanoma [As summarized above, ERK1/2 activation promotes tumor progression and metastasis. However, robust ERK1/2 inhibition by small molecule inhibitors or RKIP . One paT188 by a peptide that inhibits ERK dimerization (EDI: “ERK dimerization inhibitor”). EDI does not interfere with ERK1/2 activation and antiapoptotic ERK1/2 signaling in cardiomyocytes, but instead inhibits the induction of pERKT188 and nuclear translocation of ERK [T188 also occurs in cancer. In line with this observation, EDI was also shown to effectively inhibit pERKT188 in cancer cells and to prevent colon cancer cell proliferation in culture [In addition, a strategy was developed that allows the selective targeting of pERKn of ERK . As a co culture . Moreove culture .Like RKIP, EDI represents a potential cardio-safe intervention strategy for heart disease. In the heart, EDI has the potential to direct βAR/ERK1/2 signaling towards improving cardiac function but does not inhibit cytosolic antiapoptotic ERK signaling that acts as an additional protective shield from cardiac adverse effects such as cardiomyocyte hypertrophy and apoptosis. In cancer, RKIP inhibits Raf/MEK/ERK1/2 signaling, thereby preventing cancer metastasis, and this can be mimicked by a low-dose multidrug combination targeting the stress MAP network. EDI could function as an additional shield to prevent increased ERK1/2 signaling from promoting cancer cell proliferation. Thus, the combination of two novel intervention strategies reveals a strong “positive” interaction and may be of interest for those patients with diseases related to heart and/or cancer .A tight and well-balanced control of signaling cascades, such as the Raf/MEK/ERK1/2 cascade, is essential for the preservation of physiologically important cellular functions. Therefore, the normalization of the cascade’s activity under pathological conditions seems to be the perfect scenario. As noted above, RKIP is often missing in metastatic tumors, leading to excessive stress MAPK activation, but is also present in its phosphorylated form in the heart, enabling localized PKA activation. Here, we presented two different methods of PKA and ERK modulation that have the potential to suppress cancer progression and metastasis-related lethality while being cardio-safe or even protective against heart disease: (i) Treatment with partial RKIP “mimics”, such as low-dose drug combinations that target the stress MAPK network, could potentially suppress metastasis in tumors while enabling beneficial Raf/MEK/ERK1/2 and βAR/PKA signaling in cardiac cells. This could result in the prevention of cancer lethality and, at the same time, allow pRKIP in the heart to induce a well-tolerated positive inotropy and protect from the development of heart failure in mice; (ii) As EDI preserves cytosolic antiapoptotic and cardio-safe ERK1/2 signaling while suppressing nuclear ERK1/2 signaling, EDI treatment could result in protection from pathological cardiac remodeling and heart failure and the inhibition of cancer cell proliferation.The combination of RKIP expression in the heart with kinase inhibitors such as low-dose drug combinations or EDI would allow efficient cytosolic, antiapoptotic, and cardio-safe ERK1/2 signaling while suppressing cancer progression and protecting from pathological cardiac remodeling and heart failure. These studies suggest that there are ways to generate drugs that are effective in both cancer and heart disease, or at least which do not cause cardiotoxic side effects. Thus, sophisticated targeting of the PKA and MAPK signaling cascades may pave the way to new multi-hit drug strategies for heart disease and cancer.


Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are important zoonotic protists in humans and animals around the world, including nonhuman primates (NHPs). However, the prevalence, genetic identity and zoonotic potential of these pathogens in wild NHPs remain largely unclear.A total of 348 fecal samples were collected from wild NHPs at four locations in Yunnan, southwestern China, and analyzed for these pathogens using nested PCR targeting various genetic loci and DNA sequence analysis of the PCR products. The zoonotic potential of the pathogens was assessed by comparing the genetic identity of the pathogens in these animals with that previously reported in humans.Cryptosporidium sp., G. duodenalis and E. bieneusi, respectively. The Cryptosporidium sp. identified belonged to C. parvum subtype IIdA20G1. Both assemblages A (n = 3) and B (n = 22) were identified among G. duodenalis-positive animals. Five genotypes in zoonotic Group 1 were identified within E. bieneusi, including Type IV (n = 13), D (n = 7), Peru8 (n = 6), MMR86 (n = 2) and HNFS01 (n = 2). All genotypes and subtypes identified are known human pathogens or phylogenetically related to them.Altogether, two (0.6%), 25 (7.2%) and 30 (8.6%) samples were positive for G. duodenalis and E. bieneusi in wild NHPs in southwestern China.Data from this study suggest a common occurrence of zoonotic genotypes of  Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi, respectively, that have been reported in humans as well as domestic and wild animals. Humans can be infected with these pathogens through the ingestion of contaminated food or water, causing diarrhea in immunocompetent individuals and significant mortality in immunocompromised ones [Cryptosporidium species and 100 genotypes have been recognized, including Cryptosporidium parvum, which has a broad host range [G. duodenalis [E. bieneusi, the nearly 500 genotypes identified thus far have been classified into 11 major groups, with Group 1 genotypes having a broad host range [Cryptosporidiosis, giardiasis and microsporidiosis are three emerging zoonotic diseases caused by sed ones –3. To dast range . Similarodenalis . Among todenalis . For E. st range .Cryptosporidium spp., G. duodenalis and E. bieneusi, with the most prevalent genotypes of the pathogens also detected in humans [Nonhuman primates (NHPs), due to their high genetic homology to humans, are considered potential sources of zoonotic parasites in humans . Data frn humans . Comparen humans .Rhinopithecus bieti) are in frequent contact with local Tibetan residents [Macaca mulatta) are in close contact with monks living in temples and with pilgrims and tourists visiting the mountains [Yunnan Province in China is home to many wild NHPs . The higesidents . In the ountains . In Xishountains .E. bieneusi [Cryptosporidium spp., G. duodenalis and E. bieneusi in NHPs have also been conducted in China with captive animals [Cryptosporidium spp., G. duodenalis and E. bieneusi in these animals.In a small-scale survey of animals, captive NHPs from zoos and research laboratories in Yunnan Province were shown to be infected with several genotypes of bieneusi . A few s animals , 15–21. M. mulatta, n = 320), snub-nosed monkeys , and Assamese macaques  at four tourist attractions in Yunnan Province, China were collected from June 2019 to January 2021 . DNA was extracted from the pellet using a FastDNA Spin Kit for Soil  as previously described [Before genomic DNA extraction, approximately 300 mg of fecal material was washed with distilled water by centrifugation  rRNA gene [C. parvum detected was further subtyped by sequence analysis of the 60-kDa glycoprotein (gp60) gene [G. duodenalis was detected and genotyped by PCR and sequence analyses of the β-giardin (bg), triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes [Enterocytozoon bieneusi in the extracted DNA was detected and genotyped by PCR and sequence analyses of a rRNA fragment containing the entire internal transcribed spacer (ITS) [Cryptosporidium tyzzeri from mice, assemblage G from mice and genotype PtEb IX from dogs were used as positive controls in the PCR analysis for Cryptosporidium spp., G. duodenalis and E. bieneusi, respectively, whereas reagent-grade water was used as the negative control.RNA gene . The C. 60) gene . In conth) genes –27. Enteer (ITS) . Each sahttp://technelysium.com.au/ChromasPro.html), edited using BioEdit 7.1 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and aligned with reference sequences from GenBank using ClustalX 2.1 . Genotypes and subtypes of the pathogens were named according to the established nomenclature [E. bieneusi using MEGA 7.0.14 (http://www.megasoftware.net/) with substitution rates calculated with the general time-reversible model. The reliability of the tree was assessed using bootstrap analysis with 1000 replicates.All positive secondary PCR products were sequenced by Sangon Biotech  bidirectionally on an ABI 3730 Genetic Analyzer . The raw sequences obtained were assembled using ChromasPro 1.33  could not be generated.Of the 348 fecal samples analyzed, only two (0.6%) samples from rhesus macaques in Xishuangbanna Primeval Forest Park were positive for um Table . SequencG. duodenalis, with infection rates ranging from 2.8% (4/141) to 15.5% (11/71) among the four sampling locations . DNA sequence analysis of the bg, tpi and gdh PCR products revealed the presence of assemblages A (n = 3) and B (n = 22) in those monkeys. By animal species, only M. mulatta and R. bieti samples were positive for G. duodenalis  of the 348 fecal samples were positive for E. bieneusi in the PCR analysis of the ITS, with infection rates ranging from 1.7% (2/116) to 17.0% (24/141) among the four locations  and Shibao Mountains . Five E. bieneusi genotypes were detected in these animals, including Type IV (n = 13), D (n = 7), Peru8 (n = 6), MMR86 (n = 2) and HNFS01 (n = 2). The sequences of Type IV, D, Peru8, MMR86 and HNFS01 were identical to GenBank sequences JX683801(from humans), MK982516 (from giraffes), KF305584 (from rhesus macaques), MN399818 (from humans) and MK947105 (from flying squirrels), respectively. In the phylogenetic analysis, all were placed in zoonotic Group 1  were positive for rom 1.7% /116 to 1rom 1.7% /116 to 1M. mulatta and 37.5% (3/8) M. assamensis sampled were positive for E. bieneusi. Among these, all five subtypes  were detected in M. mulatta and two subtypes (D and Peru8) were found in M. assamensis  7/320 M. M. mulatta at Xishuangbanna Primeval Forest Park, one C. parvum-positive sample had concurrence of E. bieneusi genotype D, two samples (one with Peru8 and another with Type IV) had concurrence of assemblage B of G. duodenalis and two samples had concurrence of two E. bieneusi genotypes based on inconsistent sequencing results of the two PCR replicates (one with both Type IV and Peru8 and another with both Peru8 and D).Among five samples from Cryptosporidium sp., G. duodenalis and E. bieneusi in wild NHPs in Yunnan Province, China. These pathogens were identified in 0.6, 7.2 and 8.6% of fecal samples, respectively. The occurrence rates of these pathogens are lower than those found in captive, laboratory and zoo NHPs in previous investigations, which reported infection rates ranging from 9.1% to 46.7% for Cryptosporidium spp. [G. duodenalis [E. bieneusi [This is the first report of the occurrence of ium spp. , 31, 8.5odenalis , 32–34 abieneusi , 35–38. C. parvum is the only Cryptosporidium identified in the present study. Thus far, three Cryptosporidium species, C. hominis, C. parvum and C. muris, have been identified as common ones in NHPs in China [C. parvum in China [The zoonotic IIdA20G1 subtype of in China , 39. Unlin China , 36 and,in China –43. TherG. duodenalis in M. mulatta and R. bieti in this study [G. duodenalis.Similar to other studies in NHPs, the zoonotic assemblage B was identified as the dominant genotype of is study , 44, 45.is study . Thus, wE. bieneusi genotypes detected in NHPs in Yunnan Province all belong to Group 1, which contains most of the zoonotic genotypes of major public health concern [E. bieneusi in captive and farmed NHPs also detected mostly Group 1 genotypes [The five  concern . Among t concern , 46–48.  concern , while Henotypes , 38, 50.G. duodenalis and E. bieneusi genotypes in wild NHPs, which in turn represents a significant public health concern as tourists are coming into increasing contact with NHPs. In response, public education programs on the infective potential of pathogens from NHPs and the utility of hygiene in disease prevention should be implemented to inform park personnel and tourists as part of the One Health approach to the prevention and control of Cryptosporidium spp., G. duodenalis and E. bieneusi infections.In summary, the results of the present study show a common occurrence of zoonotic


Adenomyosis, endometriosis, endometritis, and typical endometrial hyperplasia are common non-cancerous diseases of the endometrium that afflict many women with life-impacting consequences. The mammalian target of the rapamycin (mTOR) pathway interacts with estrogen signaling and is known to be dysregulated in endometrial cancer. Based on this knowledge, we attempt to investigate the role of mTOR signaling in benign endometrial diseases while focusing on how the interplay between mTOR and eukaryotic translation initiation factors (eIFs) affects their development. In fact, mTOR overactivity is apparent in adenomyosis, endometriosis, and typical endometrial hyperplasia, where it promotes endometrial cell proliferation and invasiveness. Recent data show aberrant expression of various components of the mTOR pathway in both eutopic and ectopic endometrium of patients with adenomyosis or endometriosis and in hyperplastic endometrium as well. Moreover, studies on endometritis show that derangement of mTOR signaling is linked to the establishment of endometrial dysfunction caused by chronic inflammation. This review shows that inhibition of the mTOR pathway has a promising therapeutic effect in benign endometrial conditions, concluding that mTOR signaling dysregulation plays a critical part in their pathogenesis. Adenomyosis, endometriosis, endometritis, and typical endometrial hyperplasia are all non-cancerous conditions that originate from the endometrium and affect a substantial part of the female population. They are often associated with endocrine derangement and develop upon high levels of estrogen or estrogen dominance (estrogen unopposed by progesterone) ,2,3. AltThe molecular mechanisms that underlie the pathogenesis of benign endometrial diseases have not yet been fully elucidated. Current knowledge about their biological background brings the role of the mammalian target of the rapamycin (mTOR) signaling pathway to the fore. In fact, aside from its well-known importance in malignant endometrial disorders ,12,13, mThe mammalian target of rapamycin (mTOR) is a human serine/threonine protein kinase with multiple biological functions that determine cell growth, metabolism, proliferation, and survival . As implmTORC1 is the most well-characterized mTOR complex and the one that is sensitive to rapamycin treatment ,17. Its After being activated, mTORC1 coordinates protein synthesis through p70S6 Kinase 1 (S6K1) and eIF4E Binding Protein 1 (4EBP1) phosphorylation. Phosphorylated 4EBP1 then dissociates from the eukaryotic translation initiation factor 4E (eIF4E), allowing the latter to participate in the formation of the eIF4F complex and thus in the translation initiation . On the Apart from eIF4E and eIF4B, mTORC1 is involved in translation initiation through other eukaryotic translation initiation factors (eIFs), such as eIF2a and eIF3e. In addition to the canonical pathway previously described, mTORC1 can also regulate autophagy in a non-canonical manner by interacting with eIF2a signaling. More precisely, inhibition of mTORC1 activates the phosphatase PP6C , which iIn contrast to mTORC1, the function and regulation of mTORC2 have only been minimally characterized. mTORC2’s most important function is AKT phosphorylation, while it can also activate several members of the AGC family, and thereby control the organization of the cytoskeleton and cell migration ,30. UnliThe PI3K/AKT/mTOR pathway is involved in estrogen signal transduction, thus contributing to the adverse effects of abnormal estrogen levels on endometrial homeostasis ,33. In fEstrogen signaling plays a critical role in the pathogenesis of endometrial diseases, as it enhances the potential for proliferation, invasion, and migration of endometrial cells. ,35. ThesThe role of mTOR signaling in endometrial carcinogenesis has been extensively investigated in recent decades. PI3K/AKT/mTOR pathway derangement occurs in 80–95% of Type I or endometrioid endometrial carcinomas , with PTAdenomyosis refers to the uterine disease in which endometrial tissue invades the myometrium. It is considered an estrogen-dependent condition, as it is characterized by high estrogen levels that contribute to its pathogenesis ,32,49. EMultiple studies ,55,56,57Besides upstream effectors, downstream regulators of mTOR signaling seem to play a significant role in adenomyosis as well. Vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) are highly expressed in the ectopic endometrium of patients with adenomyosis, where they promote angiogenesis, thereby contributing to the progression of the disease . Both VEmTOR signaling may also be involved in the pathogenesis of adenomyosis by promoting epithelial to mesenchymal transition (EMT) of endometrial epithelial cells. EMT is thought to play a crucial role in the establishment of the disease, as it enhances the invasiveness of endometrial cells, thus contributing to their translocation into the myometrium ,68. MoreIt follows that targeting the PI3K/AKT/mTOR pathway may be effective for adenomyosis treatment. Interestingly, Xue et al. observed that metformin could restrain stromal cell proliferation of adenomyotic lesions by downregulating the PI3K/AKT pathway through AMPK activation .Aberrant mTOR pathway activation is also reflected in the abnormal expression of mTOR-associated eIFs in adenomyosis. Gene expression and pathway analysis applied on eutopic endometrium from adenomyosis patients revealed significant dysregulation of eIF2 as well as eIF4 signaling in comparison to normal endometrium . SpecifiEndometriosis is an estrogen-dependent disorder in which endometrial tissue develops outside the uterine cavity, most commonly in the pelvic peritoneum, ovaries, and the pouch of Douglas . The assIn recent years, the investigation of mTOR signaling in endometriosis is still evolving. Madanes et al. noted elevated PI3K expression and AKT phosphorylation as well as reduced PTEN levels in both ectopic and eutopic endometrium of endometriosis patients compared to normal endometrium. Interestingly, these findings were observed mainly in women with a minimal-mild stage of the disease, suggesting that the PI3K/AKT/mTOR pathway plays a critical role in the onset of endometriosis . SimilarAccording to other studies ,81, ectoThe frequent activation of the PI3K/AKT/mTOR pathway in endometriosis makes it an attractive therapeutic target in this disease. Therefore, scientists have attempted to evaluate the effects of inhibiting this pathway in restraining endometriosis. Leconte et al. noted a significant and dose-dependent decrease in the proliferation rate of deep infiltrating endometriotic stromal cells in both cell culture and mouse model experiments after treatment with the mTOR-inhibitor temsirolimus . In anotOther reports have emphasized the role of mTOR signaling in endometriosis-associated carcinogenesis . Cancer mTOR pathway-related eIFs exhibit differential expression between normal and ectopic endometrium. Specifically, Cai et al. observed that eIF3e is underexpressed in ovarian endometriosis tissue, thus promoting EMT through the preferential translation of Snail, as they noted a negative correlation between eIF3 and TGF-β1, Snail, vimentin, and PCNA levels . In addiEndometritis can be classified as acute or chronic, depending on the predominance of a neutrophilic or a lymphoplasmacytic infiltrate, respectively. Chronic endometritis (CE) often leads to endometrial dysfunction and therefore increases the risk of infertility and embryo implantation failure. Wang et al. demonstrated through a case-control study including women with CE and recurrent implantation failure (RIF) that mTORC1 is significantly under-expressed in inflamed endometrium . It has Endometrial hyperplasia (EH) is an irregular proliferation of the endometrium that comprises alterations in the glandular architecture and endometrial gland-to-stroma ratio. According to the simplified World Health Organization (WHO) classification, EH can be categorized as hyperplasia with or without atypia, depending on whether cytologic atypia is present or not, respectively . For theEH arises from endometrial exposure to excessive levels of estrogen that are not opposed by progesterone, as can be seen in various conditions such as post-menopause, obesity, and polycystic ovary syndrome (PCOS) ,101. ConBajwa et al. indicated the importance of mTOR signaling in advanced age-related EH. They observed an increased pS6 expression in hyperplastic endometrium collected from post-menopausal women compared to normal post-menopausal endometrium and noted a similar expression pattern of pS6 between hyperplastic and normal uterine tissue collected from aged mice . InteresmTOR signaling derangement also seems to have a pivotal role in the development of EH induced by PCOS. PCOS is a disorder characterized by chronic anovulation and aberrant androgen biosynthesis, conditions that both increase unopposed steroid levels and can therefore promote EH . The mTOGlucose metabolism in the uterus is of essential importance for normal endometrial function. Hyperinsulinemia and insulin resistance are major disorders involved in the pathogenesis of PCOS and are believed to contribute to the development of EH in women with this condition ,108. InsIt has been well established that metformin restrains EH via mTOR pathway modulation , as it aApart from exposure to unopposed estrogen, EH can also result from treatment with tamoxifen, a selective estrogen receptor modulator (SERM) used for breast cancer therapy. Upregulation of mTOR signaling might be part of the mechanism by which tamoxifen induces abnormal endometrial growth . SequestIt is noteworthy that eIF2α signaling, which is in close interaction with the mTOR pathway, seems to be involved in the pathogenesis of PCOS-induced endometrial hyperplasia. In fact, endoplasmic reticulum stress (ER) and consequent eIF2α pathway activation directly contribute to the pathogenesis of PCOS by affecting the total health of ovarian follicles and oocytes in several ways, such as induction of interstitial fibrosis, follicular atresia, and accumulation of advanced glycation end products (AGEs) in ovarian granulosa cells ,122. FurThe importance of the PI3K/AKT/mTOR signaling cascade in endometrial carcinogenesis has been well established and research has proceeded on evaluating the efficacy of inhibiting this pathway for the treatment of endometrial malignancies. Based on this knowledge, we aimed to investigate the current data about the involvement of the mTOR pathway in the development of benign endometrial conditions. From this review, it follows that mTOR and eIF signaling are deranged in adenomyosis, endometriosis, endometritis, and typical endometrial hyperplasia and may serve as druggable targets in these life-impacting diseases. However, further studies are still needed to determine the full spectrum of mTOR function in non-cancerous endometrial conditions.


Drosophila, global JNK upregulation can delay aging and extend lifespan, whereas tissue/organ-specific manipulation of JNK signaling impacts lifespan in a context-dependent manner. In this review, focusing on several tissues/organs that are highly associated with age-related diseases—including metabolic organs (intestine and fat body), neurons, and muscles—we summarize the distinct effects of tissue/organ-specific JNK signaling on aging and lifespan. We also highlight recent progress in elucidating the molecular mechanisms underlying the tissue-specific effects of JNK activity. Together, these studies highlight an important and comprehensive role for JNK signaling in the regulation of longevity in Drosophila.The evolutionarily conserved c-Jun N-terminal kinase (JNK) signaling pathway is a critical genetic determinant in the control of longevity. In response to extrinsic and intrinsic stresses, JNK signaling is activated to protect cells from stress damage and promote survival. In  Aging is characterized by the progressive decline of tissue/organ functions as well as cellular stress tolerance, which ultimately results in the death of an organism. Lifespan is therefore a robust measure of aging rate . GeneticDrosophila melanogaster has been used as a model organism to study aging and longevity for more than 100 years , and thor to advance stress defense [Jafrac1 is able to suppress Bsk activity in neurons, indicating that Jafrac1 may also act as a negative feedback regulator of JNK in specific tissues [Stress-stimulated JNK signaling is initially transduced by the JNK kinase kinases (JNKKKs), such as transforming growth factor beta activated kinase 1 (TAK1), apoptosis signal-regulating kinase 1 (ASK1), Slipper (Slpr), and MAPK kinase kinase 1 (Mekk1) ,12,13,14o stress . Alterna defense ,18. It h tissues .puc loss-of-function alleles display a significantly longer lifespan than wild-type flies, and this phenotype is reduced in hemizygotes of 1hep, a hypomorphic allele of hep [puc mutants can be reverted to wild-type levels in the background of foxo loss-of-function allele heterozygotes [l(2)efl [hsp68 [In flies, the role of JNK signaling in the regulation of longevity was initially identified through organism-wide manipulation of JNK activity. Flies with mild activation of JNK signaling due to heterozygosity of e of hep , indicatozygotes . Consist[l(2)efl  or hsp68l [hsp68 , suggestDrosophila midgut, which is functionally equivalent to the mammalian intestine, has been established as a model system to study homeostasis and aging because of its simple structure and well-defined cell lineages [The intestine is the main organ to absorb nutrients from digested food to support body metabolism. It is also an important homeostatic organ acting as the first protective barrier of defense against ingested toxins and pathogens. The adult lineages . The intlineages . Throughlineages ,22,23. Tlineages ,25. Defelineages .1hep mutants or in flies with reduced JNK activity specifically in ISCs and EB cells, the morphological and structural abnormalities of the old gut are significantly reduced [As a stress-responsive signal, JNK signaling activity is required for stress protection in intestinal cells, which induces ISC proliferation and EC apoptosis to promote intestinal epithelial turnover and thus protect adult flies from stress damage ,27,28,29 reduced ,32,33. TDrosophila intestinal tumor model caused by the loss of bone morphogenetic protein (BMP) signaling, aberrant activation of JNK signaling was observed in tumors and linked to intestinal barrier dysfunction [c-jun reduces the tumor number and size and prolongs lifespan [Age-associated ISC overproliferation and misdifferentiation increase the risk of tumorigenesis in the gut, which dramatically shortens the lifespan of flies. JNK signaling plays both anti- and pro-tumorigenic roles during tumor progression (recently reviewed in ). It inhfunction . Inhibitlifespan .Drosophila. In ISCs, increased JNK signaling activity in the aging gut promotes symmetric ISC division by regulating spindle orientation [DNbsk) can prevent age-induced planar spindle orientations in dividing ISCs. Conversely, excessive activation of JNK in ISCs by overexpressing hep or by knocking down puc is sufficient to induce planar spindle orientation. JNK signaling biases planar spindle orientation and symmetric division of ISCs through collaboration with WD40-repeat protein (Wdr62) [kif1a, a kinesin reported to inhibit symmetric divisions in brain neural stem cells [kif1a prevents JNK-induced planar spindle orientation but cannot affect spindle orientation in conditions without JNK activation [kif1a serves as a downstream step of JNK activation in the regulation of spindle orientation and ISC division. Consistent with the finding that moderate repression of JNK activity in intestinal progenitor cells extends lifespan [The molecular mechanisms underlying the effects of JNK signaling in the aging gut have been found to differ among intestinal cell lineages in entation . Planar  (Wdr62) , a spind (Wdr62) ,38. In a (Wdr62) , where iem cells . The ovetivation , indicatlifespan , suppreslifespan .Ecdysone-induced protein 93F (Eip93F) [upd3 and the growth factor gene pvf1, which promote ISC compensatory proliferation through the JAK/STAT pathway or PDGF/VEGF receptor (Pvr) signaling, respectively. As with reduced JNK signaling in the gut, loss of ets21c prolongs lifespan but renders flies sensitive to stress. Overall, JNK signaling employs distinct molecular mechanisms to modulate ISC proliferation and EC apoptosis, and the age-related upregulation of JNK signaling progressively disturbs the regenerative process and the integrity of the intestinal epithelium through these mechanisms.Unlike ISCs, ECs respond to increased JNK activation by initiating apoptosis. The transcription factor Ets21c was identified as a downstream effector of JNK function in ECs . Ets21c,(Eip93F) , which e(Eip93F) ,45. The caliban (clbn), a gene that encodes a mitochondria outer membrane protein, induced mitochondrial damage as well as typical hallmarks of aging in the intestinal epithelium: EC damage, ISC overproliferation, disruption in the intestinal barrier, and shortened lifespan. In addition, clbn mutants exhibited elevated ROS levels and elevated JNK signaling in the gut. Blocking JNK activity in clbn mutant ECs largely rescued the intestinal epithelial abnormalities and longevity phenotypes but not the defective mitochondrial morphology, indicating that mitochondrial dysfunction and ROS overproduction act upstream of JNK-induced gut defects.Aging-related upregulation of JNK signaling in the gut may be attributable to rising concentrations of reactive oxygen species (ROS). The presence of ROS induces JNK signaling in many tissues ,47,48. MDrosophila lifespan. Here, we summarize evidence that neuronal JNK exerts its effects through multiple intersecting mechanisms: regulation of neuronal proteostasis, antagonism of insulin signaling, and protection from oxidative stress.Aging is considered a primary risk factor for most neurodegenerative diseases , includipuc [puc mutants exhibit elevated JNK activity and live significantly longer than wild-type controls. Further supporting a positive role of JNK signaling for proteostasis and longevity, pan-neuronal overexpression of the JNK/FOXO target gene l(2)efl, encoding a member of the heat-shock protein family that assists in protein folding and prevents accumulation of misfolded proteins, is sufficient to extend lifespan.Optimal proteostasis is important for neuronal functions. Dysfunction in proteostasis frequently underlies neurodegenerative disease pathologies . In thespuc , a downsatg) gene result in the accumulation of protein aggregates in neurons and a shortened lifespan [atg gene in the brain is sufficient to extend the average adult lifespan [atg genes [Autophagy, a conserved lysosomal degradation pathway for damaged protein, lipids, and organelles , plays alifespan , whereaslifespan . JNK sigtg genes  and promtg genes ,60, highDrosophila genome contains seven dilp genes encoding ILPs, each expressed in a stage- and tissue-specific pattern, such as Dilp2 in IPCs and Dilp6 in fat bodies. JNK signaling is active in these IPCs, activating FOXO and restricting the expression of ILP genes to systemically antagonize IIS in peripheral tissues [The association between neuronal JNK and longevity has been well elucidated in a small cluster of neuroendocrine cells in the brain, known as insulin-producing cells (IPCs). These cells secret insulin-like peptides (ILPs) ,62, whic tissues . ConversJafrac1, a peroxiredoxin that detoxifies peroxides. Jafrac1 is expressed in the adult brain and upregulated by paraquat stress [Jafrac1 shortens, while overexpression of Jafrac1 extends, the lifespan of flies [The brain consumes a large amount of oxygen for proper function and consequently produces high levels of ROS, thus it is highly susceptible to oxidative damage . JNK sigt stress . Neuronaof flies .puc mutants, JNK signaling in brains induces the expression of G6PD (glucose-6-phosphate dehydrogenase). In turn, G6PD oxidizes glucose-6-phosphate, an intermediate of glycolysis, by transferring hydrides to NADP+, forming NADPH. The increase of cytosolic NADPH defends against oxidative stress by reducing the concentration of oxidized glutathione. Intriguingly, overexpression of G6PD specifically in neurons is sufficient to mimic the proteostatic and longevity effects of JNK gain-of-function conditions, suggesting an important contribution of G6PD in neuronal JNK-induced lifespan extension. Consistent with this hypothesis, G6PD overexpression was also shown to improve lifespan in a mouse model [Recently, Wang et al. explored another mechanism through which JNK signaling reduces oxidative stress in neurons and enhances longevity . In puc se model , althougIn addition, increasing evidence suggests a positive role of sleep in resistance to oxidative stress ,67, whicDrosophila fat body is analogous to mammalian white adipose tissue and liver, performing metabolic and immune functions [The unctions ,73, bothDrosophila and mammals. In mice, deletion of the insulin receptor in white adipose tissue results in a lean, long-lived phenotype [Drosophila, reduction of IIS pathway activity in fat bodies also extends lifespan [foxo in fat bodies is sufficient to extend lifespan [dilp6. Expression of dilp6 then systemically represses dilp2 production in brain neuroendocrine cells, ultimately reducing the IIS activity in the peripheral tissues [In fat body cells, JNK signaling facilitates lifespan control by counteracting IIS activity . The imphenotype . In Droslifespan ,76. The lifespan . JNK sig tissues ,76.Drosophila. It defends against bacteria and viruses by producing a large number of antimicrobial peptides (AMPs). Two immune pathways, the IMD (immune deficiency) pathway and the Toll pathway, are responsible for AMP gene expression in fat bodies, through the downstream NF-κB transcription factors Relish or Dif/Dorsal, respectively [Relish loss-of-function homozygotes lose the ability against infection and quickly die [Relish or the individual AMP genes globally or specifically in fat bodies induces apoptosis, elicits depolarization of the mitochondria, and significantly shortens lifespan [Relish and AMP genes chronically increases during aging. Eliminating Relish activity prevents age-dependent upregulation of AMPs and extends lifespan [The fat body is also a well-known immune tissue in ectively . Under cckly die . Howeverlifespan . Strikinlifespan . Similarlifespan , suggestRelish loss-of-function homozygotes in the Jra (c-Jun in Drosophila) heterozygous mutant background exhibit higher levels of AMP transcripts and a higher survival rate within 5 days [JNK signaling in fat body cells is a potent candidate to repress AMP expression. After bacterial infection, n 5 days . In addin 5 days . AlthougDrosophila, muscle function gradually decreases with age, resulting in impaired climbing and flight abilities [Muscle function decline is a common degenerative event during aging and is associated with many age-related diseases . In Drosbilities . Many agbilities , includiTnks mutants survive significantly shorter and show apparent decline in climbing ability as well as impaired stress tolerance. Tissue-specific analysis suggests that Tnks principally mediates lifespan via its activity in the muscle, rather than in fat bodies, intestine or neurons. Given that knockdown of Tnks strongly decreases the activity of Bsk [As in other tissues, genetic manipulation affecting the JNK pathway in muscle can influence aging and longevity in flies. Flies with reduced JNK in muscle exhibit impaired climbing ability, increased sensitivity to paraquat and starvation . Interesy of Bsk ,87, TnksAging muscles progressively accumulate damaged proteins due to defects in protein homeostasis , which ifoxo gene exhibited “old” NMJ morphology in the abdominal ventral longitudinal muscles, including larger synaptic bouton areas and shorter terminal branches. Aging neurons undergo cytoskeletal changes in order to maintain plasticity and synaptic contact to muscles. Upregulation of late endocytic vesicles indicates a disruption of motor neuron homeostasis. Birnbaum et al. found that motor neuron-specific overexpression of foxo delays these age-related changes to NMJ morphology, suggesting that FOXO activity maintains morphological and synaptic plasticity at the aging NMJ. Together, Foxo and AP-1 are both beneficial for maintaining “young” muscle and NMJ.Accurate and fast muscle function requires crosstalk between muscles and motor neurons. The neuromuscular junction (NMJ) serves as the synaptic interface between the branched terminals of motor neurons and the muscle fibers. Fos and Jun, two components of AP-1, are abundantly expressed in motor neurons. Induction or inhibition of AP-1 in motor neurons is necessary and sufficient to modulate NMJ size and strength ,90. MostDrosophila and mammalian systems. However, the role of JNK signaling in normal physiological aging processes and in longevity remains elusive in mammals. The long lifespan of mammalian models may pose a major obstacle to such studies.JNK signaling is evolutionarily conserved in eukaryotes. Its conserved functions of inducing apoptosis and/or cytoprotective responses have been demonstrated in both As an alternative, the age-related disease models have been repeatedly used to understand the function of JNK signaling. For instance, in mouse models of Alzheimer’s disease (AD), the most frequent neurodegenerative disorder, JNK3 phosphorylation levels correlate with the progression of AD, thus, it serves as a biomarker for AD. Mechanically, JNK3 phosphorylates the amyloid precursor protein (APP), causing amyloidogenic proteolytic processing and Aβ production in the brain . In mousJNK signaling has also been examined in mouse models of disease outside of the nervous system. In a mouse model of liver injury caused by acetaminophen overdose, JNK promotes hepatocyte death. Upregulation of JNK activity by knocking out MAPK phosphatase 1 (Mkp-1), a negative regulator of JNK, exacerbated hepatic injury and shortened survival time . SimilarDrosophila. Ubiquitous elevation of JNK signaling activity in the whole organism can delay aging and extend lifespan. Moreover, manipulating JNK signaling in a specific tissue/organ, such as intestine or neurons, is sufficient to alter fly lifespan; however, these effects may be positive or negative depending on the context. For example, lifespan is extended when JNK is activated in the brain, whereas moderate activation of JNK in midgut ISCs and EBs leads to a significant shortening of lifespan. Therefore, efforts to dissect the mechanism underlying JNK function in the determination of longevity should be conducted in a tissue/organ-specific manner.JNK signaling is a significant genetic determinant of aging and longevity in Drosophila mitigates the effects of aging and prolongs lifespan [The classic function of JNK signaling is to trigger apoptosis for the clearance of abnormal or needless cells ,97,98,99lifespan .In addition to its proapoptotic function, JNK signaling functions as a stress-activated signal to induce cytoprotective cellular responses, including autophagy, DNA repair, and antioxidant enzyme induction. In aging tissues/organs where increased apoptosis gradually accelerates cell loss, JNK activity shifts toward a cytoprotective role rather than a proapoptotic role, promoting cell repair and restricting apoptosis to improve tissue/organ homeostasis. In the aging fat bodies and neurons, JNK antagonizes the insulin signaling and activates FOXO, which in turn switches on the expression of a subset of cytoprotective genes to protect cells from various stress challenges. In addition, JNK signaling in muscles and neurons improve proteostasis to delay aging. In contrast, in tissues with a high rate of cell turnover, such as the intestinal epithelium, age-induced JNK activity persists to trigger cell death and subsequent regenerative cell proliferation, mainly contributing to a decline in homeostasis. Outside of these limited exceptions, however, increasing JNK signaling in aging tissues/organs most often protects cells from death and extends lifespan.To understand the different outcomes of JNK signaling during aging, that is, its proapoptotic effects in young organisms and its cytoprotective effects in aging organisms, the upstream signaling is an important consideration. Numerous studies have revealed that JNK signaling during development and young adulthood can be activated by developmental cues as well as extrinsic or intrinsic stressors ,104,105.Drosophila. Studies in C. elegans have also revealed the significant contributions of JNK signaling in longevity and survival following bacterial infections [In the past decades, researchers have achieved significant progress in unraveling the molecular mechanisms and consequences of JNK signaling on aging and lifespan in fections ,107. The


To prospectively validate three quantitative single-energy CT (SE-CT) methods for classifying uric acid (UA) and non-uric acid (non-UA) stones.Between September 2018 and September 2019, 116 study participants were prospectively included in the study if they had at least one 3–20-mm urinary stone on an initial urinary tract SE-CT scan. An additional dual-energy CT (DE-CT) scan was performed, limited to the stone of interest. Additionally, to include a sufficient number of UA stones, eight participants with confirmed UA stone on DE-CT were retrospectively included. The SE-CT stone features used in the prediction models were (1) maximum attenuation (maxHU) and (2) the peak point Laplacian (ppLapl) calculated at the position in the stone with maxHU. Two prediction models were previously published methods (ppLapl-maxHU and maxHU) and the third was derived from the previous results based on the k-nearest neighbors (kNN) algorithm (kNN-ppLapl-maxHU). The three methods were evaluated on this new independent stone dataset. The reference standard was the CT vendor’s DE-CT application for kidney stones.Altogether 124 participants  with 106 non-UA and 37 UA stones were evaluated. For classification of UA and non-UA stones, the sensitivity, specificity, and accuracy were 100% (37/37), 97% (103/106), and 98% (140/143), respectively, for kNN-ppLapl-maxHU; 95% (35/37), 98% (104/106), and 97% (139/143) for ppLapl-maxHU; and 92% (34/37), 94% (100/106), and 94% (134/143) for maxHU.A quantitative SE-CT method (kNN-ppLapl-maxHU) can classify UA stones with accuracy comparable to DE-CT.Single-energy CT is the first-line diagnostic tool for suspected renal colic.• A single-energy CT method based on the internal urinary stone attenuation distribution can classify urinary stones into uric acid and non-uric acid stones with high accuracy.• This immensely increases the availability of in vivo stone analysis.•  Urinary stone disease continues to be an increasing reason for health care admissions worldwide, with an incidence of 7% among women and 11% among men in the USA in 2010 .Distinguishing UA from non-UA stones is of particular interest for the urologist, as the former can be treated with alkalization of the urine, allowing for secondary prophylaxis after ex vivo composition analysis of a passed or surgically removed stone –7. IdealNon-enhanced single-energy CT (SE-CT) is the first-line diagnostic tool for suspected renal colic and is able to detect nearly all urinary stones with high specificity. It is highly reproducible for measuring stone size and useful for predicting spontaneous stone passage , 14, 16.In a recent exploratory study on 126 urinary (22 UA and 104 non-UA) stones, Lidén correlated quantitative CT parameters in the stones to the chemical composition . The higAdditional analysis of the UA- and non-UA stone data in the previous study suggested that machine learning, avoiding the sharp cutoff lines in the ppLapl-maxHU method, might perform even better than the original method . K-neareUsing the same data as used in the recently developed ppLapl-maxHU method , a kNN aThe purpose of the present study was to prospectively validate two previously published (ppLapl-maxHU and maxHU), and one derived (kNN-ppLapl-maxHU) quantitative single-energy CT methods for classifying uric acid stones on a separate, previously unseen stone dataset.This study was approved by the Regional Research Ethics Board. Written informed consent was obtained from all prospectively and retrospectively included participants.Between September 2018 and September 2019, 116 study participants planned for elective urinary stone CT examination on our DE-CT scanner, with at least one 3–20-mm urinary stone, were prospectively included in the study to create a test dataset for validating the index tests described below . Because of the known low prevalence of UA stones in our patient population, all eligible UA stones were included, but to keep the heterogeneity of the non-UA stones as high as possible, only one non-UA stone per participant. For inclusion and exclusion criteria, see Fig. A preliminary reading of the CT scans was performed in patients accepting participation. Once the radiographer had established that there was at least one urinary stone fulfilling the criteria, the patient was included and a reference DE-CT was performed. The definite decision about eligibility for inclusion was later made by a radiologist with 15 years’ experience in reading abdominal CTs (J.J.).Based on the expected low number of UA stones in our patient population, we made a pre-study-planned, additional retrospective inclusion of UA stones demonstrated on DE-CT between August 2016 and August 2018, where a SE-CT scan was available. One DE-CT stone analysis examination during the prospective inclusion period was missed and therefore retrospectively included. In total, eight (73%) of the retrospectively eligible participants were included.The sample size of ≥ 35 UA and 100–150 non-UA stones was estimated with the objective of reaching the lower limit of a 95% confidence interval (CI) of 80%, for the hypothesized sensitivity of 95%, and the lower limit of 95% for the hypothesized specificity of 97%.All study participants were examined on a 2 × 128 channel dual-source system  using our local routine single-energy and a dual-energy protocol, for scan parameters, see Table The SE-CT and DE-CT reformats were pseudonymized and saved with different keys in separate folders in the local picture archiving and communication system .The three index tests were created using the same data from a previous study consisting of 126 stones with known pure UA/non-UA composition . While tThe two quantitative variables used for stone type prediction were (1) the highest attenuating voxel in the stone (maxHU) and 2) the value at the same position as for maxHU in a scaled Laplacian filtered image (ppLapl). An interpretation of the ppLapl is a computation of the attenuation difference between the highest attenuating voxel value and the weighted mean of the surrounding 26 voxels . The pse the valuThe kNN machine learning prediction method is based on the ppLapl-maxHU method. The prediction model was created in MATLAB using the nine nearest neighbors, a standard setting, and Euclidean distance. The method thereby uses the maxHU and ppLapl values of all the known stones from the previously annotated dataset, compares them with the position of an unknown stone in a scatter plot, and performs a “majority vote” between the nine stones closest to the unknown stone. With only two variables in the kNN, the model can be illustrated as a curved line separating the predicted UA from non-UA stones in a scatter plot Fig. . The difThe third index test used only the optimal maxHU, 745 HU, defined previously  Fig. 4c4c.n = 7) were considered UA/non-UA mixed stones and removed from further analysis, since the DE-CT has lower reliability for mixed stones and consequently is insufficient as a reference test [The stone classification (UA vs. non-UA) in DE-CT was used as ground truth for each included stone, as DE-CT can reliably differentiate pure UA stones from non-UA stones –13. The nce test –22.The largest diameter of each stone was measured manually in the axial plane 3-mm slice using the caliper tool in the PACS workstation with a soft window setting of C50/W400 and a zoom level of pixel-to-pixel × 8.Analyses were performed using IBM SPSS for Mac OS, v26.0.0.0 (SPSS Inc.), and MATLAB.Sensitivity and specificity for, and accuracy of the prediction of UA stones, using binomial distribution and 95% CIs were calculated for the SE-CT classification methods .This analysis was made primarily for all stones grouped together. Secondary analyses were performed: (1) prospectively included stones and (2) including only one UA  or non-UA stone per patient.p < 0.05).Statistical significance of the difference between the index methods was tested using McNemar’s test  with 106 non-UA and 37 UA stones were evaluated. Thirteen men had UA stones (14%) and 78 had non-UA stones (86%). Five women (15%) had UA stones, whereas 28 (85%) had non-UA stones. None of these stones has been used for the development of the index methods. Table Cross-tabulations of the results of the kNN-ppLapl-maxHU, ppLapl-maxHU, and maxHU methods by the results of the reference DE-CT are shown in Table p = 0.06) according to the McNemar test, there was a tendency towards higher accuracy for the kNN-ppLapl-maxHU method (98%) compared to the maxHU method (94%). The 95% CI for the difference was - 0.5 to 9.5 percentage points.The area under the receiver operating characteristics (ROC) curve (AUC) for the kNN-ppLapl-maxHU, as well as the maxHU, was 0.99. The AUC cannot be computed for the ppLapl-maxHU method with two static cutoffs. Although not statistically significant  were developed by Lidén  using thConsequently, a main strength of the present study compared to the previous is that the three tested algorithms were predefined, thereby avoiding the problem of overfitting. Although thirteen UA stones were retrospectively included, the results were similar when using only the prospectively included stones.p = 0.06)).The kNN-ppLapl-maxHU model uses a previously acquired dataset  to classTo our knowledge, this is the first prospective validation study of a SE-CT method for in vivo classification of UA and non-UA urinary stones. The results of this study are comparable to previously published results for DE-CT –13.This study has limitations. Three stones in the retrospective and four in the prospective subgroup were of mixed composition  and were removed from the analysis. Although a recent phantom study showed promising results in demonstrating the main stone component, using a machine learning algorithm on spectral detector DE-CT , no prevThirteen UA stones in eight patients were included retrospectively to achieve a sufficient number of UA stones. The index tests are purely quantitative and the radiologist performing them was blinded to the results of the reference standard. The risk for bias by retrospectively including eight participants with confirmed UA stone was considered low. In addition, the sensitivity and specificity were virtually unchanged when only the prospectively included stones were analyzed; merely the CIs became broader. Because of the known low prevalence of UA stones in our population, all UA stones in a patient meeting the inclusion criteria were included, similar to previous studies , 33–36 wThe validation is made for the current settings in the CT scanner family used in this study. Validation tests on scanners from various CT providers need to be performed before the method can be generalized to other CT manufacturers . This isIn conclusion, this study demonstrates that a purely quantitative single-energy CT method can classify uric acid (UA) and non-UA stones in a previously unseen dataset, with accuracy comparable to dual-energy CT, enabling immediate stone classification when a urinary stone is detected. Considering the lower cost, better availability, and lower radiation exposition, this is a promising alternative to dual-energy CT for in vivo characterization of urinary stones.


Salmonella Enteritidis (S. Enteritidis) being one of the most prevalent foodborne pathogens worldwide poses a serious threat to public safety. Prevention of zoonotic infectious disease and controlling the risk of transmission of S. Enteriditidis critically requires the evolution of rapid and sensitive detection methods. The detection methods based on nucleic acid and conventional antibodies are fraught with limitations. Many of these limitations of the conventional antibodies can be circumvented using natural nanobodies which are endowed with characteristics, such as high affinity, thermal stability, easy production, especially higher diversity. This study aimed to select the special nanobodies against S. Enteriditidis for developing an improved nanobody-horseradish peroxidase-based sandwich ELISA to detect S. Enteritidis in the practical sample. The nanobody-horseradish peroxidase fusions can help in eliminating the use of secondary antibodies labeled with horseradish peroxidase, which can reduce the time of the experiment. Moreover, the novel sandwich ELISA developed in this study can be used to detect S. Enteriditidis specifically and rapidly with improved sensitivity.S. Enteritidis, respectively, was developed and found to possess a detection limit of 5 × 104 colony forming units (CFU)/mL. In the established immunoassay, the 8 h-enrichment enabled the detection of up to approximately 10 CFU/mL of S. Enteriditidis in milk samples. Furthermore, we investigated the colonization distribution of S. Enteriditidis in infected chicken using the established assay, showing that the S. Enteriditidis could subsist in almost all parts of the intestinal tract. These results were in agreement with the results obtained from the real-time PCR and plate culture. The liver was specifically identified to be colonized with quite a several S. Enteriditidis, indicating the risk of S. Enteriditidis infection outside of intestinal tract.This study screened four nanobodies from an immunized nanobody library, after four rounds of screening, using the phage display technology. Subsequently, the screened nanobodies were successfully expressed with the prokaryotic and eukaryotic expression systems, respectively. A sandwich ELISA employing the SE-Nb9 and horseradish peroxidase-Nb1 pair to capture and to detect S. Enteriditidis in the spike milk sample and to analyze the colonization distribution of S. Enteriditidis in the infected chicken. These results demonstrated that the developed assay is to be applicable for detecting S. Enteriditidis in the spiked milk in the rapid, specific, and sensitive way. Meanwhile, the developed assay can analyze the colonization distribution of S. Enteriditidis in the challenged chicken to indicate it as a promising tool for monitoring S. Enteriditidis in poultry products. Importantly, the SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time. In short, the developed sandwich ELISA ushers great prospects for monitoring S. Enteritidis in food safety control and further commercial production.This newly developed a sandwich ELISA that used the SE-Nb9 as capture antibody and horseradish peroxidase-Nb1 to detect The online version contains supplementary material available at 10.1186/s12951-022-01376-y. Nanobodies (Nbs) are single-domain antibodies (sdAbs) derivatives of the immunoglobulin heavy chain in camelids or sharks, which represent the smallest antibody unit with complete antigen-binding characteristics owing to about 15 kDa molecular mass –4. SeverSalmonella Typhimurium (S. Typhimurium) and Salmonella Enteritidis (S. Enteritidis) are well-known to be the most harmful zoonotic pathogens [S. Enteritidis initiate significant cases comprising 1.35 million infections, 26,500 hospitalizations, and 420 deaths annually in America [S. Enteritidis should compulsorily be undertaken in foodborne products [S. Enteritidis. Such an advanced detection technology would provide a significant measure for reducing the prevalence of S. Enteritidis and the transmission risk to humans, as well as prevent and avoid the foodborne infectious diseases [S. Enteritidis.Among all the foodborne pathogens, athogens . Studies America . Hence, products , 18. As diseases . NanobodThere are many reported studies where nanobodies-fused reporter proteins have been used as detection agents for detecting target antigen by developing immunoassays to develop the immunoassays like the competitive ELISA and sandwich ELISA –22. The S. Enteritidis by developing an improved nanobody-horseradish peroxidase-based sandwich ELISA. The phage display technology was used to screen the specific nanobodies against S. Enteritidis obtained from an immunized Bactrian camel . The complete Freund’s adjuvant, incomplete Freund’s adjuvant and Amicon UltraCentrifugal Filter Units were procured from Sigma Aldrich . All the restriction enzymes utilized in the study were procured from New England Biolabs (Beijing) LTD . The 96-well microplates were purchased from Corning . The PCR Purification Kit, Gel Extraction Kit and TIANprep Mini Plasmid Kit used for plasmid preparation were obtained from TIANGEN . All the reagents utilized in this study were analytical grade, unless otherwise indicated.S. Enteriditidis FY-04 (GenBank accession JAKIRO000000000), S. Enteriditidis ATCC13076, S. Typhimurium ATCC23566, S. Pullorum ATCC13036, S. gallinarum CICC21510, Staphylococcus aureus ATCC25923, Klebsiella pneumoniae (isolated by Laboratory), Campylobacter jejuni (isolated by Laboratory), Listeria monocytogenes ATCC19115, Escherichia coli ATCC 25922. The pET-25b vector  was utilized to express the recombinant nanobody fusions against S. Enteriditidis in the E. coli system. The pMECS vector and M13K07 helper phage were kindly gifted by Ph.D. Qizhong Lu from the Stata Key Laboratory of biotherapy, Sichuan University, and were used to construct the VHH library. The pEGFP-N1 vector  stored at in our laboratory was used as a backbone to construct the pCMV-N1-HRP vector.The HEK293T cell lines were cultured in the Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 100 IU/mL penicillin‒streptomycin solution (Gibco USA). All the strains were from our laboratory preserved strains  including S. Enteritidis FY-04 was inactivated with formaldehyde as previously described [S. Enteriditidis was cultured overnight in LB media and harvested by centrifugation at 8000 rpm for 10 min and resuspended in PBS supplemented with 0.2% (v/v) formaldehyde, and placed at 37℃ for 48 h. The inactivated S. Enteriditidis was adjusted to 1 × 109 CFU/mL of the final concentration. Finally, the inactivation efficacy was evaluated using the plating count. There were no colonies on the plate, suggesting that the strains were completely inactivated and could be used to inject the Bactrian camel for the experiment.To ensure the safety of the immunized Bactrian camel, the pathogenic escribed . BrieflyS. Enteriditidis FY-04, a previously described protocol was used [S. Enteritidis FY-04 mixed with equal volumes of adjuvant. Freund’s complete adjuvant was used for the first immunization, four times followed with Freund’s incomplete adjuvant at an interval of 2 weeks. To evaluate the titration of serum from the last immunized camel, an indirect ELISA with HRP-conjugated rabbit anti-camel IgG  was performed where the inactivated S. Enteritidis as a coated antigen having a concentration of 1 × 109 CFU/mL was used.To obtain a specific VHH library against was used , 25. BriPstI and NotI). Then, the recombinant phagemids (pMECS-VHHs) were electroporated into the pre-prepared E. coli strain TG1 electroporation-competent cells. The capacity of the VHH library constructed here was analyzed through the plate count method, and the LB plates were supplemented with 2% (w/v) final concentration of glucose and 100 µg/mL ampicillin. The randomly-selected 48 colonies were determined using the PCR amplification with the primer pair MP57 and GIII  library using the procedures presented in Scheme S. Enteriditidis across four rounds of bio-panning using indirect ELISA (iELISA) as described previously [-S. Enteriditidis of 1 × 108 CFU/well in NaHCO3 buffer  at 4℃ overnight. The coated wells were washed three times with PBS containing 2% tween-20 , and then blocked with 3% skimmed milk (w/v). Then, 5 × 1011 PFU of the rescue phage was added and incubated at 37℃ for 1 h. Then, each well was washed ten times with PBST, 100 μL TEA solution  was added and incubated at RT for 10 min to elute specific phage particles, which was immediately neutralized with 100 μL of 1.0 M Tris–HCl (pH = 7.4). Subsequently, for the next round of selection, the eluted phage particles were transferred to infect the TG1 cells for titration evaluation and amplification. The population of TG1 cells infected was counted to quantify the input and output phages and the enriched phage particles were detected using iELISA with an anti-M13 antibody . After four rounds of screening, 96 individual clones were randomly selected from the third, fourth round of eluted phages. They were then separately induced with 1 mmol/L IPTG for expressing the soluble nanobodies in the E. coli periplasm in the 96-well plates. Several freeze–thaw cycles yielded the periplasmic extract (PE) which consisted of Nbs with Hemagglutinin (HA) and His tags. Furthermore, the presence of specific nanobodies against S. Enteriditidis were determined using iELISA with mouse anti-HA monoclonal antibody . Finally, sequencing based on their complementary determining regions (CDRs) amino acid sequence were able to identify and classify positive colonies (P/N > 3.0).The specific nanobodies were selected against eviously , 27. BriS. Enteriditidis, the pET-25b+ vector with a signal sequence for expressing C-terminally HSV-tagged and His-tagged proteins in the periplasm was utilized for expressing high-yield nanobodies [+-VHHs vector, the PCR products were digested and ligated into the pET-25b+ vector at the same restriction site. The E. coli BL21 (DE3) transformed positive vectors were induced with 0.2 mM IPTG for 16 h at 16 °C to obtain the nanobodies against S. Enteriditidis. Moreover, to evaluate the binding capacity of periplasm extract, an iELISA with anti-HSV tag monoclonal antibody  was used. The Nbs were purified from periplasm extract using Ni-IDA 6FF Sefinose (TM) Resin Kit and imidazole were removed using the Sephadex DeSalting Gravity Column . The expression and purification of the nanobodies was analyzed using SDS-PAGE and western blot. The binding capacity, specificity and cross-reactivity of the purified nanobodies, for the strains which include one S. Enteriditidis, and the other three Salmonellas, and five non-Salmonellas were confirmed by iELISA using the anti-HSV tag monoclonal antibody as a detection antibody.To obtain nanobodies against nobodies , 28. TheNheI and XbaI before it was ligated into the commercial vector pEGFP-N1 (cut with the same enzymes) to create the pCMV-N1-vHRP vector. To determine whether inserting an exogenous gene could successfully express upon recombination pCMV-N1-vHRP vector in HEK293T cells, the EGFP coding sequence was amplified as a positive control using the primer pairs EGFP-F and EGFP-R . After 48 h of transfection, the EGFP-vHRP fusion was directly observed using fluorescence microscopy .The pCMV-N1-vHRP vector was developed using the pEGFP-N1 vector as a backbone. This vector is designed to express nanobody fusions including the human Ig kappa chain signal peptide, HA tag, nanobodies coupled with the codon-optimized HRP, and the His tag in the HEK293T cells , 21, 23.g for 5 min to remove the cell debris. The secreted nanobody-HRP fusions supplemented with 0.02% NaN3 (w/v) were then stored at 4℃ for direct use. Then, the expressions of the nanobody-HRP fusions in the HEK293T cells and culture supernatant were separately determined using indirect immunofluorescence assay (IFA) and Western blot assay. In addition, the anti-HA monoclonal antibody was not only treated as the first antibody with the FITC- and HRP-labeled goat anti-mouse antibodies  but also as the second antibody in the two assays above. To evaluate the specificity, titers and cross-reactivity of the nanobody-HRP fusions in the cultured supernatant, the iELISA was performed using the anti-HA monoclonal antibody as the second antibody.The nanobody-HRP fusions were expressed in the HEK293T cells as described above. Briefly, the VHH coding genes were amplified by PCR using the Nb‑vHRP-F and Nb‑vHRP-R primers  were coated as antigens in the 96-well plate overnight at 4 ℃, where the NaHCO3 buffer was used as a negative control. After blocking with 3% skimmed-milk, the primary antibodies  were added to the plates and incubated for 1 h at 37 ℃. For directly detecting the titer of the serum samples, the HRP-conjugated rabbit anti-camel antibody was employed as a detection antibody for direct use. The anti-HA and anti-HSV monoclonal antibodies were separately treated as the second antibody in the specific nanobodies screening and characterization, which were both followed by treatment with the HRP-conjugated goat anti-mouse IgG at 37℃ for 1 h. However, in the case of the nanobody-HRP fusions, no secondary and detection antibodies were used. After washing five times with PBST, 100 μL/well of TMB  was added and incubated in the dark at 37 °C for 15 min for a colorimetric reaction, followed by stopping with 2 mol/L H2SO4 (50 μL/well). Finally, the optical density was measured at 450 nm using a Multiskan FC microplate reader .The iELISA being a common immunoenzyme technique was frequently used in this studying for detecting the titer in the camel blood samples, screening of the specific nanobodies, and the specific binding and cross- reactivity of the nanobodies and nanobody-HRP fusions. Briefly, the inactivated The procedure for IFA was modified based on a previously reported assay . After 4S. Enteriditidis, where the His-tagged Nbs and nanobody-HRP fusions were used as for capturing and detecting antibodies, respectively. The orthogonal assay was designed to select the best pair, procedure based on a previously reported procedure [S. Enteriditidis (1 × 108 CFU/well) was added and incubated in the plates for 1 h at 37 °C, an equal volume of NaHCO3 buffer as the negative control. To this, 100 μL of different nanobody-HRP fusions  were added to the plates and incubated for 1 h at 37 °C. The TMB (100 uL/well) which was used as a substrate of HRP was added to the plates and incubated in dark for 15 min at 37 °C. The color reaction was then stopped by adding 2 mol/L H2SO4 (50 μL/well) and the OD450 nm value of each well was measured with a microplate reader . Henceforth, the best pair of nanobodies with the highest P/N value was selected. The optimal concentration for the capturing and detecting Nbs was determined for the different concentrations of the coated antigen by searching for the conditions with the highest P/N value as previously described [S. Enteriditidis was employed as the positive control while NaHCO3 buffer served as the negative control. Then, when the P/N ratio was the highest, the optimal amount of the capture nanobody and detection nanobody-HRP fusions were determined. To characterize the specificity and cross-reactivity of the double nanobody‑based sandwich ELISA, one S. Enteriditidis, the other three Salmonella and five non-Salmonella as mentioned above were used to evaluate. The standard curves were determined to quantify and determine the limit of detection (LOD) of the developed method. It used 3 aliquots of gradient dilution of S. Enteriditidis as detection antigen, with a primary concentration of 3.0 × 109 CFU/mL.The double nanobody‑based sandwich ELISA was established for detecting rocedure , 29. Briescribed . BrieflyS. Enteriditidis using the plate counting method. The spiked milk sample was prepared by adding different concentrations of the S. Enteritidis FY-04 to the milk to attain final concentrations of 1 × 106, 1 × 107 and 1 × 108 CFU/mL, respectively. The prepared samples were collected by centrifugation at 5000 rpm for 10 min. And after washing the pellet with PBS, the established sandwich ELISA was used to analyze the recovery rate. For detecting the enriched bacteria, 1 mL spiked milk with S. Enteritidis around 10 CFU were mixed with 9 mL LB liquid medium and enriched at 37 °C for 6 h, 8 h, 10 h, 12 h, 14 h at 37 °C, respectively. The samples were analyzed using the same method as described above, without adding S. Enteritidis to the milk sample as a negative control.To validate the effectiveness of the double nanobody‑based sandwich ELISA, the skimmed milk from local supermarket ensured to be free of 8 CFU of S. Enteritidis FY-04, while group B was treated with the same volume of PBS as the blank control. On the fourth day after the oral challenge, all the chickens from each group were dissected to collect the tissues like the heart, liver, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, pancreas and kidney. For bacterial enrichment of these selected tissues, 1 g of every sample was cut and mixed with 25 mL pre-prepared buffered peptone water (BPW) and enriched at 37 °C for 12 h. Subsequently, the samples were centrifuged at 5000 rpm for 10 min and then were resuspended in PBS. The samples containing S. Enteritidis were determined using the established sandwich ELISA, qPCR detection and plate culture.The neonatal chickens were randomly divided into two groups under feeding conditions where Group A was orally challenged with 1.0 × 10S. Enteritidis ATCC 13,076 was used as a positive template and sterilized water as a negative control.The bacteria genomic DNA was extracted using TIANamp Bacteria DNA Kit  following the manufacturer's instructions. The real-time PCR reactions were executed according to previous descriptions . Precisehttp://www.spss.com.cn).All the assays were independently repeated at least three times. The data presentation was represented using GraphPad Prism version 7.0 . The Kappa values were calculated to estimate the coincidence between the developed sandwich ELISA assays, and the real-time PCR using the SPSS software  was found to increase from 54 to 1.2 × 104  and the fourth round (48 clones). The results revealed that the periplasmic extract of 96 clones could specifically bind with S. Enteriditidis , with the Y-axis and X-axis represented the absorbance value at 450 nm and the concentration of bacteria .The double nanobody‑based sandwich ELISA was developed using SE-Nbs to capture antigen and SE-Nbs‑vHRP to detect the captured antigen. The result of the orthogonal assay was presented in Additional file dis Fig. b and theS. Enteriditidis spiked in the dairy product of skimmed milk were designed . The recovery assay revealed the recovery value to range from 97.02% to 108.59%, indicating that the developed sandwich ELISA was feasible for detecting S. Enteriditidis in the milk samples as shown in Table S. Enteriditidis could be detected in the milk using the developed sandwich ELISA with no reactivity in the control well as evident in Table S. Enteriditidis in skimmed milk with good sensitivity and specificity.To verify the credibility of the established sandwich ELISA, the procedures monitoring the various concentrations of S. Enteriditidis was investigated in the intestinal tract and organs of the neonatal chicken with an oral challenge using the established sandwich ELISA. After 8 h of enrichment, all the selected organs and intestinal tract in the chickens were assessed by the developed ELISA assay, indicating that the S. Enteriditidis was maintained in almost all parts of the intestinal tract, including the duodenum, jejunum, ileum, caecum, colon, rectum and pancreas, but not in the stomach (Fig. S. Enteriditidis in the other organs were also detected using the same method. The liver was particularly found to colonize with some S. Enteriditidis presenting a high value at 450 nm, which suggested that the liver was an organ with a risk of colonization and transmission of S. Enteriditidis. Moreover, there was no S. Enteriditidis in heart and kidney (Fig. S. Enteriditidis in the positive sample as shown in Table The colonization of ach Fig. a. In addney Fig. b. The reS. Enteritidis as a major zoonotic pathogen cause due to the transmission of bacteria through the ingestion of contaminated food including dairy products, meats, eggs, seafood, vegetables and many other foods, which lead to human gastroenteritis [S. Enteritidis are labor-intensive, time-consuming, low throughput and lack efficiency [nteritis –33. To dficiency . Althougficiency , such asficiency , real-tificiency , nucleicficiency , loop-meficiency , recombificiency , recombificiency , rollingficiency , and itsficiency , 41. FurThe polyclonal and monoclonal antibodies based on immunoassays are determined by the capacity of the particular antibody to bind to its specific antigen. Nonetheless, the preparation of antibodies is troublesome, with poor stability between the batches and the half-life and tolerance worried in extreme conditions . Hence, S. Enteritidis rapidly with sensitivity. The capture antibody of SE-Nb9 was easily expressed in the high-yield E. coli system with purification of convenient compared to conventional antibodies. The SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time in the sandwich ELISA. Moreover, the HEK293T cell lines which stably express the SE-Nb1-vHRP can be modulated into a stable cell line by cell domestication technology, with reducing the cost and simplifying the production.Nanobodies are endowed with numerous excellent characteristics like high affinity, thermal stability, easy modification and production. This study developed an improved nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella-specific polyclonal antibody as the capture and nanobody-13 as detection antibody to establish the sandwich immunoassay for detecting S. Enteritidis in milk sample, in which the LOD reach to 1.4 × 105 CFU/mL S. Enteritidis and require the recognition of HA tag on nanobody with HRP conjugated anti-HA antibody for color reaction. Herein, the double nanobodies based immunoassay with the LOD of 5 × 104 CFU/mL is more sensitive than above methods and it does not require the commercial horseradish peroxidase labeled secondary antibody. The fact that this assay was utilized to detect S. Enteritidis is its biggest strength, which implied that both time and cost could be saved, as well as the shelf life could be prolonged with and the ability to tolerate extended working periods in harsh environments [S. Enteriditidis was explored in infected chicken. We found that S. Enteriditidis could survive practically in all the sections of the intestinal tract and the liver with a variety of strains. Moreover, the results were found to be realistic, reproducible, and aligned well with that of the real-time PCR. The plate culture approach may be used to successfully isolate S. Enteriditidis from positive samples of the challenged chicken, with a 100% match to result of the sandwich ELISA. The sandwich assay speculated to be a supplementary technology for effective isolating S. Enteriditidis from the poultry clinical samples. Of course, larger numbers and more complex clinical samples are needed to verify this method. These features make this strategy suitable for the on-site detection for risk assessment of S. Enteriditidis associated with them. At last, the developed sandwich ELISA based on the double nanobodies believed to have great potential for monitoring S. Enteriditidis in milk and poultry products, and can extend its application.Compared with the other immunoassays reported, the developed sandwich assay was found to show much superiority in terms of sensitivity , 43, 44.ronments . Using tS. Enteriditidis using the phage display technology. The SE-Nb1 and SE-Nb9-vHRP are employed as the capture and detection reagents respectively for developing a sandwich ELISA to detect S. Enteriditidis. This newly developed sandwich ELISA offers a simple alternative method with low-cost production, high throughput, and rapid detection. This method can be used to detect S. Enteriditidis in milk as low as 5 × 104 CFU/mL and approximately 10 CFU/mL of S. Enteritidis can be detected after 8 h of enrichment step. In addition, the developed sandwich can analyze the colonization of S. Enteriditidis in the infected chicken for risk assessment. Therefore, the developed sandwich ELISA is applicable for determining S. Enteritidis in spiked milk and has immense prospects in the poultry industry for controlling food safety.In summary, this study screened four specific nanobodies against Additional file 1. Additional figures and tables.