Giardia duodenalis infection is distributed worldwide and can achieve prevalence around 60%, especially in developing countries. This protozoan is divided into eight assemblages, in which A and B have high zoonotic potential, whereas C to H are host-specific. This scenario is changing as molecular studies progress, highlighting that knowledge on host-specificity still has a long way to go. Understanding the players involved in transmission routes enables rational designs of control strategies. Considering the high prevalence of giardiasis, this review aims to gather together the data on available studies on the distribution of G. duodenalis assemblages in Brazil until September 2020. Giardia is an intestinal protozoan found in a wide spectrum of animal hosts . In mammals the infection occurs by G. duodenalis, synonymous with G. lamblia and G. intestinalis. Although there is no consensus on the most appropriate name, in this review we will use G. duodenalis.G. duodenalis present worldwide distribution, showing prevalence of 2-7% in developed countries and can reach over 30% in low and middle-income countries.G. duodenalis transmission in outbreaks, in endemic areas direct transmission has great epidemiological relevance.G. duodenalis species meant that its subspecies were initially organised into groups, which are currently called assemblages or genotypes. The first divisions in G. duodenalis assemblages were carried out according to specificity by the host from which the isolate originated.,,G. duodenalis.,The great genomic diversity found in the G. duodenalis species is phylogenetically divided into eight assemblages, classified from A to H.,,,,Nowadays, the ,,,,,,,When symptomatic, the most frequent symptoms of giardiasis are consequence of acute or chronic diarrhoea, such as abdominal colic, flatulence, dehydration, nausea, vomiting and fatigue.G. duodenalis assemblages, especially in regions with high frequencies of infection.The different molecular characterisation tools have played an important role not only to better understand the characteristics of the assemblages, but also to understand the zoonotic cycles of transmission. Further genotyping studies are needed in order to elucidate the complexity involved in the transmission dynamics of G. duodenalis frequency in children can be higher than 60%.,,,,G. duodenalis assemblages could contribute to reducing giardiasis. In this context, this review aimed to describe the distribution of G. duodenalis assemblages in Brazil between 2007-2020.Brazilian studies about the epidemiology of giardiasis indicates that the Giardia duodenalis assemblages circulating in Brazil”. The electronic database, Medical Literature Analysis and Retrieval System on-line (Medline), was used.A review was carried out based on an exploratory and descriptive bibliographic survey in September, 2020, with the main theme: “Giardia lamblia) OR  OR  AND assemblage] AND Brazil; (3) [(Giardia) AND (genotype)] AND Brazil; (4) [ OR (Giardia lamblia) OR  AND (genotype)] AND Brazil  terms were used to define the search for: (1) etiological agent: “Giardia”; (2) characteristic of the etiological agent: “assemblage” or “genotype”; (3) geographical area: “Brazil”. To search with the aforementioned descriptors, the Boolean operator “and” was used. Thus, we obtained the following research strategy: (1) [(Giardia) AND assemblage] AND Brazil; (2) [(D Brazil .Giardia isolates (n = 5); studies that performed analysis in silico or from data available from databases (n = 2); studies that were not performed with isolates from Brazil (n = 2); in vitro and experimental model studies that did not perform genotyping of original isolates (n = 4); studies that used DNA from single cysts of G. duodenalis separated using a micromanipulation technique (n = 1); studies on archaeological material (n = 2); studies that did not use original G. duodenalis genotyping results and that had already been addressed in previous papers (n = 2); studies review (n = 1).The following inclusion criteria were used for the selection of manuscripts: articles retrieved in full, published in English until the year 2020. The following were excluded: studies that did not perform genotyping of the Frequency and distribution of genotyping studies in BrazilG. duodenalis in the country makes it difficult to implement strategic control actions that are targeted to potential sources of transmission. The scarcity of studies associated with the absence of a national infection survey makes it hard to understand the evolution of parameters related to the frequency of giardiasis in the population. Currently, it is not possible to say whether the G. duodenalis distribution in Brazil is changing upwards or downwards.Knowledge of the geographic distribution of parasitosis, such as giardiasis, is essential to target specific control measures. In Brazil, giardiasis is a public health problem with an unknown prevalence. Most of the studies in this field results from research by individual groups, and are usually sectional and aimed at local scope parasitological surveys. The lack of information about the transmission dynamics of G. duodenalis infection (over 40%), all genotyping studies are relatively recent, with the first one dating back to 2007.Although there are many areas with high frequencies of G. duodenalis genotyping, while two others (North and Northeast) share only two studies, each carried out from human samples. Most of the studies were developed in the Southeast region (30/41). This region is responsible for the main share of the country’s gross domestic product (GDP), and despite its small geographic dimension, it concentrates a large number of research institutes and postgraduate courses (48.45%).Brazil is a country of large continental proportions , composed of 27 states and a federal district divided into five regions . Of the Brazilian regions, one of them (Midwest) does not present any studies of Giardia and its genotypes in wild animal studies. All studies were carried out using samples of wild animals that had some proximity to humans  which would facilitate transmission.,,G. duodenalis assemblages in these animals in a natural environment.Brazil has the greatest biodiversity on the planet. It is estimated that about 11,000 known vertebrate animal species and 210 species of mammals have already been observed.G. duodenalis genotyping are carried out with samples of humans, domestic animals or farm animals  of  animals . Up to nGiardia in humans, and the largest diversity of animals in the world, the Brazilian contribution to the understanding of G. duodenalis diversity is scarce. In relation to developed countries, such as Portugal, Spain, Italy and China, Brazil has few studies of G. duodenalis genotyping, and most of them are concentrated in a single region. Further studies on the topic should be promoted, both in humans and in domestic, farm and wild animals, in order to assist in understanding the dynamics of transmission of G. duodenalis.Despite the high prevalence of G. duodenalis in BrazilGenotyping strategies of Giardia and/or discriminate G. duodenalis assemblages.G. duodenalis typing, the genes most widely used are those that encode the proteins Triose Phosphate Isomerase (tpi),gdh)G. duodenalis genotyping studies carried out from 2007-2019 (29/35) used the sequencing of regions of classically-used genes. βg, gdh and/or tpi were used in all studies, with the exception of one research study that used only the target SSU-rRNA.bg, gdh or tpi  is suggested for genotyping;gdh and tpi markers, but orfC4 and SSu-rRNA were also observed.,,,,,,gdh, tpi, orfC4; PCR-RFLP: βg, gdh).tpi target, when compared to gene sequencing.In Brazil, assemblage-specific PCR was used in four studies, mainly with the ,,,,,,,,,gdh, and sometimes appeared combined with the target βg.,,,,,,,βg target for genotyping in PCR-RFLP.PCR-RFLP was used for genotyping in ten studies, most of which were accompanied mainly by gene sequencing (8/10).G. duodenalis isolates is undeniable; however, all other sequencing brings an extremely relevant contribution to the scientific literature. The choice of the genotyping tool must be determined according to the investigation focus. PCR-RFLP proved to be a good strategy for an initial survey of circulating assemblages. Assemblage-specific PCR can be of great value in places where infectious assemblages are already known. Due to the divergences between the gene targets used in the sequencing, the use of more than one marker is recommended, as data from multi-locus sequencing brings more robustness to understanding the assemblage transmission dynamics. However, new gene markers and new financially viable tools are urgently needed to stimulate the expansion of genotyping studies and, consequently, enable a better understanding of the real zoonotic potential of G. duodenalis assemblages.The relevance of the whole genetic sequence for a better understanding of G. duodenalis assemblages in environmental samples from BrazilCirculation of Giardia, for more than 5,300 years.,G. duodenalis isolates circulating at that time.The presence of intestinal parasites dates from antiquity. In Brazil, the findings in coprolites and mummies point to the existence of these infections for more than 7,000 years and the existence of Giardia in water and food is associated with contamination by faeces from humans and/or animals, so different species and assemblages can be found. The main form of G. duodenalis infection, reported mainly in cases of outbreaks, is via waterborne transmission.The occurrence of G. duodenalis in water are basically concentrated in the State of São Paulo,,,,,,,G. duodenalis assemblages A, B, C, D and E suggests the contamination of water sources by faeces from different groups of hosts.In Brazil, genotyping studies of G. duodenalis isolates from sewage samples identified the presence of assemblages A, B and C  Is it possible that the circulation of these assemblages has always occurred and the scarcity of studies has hindered their previous identification? (2) Is it possible that the high frequency of interspecific contact could increase the pressure of infection and consequently select individuals capable of infecting this new host?Although the division of ,,,,,,,,,,,,,,,,,,,,,,,,In Brazil, assemblages A, B, C and E have already been identified in isolates from human faeces samples distributed in 21 studies carried out in the nine states.G. duodenalis zoonotic transmission, mainly anthropozoonotic. The two genotyping studies of G. duodenalis in cats carried out in São Paulo,Domestic animals have great relevance in ,,,,,,tpi and gdh). This could explain the identification of many different assemblages in these dog samples,In dogs, infection by host-specific assemblages (C and D) was observed in Minas Gerais, São Paulo, Santa Catarina and Paraná  Fig. 3)Fig. 3).,,,,In Rio de Janeiro, only assemblage A was identified in dogs.G. duodenalis isolates from these domestic animals still remains an unknown field.In Brazil, all assemblages  were identified in domestic animals, demonstrating the epidemiological importance of pets in maintaining cysts in the environment. The identification of assemblages A or B circulating in pets suggests that hosts other than dogs and cats may be involved in the transmission cycles. However, the genotyping of G. duodenalis in infecting wild animals, assemblages with high anthropozoonotic potential (A and B) were already identified in these animals .,,,Even considering the scarce knowledge of ,,,,,In farm animals , the assemblages A, B and E were reported, as expected -II Fig..40,In Brazil, assemblages G and H were not yet identified, probably due to the scarcity of studies in rodents and marine mammals. From marine waters, only isolates found in oysters were phylogenetically classified as assemblage A.Final considerationsG. duodenalis assemblages circulating in the country, and most of the regions still need to be explored. G. duodenalis genotyping is an important strategy to learn about the genetic variability of the species, mainly, to help understand potential transmission cycles. Despite the high frequency of this protozoa, especially in Brazilian children, little has been explored about the epidemiology of assemblages in humans and animals. In wild animals, we do not know whether G. duodenalis infection occurs in the natural environment and which circulating assemblages would be involved in this case. By sequencing or PCR strategies, and its variations, the circulation of assemblages A, B, C, D, E and F are being reported. Assemblage A in particular, and also assemblage B, have a high anthropozoonotic potential, so their occurrence is expected in different host species. The others assemblages (C-H) are considered host-specific, but can cause the infection of different, atypical hosts, as observed in human infection by assemblage C (host-specific for dogs) and E , in canine infection by assemblage E and in toucan infection by assemblage A. Despite these data, given that few areas of the country were studied and also that few hosts were investigated, knowledge about the real epidemiology of G. duodenalis assemblages in Brazil is still a long way to go.Brazil is a country with extensive territorial proportions, which means that it presents geographical, climatical biomes with cultural and GDP differences. However, there is a lack of knowledge about the


Viral infection is controlled by host innate immune cells that express specialized receptors for viral components. Engagement of these pattern recognition receptors triggers a series of signaling pathways that culminate in the production of antiviral mediators such as type I interferons. Mitochondrial antiviral-signaling protein (MAVS) acts as a central hub for signal transduction initiated by RIG-I-like receptors, which predominantly recognize viral RNA. MAVS expression and function are regulated by both post-transcriptional and post-translational mechanisms, of which ubiquitination and phosphorylation play the most important roles in modulating MAVS function. Increasing evidence indicates that viruses can escape the host antiviral response by interfering at multiple points in the MAVS signaling pathways, thereby maintaining viral survival and replication. This review summarizes recent studies on the mechanisms by which MAVS expression and signaling are normally regulated and on the various strategies employed by viruses to antagonize MAVS activity, which may provide new insights into the design of novel antiviral agents. The innate immune system is the first line of host defense against pathogens. Innate immune cells express specific pattern-recognition receptors that recognize microbial components known as pathogen-associated molecular patterns  and activate intracellular signaling pathways leading to the antiviral response . The majRLRs have been extensively studied as sensors of cytoplasmic viral dsRNA and their importance in controlling RNA virus infection is now clear. The RLR family has three members: retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology protein 2 (LGP2). RIG-I and MDA5 are typical pattern recognition receptors, whereas LGP2 is considered to be a regulator of RIG-I- and MDA5-mediated signal transduction , 17. UpoMAVS is a 540-amino acid protein encoded by the nuclear genome . MAVS isUpon binding of viral components, RIG-I and MDA5 undergo a conformational change that exposes their N-terminal tandem CARDs and forms the tetramer via CARDs , which aThe expression and function of MAVS are tightly regulated at the post-transcriptional and post-translational levels, which ensures that RLR signaling pathways are not only rapidly activated upon viral recognition but also curtailed in a timely manner upon viral clearance to avoid potentially harmful tissue damage.MAVS mRNA is polycistronic, and the key feature of polycistron is that protein translation can start not only from the N-terminal methionine, but also from the internal methionine, thus encoding for isoforms by alternative translation of distinct start sites , 31. MAVIt is well-known that mRNA contains a number of regulatory elements in the 3′ and 5′ UTRs. Binding of regulatory factors such as proteins and complementary small RNA molecules to these elements plays an important role in controlling protein translation . For exaThe DEAD-box (DDX) family of highly conserved RNA helicases play important roles in RNA synthesis, processing, transport, and degradation . A recenMAVS can undergo a number of post-translational modifications  that infViral infection leads to K63-linked ubiquitination of MAVS at the mitochondrial outer membrane, which in turn induces MAVS aggregation, a marker of its activation. At present, ubiquitination by the E3 ubiquitin ligase TRIM31 is the only known initiator of MAVS aggregation and activation . NotablyK27-linked ubiquitination by the E3 ubiquitin ligase TRIM21 also has a positive regulatory effect on MAVS. Viral infection upregulates the expression of TRIM21, thereby promoting the recruitment of TBK1 to MAVS and enhancing downstream signaling  41)..41).Phosphorylation is another post-translational modification that plays a key role in regulating MAVS signaling. After activation of TBK1 complexes by MAVS, the complex components TBK1 and IKK kinases phosphorylate MAVS, which then gains the ability to recruit IRF3. Recruited IRF3 is then phosphorylated by TBK1, which induces IRF3 homodimerization, translocation to the nucleus, and promotion of IFN transcription  53)..53).It is well-known that K48-linked ubiquitination of MAVS promotes its proteasomal degradation , and proCollectively, these studies have revealed that MAVS signaling is regulated via three main post-translational modifications that act in concert: (1) TRIM31-mediated K63-linked ubiquitination activates MAVS; (2) TRIM21-mediated K27-linked ubiquitination promotes the recruitment of TBK1 to MAVS; and (3) TBK1- and IKK-mediated MAVS phosphorylation promotes the recruitment of IRF3 to MAVS. In addition, inhibition of K48-linked ubiquitination and degradation is necessary to maintain an adequate cellular level of MAVS to ensure antiviral immunity.At present, the negative post-translational regulation of MAVS is mainly manifested in K48-linked ubiquitination modification, signal blocking, autophagy, and apoptosis .As noted above, K48-linked ubiquitination of MAVS triggers its proteasomal degradation and thus limits RLR signaling. A number of E3 ubiquitin ligases have been shown to mediate MAVS K48-linked ubiquitination, including TRIM25, AIP4, Smurf1, Smurf2, MARCH5, and RNF5 see also  44). Re. Re44). Several proteins negatively regulate antiviral innate immunity by inhibiting TRIM31-mediated K63-linked ubiquitination and activation of MAVS. One is Fas-associated factor 1 (FAF1), a protein that enhances Fas-induced apoptosis and was recently shown to be involved in the MAVS response. Under physiological conditions, the aggregates of FAF1 formed by its UBL domain, bind and antagonize MAVS by competing with TRIM31 for MAVS interaction, thereby inhibiting the MAVS aggregation. However, after viral infection, FAF1 is phosphorylated by activated IKKε, which promotes its depolymerization and lysosomal degradation and thus relieves MAVS inhibition . Rac1 isCollectively, these studies suggest that MAVS K48-linked ubiquitination under physiological conditions serves to promote MAVS degradation and prevent spontaneous activation of the immune response. The same event occurring in the late stages of viral infection serves to restore cellular basal activity and prevent excessive production of potentially harmful immune mediators. Similarly, inhibition of TRIM31-mediated K63-linked ubiquitination of MAVS prevents its aggregation and activation, and also serves to maintain MAVS homeostasis.Another mechanism by which MAVS signaling is held in check under physiological conditions is via direct binding of tubulin-tyrosine ligase-like protein 12 (TTLL12) to MAVS, TBK1, and IKKε . During Lactate, the end product of anaerobic glycolysis, has long been considered a metabolic waste product, but recent studies have shown that it has an important role in immunomodulation. Indeed, lactate acts as a key negative regulator of RLR signaling via direct binding to the MAVS TM domain, which prevents MAVS mitochondrial localization, aggregate formation, and signaling function. It is known that lactate level is directly related to glycolysis. Under physiological conditions, the interaction between hexokinase II (HK2), a key rate-limiting enzyme for glycolysis and located on the mitochondrial outer membrane, and MAVS maintains HK2 activity in its basal state, which ensures the operation of glycolysis metabolism and the suppression of MAVS mediated by lactate. However, during viral infection, binding of activated RIG-I to MAVS causes dissociation of HK2, leading to a reduction in glycolysis and lactate production, attenuation of lactate-mediated MAVS inhibition, and upregulation of downstream signaling for IFN production , 63. It During the later stages of viral infection, MAVS activity is negatively regulated by UBX-domain-containing protein 1 (UBXN1), a member of the ubiquitin-binding protein family. The expression of UBXN1 is strongly upregulated late during viral infection and it competes with TRAF3/TRAF6 for binding to MAVS (amino acids 455–460), thus blocking MAVS signaling , 65. GpaWhen other mechanisms fail, apoptosis can be used as a strategy to inhibit viral replication. However, the cell has also evolved various feedback mechanisms to ensure timely inhibition of immune signaling to avoid excessive tissue damage. For example, NLRP11, a type I IFN-induced Nod-like receptor, is upregulated after viral infection and can inhibit type I IFN production. Recent studies demonstrated that part of NLRP11 translocates from cytoplasm to mitochondria and interacts with MAVS, but NLRP11 neither disrupts the interaction between MAVS and RIG-I nor the ubiquitination of MAVS. However, NLRP11 strongly interacts with TRAF6 and promotes K48-linked ubiquitination degradation of TRAF6, thereby inhibiting the RLR signaling. Interestingly, overexpression of NLRP11 inhibited the cleavage of poly-ADP-ribose polymerase (a molecule involved in apoptosis related to DNA damage) in cells while knockout of TRAF6 abrogated NLRP11-mediated inhibition . BecauseAutophagy, a cellular degradation process, also acts as an antiviral defense mechanism by clearing intracellular microorganisms and interacting with the innate immune response. For example, Tetherin  is an IFN-induced membrane protein that is upregulated following viral infection. Tetherin induces K27-linked ubiquitination of MAVS by recruiting the E3 ubiquitin ligase MARCH8. The autophagy protein NDP52 then binds to K27-linked ubiquitinated MAVS, leading to lysosomal degradation. Tetherin-mediated autophagic degradation of MAVS can thus be considered another negative feedback mechanism that suppresses excessive signaling .Additional negative regulatory mechanisms in MAVS-mediated antiviral immunity include phosphorylation of MAVS by Nemo-like kinase, which occurs in the later stage of viral infection and induces MAVS degradation . The mitViruses can escape the host antiviral immune response by promoting the cleavage or degradation of MAVS and by directly interfering with RLR-activated signaling pathway components. Notably, there is little evidence that viral proteins play a role in enhancing the host innate immune response.pro, which cleaves MAVS at Gln148 and inhibits its activity  are particularly noteworthy. 3ABC produced by bat and human HAV cleave human MAVS at Glu463 and Gln427, respectively, highlighting the possibility that cross-species interference with MAVS signaling may promote the transfer of HAV between species , a serum marker of liver disease and hepatocellular carcinoma, and promotes the coiled-coil domain of TRAF6 to recruit GP73 to MAVS. GP73 binding leads to the degradation of MAVS and TRAF6 through a proteasome-dependent pathway, thereby supporting HCV infection . HCV infpro proteins of coxsackievirus (CV)-A16, CV-A6, and EV-D68 , the most common type of influenza virus, has caused many worldwide pandemics because of its variability and high pathogenicity. The H5N1 strain of IAV encodes PB1-F2 protein and PB2Δ, a defective but functional polypeptide, which play opposing roles in MAVS-mediated antiviral innate immunity. PB1-F2 binding to the MAVS TM domain reduces the mitochondrial membrane potential and disrupts MAVS signaling, whereas PB2Δ directly interacts with MAVS to promote signaling for type I IFN production, thereby inhibiting virus replication  101, 10. IAV NS1activity .Viral proteins can also promote intracellular proteins to inhibit MAVS activity . For exaAlthough most current studies focus on hepatitis viruses  and respiratory viruses , increasing attention is being paid to viruses such as EV71, CV-A16, CV-A6, Nipah virus, Seneca Valley virus, and Zika virus. This work will undoubtedly add to our understanding of the mechanisms by which viruses evade the host antiviral response.Recognition that MAVS plays a pivotal role in antiviral immunity has led to a surge in research on this molecule. It is now clear that cells and viruses regulate MAVS at both the post-transcriptional and post-translational levels using multiple mechanisms. Among them, ubiquitination plays a particularly crucial role in initiating, maintaining, and curtailing RLR-mediated signaling pathways, with K27-, K48-, and K63-linked ubiquitination acting in concert to regulate MAVS signaling. Whether ubiquitination of other types  can affect the biological functions of MAVS is unclear. The manner in which MAVS is affected by other modifications, such as phosphorylation and glycosylation as well as products of anaerobic glycolysis (lactate) and components of the apoptosis and autophagy pathways are attracting increasing interest. Viral replication requires a considerable uptake of nutrients from the host organism, and whether other components of carbohydrate and protein metabolism can regulate the antiviral immune response is another new field of study. As the key adaptor protein of RLR-mediated signaling, MAVS clearly plays a crucial role in the innate immune response. Despite significant advances in recent years, as illustrated in this review, the mechanisms regulating MAVS-mediated signaling in other species remains unclear. Further work in this area is expected to provide a clearer understanding of MAVS activity in antiviral immunity and to fuel the discovery of new drugs for the treatment of viral infection.ZR carried out literature search and designed idea. TD sorted out important background information and drafted the manuscript. ZZ and ZX critically revises the content of the manuscript. JD and ZW performed the manuscript review. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The electrochemical-based detection of uric acid (UA) is widely used for diagnostic purposes. However, various interfering species such as ascorbic acid, dopamine, and glucose can affect electrochemical signals, and hence there is an outstanding need to develop improved sensing platforms to detect UA with high selectivity. Herein, we report a pentagonal mediator-based non-enzymatic electrochemical biosensing platform to selectively measure UA in the presence of interfering species. The working electrode was fabricated by electrodepositing polymerized 1-vinylimidazole (PVI), which has an imidazole ligand, onto indium tin oxide (ITO), and then conjugating nickel ions to the PVI-coated ITO electrode. Electrode performance was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements and integrated together with pentacyanoammineferrate, which can bind to the amine groups of UA and function as an electron transferring mediator. The experimental results showed a wide linear range of UA concentration-dependent responses and the multi-potential step (MPS) technique facilitated selective detection of UA in the presence of physiologically relevant interfering species. Altogether, these findings support that pentacyanoammineferrate-based non-enzymatic electrodes are suitable biosensing platforms for the selective measurement of UA, and such approaches could potentially be extended to other bioanalytes as well. Uric acid (UA), also named 2,6,8-trihydroxypurine, is the final product of purine metabolism that is related to various diseases such as gout, hyperuria, obesity, diabetes, hypertension, and kidney and heart problems . UA can The electrochemical measurements have been mainly used for diagnostic sensors in the medical field ,14,15,16For these reasons, many researchers have recently been interested in developing the non-enzymatic electrochemical sensors  to increIn this regard, the metal hexacyanoferrates-based electrodes are promising . Nickel In this study, we developed a metal pentacyanoammineferrate-based non-enzymatic UA sensor with minimized interferences and thus increased selectivity. To fabricate the enzyme-free UA sensor, the pentacyanoammineferrate was conjugated onto the indium tin oxide (ITO) electrode modified with nickel (II) ion-absorbed polyvinyl imidazole (PVI). Our electrode is disposable, and we have characterized its performance by the following electrochemical potentiostat techniques: Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and multi-potential step (MPS). We found optimized pentacyanoammineferrate composition and system to measure UA. Our findings can be used to support the development of UA biosensor for healthcare applications.2PO4), 15.1 mM sodium hydrogenphosphate (Na2HPO4), and 140 mM NaCl) and all other solutions were prepared using deionized (DI) Milli-Q water . Poly(1-vinylimidazole) (PVI) was synthesized as previously described  [5-Ni-PVI-ITO electrodes were investigated in 0.5 M KCl solution containing 2.0 mM K3[Fe(CN)6]/K4[Fe(CN)6]. As shown in etR of PVI-ITO (red circle) dramatically increased over that of bare ITO (black square), from 687 to 4092 Ω. On the other hand, the etR of Ni-PVI-ITO (green triangle) was 3950 Ω, which is smaller than that of PVI-ITO due to the effect of nickel metal ion. The Fe(CN)5-Ni-PVI-ITO (blue inverted triangle) had an even smaller etR of 1666 Ω due to the conductive redox mediator of ferrate. This result suggests that the PVI polymer blocks the electron transfer of the electrode, while Fe(CN)5 and nickel ion help increase the conductivity of the electrode.The interface properties of the modified electrodes were confirmed electrochemically. In a Nyquist plot of EIS data, the semicircular part at higher frequencies corresponds to an electron-transfer-limited process, and the linear part at lower frequencies corresponds to a diffusion-controlled process. The semicircle diameter indicates the electron-transfer resistance (t) [Ret] . The impThe optimized Ni-PVI-ITO electrode was used for the determination of UA (1.0 mM) with pentacyanoammineferrate. As shown In addition, the four amines of UA likely coordinate with pentacyanoammineferrate and get oxidized on the Ni-PVI-ITO electrode. When the interference species  were added, the measurement signals were insignificant for all the tested electrodes  without pentacyanoammineferrate a–c in boBesides the buffer condition, it is noteworthy that the pentacyanoammineferrate has the unstable pentagonal structure and thus can play a role in UA oxidation. To investigate this role, we performed the same experiments with hexacyanoferrate, which has the stable hexagonal structure and found that UA measurement was not selective in both phosphate a and borDifferent UA concentrations of 0.00, 0.10, 0.25, 0.50, 0.75, 1.00, 2.00, 5.00, and 10.00 mM were tested by CV and MPS for the Ni-PVI-ITO electrode with pentacyanoammineferrate in borate buffer (pH 9.0). The cyclic voltammograms of UA a show th3+ to Fe2+. The electrons from reduced pentacyanoammineferrate are then moved to the electrode for making catalytic currents. 2 of 0.97771) than at high UA concentration range between 1.00 and 10.00 mM (with a slope of −4.88 μA/mM and a correlation coefficient R2 of 0.94200). The limit of detection (LOD) was 0.05 mM (n = 5) and thus, our system has good performance for quantitative UA measurements for diagnosis.The electrons from UA oxidation are transferred to pentacyanoammineferrate to reduce Fe2 = 0.97513) than at 1.00–10.00 mM , indicating that the sensitivity is greater at low UA concentrations. Overall, the Ni-PVI-ITO electrode can be used for selective UA sensing in the presence of the interfering species.The reliability of the fabricated Ni-PVI-ITO electrode in the presence of physiologically relevant interfering species—0.1 mM AA, 0.1 mM DA, 5.5 mM glucose, 50.0 mM urea, 100.0 mM KCl, and 269.0 mM NaCl—was analyzed. n = 5) to confirm reproducibility. The results of CV and MPS measurements of UA-spiked urine samples are shown in Electrochemical signals were measured by spiking UA to healthy adult urine samples in the prepared Ni-PVI-ITO electrode. Measurements were carried out by adding 20 μL of 0.2 mM pentacyanoammineferrate, 10 μL of unpurified urine sample, and 10 μL of UA (0.00 to 2.50 mM) in borate buffer (pH 9.0). Urine samples were provided by two healthy adults, and experiments were conducted with five sets of electrodes (n = 5). In addition, the MPS data showed that the maximum electrochemical currents of UA were detected at 0.4 V in the presence of interfering substances. The measurement signals obtained with urine samples spiked with different concentrations of UA were also linearly proportional to spiked UA concentration, which indicates the practical utility of our platform for real biological samples. Taken together, we have demonstrated that our system can be used for the development of enzyme- and label-free electrochemical diagnostic sensors to quantify UA, while its reusability remains to be improved.UA is the primary end product of purine metabolism, and quantitative analysis of UA in body fluids is an important indicator associated with the prevention of diseases. Moreover, the development of an electrochemical UA sensor, which provides easy sensing and reliable quantitative results, is very important. In this study, we developed a non-enzymatic UA sensor. We first optimized the electrode fabrication conditions regarding PVI concentration, nickel ion concentration, electrodeposition cycles of PVI, and reaction time of nickel, and found them to be 5 mg/mL, 5 mg/mL, 10 cycles, and 5 min, respectively. Moreover, we analyzed the measurement of UA in the presence of various interfering species  with pentacyanoammineferrate in borate buffer. We found that the selectivity for UA is due to the pentagonal structure of pentacyanoammineferrate as well as the basic condition of borate buffer, allowing the oxidation of UA at the electrode. The key role of the pentagonal structure in conferring selectivity was evaluated against the hexacyanoferrate. The oxidative catalyst currents produced by UA were linearly proportional to UA concentration, showing good performance of our system as an electrochemical sensor for the quantification of UA from 0.00 to 10.00 mM with LOD of 0.05 mM (


The mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr protein kinase that senses multiple upstream stimuli to control cell growth, metabolism, and autophagy. mTOR is the catalytic subunit of mTOR complex 1 (mTORC1). A significant amount of research has uncovered the signaling pathways regulated by mTORC1, and the involvement of these signaling cascades in human diseases like cancer, diabetes, and ageing. Here, we review advances in mTORC1 regulation by upstream stimuli. We specifically focus on how growth factors, amino acids, G-protein coupled receptors (GPCRs), phosphorylation, and small GTPases regulate mTORC1 activity and signaling. In 1964, a scientific expedition ventured to Rapa Nui  to collect soil and plants samples ,3. ThesemTOR is an evolutionarily conserved Ser/Thr protein kinase that is classified in the phosphatidylinositide 3 kinase (PI3K)-related kinase family within the human phylogenetic kinome tree. mTOR functions as the catalytic subunit of two distinct complexes, referred to as mTORC1 and mTORC2. Rapamycin and rapalogs inhibit mTORC1 activity allosterically, while mTORC2 demonstrates short-term rapamycin insensitivity ,12,13. TmTORC1 consists of three main core components: mTOR, regulatory protein associated with mTOR (Raptor) and mammalian lethal with Sec13 protein 8  , Left 1,18,19. RWhen localized to the lysosome, mTORC1 directly interacts with and is activated by the small GTPase Ras homolog enriched in brain (Rheb) ,31. HowemTORC1 regulates a multitude of cellular processes, such as protein translation, autophagy, lysosome biogenesis, lipid synthesis, and growth factor signaling . mTORC1 Similar to mTORC1, mTORC2 consists mTOR and mLST8. However, mTORC2 contains rapamycin insensitive companion of mTOR (Rictor) as the substrate recognizing component , Right ,13. AddimTORC2 has been observed in multiple locations throughout the cell. Using a reporter of endogenous mTORC2 activity, a study showed mTORC2 associates with the plasma membrane, mitochondria, and on endosomal vesicles . mTORC2 mTORC2 regulates physiological processes through the phosphorylation and activation of downstream substrates like the protein kinase A, G and C (AGC) family. Protein kinase C α (PKCα) at Ser 657 was the first identified substrate of mTORC2 . Other PmTORC1 activation is controlled by growth factors, nutrients, energy status, and stress . Growth Low energy levels during glucose deprivation activate AMPK ,82,83 thAmino acids promote the lysosomal translocation and activation of mTORC1 ,91. OnceThe discovery of the Rag GTPases in 2008 advanced our understanding of how amino acid activate mTORC1 A 90,97],9790,97]The guanine nucleotide loading of the Rag GTPases is controlled by guanine nucleotide exchange factors (GEFs) and GAPs. The Ragulator and solute carrier family 38 member 9 (SLC38A9) have been suggested to act as GEFs for RagA/B ,102. RagSome amino acid sensors for the Rag GTPase-dependent pathway have been identified. For example, two Arg sensors for mTORC1 have been identified for mTORC1, cellular Arg sensor for mTORC1 (CASTOR1) and SLC38A9 ,115,116.As mentioned above, SLC38A9 has been identified as part of the lysosomal machinery to control mTORC1. Other SLC transporters have been shown to regulate mTORC1 activity. For example, SLC15A4 was shown to transport His and oligopeptides out of the lysosome into the cytosol . In B lyThe Rag GTPase pathway was once considered the main mechanism for activation of mTORC1 by amino acids. However, it is now understood that a Rag GTPase-independent pathway exists, where Gln and Asn can activate mTORC1 B 99]. I. I99]. ILeu, Arg, Met, and Gln have previously been shown to stimulate mTORC1 activity. However, a recent study from our group demonstrated that other amino acids are capable of activating mTORC1  132]. U. U132]. With the advancement of cryo-electron microscopy (cryo-EM) technology, the structural understanding of components involved in amino acid signaling to mTORC1 have been better elucidated. Two different structures of mTORC1 with subunits mTOR, Raptor, and mLST8 were solved at a global resolution of 5.9 Å  and 4.4 s, Gαi/o, Gαq/11 and Gα12/13 [s and Gαi/o regulate adenylate cyclases (ACs). Gαs stimulates AC activity through binding, while Gαi/o inhibits AC activity. Activation of ACs increase production and accumulation of intracellular 3′,5′-cyclic adenosine monophosphate (cAMP). Elevated cAMP levels regulate protein kinase A (PKA), exchange protein activated by cyclic AMP (EPAC), and cyclic-nucleotide-gated ion channels. Negative regulators of Gαs signaling are cyclic nucleotide phosphodiesterases (PDEs), which can degrade cAMP [q/11 activates phospholipase C (PLC) and PLC can convert PIP2 to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) [12/13 targets GEFs for the small GTPase Ras homolog gene family (Rho), which has a role in actin cytoskeleton regulation [G-protein-coupled receptors (GPCRs) are seven-transmembrane pass proteins that are activated by an upstream ligand or agonist, resulting in the activation of multiple downstream signaling cascades . GPCRs a Gα12/13 . Gαs andade cAMP . Gαq/11 ol (DAG) . IP3 gatgulation . Additiogulation ,154.s signaling can regulate mTORC1 [s-coupled GPCRs increase cAMP to activate PKA and inhibit mTORC1 through the phosphorylation of Raptor on Ser 791  family are scaffolding proteins that tether PKA to unique subcellular locations through the PKA regulatory domain . AKAPs amTORC1 activity is regulated through phosphorylation by multiple upstream kinases . StudiesRaptor is also phosphorylated on multiple sites. mTOR itself has been reported to phosphorylate Raptor in vitro and in vivo on Ser 863, where Ser 863 phosphorylation promotes mTORC1 activity . In a suRaptor also has multiple phosphorylation sites that lead to negative regulation. Raptor is a direct substrate of AMPK, with target sites on Ser 722 and Ser 792 . This leIn the Rag-dependent amino acid  signaling pathway to mTORC1, RagA/RagB and RagC/RagD form a heterodimer to shuttle mTORC1 to the lysosome . In the Ras-related protein Ral-A  has been shown to induce mTORC1 signaling, downstream of Rheb . The stuThe Ras-related protein Rab (Rab) family of GTPases have also been demonstrated to regulate mTORC1 activity. The Rab proteins are canonically known for their role in regulating the process of vesicular trafficking . HoweverThe Rho GTPases are known for their function in cytoskeleton mediation, cell migration, proliferation, and transcription . The thrA recent study reported that when amino acids are limited, Ras related protein 1 (Rap1) changes lysosome abundance which leads to the suppression of mTORC1 signaling . Under aResearch continues to uncover new components and molecular mechanisms of how growth factors and amino acids filter through upstream regulators in order to control mTORC1. Recent identification of new individual amino acids that regulate mTORC1 signaling could indicate the existence undiscovered upstream sensors involved in mTORC1 activation. Additionally, newly found amino acids that potently activate mTORC1 warrant further exploration. The identification of additional components involved in the Rag-independent pathway will provide more molecular insight to Gln- and Asn-induced mTORC1 activation. Why there are two distinct amino acid signaling pathways to mTORC1 and the difference in the signaling kinetics of these pathways to mTORC1 is currently not understood.s pathway, such as AKAPs, PKA regulatory subunits, and phosphodiesterases involved in mTORC1 regulation, would also be extremely beneficial. For example, PKA phosphorylation of Ser 791 on Raptor leads to activation or inhibition of mTORC1 depending on the cell type. Therefore, utilizing FDA approved drugs (agonists and antagonists) that target GPCR-Gαs, could modulate mTORC1 activity. Different tissues contain mixtures of GPCR populations [Understanding the mechanistic details of mTORC1 signaling provides major advances to the research community. However, we should not lose sight of the larger goal: Elucidating how mTORC1 signaling affects cellular physiology and how this pathway can be targeted for therapeutic benefit. The dysregulation of mTORC1 can be found in many diseases ranging from diabetes, cancer, neurodegeneration and so forth . Revealiulations ,180. Repulations . Furtherulations . The faimTORC1 phosphorylation has provided more insight into the mechanism of mTORC1 regulation by upstream stimuli. Furthermore, studying the small GTPases involved in mTORC1 regulation contribute to another important area of research. Lysosomal translocation and activation of mTORC1 can be regulated by the Rag GTPases. However, the localization of mTORC1 at the lysosome stems multiple questions. Where are mTORC1 substrates phosphorylated? Some known substrates are not localized to the lysosome. How is mTORC1 trafficked to the lysosome, and where is mTORC1 located when it’s not at the lysosome? One possibility could be the regulation of mTORC1 by other small GTPases, many of which are associated with cellular trafficking. Additionally, other small GTPases could regulate mTORC1 at different subcellular locations. How these small GTPases function in accordance with amino acid and growth factor signaling will provide more insight into mTORC1 regulation.


The vascular endothelium is the innermost layer of blood vessels and is a key regulator of vascular tone. Endothelial function is controlled by receptor signaling through G protein-coupled receptors, receptor tyrosine kinases and receptor serine-threonine kinases. The β-arrestins, multifunctional adapter proteins, have the potential to regulate all of these receptor families, although it is unclear as to whether they serve to integrate signaling across all of these different axes. Notably, the β-arrestins have been shown to regulate signaling by a number of receptors important in endothelial function, such as chemokine receptors and receptors for vasoactive substances such as angiotensin II, endothelin-1 and prostaglandins. β-arrestin-mediated signaling pathways have been shown to play central roles in pathways that control vasodilation, cell proliferation, migration, and immune function. At this time, the physiological impact of this signaling has not been studied in detail, but a deeper understanding of it could lead to the development of novel therapies for the treatment of vascular disease. Endothelial cells are a key modulator for vascular homeostasis. Comprising the innermost layer within blood vessels, these cells act not only as a barrier between tissue and blood moieties, but also a regulator of vascular tone based off of physical and chemical signaling inputs from the environment ,2. A majIn the normal vasculature, NO maintains the vascular wall in a dormant state through inhibition of inflammation, cell proliferation, and thrombosis. This NO-dominant phenotype is believed to maintained via laminar shear stress and is assisted via S-nitrosylation of cysteine residues in proteins such as NFκB and other cell cycle proteins . DysreguA central component to endothelial function is the receptors which respond to the large variety of environmental signals such as hormones, other cells within the vasculature (i.e smooth muscle cells), neurotransmitters, pericytes, cytokines and oxygen tension. These include but are not limited to G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and receptor serine-threonine kinases (RS/TKs). Regulating their downstream signaling, β-arrestins have emerged as a crucial effector for several of these receptors.In this review article, we will focus on the roles of β-arrestin as a key partner in endothelial cell mediated signaling and function. We will summarize recent findings of major signaling pathways mediated by β-arrestin within chemokine receptors, as well as other pertinent GPCRs, RTKs and RS/TKs. Furthermore, we will discuss the impact of these on vascular endothelial cell function and their application to potential therapeutics for vascular disease.The β-arrestins (β-arrestin1 and 2) are intracellular adapter proteins best known for their regulation of GPCR signaling and can perform a wide array of functions in the cell  5]. Wit. Wit5]. Though β-arrestins are classically understood to be a part of the machinery required for GPCR trafficking, it is now appreciated that they also act as signal transducers and transactivators for GPCRs and RTKs  14]. Af. Af14]. An additional level of complexity in β-arrestin signaling stems from the function divergence of the two β-arrestin isoforms . In someStudies have implicated β-arrestins in endothelial and vascular function. For example, β-arrestin are reported to interact with caveolin-1, an abundant membrane protein in endothelial known present within caveolae, influence several endothelial cell functions, such as a membrane protein endocytosis of GPCRs and modulation of intracellular signaling events, extracellular matrix organization, and mechanotransduction . The β-aIn addition to producing a broad range of chemokines in response to proinflammatory stimuli, endothelial cells also express a variety of chemokine receptors . ChemokiWithin endothelial cells, C-X-C chemokine receptor type 3 (CXCR3) serves as a prime regulator for pro-angiogenic and anti-angiogenic signals. CXCR3 is expressed as 3 reported splice variants  and primarily binds to the ligands CXCL4, CXCL9, CXCL10, CXCL11. CXCR3 activation by CXCL10 promotes cAMP production and protein kinase a (PKA) activation to inhibit VEGF-mediated endothelial tube formation . CXCL10 C-X-C chemokine receptor type 4 (CXCR4) has long been implicated in promotion of angiogenesis through its primary cognate ligand, CXCL12 ,61,62. CAtypical chemokine receptor 3  is co-expressed with CXCR4 in diverse cell types. These include vascular smooth muscle cell (VSMCs)  and vascThe angiotensin II type 1 receptor (AT1R) is important in the regulation of vascular tone, and in proliferation and chemotaxis of vascular smooth muscle cells (VSMCs) . StimulaThe apelin receptor (APJ) is chiefly found in the endocardium of the heart and the endothelium of the vasculature . It is i19F-NMR examining the active conformation of the β2AR has shown that full agonists (such as isoproterenol) produce significant changes in the conformations of helices VI and VII, while β-arrestin-biased agonists (such as carvedilol and isoetharine) only impact helix VII; this suggests a structural basis for a separate, but parallel, nature of G protein and β-arrestin signaling at the β2AR [The β2-adrenergic receptor (β2AR) is important in the regulation of vascular tone . 19F-NMRthe β2AR . Additiothe β2AR . When ththe β2AR , where ithe β2AR . The resthe β2AR . β-arresthe β2AR . Furtherthe β2AR . Howeverthe β2AR . Additiothe β2AR . This suthe β2AR .Sphingosine-1-phosphate receptor 1, also known as S1P1R is a high affinity receptor for the bioactive lipid -Sphingosine 1-phosphate ,81. AlthProtease-Activated Receptor 1 (PAR1) contains a tethered ligand that is exposed after cleavage by thrombin and can increase vascular permeability . ActivatAR) and B (ETBR) [AR is expressed primarily in smooth muscle cells and exerts vasoconstrictive and cardiotonic effects, while the ETBR is expressed primarily in endothelial cells and seems to antagonize ETAR signaling. ET-1 receptors are phosphorylated primarily via GRK2, however phosphorylation is not an absolute requirement for β-arrestin binding, desensitization, or internalization [A/BR signaling [AR to β-catenin signaling [Endothelin-1 (ET-1) was originally extracted from aortic endothelial cells and was identified as a strong vasoconstrictor  and to pB (ETBR) . ETAR islization . The knoignaling . Epidermignaling . Notablyignaling ,54, Akt ignaling , and NFκignaling , consistGPCRs can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration . The AT1Vascular endothelial growth factor receptors (VEGFRs) belong to the family of receptor tyrosine kinases and play a central role in endothelial function, including cell proliferation and survival, angiogenesis, and lymphangiogenesis . VEGFRs β-arrestins have been shown to exert antiapoptotic and cardioprotective effects through transactivation of epidermal growth factor (EGF) receptor (EGFR) via Src, matrix metalloproteinase, heparin-binding EGF-like growth factor (HB-EGF) . β-arresThe type III TGFβ receptor (TβRIII or betaglycan) is a coreceptor expressed in endothelial-specific heteromeric complexes . β-arresBone morphogenic receptors (BMPRs) are members of the transforming growth factor-superfamily and play a multifunctional role in aiding development that regulates cell proliferation, differentiation, and apoptosis in various tissue types . The majEndothelial dysfunction, characterized by an imbalance in signaling between endothelium-derived vasodilators  and vasoconstrictors , has been associated with many cardiovascular diseases . EndotheSeveral studies have cited CXCR4 signaling to be implicated in vascular diseases. Lung tissue and animal models for PAH have shown increased CXCR4 expression . AMD3100ACKR3 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through β-arrestins. Injured coronary arteries from both humans and angioplasty wire injury mice exhibited endothelial ACKR3 expression. Conditional endothelial ACKR3 deletion promoted neointimal formation after endothelial injury and exacerbated heart functional impairment after myocardial infarction (MI). Both ACKR3 gene delivery via left ventricular injection and treatment with a CXCR7 agonist offered cardiac protection after MI . ACKR3 iCurrent clinical molecules that have been developed to target S1P1R provide therapeutic benefits in autoimmune disorders given S1P1R’s role in lymphocyte trafficking and endothelial cell function. Given the deleterious effects of prolonged β-arrestin mediated desensitization of S1P1R in endothelial cell survival which pose, a recent study developed a G-protein biased compound, SAR247799 to see if the damaging side effects of S1P1R desensitization could be selectively inhibited . SAR2477Activation of PAR1 by thrombin and APC leads to paradoxical functions where thrombin promotes cell death and barrier disruption, while APC promotes cell protection and barrier protection . CurrentET-1 is a strong vasoconstrictor that plays important roles in the pathogenesis and progression of cardiovascular remodeling. ET receptor antagonists (ERAs) are FDA-approved for the treatment of PAH by preventing vasoconstriction and abnormal smooth muscle cell proliferation . NotablyVefgr3 in mice causes exacerbation of chronic hypoxia-induced PAH, suggesting a role for VEGFR3 signaling in endothelial cells in the pathogenesis of PAH [VEGFR signaling a common target for tumor angiogenesis ,124. Bres of PAH . Recent s of PAH . TherefoEndothelial cell dysfunction is a hallmark of several vascular diseases. Over a decade of research has implicated chemokine and other receptor signaling in endothelial cell physiology through their effects on growth, migration, and immune function. In this review, we highlighted various ways in which β-arrestin modulates key aspects of endothelial cell function through their regulation of GPCRs, RTKs and RS/TKs. At this time there have only been limited studies on how β-arrestins can act as nodes for integrating signaling from these different signaling axes. With additional studies that focus on such cross-talk and on the physiological implications of this signaling, we may develop novel insights into how targeting β-arrestin-mediated receptor signaling in the vasculature can be useful as a therapeutic strategy in vascular disease.


Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases. Sphingolipids are both structural lipids and signaling molecules with significant physiological functions. Sphingolipids are found associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated to maintain homeostasis ,2. A claThere is growing evidence that sphingolipid metabolism is altered in diabetes ,11; howeCeramides are an integral part of cell membranes associated with macrodomains regulating surface receptors and signaling pathways ,21,22,23w/v), chloramphenicol (20 µg/mL), gentamycin sulfate (50 µg/mL), epsilon amino-caproic acid (0.13% w/v), and dithiobisnitrobenzoic acid (0.04% w/v) to inhibit the activity of lecithin-cholesterol acyltransferase, a major enzyme on HDL and LDL that converts free cholesterol into cholesteryl ester. Blood was then centrifuged, plasma was obtained, and lipoproteins fractioned by preparative ultracentrifugation as described previously  was prepared  and usedDuplicate aliquots from each lipoprotein  were analyzed at the Analytical Unit of the MUSC Lipidomics Shared Resource for sphingolipid content as previously described . All prePodocytes were differentiated as described above. Media were changed 24 h before the start of the experiment, then lipoproteins with and without enrichment were added to a final concentration of 200 μg/mL. After 7 h of incubation at 37 °C, 5% CO2, the plates were washed with PBS, and stored at −80 °C wrapped with aluminum foil.RNA was extracted from frozen cells in the stored plates using Qiagen’s RNeasy kit according to manufacturer protocol and concentration quantified. An amount of 100–200 ng of total RNA was used to prepare RNA-Seq libraries using the TruSeq RNA Sample Prep Kit V2 , following the protocol described by the manufacturer. High-throughput single read (1 × 50 cycles) sequencing (HTS) was performed using an Illumina HiSeq2500 (v4 chemistry) with each sample sequenced to a minimum depth of ~50 million reads. Data were subjected to Illumina quality control (QC) procedures (>80% of the data yielded a Phred score of 30). Secondary analysis was carried out on an OnRamp Bioinformatics Genomics Research Platform   ,35. OnRap-value or q-value, which is the smallest false discovery rate (FDR) at which a transcript is called significant. FDR is the expected fraction of false positive tests among significant tests and was calculated using the Benjamini–Hochberg multiple testing adjustment procedure. The statistical analysis of pathways and gene ontology terms were carried out using this sorted transcript list as described previously . In. InFLNA)ched LDL . The dowegulated . The effTUBA), ACTN and lamin (LMNA) (FasLG) gene, which drives cells into apoptosis [It is well-known that podocyte effacement is a major outcome of glomerular diseases and is associated with podocyte loss leading to renal dysfunction. Whether programmed cell death mechanisms contribute to podocyte loss remains contentious, in part because the detection of apoptosis and other pathways of programmed cell death in podocytes has been technically challenging. Our data show that C16 ceramide-enriched LDL induced the upregulation of the apoptosis-related genes encoding α-tubulin (n (LMNA) , which apoptosis , was dowThe mTOR signaling pathway is a well-described pathway in kidney cell biology, and the phosphorylation of its intermediates is a key step in the activation of the pathway. To further validate our RNAseq data, we employed multiplex technology to detect the regulation (phosphorylation) of mTOR signaling intermediates in response to C16 ceramide-enriched LDL, HDL2 and HDL3 treatments of podocytes. Unlike the qualitative/semi quantitative immunoblot system, the multiplex assay used here offered a quantitative determination of phosphoproteins detected. Each antibody used in this assay was validated and all the antibodies in the provided kits do not cross-react with each other. We found that podocytes treated for two hours with C16 ceramide-enriched HDL2 resulted in downregulation of all phosphorylated intermediates of the mTOR signaling pathway except IGFR1, AKT, and insulin receptor (IR), in comparison to treatment with native HDL2 A. In conThe C16 ceramide-enriched LDL treatment induced downregulation of phosphorylated GSK3a and GSK3B signaling molecules in comparison to treatment with native LDL C. The daIntriguingly, 2OH C16 ceramide-enriched HDL3 induced upregulation of IR phosphorylation compared to native HDL3, whereas 2OH C16 ceramide-enriched LDL induced upregulation of phosphorylated AKT, mTOR and IR. The pathophysiological relevance of 2OH C16 ceramide modification of lipoproteins is currently unknown.The transcriptomics findings of this study suggest that the analyses of sphingolipid profiles of lipoprotein particles, although more laborious, have the potential for an added value over plasma/serum sphingolipidomics in providing more specific information about not only biomarkers of disease but also mechanistic insights about the pathology of the disease and potential drug target molecules. Sphingolipids are non-soluble molecules and the cell membrane is impermeable to these molecules, and in the circulation they are carried and transported by lipoproteins. Mechanistically, our findings suggest that whereas LDL particles—including their bioactive sphingolipid cargo—are likely internalized and metabolized upon their contact with podocytes, HDL particles (mainly HDL2) remain in contact with the cell surface capable of altering the structure of membrane signaling domains and triggering downstream signaling pathways .For future research on the effect of sphingolipid-altered lipoproteins on the functionality of renal cells , other ceramide or sphingolipid species could be incorporated into lipoproteins in vitro, as described here. For sphingolipids containing very long-chain fatty acids (and therefore not easily incorporated in lipoproteins in vitro), fabricated lipoprotein model particles with incorporated sphingolipids could be utilized to investigate the potential for defined sphingolipid species to modify cell membrane sphingolipid composition and alter the cell signaling cascades involved with the pathophysiology of renal cells. Uncovering the role of lipoprotein sphingolipids in the development and progression of kidney disease could completely change the paradigm of intervention to prevent/delay the development or reduce the progression of nephropathy.


Objective: To explore the mechanism of Radix Rhei Et Rhizome  intervention in intracerebral hemorrhage (ICH) based on systematic pharmacology and proteomics strategy.Methods: The systematic pharmacological strategies were utilized to find the bioactive compounds of Radix Rhei Et Rhizome, predict its potential targets, and collect ICH’s disease genes; then, the Cytoscape 3.7.1 software was applied for network construction and network topology analysis. After that, in-depth analysis of the proteomics data of Radix Rhei Et Rhizome intervention in ICH was performed to complement and validate the results of systematic pharmacological predictions.Results: A total of three major networks were constructed in the present study: (1) compound–compound target network of Radix Rhei Et Rhizome, (2) DH-ICH PPI network, (3) proteomics proteins’ PPI network. These three major networks have been analyzed by network topology, and several small networks derived . The enrichment analysis showed that Radix Rhei Et Rhizome can intervene in several biological process , signaling pathway , and reactome pathway .Conclusion:Radix Rhei Et Rhizome may intervene in ICH-related biological process, signaling pathway, and reactome pathway found in this research so as to achieve the effect of treating ICH related injuries. Intracerebral hemorrhage (ICH) refers to bleeding result from non-traumatic rupture of blood vessels in the brain parenchyma, accounting for 25–30% of all strokes. However, its acute mortality rate is 30–40%, and the survivor’s invalidism rate is as high as 70%, which can leave severe complications such as paralysis, aphasia, epilepsy, and dementia, affecting the quality of life of patients ,2. The cRadix Rhei Et Rhizome  is the dried roots and rhizomes of Rhympalmatum L, Maximum.exBalf or Rhumofificinal Bail  were used to screen biologically active compounds, and a total of nine active compounds were obtained [With “edu.cn/)  and TCM@/zh-tw/) . After sobtained ,22–25. Hobtained , in ordehttp://scifinder.cas.org) and PubChem (https://pubchem.ncbi.nlm.nih.gov/) were used to search the standard molecular structure of those potential targets, and they were drawn in ChemBioDraw 14 and saved in “mol2” format.Eventually, combined with reference ,28, totahttp://lilab-ecust.cn/pharmmapper/) for potential targets prediction [Z scores of the docking scores were arranged in descending order. The top 300 targets of each compound were selected for subsequent research. The UniProtKB (http://www.uniprot.org/), a database contains the accurate annotation of proteins and so on, was used for the correction of protein’s names and the collection of official symbols with the species limited to  (Supplementary Table S1) or “Rattus norvegicus” (for proteomics data) (Supplementary Table S2). The ICH genes were collected from GeneCards database (http://www.genecards.org/) [http://www.ncbi.nlm.nih.gov/omim) [The “mol2” format files of the potential compounds were input into PharmMapper server platform (ediction . Set “seov/omim) . A totalRadix Rhei Et Rhizome and CI genes were imported into String 11.0 (https://string-db.org/), and the species was restricted to “Homo sapiens”  or “Rattus norvegicus” (for proteomics data) to obtain protein–protein interaction (PPI) data [The potential targets of PI) data .http://www.cytoscape.org/) [Radix Rhei Et Rhizome, DH-ICH PPI network, proteomics proteins’ PPI network, and other small networks derived from these major networks. In the network, nodes represent genes, proteins, or molecules; the connections between nodes are represented by edges, which stands for the interactions among these biological molecules [Radix Rhei Et Rhizome plays the role of regulating the biological network, namely Clusters [The active potential compounds, potential targets, ICH genes, and proteomics data were introduced into Cytoscape 3.7.1 (pe.org/)  softwareolecules . Degree olecules . In the Clusters . The CytClusters ,33.https://david-d.ncifcrf.gov) [https://reactome.org/) [P value <0.05 were collected for analysis.The Gene Ontology (GO) enrichment analysis and KEGG signaling pathway enrichment analysis were performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (crf.gov) . The reame.org/) . The bioAfter introducing 14 potential compounds into pharmmapper for prediction, 425 potential targets were obtained. (-)-Catechin gets 295 potential targets; Aloe-emodin gets 294 potential targets; beta-sitosterol has 217 potential targets; Chrysophanol has 249 potential targets; Danthron has 172 potential targets; Daucosterol has 216 potential targets; Emodin has 293 potential targets; Eupatin has 293 potential targets; Mutatochrome has 251 potential targets; Palmidin A gets 297 potential targets; Physcion gets 294 potential targets; Rhein gets 296 potential targets; Sennoside A gets 297 potential targets; Toralactone gets 296 potential targets. Meanwhile, 423 ICH-related genes with relevance score> 3 were selected for research. There is overlap between the potential target set and the ICH gene set .Radix Rhei Et Rhizome were input into Cytoscape 3.7.1 to construct compound–compound target network of Radix Rhei Et Rhizome. This network consists of 14 compound nodes, 425 compound target nodes, and 3760 edges. In this network, some targets can be regulated by most compounds, such as: ZAP70, TYMS, TTR, TGM3, SYK, SULT2A1, SRC, RXRA, REN, PTPN1, PRKACA, PLA2G2A, PIM1, PDPK1, PDE5A, PDE4D, PDE4B, NR3C1, NR1H4, NQO1, NOS3; other targets can only be regulated by a small number of compounds (or even only one compound), such as SIRT5, RTN4R, QPCT, PLAT, NPR3, NNT, MAPK12, IGLV2-8, HSPA1A, GLTP, GLRX, FGF1, FCAR, DDX6, CTSL, CRYZ , and their PPI data were input into Cytoscape 3.7.1 to establish the DH-ICH PPI network. This network is composed of 331 ICH gene nodes, 370 potential target nodes, 46 DH-ICH target nodes, and 15458 edges. The top 20 targets for degree in this network are: (1) DH targets: MAPK1 (206 edges), SRC (197 edges), EGFR (197 edges), MAPK8 (167 edges), HRAS (158 edges), ESR1 (156 edges); (2) ICH genes: INS (332 edges), IL6 (265 edges), VEGFA (245 edges), TNF (239 edges), FN1 (214 edges), APP (176 edges), AGT (161 edges); (3) DH-ICH targets: ALB (316 edges), AKT1 (279 edges), IGF1 (191 edges), MMP9 (178 edges), CASP3 (174 edges), NOS3 (166 edges), ACE (154 edges) (Four hundred and twenty-three (423) ICH genes, 524 potential targets of 4 edges) .The DH-ICH PPI network was analyzed by the MCODE, and 20 clusters were returned . The genCluster 1 is related to inflammation, smooth muscle proliferation, platelet activation, blood pressure regulation, angiogenesis, hypoxia, inflammatory response of leukocytes, vascular endothelial cells, vasodilation, vascular remodeling, and neuronal apoptosis. Cluster 2 is associated with the positive regulation of cytosolic calcium ion concentration, hypoxia, angiogenesis, vasoconstriction, blood pressure regulation, leukocyte activation and migration, platelet activation, axonal injury, synaptic transmission, neuronal apoptosis, and iron metabolism. Cluster 3 is mainly involved in platelet activation, blood pressure regulation, and glucose homeostasis. Cluster 4 is associated with platelet degranulation, hypoxia, blood pressure regulation, synaptic transmission, vasodilation, redox, and endothelial apoptosis. Cluster 5 is involved in angiogenesis. Cluster 10 is associated with coagulation, fibrinolysis, blood pressure regulation, redox, and hypoxia. Clusters 6, 7, 8, and 9 failed to return any ICH-related biological processes. The details were shown in Supplementary Table S4.As the biological process of cluster 1 is more typical, it is shown as an example in P-value, fold enrichment, and count of those signaling pathways were shown in The potential targets and ICH genes in DH-ICH PPI network were input into DAVID for signaling pathway enrichment analysis, and 28 ICH-related signaling pathways were obtained . The P-vThe potential targets and ICH genes in DH-ICH PPI network were input into Reactome for reactome pathway enrichment analysis, and a lot of reactome pathways were returned . After sP value and descending order of Count. The top 10 pathways are: (R-HSA-449147) Signaling by Interleukins, (R-HSA-6785807) Interleukin-4 and Interleukin-13 signaling, (R-HSA-383280) Nuclear Receptor transcription pathway, (R-HSA-76002) Platelet activation, signaling and aggregation, (R-HSA-1280215) Cytokine Signaling in Immune system, (R-HSA-9006934) Signaling by Receptor Tyrosine Kinases, (R-HSA-6798695) Neutrophil degranulation, (R-HSA-2219528) PI3K/AKT Signaling in Cancer, (R-HSA-168256) Immune System, (R-HSA-1592389) Activation of Matrix Metalloproteinases. The P-value, FDR, and count of those signaling pathways were shown in These reactome pathways were sorted in ascending order of Radix Rhei Et Rhizome and found that the core targets that it can regulate are: ZAP70, TYMS, TTR, TGM3, SYK, SULT2A1, SRC, RXRA, REN, PTPN1, PRKACA, PLA2G2A, PIM1, PDPK1, PDE5A, PDE4D, PDE4B, NR3C1, NR1H4, NQO1, NOS3, et al. The results of the biological module  analysis showed that Radix Rhei Et Rhizome can interfere with ICH-related biological processes such as: is related to inflammation, smooth muscle proliferation, platelet activation, blood pressure regulation, angiogenesis, hypoxia, inflammatory response of leukocytes, vascular endothelial cells, vasodilation, vascular remodeling, neuronal apoptosis, and so on. The signaling pathway enrichment analysis also shows that Radix Rhei Et Rhizome can regulate many ICH-related signaling pathways, such as: FoxO signaling pathway, Complement and coagulation cascades, cGMP-PKG signaling pathway, Rap1 signaling pathway, HIF-1 signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway, Neurotrophin signaling pathway, Estrogen signaling pathway, TNF signaling pathway. The results of reactom pathway enrichment analysis show that the reactome pathway regulated by Radix Rhei Et Rhizome is related to interleukin signaling and its signaling pathway, platelet activation, neutrophil degranulation, blood coagulation and fibrinolysis, FOXO-mediated oxidative stress, metabolism and transcription of neuronal genes, the VEGFA–VEGFR2 pathway, dopamine receptor, MyD88/Toll signaling pathway; PI3K/AKT signaling pathway and so on. Experimental studies also showed that Radix Rhei Et Rhizome can regulate oxidative stress, programmed cell death , and neurotrophic biological modules [The present study analyzed the network related to ICH intervention by  modules .Radix Rhei Et Rhizome or its active compound  can attenuate the destruction of the blood–brain barrier by increasing the expression of zonal closure protein-1 in rats with ICH [Radix Rhei Et Rhizome can down-regulate MMP-9 and up-regulate ZO-1 by inhibiting the ERK signal pathway [Radix Rhei Et Rhizome have similar effects, and the target sets between those compounds also overlap, which may be related to the main active compound being anthraquinones. For example, chrysophanol and rhein have almost the same molecular structure, except that one methyl group of chrysophanol is replaced by a carboxyl group; they can reduce the expression of caspase-3 and increase the activity of SOD in cerebral ischemic stroke models [Radix Rhei Et Rhizome to treat ICH, and to find the best combination of anthraquinone monomers.Recent research also confirmed some of the findings of the present study. In terms of inhibiting inflammation, emodin can promote microglia apoptosis by inhibiting the levels of IL-1 β, TNF-α, and increasing caspase 3 and 7 . In termwith ICH . Rhubarbwith ICH . Radix R pathway . In term pathway . Interese models ,49. Samee models . The preRadix Rhei Et Rhizome’s intervention in ICH has been predicted above using network pharmacology strategies. In order to verify the above results and further explore the molecular mechanism of Radix Rhei Et Rhizome treatment of ICH, the previous proteomics data will be analyzed in depth below. The proteomics data come from reference [The mechanism of eference .The proteomics protein of reference  were shohttp://metascape.org/gp/index.html#/main/step1) for enrichment analysis. The biological processes, signaling pathways, and reactome pathways were shown in Supplementary Table S7 and Radix Rhei Et Rhizome can interfere with multiple disease modules of ICH. For example: (1) Nerve cell transmission information module: chemical synaptic transmission, regulation of neurotransmitter levels, modulation of chemical synaptic transmission, regulation of trans-synaptic signals, activation of NMDA receptors and post-synaptic events, synaptic vesicle circulation, and so on. (2) Neuron module: regulation of neuronal projection projection development, regulation of neuronal differentiation, positive regulation of neuronal projection development, and so on. (3) Calcium ion module: calcium ion response, response to elevated platelet cytosolic Ca2+, and so on. (4) Excitatory and inhibitory amino acid modules: glutamate synapse, release NMDA receptor, glutamate binding and activation, glutamate receptor signaling pathway, and so on. (5) Coagulation module: platelet activation, coagulation, and so on. (6) Fibrinolytic module: fibrinolytic. The results of signaling pathway enrichment analysis showed that Radix Rhei Et Rhizome can regulate (rno04721) Synaptic vesicle circulation, (rno04724) glutamate synapse, (rno046100) complement and coagulation cascade, and so on. The results of reactome enrichment analysis showed that Radix Rhei Et Rhizome can regulate (R-RNO-112316) Neuronal System, (R-RNO-112315) Transmission across Chemical Synapses, (R-RNO-76005) Response to elevated platelet cytosolic Ca2+, (R-RNO-114608) Platelet degranulation, (R-RNO-109582) Hemostasis, (R-RNO-76002) Platelet activation, signaling and aggregation, (R-RNO-442755) Activation of NMDA receptors and postsynaptic events, (R-RNO-382551) Transport of small molecules, (R-RNO-111997) CaM pathway, (R-RNO-111933) Calmodulin induced events, (R-RNO-1489509) DAG and IP3 signaling, (R-RNO-422475) Axon guidance, (R-RNO-442729) CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde, (R-RNO-9619229) Activation of RAC1 downstream of NMDARs, (R-RNO-111932) CaMK IV-mediated phosphorylation of CREB, (R-RNO-112314) Neurotransmitter receptors and postsynaptic signal transmission, (R-RNO-373760) L1CAM interactions, (R-RNO-438066) Unblocking of NMDA receptors, glutamate binding and activation, (R-RNO-112310) Neurotransmitter release cycle, (R-RNO-3858494) β-catenin independent WNT signaling, (R-RNO-5578775) Ion homeostasis. Experimental studies have also shown that Radix Rhei Et Rhizome can regulate nerve-related modules, oxidative stress modules, and extracellular matrix-related modules [All proteomics proteins were input into DAVID and metascape , intracellular signaling by second messengers, innate immune system, FLT3 Signaling, signaling by VEGF, post-translational protein phosphorylation, MAPK1/MAPK3 signaling, MAPK family signaling cascades, and RAF/MAP kinase cascade. In addition, proteomics enrichment analysis also revealed more biological processes, signaling pathways, and reactome pathways, see Supplementary Table S7.Compared with the predicted results, it can be found that the two have common biological processes with high enrichment, such as coagulation module, fibrinolytic module, neuronal synaptic plasticity, inflammatory factors and inflammatory cells, calcium ion module, oxidative stress, and iron metabolism. The common signaling pathways are: complement and coagulation cascades, Rap1 signaling pathway, estrogen signaling pathway, cAMP signaling pathway, and calcium signaling pathway. The common reactome pathways are: platelet activation, signaling and aggregation, signaling by receptor tyrosine kinases, neutrophil degranulation, hemostasis, platelet degranulation, response to elevated platelet cytosolic CaRadix Rhei Et Rhizome may intervene in biological process , signaling pathway , and reactome pathway , so as to achieve the effect of treating ICH-related injuries.Click here for additional data file.


Drosophila as a genetic model, we characterized the role of Toll signaling in apoptotic cell death.Apoptosis plays pivotal roles in organ development and tissue homeostasis, with its major function to remove unhealthy cells that may compromise the fitness of the organism. Toll signaling, with the ancient evolutionary origin, regulates embryonic dorsal–ventral patterning, axon targeting and degeneration, and innate immunity. Using We found that gain of Toll signaling is able to trigger caspase-dependent cell death in development. In addition, JNK activity is required for Toll-induced cell death. Furthermore, ectopic Toll expression induces the activation of JNK pathway. Moreover, physiological activation of Toll signaling is sufficient to produce JNK-dependent cell death. Finally, Toll signaling activates JNK-mediated cell death through promoting ROS production.Drosophila to human, this study may shed light on the mechanism of mammalian Toll-like receptors (TLRs) signaling in apoptotic cell death.As Toll pathway has been evolutionarily conserved from  Drosophila for its role in establishing the dorsal–ventral axis at the early embryonic stage [Drosophila, Toll is activated by the cleaved cytokine Spätzle, and proceeds to the phosphorylation and degradation of the IκB factor Cactus through the MyD88-Tube-Pelle complex, eventually results in the release and translocation of two NF-kB factors Dorsal and Dorsal-related immunity factor (Dif) from cytoplasm to nucleus to activate the transcription of target genes [The type I trans-membrane receptor Toll was first identified in ic stage , and wasic stage . To dateic stage –6. In Dret genes . Dorsal et genes .basket (bsk) encodes the sole Drosophila JNK that is phosphorylated and activated by the conserved upstream MAPK cascade, including the JNKK kinase dTAK1 and the JNK kinase Hemipterous (Hep) [puckered (puc), a target gene of JNK signaling, encodes a phosphatase that dephosphorylates and negatively regulates JNK activity [reaper (rpr), head involution defective (hid) and grim [Drosophila IAP-1) to release the initiator caspase Dronc (Drosophila NEDD2-like caspase) [The c-Jun N-terminal kinase (JNK) is a member of the highly conserved MAPKs family that plays pivotal roles in various cellular processes including apoptosis –15. baskus (Hep) , 17. pucactivity . In fly,activity , which iand grim , whose pcaspase) , which icaspase) . JNK sigcaspase) –24, whiccaspase) .Drosophila as an in vivo system and characterized that Toll signaling induces JNK-dependent apoptotic cell death via ROS production. Firstly, activation of Toll signaling induces apoptotic cell death in the developing wings and eyes. Secondly, depletion of JNK signaling suppresses Toll-induced apoptosis. Moreover, Toll signaling is able to trigger JNK pathway activation. Finally, Toll elicits JNK-dependent apoptosis via promoting ROS production.Besides the well-documented functions of Toll/NF-kB signaling in development and immunity, several reports suggest that Toll pathway is also required for cell death triggered by tumor necrosis factor (TNF)  or chrom10B, an activated form of Toll, driven by patched (ptc)-Gal4 along the A/P compartment border  produces a loss of anterior cross vein (ACV) phenotype in the adult wings -Gal4 , and is mediated by the cleavage and activation of a group of cysteine proteases, termed caspases [ptc>Toll10B triggers apoptosis visualized by anti-cleaved caspase-3 (CC-3) antibody staining  -Gal4 up-regulates the transcription of the pro-apoptotic gene rpr, revealed by X-gal staining of a rpr-LacZ reporter, accompanied by enhances caspase activity  or Puc, an inhibitor of JNK kinase activity [Sd>Toll10B-induced adult wing blade reduction and apoptotic cell death in 3rd instar larval wing discs were soundly suppressed by expressing BskDN or Puc -Gal4, which expresses in all cell types posterior to the morphogenetic furrow (MF) in the developing eye , which encodes a detoxification enzyme that responds to oxidative stress [10B was sufficient to increase the gstD1 mRNA level as measured by qRT-PCR, which was suppressed by expressing Sod, but not BskDN -mediated oxidative stress in osophila . To addrrol Fig. a, Toll10ion Fig. i, which ion Fig. c, a supe DN Fig. d, suggese stress , 41. We Sod Fig. j, while rol Fig. h. CollecDrosophila has been widely accepted as an excellent model organism to dissect the roles of various signaling pathways in regulating the apoptotic program for the last two decades [Drosophila suggest that Toll signaling, consisting of the Toll ligand Spätzle, several Toll-related receptors and NF-kB factors, is required for apoptotic cell death of loser cell elimination during epithelial cell competition, which up-regulates the expression of pro-apoptotic genes, yet this function of Toll signaling is independent of the JNK activity [Drosophila neurotrophin (NT) ligands , combined with distinct Toll receptors, can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism [Drosophila larval tissue [ decades , 43. Whi decades . In vitr decades , 46. Int decades . Recent activity –51. In aactivity . Drosophechanism . For exal tissue , yet wheStaphylococcus aureus (S. aureus), a Gram-positive bacterium , yet the mechanism by which Toll signaling activates ROS production remains unclear, which merits further investigation. Consistent with our findings, mammalian TLRs was reported to induce the production of pro-inflammatory mediators upon pathogen invasion, which act as secondary messengers to regulate oxidative stress [Toll signaling can be activated in response to Gram positive bacteria, fungi and viruses in both Drosophila and mammals , 54–58. e stress . Up-regue stress . Given te stress .In this study, we report that Toll signaling induces ROS-mediated JNK-dependent apoptotic cell death in vivo. First, we characterized a physiological function of the Toll/NF-kB signaling in developmental cell death. In addition, Toll-induced cell death depends on JNK activity. Furthermore, Toll signaling is sufficient to trigger JNK pathway activation. Finally, we provide evidence that Toll activates JNK-mediated apoptosis through ROS production. Thus, our study provided the first in vivo evidence that Toll signaling promotes JNK-dependent apoptosis via ROS production.ptc-Gal4, Sd-Gal4, GMR-Gal4, UAS-GFP [act-Gal4, UAS-GFP-IR [UAS-Toll10B [UAS-DroncDN, UAS-BskDN, UAS-Puc, pucE69 and rpr-LacZ [UAS-dorsal-IR (27650) and UAS-Sod1 (24750) were obtained from the Bloomington stock center, UAS-dorsal-IR (45998) was obtained from the VDRC center, UAS-cactus-IR-1 (5848R-3) and UAS-cactus-IR-2 (5848R-1) were obtained from the NIG-FLY center, and dld05894 was obtained from the Exelixis collection at Harvard.Flies were raised on a standard cornmeal and ager medium at 25 °C unless otherwise indicated. Fly strains used in this work include:  UAS-GFP , act-GalS-GFP-IR , UAS-Tol-Toll10B , UAS-Drorpr-LacZ  were preStaphylococcus aureus (S. aureus) strain was obtained from Prof. Wei Zuo at Tongji University. For experiment, the bacterial culture diluted with PBS to 1 × 1013 cells/ml. Third instar larvae were washed with PBS. l μl bacterial suspension of S. aureus was injected into the larva body with a sharp needle. Treated larvae were placed on the medium at 25 °C. mRNA level was monitored by qRT-PCR 4 h post infection.Eastep Super (Shanghai Promega) was used to isolate total RNA from ten third instar larvae of indicated genotypes, and qRT-PCR was performed using SYBR Green PCR Premix Kit (TaKaRa). Primers used were as follows:rp49 FP: TCTCCTTGCGCTTCTTGGAFor RP: TACAGGCCCAAGATCGTGAAdl FP: ATCCGTGTGGATCCGTTTAAFor RP: AATCGCACCGAATTCAGATCcactus FP: CTCACTAGCCACTAGCGGTAAFor RP: CCCGAATCACTGGTTTCGTTTToll FP: AATCCCACGTTTAGGCTAACCAFor RP: CCTCACCGATCCGCAACTTgstD1 FP: CGCGCCATCCAGGTGTATTTFor RP: CTGGTACAGCGTTCCCATGT−5 M AO for 5 min at room temperature prior to imaging [Eye and wing discs were dissected from third-instar larvae in 0.1% PBST  + 0.1% Tween-20) and incubated in 1 × 10 imaging .Antibody staining was performed by standard procedures for imaginal discs . Rabbit Wing discs were dissected from third instar larvae in PBST (PBS + 0.1% Tween-20) and stained for β-galactosidase as described .The level of ROS was measured by CellROX . Wing discs were dissected from third instar larvae, incubated in 5 μM CellROX for 30 min at 37 °C, rinsed in PBS, fixed in 3.7% formaldehyde for 5 min, and mounted in PBS for imaging .Images of fly eyes and wings were prepared as described . BrieflyAdditional file 1: Figure S1. The expression patterns of ptc-Gal4, Sd-Gal4 and GMR-Gal4. Fluorescence micrographs of third instar larval wing  and eye discs (c) are shown. Expression region of ptc-Gal4 (a), Sd-Gal4 (b) and GMR-Gal4 (c) are labeled by the UAS-GFP reporter. Scale bar: 100 μm. Figure S2. The knock-down efficacies of dorsal and cactus RNAi lines. (a and b) Expression of two independent dorsal RNAi and cactus RNAi significantly decrease the level of dorsal mRNA and cactus mRNA, as measured by qRT-PCR. Total RNA of Drosophila third instar larval wing discs  was extracted and normalized for cDNA synthesis. Error bar indicates standard deviation. One-way ANOVA test was used to compute P-values, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Figure S3. JNK is required for Toll/NF-kB signaling impaired ACV development. Light micrographs of Drosophila adult wings (a-f) are shown. Compared with the ptc-Gal4 control (a), Toll10B-induced loss-of-ACV phenotype in adult wings (b), is blocked by expressing BskDN (c) or Puc (d). Depletion of cactus also produces a loss-of-ACV phenotype (e), which is suppressed by expressing BskDN (f). The lower panels show high magnification view of the boxed areas in upper panels (a-f). (g) Statistical analysis of ACV phenotype in adult wings (n = 45 for each genotype) is shown. Error bar indicates standard deviation. One-way ANOVA test was used to compute P-values, ****P < 0.0001. Scale bar: 100 μm. Figure S4. JNK pathway is up-regulated by physiological activation of Toll signaling. Light micrographs of third instar wing discs with X-Gal staining (a-c) are shown. Compared with the Sd-Gal4 control (a), elevating endogenous Toll signaling by knockdown of cactus (b and c) up-regulates puc-LacZ expression. (d) Statistical analysis of X-Gal staining (n = 8) is shown. One-way ANOVA was used to compute P-values, ****P < 0.0001. Scale bar: 100 μm. Figure S5. Toll regulates the stress response gene gstD1. (a) Histogram showing the level of gstD1 mRNA as measured by qRT-PCR. Error bar represents standard deviation from three independent experiments. One-way ANOVA was used to compute P-values, ***P < 0.001, ns indicates not significant. Figure S6. Evaluate the level of Toll expression. (a) Histogram showing the level of Toll mRNA as measured by qRT-PCR. Error bar represents standard deviation from three independent experiments. One-way ANOVA was used to compute P-values, ****P < 0.0001, **P < 0.01.