Tissue repair is a complex process that requires effective communication and coordination between cells across multiple tissues and organ systems. Two of the initial intracellular signals that encode injury signals and initiate tissue repair responses are calcium and extracellular signal‐regulated kinase (ERK). However, calcium and ERK signaling control a variety of cellular behaviors important for injury repair including cellular motility, contractility, and proliferation, as well as the activity of several different transcription factors, making it challenging to relate specific injury signals to their respective repair programs. This knowledge gap ultimately hinders the development of new wound healing therapies that could take advantage of native cellular signaling programs to more effectively repair tissue damage. The objective of this review is to highlight the roles of calcium and ERK signaling dynamics as mechanisms that link specific injury signals to specific cellular repair programs during epithelial and stromal injury repair. We detail how the signaling networks controlling calcium and ERK can now also be dissected using classical signal processing techniques with the advent of new biosensors and optogenetic signal controllers. Finally, we advocate the importance of recognizing calcium and ERK dynamics as key links between injury detection and injury repair programs that both organize and execute a coordinated tissue repair response between cells across different tissues and organs.Models of Systems Properties and Processes > Mechanistic ModelsBiological Mechanisms > Cell SignalingLaboratory Methods and Technologies > ImagingModels of Systems Properties and Processes > Organ, Tissue, and Physiological ModelsThis article is categorized under: Intracellular signaling dynamics are critical for detecting injury, encoding information about an injury, and coordinating a tissue‐wide repair response. These signaling dynamics can now be spatiotemporally quantified and controlled using new technologies, linking them directly to the repair processes they coordinate. Phospholipase C is activated when adenosine triphosphate (ATP) released from damaged cells near the wound edge binds to P2y purinergic G‐coupled protein receptors  to be secreted and cleave tethered EGF. This cleaved EGF acts as a paracrine signal, binding receptors on nearby cells, activating the MAPK signaling cascade, and culminating in the activation of ERK , can also induce IP3  cells. Using immunofluorescence staining for phosphorylated ERK, distinct patterns of ERK activation were observed after wounding. Specifically, two waves of ERK activation were present; the first wave started immediately after injury and disappears within 10 min, and the second was observed to begin an hour after injury and was sustained until inactivation upon gap closure  may be a key mechanism for calcium and ERK to activate specific repair response programs.6.12O2. Specifically, calcium activates the enzyme DUOX, an NADPH oxidase, which increases H2O2 production and secretion, recruiting immune cells within minutes of activation , via the phosphatase calmodulin  and the second being the nuclear factor kappa‐light‐chain‐enhancer of activated B cells  or mutant models to modulate cellular signaling. While potent, both of these methods provide little to no spatial or temporal control of cellular signaling. For example, while ionophores such as ionomycin or drugs such as histamines can reliably induce a rise in intracellular calcium, it is difficult to recreate the spatial calcium waves seen in epithelia or the oscillatory calcium spikes seen in fibroblasts using these drugs. The development of light sensitive genetically encoded tools (optogenetics) now enables precise control over both the time and location of cellular signaling. Highlighted below are several optogenetic tools which could be used to explore how calcium and ERK dynamics control the tissue repair response.7.1The biggest advance in controlling intracellular calcium dynamics has come from the discovery of light controlled ion channels, which gave rise to the field of optogenetics (Tye & Deisseroth, 7.2Control over ERK activity in cells has been enabled by various light‐inducible systems for protein dimerization, such as the protein pairs Phy and PIF, Cry2 and CIBN, and the iLID system consisting of SsrA caged by a LOV domain and SspB (Guntas et al., 8In this review, we have highlighted the roles that intracellular calcium and ERK signaling play in sensing tissue injury and initiating epithelial and stromal tissue repair. Both calcium and ERK integrate multiple biochemical and mechanical injury signals and initiate multiple different injury repair programs, making it difficult to connect the input injury signals to their corresponding output cellular behaviors. Here we propose that the spatiotemporal dynamics of calcium and ERK signals encode information that is the key to relating injury stimuli to downstream cellular repair programs. This section highlights three major questions for future studies to test this hypothesis.8.1One of the challenges in studying cellular signaling's role in tissue repair is relating specific input injury signals to specific output injury repair behaviors. Fortunately, given the recent development of optogenetic controllers, it is now possible to perturb intact cellular signaling networks with precise spatiotemporal control and visualize the resulting intracellular calcium and ERK signaling dynamics in real‐time using fluorescent biosensors (Aoki et al., 8.2Equally important to the injury response process is how these cellular repair programs are coordinated both spatially and temporally across cells in a tissue. Tissue‐level injury responses could be predicted by combining known injury signals with the single cell input–output relationships measured in Section 8.3After observing intracellular signaling and injury repair responses, it is vital to determine if the observed cellular signaling directly causes the injury repair response or if they are merely correlated. In many cases, causality can be observed by using an inhibitor drug or knock‐out mutants to block intracellular signaling, and seeing if the correlated cellular behavior still occurs. However, in the case of critical second messengers and master regulators such as calcium and ERK, simply blocking calcium and ERK signaling with a drug will inhibit a host of different cellular programs (see Sections 8.4The majority of studies measuring cellular signaling have looked at a single tissue in isolation generating valuable insights into the signaling molecules and dynamics underlying injury detection and repair (de Roos et al., The authors have declared no conflicts of interest for this article.Samuel Ghilardi: Conceptualization; visualization; writing‐original draft; and writing‐review and editing. Breanna O'Reilly: Conceptualization; visualization; writing‐original draft; and writing‐review and editing. Allyson Sgro: Conceptualization; funding acquisition; visualization; writing‐original draft; and writing‐review and editing.https://doi.org/10.1002/wsbm.108https://doi.org/10.1002/wsbm.110https://doi.org/10.1002/wsbm.1236https://doi.org/10.1002/wsbm.1261https://doi.org/10.1002/wsbm.16


Rf4, but different fertility segregation ratios are found in several F2 populations derived from crosses between isocytoplasmic allonucleus CMS-C lines and A619. In the present study, the segregation ratios of fertile to sterile plants in the (CHuangzaosi × A619) F2 and BC1F1 populations  did not follow a typical monogenic model of inheritance, which suggested that some F2 and BC1F1 plants displayed restored fertility even without Rf4. To determine the hidden locus affecting fertility restoration, next-generation sequencing-based QTL-seq was performed with two specific extreme bulks consisting of 30 fertile and 30 sterile rf4rf4 individuals from the F2 population. A major QTL related to fertility restoration, designated qRf8-1, was detected on the long arm of chromosome 8 in A619. Subsequently, qRf8-1 was further validated and narrowed down to a 17.93-Mb genomic interval by insertion and deletion (InDel) and simple sequence repeat (SSR) marker-based traditional QTL mapping, explaining 12.59% (LOD = 25.06) of the phenotypic variation. Thus, using genetic analyses and molecular markers, we revealed another fertility restoration system acting in parallel with Rf4 in A619 that could rescue the male sterility of CHuangzaosi. This study not only expands the original fertility restoration system but also provides valuable insights into the complex genetic mechanisms underlying the fertility restoration of CMS-C.C-type cytoplasmic male sterility (CMS-C), one of the three major CMS types in maize, has a promising application prospect in hybrid seed production. However, the complex genetic mechanism underlying the fertility restoration of CMS-C remains poorly understood. The maize inbred line A619 is one of the rare strong restorer lines carrying the restorer gene  Rf), can counteract the male sterile effect of the corresponding specific cytoplasm. CMS/Rf systems thus provide a useful genetic tool for the utilization of heterosis as well as a unique model for the elucidation of nuclear-cytoplasmic interactions in plants. To date, diverse Rf genes have been identified and characterized in various plants, and at least 15 Rf genes have been isolated from crop plants, which not only largely improves our knowledge of nuclear-cytoplasmic interactions but also facilitates the efficient utilization of CMS in practice , a maternally inherited trait, is characterized by the inability to produce functional pollen and occurs widely in higher plants. Certain nuclear genes, termed restorers-of-fertility  transcription factor, and a single amino acid substitution controls fertility restoration  to generate BC1F1, BC2F1, BC3F1 and BC6F1 populations.Five isocytoplasmic allonucleus CMS-C lines, namely, CHuangzaosi, C478, CMo17, C698-3 and C48-2, and five isonucleus allocytoplasmic CMS-C lines, namely, G48-2, EC48-2, ES48-2, RB48-2 and Lei48-2, were crossed with A619 to produce the respective FThe degree of fertility was ranked on a scale of I to V using a modified Beckett’s scale  as follo1 hybrid was evaluated in three individual plants using the iodine-potassium iodide (I2-KI) staining method. Three non-dehiscent anthers were taken from the upper, middle and bottom portions of the tassel, and then the anthers were squashed and stained with 1% I2-KI solution on a glass slide. The pollen grains were subsequently observed under a light microscope (Zeiss Axio Imager M2), and three images of each replicate were taken for analysis.In addition, the pollen fertility of the F2 and BC1F1 progenies were investigated in the spring of 2018 at Chengdu and in the fall of 2018 at Jinghong. Fertility ratings of the F1 hybrids and BC2F1 progenies were investigated in the fall of 2018 at Jinghong and in the spring of 2019 at Chengdu. Fertility ratings of the F2:3, BC3F1 and BC6F1 progenies were investigated in the spring of 2019 at Chengdu. All of the F1 hybrids and segregating populations were grown in a field, and field management was applied according to common agricultural practices.Fertility ratings of the FRf4 in A619 and rf4 in CHuangzaosi. Polymerase chain reaction (PCR) amplification was performed using KOD FX (TOYOBO) and the following recommended program: initial denaturation at 94° for 2 min, followed by 35 cycles of denaturation at 98° for 10 s, annealing at 58° for 30 s, extension at 68° for 2 min 30 s, and final extension at 68° for 5 min. Sequence alignment was conducted using DNAMAN software . Based on sequence variations in the third intron, the InDel marker B4-2 with primers F: 5′-CCTTGACCTCGTCCACCT-3′ and R: 5′-CATGGTAGCCTAGTACTCGT-3′ was designed to detect the genotype at the Rf4 locus. PCR amplification was performed using 2× Taq Master Mix (TSINGKE) and the following program: initial denaturation at 95° for 5 min, followed by 35 cycles of denaturation at 95° for 30 s, annealing at 57° for 30 s, extension at 72° for 40 s, and final extension at 72° for 5 min. Amplified fragments were visualized in a 3.5% agarose gel with 0.5× TBE buffer and GoldView staining.According to the genome sequence of GRMZM2G021276, primers F1/R1  were designed to amplify 2 population, two contrasting bulks, a fertility restored bulk (FR-bulk) and a sterility maintained bulk (SM-bulk), were constructed by mixing equal amounts of leaf tissues of 30 fully fertile rf4rf4 plants (grade V) and 30 sterile rf4rf4 plants (grade I or II) from the 2018 fall experiment. Total genomic DNA was isolated from two bulks as well as the parents, CHuangzaosi and A619, using a modified cetyltrimethylammonium bromide (CTAB) method , the quality of the clean reads was further checked using the FastQC program (v. 0.19.7). The high-quality clean reads were subsequently aligned to B73_RefGen_v4 (www.maizegdb.org) using Burrows-Wheeler Aligner (BWA) software, and duplicates were marked using SAMtools  than the threshold were selected as candidate intervals associated with fertility restoration.Homozygous SNPs between parental lines with high quality (sequence read depth > 5×) were selected for SNP-index analysis. The SNP-index, which represented the proportion of reads harboring the A619 genotype at every SNP site, was calculated for the FR and SM bulks (2 population. SSR markers in and around the predicted QTL region of chromosome 8 (www.maizegdb.org) were employed for polymorphism screening between the two parental lines. Additional InDel markers in the same region were developed using the whole-genome resequencing data of the two parental lines. Polymorphic markers with strong specificity were applied to genotype the 949 rf4rf4 individuals of the F2 population. Based on the genotype data of the F2 population, a genetic linkage map was constructed using QTL IciMapping V4.1 (http://www.isbreeding.net/). QTL analysis was conducted using the Inclusive Composite Interval Mapping of Additive (ICIM-ADD) module within QTL IciMapping V4.1 (P < 0.05).To verify the QTL associated with fertility restoration identified by QTL-seq, traditional QTL analysis was performed using fertility rating data obtained from the Fhttps://doi.org/10.25387/g3.12277268.The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures and tables. Supplemental material available at figshare: 1 hybrids obtained from the crosses between ten CMS-C lines and A619 were fully male-fertile with an identical fertility grade of V. The pollen stainability of the F1 hybrids ranged from 93.68 to 98.82% in different environments  mapped on the short arm of chromosome 8  and BC1F1 (Chengdu: 2.34:1 and Jinghong: 2.40:1) populations. The fertility segregation ratios did not follow a common monogenic or digenic pattern of inheritance of fertility restoration to CMS-C. Rf4 could not explain every scenario of fertility restoration to CMS-C in this study. Interestingly, only 102 of the 3,853 plants in the F2 population were sterile, whereas the sterile plants accounted for 29.78% of the total plants in the BC1F1 population.A619 is known as a CMS-C restorer line with a dominant restorer gene  and 1:1 segregation ratio in the BC1F1 populations  for a single gene model. A total of 2,901 Rf4-containing (Rf4Rf4 or Rf4rf4) plants were detected in the F2 population, of which 2,891 plants were fully fertile (grade V) and the other ten plants were partially fertile (grade III or IV). While 952 rf4rf4 plants in the F2 population displayed varying degrees of fertility, of which 758 plants were fully fertile (grade V), 92 plants were partially fertile (grade III or IV) and 102 plants were sterile (grade I or II). For the BC1F1 population, there were 384 Rf4rf4 plants, of which 383 plants were fully fertile (grade V) and only one plant was partially fertile (grade IV). While 328 rf4rf4 plants displayed varying degrees of fertility, of which 59 plants were fully fertile (grade V), 57 plants were partially fertile (grade III or IV) and 212 plants were sterile (grade I or II). Completely and partially restored rf4rf4 plants were frequently observed in the F2 and BC1F1 populations. Notably, approximately 79.62% of the rf4rf4 plants in the F2 population were fully fertile, whereas the percentage of fully fertile rf4rf4 plants in the BC1F1 population was considerably reduced (17.99%). Thus, the above results imply that another fertility restoration system acting in parallel with Rf4 in A619 is capable of fully or partially restoring the fertility of CHuangzaosi independently.Based on three deletions  found in the third intron of ker B4-2 . The InD2 and BC1F1 populations were systemically analyzed. Rf4rf4 plants with fertility grade V and rf4rf4 plants with fertility grades II to IV in the BC1F1 population were randomly selected to backcross with Huangzaosi, and the fertility of the BC2F1 progenies was evaluated during 2018-2019 (Table S2). For BC2F1 progenies derived from fully restored Rf4rf4 plants, one ear gave a good fit to a 1 fertile:1 sterile ratio . However, the other ear deviated from the 1:1 ratio , probably due to the effect of the other fertility restoration system. By contrast, 490 of 561 BC2F1 individuals (approximately 87.34%) derived from fully restored rf4rf4 plants were sterile. Only five BC2F1 individuals derived from fully restored rf4rf4 plants were rated grade V, and 66 BC2F1 individuals derived from fully restored rf4rf4 plants were rated grade III or IV. Approximately 35.37% of rf4rf4 individuals in the BC1F1 population were fully or partially fertile, but the proportion of fertile individuals in the BC2F1 population derived from fully restored rf4rf4 plants was considerably reduced (approximately 12.66%). BC2F1 progenies derived from partially restored rf4rf4 plants were partially fertile or sterile, and partially fertile individuals accounted for only 3.65% of the total individuals in this population. All BC2F1 progenies derived from sterile rf4rf4 plants maintained male sterility. Additionally, BC3F1 and BC6F1 progenies derived from Rf4rf4 plants gave a satisfactory fit to a 1 fertile:1 sterile ratio. Furthermore, we found many fully or partially restored F2:3 progenies generated from rf4rf4 plants (Table S2). All these results collectively suggest that a number of QTL are probably involved in this complex genetic system.To further characterize the inheritance pattern of the novel fertility restoration system, a set of progenies derived from the (CHuangzaosi × A619) FRf4, the SNP-index of each SNP locus was calculated using high-confidence SNPs. The average SNP-index across a 5-Mb genomic interval was computed individually in FR-bulk and SM-bulk using a 10-kb increment, and SNP-index graphs were generated by plotting the average SNP-index against all ten chromosomes of the B73 reference genome  from CHuangzaosi (average depth 10.53×), A619 (average depth 9.71×), FR-bulk (average depth 31.22×) and SM-bulk (average depth 34.09×), respectively, covering 84.31–93.10% of the B73 reference genome . The come genome . Similare genome . A genommosome 2 .rf4rf4 plants selected from the F2 population in the 2018 spring and fall experiments. Based on the inclusive composite interval mapping (ICIM) method, a major QTL was mapped in the interval of bnlg2181 to M8-155 . We found that 82 of 116 completely or partially restored plants possessed A619 alleles in this population. These results further confirm the effects of qRf8-1 on fertility restoration and could be useful in the marker-assisted selection of new male sterile and restorer lines with the C-type cytoplasm.To check the accuracy of the QTL for fertility restoration detected by QTL-seq, we performed traditional biparental QTL mapping. A total of 11 markers, including five SSR and six InDel markers (details in Table S3), were applied to genotype 949 o M8-155 , which wRf4 and Rf5, can fully restore the fertility of CMS-C  in A619  graph, minor-effect QTL might exist on chromosomes 2 and 9, which needs to be confirmed in further studies. It is worth noting that the fertility restoration of CMS-C displays many features of quantitative traits, but it is not exactly the same. Moreover, the fertility restoration of CMS is controlled not only by nuclear genes but also by cytoplasm genes. These features might influence the efficiency of QTL detection. In the future, it is imperative to identify and locate more restorer genes, which would be of great use in the development of high-quality male sterile, maintainer and restorer lines of maize. In summary, these results not only expand the original fertility restoration system but also contribute to a better understanding of the complexity of the fertility restoration mechanism for CMS-C.The novel fertility restoration system is much more complex than the previously well-known fertility restoration system controlled by dominant main restorer genes. In this system, multiple loci might be involved in the fertility restoration of CMS-C, and only a certain combination of QTL could lead to the full or partial restoration of fertility. In this study, we employed QTL-seq (


Giardia duodenalis is an intestinal protozoan with a high prevalence in children of developing countries. Molecular studies revealed a great genetic diversity of G. duodenalis, with assemblages A and B found mainly in humans. Despite its importance, the information on the molecular epidemiology of human giardiasis is still limited in Brazil. G. duodenalis molecular isolates in children from Salvador, Bahia, Brazil.To characterize Giardia duodenalis positive fecal samples were obtained from 71 children from two day care centers and 39 users of a clinical analysis laboratory. Samples were analyzed by PCR-RFLP of the glutamate dehydrogenase (gdh) and beta-giardin genes and by the sequencing of beta-giardin.G. duodenalis samples, 80 (72.7%) amplified one or both target genes. Of these, 62 (77.5 %) were identified as assemblage A and 18 (22.5%) as assemblage B. The subassemblage AII was identified in 58.8% (n=47) of isolates followed by the sub-assemblage AI , BIV , and BIII . The AII sub-assemblage was the most frequent in children of both day care centers whereas AI was found only in the group attended at the clinical laboratory. Sub-assemblage AII predominated in children under two years. Of the 110 Giardia may occur during child growth.The higher frequency of AII sub-assemblage suggests that anthroponotic transmission is more common in Salvador, but that zoonotic transmission pathways are also present and a change in susceptibility to different molecular patterns of  In countries such as Bangladesh . However, a higher prevalence of human infections by assemblage B was observed in Austria  supported by philanthropic institutions and from 39 children under 14 years of age attending the clinical analysis laboratory of the Faculty of Pharmacy at the Federal University of Bahia. All children were users of health public services and came from low-income families. Positive samples were identified by centrifugalsedimentation in water Giardia™, R-Biopharm AG, Germany). To compare the frequencies of specific protozoa assemblages and sub-assemblages, children infected with G. duodenalis were divided according to their age and gender.DNA extraction from feces and PCR conditions. DNA from G. duodenalis cysts was purified using QIAamp DNA Stool Mini Kit™  following the manufacturer's instructions with some modifications. For example, the time and temperature of the cell lysis step were increased to 10 min at 95°C and the DNA elution volume was reduced to 100 μl of the buffer.beta-giardin gene was amplified using forward primer G7 and reverse primer G759 2, 200 μl of each dNTP, 10 pmol of each primer, 2.5 units of Taq DNA polymerase (Invitrogen), and 1 μl of purified DNA in a final volume of 25 μl. The PCR reactions were performed as follows: An initial denaturation step of 5 min at 94°C for the first PCR and 15 min at 95°C for the nested-PCR followed by 35 cycles of 30 sec at 94°C, 30 sec of annealing (65°C for the primary beta-giardin PCR and 55°C for the nested PCR), and 60 s at 70°C with a final extension of 7 min at 72°C.A 753-bp fragment of the G. duodenalis isolates identified as genotype A through the analysis of the beta-giardin gene were subjected to a semi-nested PCR (sn- PCR) for amplification of the 384-bp fragment using the direct primers G376 and reverse G759 under the same PCR conditions used for the amplification of the 753-bp beta-giardin fragment Additionally, gdh gene was amplified using semi-nested PCR as previously described GDHeF and reverse primer GDHiR. In the sequential semi-nested PCR reaction, a 432-bp fragment was amplified using forward primer GDHiF and reverse primer GDHiR. In all cases, the PCR mixture consisted of 1X buffer containing 2 mM MgCl2, 200 μM of each dNTP (GC:TA = 3:1), 12.5 pmol of each primer, 1 unit of Taq DNA polymerase (Invitrogen), and 1 μl of purified DNA in a final volume of 25 μl for the primary PCR and 50 μl for the sn-PCR. The PCR reactions were performed as follows: An initial denaturation step of 5 min at 94°C followed by 40 cycles consisting of 30 s at 94°C, 20 s of annealing at 65°C and 45 s at 72°C with a final extension of 7 min at 72°C. All PCR products were analyzed by electrophoresis on ethidium bromide-stained 1% agarose gels.A 432-bp fragment of the Amplicon analyses by RFLP and sequencing. For the characterization of Giardia assemblages, 10 μl of the 511 bp beta-giardin amplicon were digested overnight with 10 U of HaeIII in a final reaction volume of 32 μl at 37°C (7). For identification of A sub-assemblages , the 384 bp fragment produced by snPCR was digested with the endonuclease HhaI as described above gdh gene was digested overnight at 37°C using 10 μl of the 432 bp amplicon of the snPCR and 10 U of the enzyme NlaIV (BspLI) in a final volume of 32 μl. Samples indicating the presence of assemblage B had the amplicons also digested with a second endonuclease, the RsaI, under the same conditions to specify sub-assemblages BIII and BIV beta-giardin gene. PCR products were purified and sequenced by the Macrogen Inc. sequencing service . Nucleotide sequences and electropherograms were analyzed and edited using the program CLC Main Workbench™, version 8.0 . To determine the genotype of each sample, the tree phylogenetic analysis was performed using the neighbor-joining method using the MEGA 6 software Beta-giardin gene references corresponding to the different G. duodenalis assemblages or sub-assemblages were obtained from GenBank . Sequences were deposited in GenBank under accession numbers MG845536 to MG845549.The isolates with mixed genotype patterns or inconclusive RFLP results were submitted to amplicon sequencing of the ™ software for Windows and the statistical analyses were performed with the GraphPad Instat™ program . The chi-squared test was used to compare the frequency of G. duodenalis assemblages and subassemblages according to the age and gender of children while the Kruskal- Wallis followed by Dunn post-test was performed to compare numerical variables. A probability of less than 0.05 was considered significant.The data were analyzed using the IBM SPSSThe Ethics Committee of the Nursing School at the Federal University of Bahia, Brazil, approved the study .Children whose parents agreed to participate in the study and signed an informed consent form were enrolled during the research period. Children over 8 years of age were also informed about the research and signed a consent form. All parasitological test results were sent to the children's parents and individuals with parasitic infections were adequately treated by pediatricians when necessary. G. duodenalis, 80 (72.7%) had the DNA successfully amplified in one or both genes  only gdh  of the isolates (p<0.05). Assemblage B was identified in 22.5% (18/80) of the G. duodenalis samples  in samples from day care center 1 and laboratory users, whereas in day care center 2 there was no statistical difference in the occurrence of these two genetic types . AssemblG. duodenalis samples identified as assemblage B, 13 were successfully sub-classified as BIII (5.0%) and BIV (11.2%)  followed by AI . Of the 18  (11.2%) .Giardia duodenalis sub-assemblage distribution also differed among groups: AI was found only in children's samples from the routine laboratory and 10 of these 15 isolates (66.7%) were from children under six years of age, i.e., in the same age range as children from the day care centers. A significant predominance of AII sub-assemblage  was observed in day care center 1. On the other hand, although AII was the most frequent (54.2%) type in day care center 2, no significant difference was found compared to assemblage B frequency (45.8%). In both day care centers, only AII was detected among G. duodenalis isolates identified as assemblage A.G. duodenalis sub-assemblage the distribution as regards children's gender but there was a difference regarding their age: AI sub-assemblage was more frequently detected in children between 3 and 10 years of age while AII was predominant in children under 2 years  but only A and B are regularly found in humans, although they can be detected in other domestic and wild animals ,,,Advances in molecular biology studies of G. duodenalis. It is rare to find reports from the northeastern region of the country and there are no data of assemblage distribution in the state of Bahia.Despite the high prevalence of giardiasis in Brazil, there are few studies on the genetic diversity of G. duodenalis from children living in Salvador who were divided into two groups: children who attended day care centers and those who were seen in a public clinical laboratory. All the isolates were subjected to PCR to amplify beta-giardin and gdh gene fragments. Eighty (72.7%) samples were successfully amplified in at least one of the genes analyzed with slightly greater success in the amplification rate of gdh (67.2%) than in beta-giardin (53.6%). These genes are often used to detect and/or genotype Giardia isolates from fecal samples but differences in their amplifications have been reported ,,In our study, we performed the molecular characterization of 110 isolates of ,Thirty (27.3%) of the isolates in this study did not amplify any of the genes tested. The negative PCR results could be explained by the presence of fecal DNA polymerase inhibitors, such as bilirubin, bile salts, hemoglobin, phenolic compounds, and complex polysaccharides, which are co-purified during the extraction of genomic DNA ,In this study, we detected assemblages A (77.5%) and B (22.5%) with a significant predominance of the former, as found in Spain G. duodenalis assemblages varies between regions. Recent studies in day care children in Sao Paulo showed a predominance of assemblage B G. duodenalis isolates ,,,-G. duodenalis from different groups of children were analyzed.In Brazil, due to the huge territorial dimension of the country, the prevalence of G. duodenalis assemblage distribution between groups: In day care center 1, assemblage A was the most prevalent  while no significant differences between A (54.2%) and B (45.8%) occurrence were observed in day care center 2. The dissemination of G. duodenalis cysts through person-to- person contact, common in day care centers, can promote the concentration of certain molecular isolates Regarding our sampling, we found differences in ,,,,,,Regarding sub-assemblage characterization, AII was the most frequently detected in 58.8% of cases (47/80) followed by AI (18.8%), BIV (11.2%), and BIII (5.0%). Similarly, studies in other countries have reported a predominance of AII sub-assemblage in children ,,When we specifically analyzed the distribution of the sub-assemblages in the samples from the two day care centers, there was a higher occurrence of AII followed by BIII and BIV. The detection of these subtypes corroborates reports of the role of person-to-person transmission of giardiasis due to agglomeration of individuals in childcare centers since they are predominantly found in humans G. duodenalis molecular isolates has been reported in previous studies with rates varying from 2 to 21% and higher in developing countries ,,,,,beta-giardin gene sequencing. The occurrence of mixed infections by various G. duodenalis assemblages/sub-assemblages reflects the complex circulation of the parasite in the environment, the exposure of this population to multiple sources of infection, and the lack of cross-immunity between different molecular isolates et al.G. duodenalis genome project.The occurrence of mixed human infections involving different G. duodenalis sub-assemblage frequency by gender in this study, which agrees with previous reports ,G. duodenalis molecular type B as the most frequent in females ,G. duodenalis molecular type due to active immunological memory against a previously eliminated isolate. In fact, studies conducted in Rio de Janeiro at different periods suggest the substitution of one G. duodenalis genetic isolate for another in the population ,There were no significant differences in G. duodenalis did not have diarrhea or relevant gastrointestinal symptoms at the time of fecal analysis. Among the giardiasis cases analyzed, out of 9 children (8.2%) seen at a clinical analysis laboratory seven were symptomatic and had characterized while only two of them had diarrhea (both infected with AI sub-assemblage). The other five had other gastrointestinal symptoms . Given the limited number of symptomatic individuals in our study, it was not possible to evaluate associations between molecular isolates and symptoms. However, it is important to emphasize that asymptomatic children play a role as cyst disseminators in day care centers and in the environment.In our study, 91.8% of the children infected with G. duodenalis carriers studied were asymptomatic (predominantly of assemblage A), it is important to highlight the similar distribution of A and B molecular groups in one of the day care centers suggesting that factors intrinsic to the host  are more relevant in triggering disease than the parasite molecular type involved.Although most G. duodenalis genetic characterization undertaken in northeastern Brazil and the first one in the state of Bahia. The results show that although AII sub-assemblage predominated in the analyzed population suggesting that anthroponotic transmission is more common in our environment, there is a high molecular variability of G. duodenalis isolates, which evidences that zoonotic transmission routes can also be present. Apparently, in early childhood, there is a preferential susceptibility to AII G. duodenalis sub-assemblage, which changes to AI and possibly BIV in children over three years of age and is maybe related to the development of a subassemblage-specific immune response.This is one of the few studies of G. duodenalis with a variety of clinical conditions are necessary for a better understanding of the molecular epidemiology of giardiasis.More studies analyzing different groups parasitized by


ANXA1 gene expression may be dysregulated in HTLV-1-infected HAM/TSP patients.Human T-lymphotropic virus 1 (HTLV-1) is etiologically associated with the chronic inflammatory neurodegenerative disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) Annexin A1 (AnxA1) is an anti-inflammatory protein with proposed neuroprotective and anti-neuroinflammatory functions. We hypothesized that ANXA1 and formyl peptide receptor  expression and HTLV-1 proviral load (PVL) in peripheral blood cells were evaluated by real-time quantitative PCR (qPCR), and plasma AnxA1 levels were determined by enzyme-linked immunosorbent assay (ELISA).This study involved 37 individuals infected with HTLV-1, including 21 asymptomatic (AS) carriers and 16 with HAM/TSP, and a control group of 30 individuals negative for HTLV-1 and HTLV-2. For AS HTLV-1-positive and HAM/TSP patients, ANXA1 gene expression was increased in the AS group compared with the HAM/TSP and control groups, but the differences were not statistically significant. FPR1 gene expression was higher in patients with HTLV-1 than in controls . Plasma AnxA1 levels were higher in the AS group than in the HAM/TSP group (p = 0.0045), and PVL was higher in patients with HAM/TSP than in AS individuals (p = 0.0162). The use of a combined ROC curve using Annexin 1 levels and proviral load significantly increased the sensitivity and specificity to predict progression to HAM/TSP .Our results suggest that AnxA1 may be dysregulated in HAM/TSP patients. Serological detection of AnxA1 in association with proviral load may provide a prognostic biomarker for HTLV-1-associated neurodegenerative disease.The online version contains supplementary material available at 10.1186/s12879-021-05917-y. Retroviridae, subfamily Orthoretrovirinae, genus Deltaretrovirus [+ T cell neoplasms, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic and progressive neurodegenerative disease [Human T-lymphotropic virus 1 (HTLV-1) is a member of the family trovirus . HTLV-1  disease , 4.HAM/TSP is a chronic, progressive, demyelinating disease that affects the spinal cord and brain white matter, leading to the onset of a severe clinical syndrome involving motor impairment of the lower limbs . The clitax gene, which is involved in transactivation of the interleukin-2 (IL-2) and IL-2 receptor genes, among others [Several studies have identified Tax regulatory protein as the main target of the immune response against HTLV-1, as this antigen is most efficiently recognized by cytotoxic T lymphocytes –9. HTLV-g others . Indiscrg others .Based on the recognition that a sustained systemic inflammatory response contributes to chronic neurodegenerative disorders, we hypothesized that the anti-inflammatory response may be dysfunctional in HAM/TSP patients. Among many anti-inflammatory molecules, annexin A1 (AnxA1) likely plays an important role in modulating neuroinflammation triggered by both resident glial cells as part of the innate immune system in the brain and circulating leukocytes that breach the blood–brain barrier . AnxA1, ANXA1 gene expression profiles and protein levels with the development of HAM/TSP.Thus, considering that HAM/TSP is a systemic immune disorder caused by Th1 cell activation and increased levels of proinflammatory cytokines, we hypothesized that AnxA1 is dysfunctional in this context, which may impact its neuroprotective immunomodulatory role. Therefore, the present work investigated the associations of A total of 57 individuals participated in this study and were divided into the following groups:Group 1 : 21 AS HTLV-1 carriers who were positive by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (qPCR).Group 2 (HAM/TSP): 16 HTLV-1 carriers who were by positive ELISA and real-time PCR with a clinically confirmed HAM/TSP diagnosis.Group 3 : 20 individuals negative for HTLV-1 and HTLV-2 by serological and molecular tests.The clinical classification of patients was performed by a neurologist at the Tropical Medicine Center (NMT) in Belém, State of Pará, Brazil, following the protocol of De Castro-Costa et al. . The maiRNA was extracted from whole blood cells using TRIzol® reagent  following the manufacturer’s protocol. RNA was quantified in the SpectraMax® i3 Multi-Mode Detection Platform, which uses a 24-well microplate containing 2 μL of elution buffer in one well as a blank and 2 μL of each sample in the other wells. The absorbance of the samples was read at a wavelength of 260 nm. Then, 20 ng of deoxyribonuclease I (DNase I)-treated RNA was used for the reverse transcription of messenger RNA (mRNA) into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit , following the manufacturer’s technical recommendations. The cDNA was stored at − 20 °C prior to use.-ΔΔCT analysis method. All assays showed the expected efficiency  with β-actin as the reference gene. The reactions were carried out in the StepOne PLUS Sequence Detector . The primer sequences are provided in Table cDNA was analyzed using real-time quantitative PCR (relative quantification (RQ) by the ΔΔCT method). The qPCR results for endogenous genes and targets were standardized to calculate the efficiency of the amplification reactions. Different concentrations of cDNA were tested . Reactions were performed in triplicate wells and analyzed simultaneously using the same cDNA (at different dilutions) with different probes to construct an efficiency curve and validate the 2eference . ANXA1, Plasma AnxA1 levels were measured by ELISA  with specific polyclonal anti-human AnxA1 antibodies. The assays were conducted according to the manufacturer’s recommendations.3, and the final results are presented as DNA proviral copies/mm3.Proviral load (PVL) was quantified by qPCR using three target sequences synthesized using the TaqMan® system  according to a previously described protocol . The resANXA1-expressing immunoinflammatory cells were compared among groups using the nonparametric Kruskal-Wallis test. Significant results in the Kruskal-Wallis test were subjected to multiple comparisons analysis by Dunn’s post test. Plasma AnxA1 levels and PVL were compared between the HAM/TSP and AS groups by the Mann-Whitney test. Receiver operating characteristic (ROC) curves were made to investigate diagnostic accuracy in the PVL, AnxA1 and PVL + AnxA1 tests in relation to sensitivity and specificity. The area under the ROC curve (AUC) represents the ability of the test to correctly classify participants with HAM/TSP and progression to disease. The AUC values vary between 1 (diagnosis correctness) and 0 (diagnosis error). The tests were performed using BioEstat 5.3 software [p < 0.05 were considered significant.Normality analysis of the sample distribution was performed using the Kolmogorov-Smirnov test. Target gene expression levels and the percentages of software  and the ANXA1, FPR1, FPR2 and FPR3 gene expression levels in the investigated groups showed lower ANXA1 mRNA levels in the controls than in the HTLV-1-infected individuals, but these differences were not significant. Among the infected patients, those with HAM/TSP expressed lower ANXA1 levels  in the HAM/TSP patients than the AS individuals  and PVL are important risk factors for the development of TSP/HAM [The host genotype (determined mainly by the human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptor (KIR) loci), quality of the cytotoxic T cell (CTL) response against HTLV-1-infected cells, T regulatory cell (Treg) frequency (forkhead box protein 3 (FOXP3) TSP/HAM –29. An i TSP/HAM , 31. AS  TSP/HAM , 33.ANXA1 and its receptors (FPRs) in peripheral blood cells. The results showed that AS individuals had higher ANXA1 mRNA levels, which might suggest that AnxA1 is an important factor controlling the inflammatory response that triggers HTLV-1-associated neurodegenerative disease. A previous study suggested that AnxA1 is a potential biomarker for the advanced stage of cell transformation, which is directly linked to the uncontrolled growth of infected cells, accumulation of genetic defects and development of symptoms of HTLV-1-associated diseases [Given the debate regarding the factors that affect HAM/TSP pathogenesis, in the present study, we investigated the potential association of AS HTLV-1 infection or infection with HAM/TSP symptoms and the gene expression of diseases . HoweverANXA1 mRNA transcription and HTLV-1 infection, a relationship was observed between infection and FPR mRNA levels. The interactions of these receptors with other ligands (both anti- and proinflammatory) enable the regulation of several pathological conditions, including tumorigenesis [In addition to the association between igenesis , 36, infigenesis  and someigenesis .FPR1 mRNA levels than HTLV-1-infected AS subjects and controls. Although these data suggest a relationship between FPR1 and the pathogenesis of HAM/TSP in HTLV-1-infected patients, the evidence remains preliminary, and further mechanistic studies are necessary. Additional studies must be performed to confirm whether the higher viral load somehow triggers increased FPR1 levels through another receptor or if the lower AnxA1 levels induce FPR1 expression.We observed that the HTLV-1 carriers with HAM/TSP symptoms had higher We evaluated serum AnxA1 levels to assess whether the mRNA expression profile reflects free annexin levels in plasma. The quantification showed high AnxA1 plasma levels in AS patients than in HAM/TSP patients, inverse to the PVL results. Although a direct correlation between annexin and PVL (data not shown) was not observed in the present study, AnxA1 seems to be a potential new biomarker that, in addition to PVL, could contribute to the follow-up of HTLV-1-infected subjects, but longitudinal data are required among people with asymptomatic HTLV-1 infection to confirm our hypothesis.Although the study was cross-sectional and does not do a mechanistic analysis, the results seem to suggest an anti-inflammatory role of AnxA1 in HTLV-1 infection, what if confirmed, could be neuroprotective, since patients with higher levels of this protein could better control the development of HAM/TSP. Thus, the possible antiviral activity of AnxA1 against HTLV-1 must be more thoroughly investigated.In conclusion, taken together the quantification of Anexin and proviral load quantification, could be an important laboratory tool to aid in the diagnosis of HAM/TSP. Even though the sample used herein was small, which is always a big problem in the study of HTLV-1, the periodic clinical assessment of HTLV-1 carriers associated with the quantitative analysis of Annexin and proviral load, as used in the present study, seems to be of relevance in the prognostic prediction of the progression of asymptomatic infection to HAM/TSP, considering the high sensitivity and specificity values found. However, we highlight the need of population-based and longitudinal cohort studies aiming to define cut-offs for serum AnxA1 levels among different groups as differential diagnostic criteria and so to confirm our hypothesis.Additional file 1.


Toll-like-receptor (TLR) family members were detected in the central nervous system (CNS). TLR occurrence was noticed and widely described in glioblastomamultiforme (GBM) cells. After ligand attachment, TLR-4 reorients domains and dimerizes, activates an intracellular cascade, and promotes further cytoplasmatic signaling. There is evidence pointing at a strong relation between TLR-4 signaling and micro ribonucleic acid (miRNA) expression. The TLR-4/miRNA interplay changes typical signaling and encourages them to be a target for modern immunotherapy. TLR-4 agonists initiate signaling and promote programmed death ligand-1 (PD-1L) expression. Most of those molecules are intensively expressed in the GBM microenvironment, resulting in the autocrine induction of regional immunosuppression. Another potential target for immunotreatment is connected with limited TLR-4 signaling that promotes Wnt/DKK-3/claudine-5 signaling, resulting in a limitation of GBM invasiveness. Interestingly, TLR-4 expression results in bordering proliferative trends in cancer stem cells (CSC) and GBM. All of these potential targets could bring new hope for patients suffering from this incurable disease. Clinical trials concerning TLR-4 signaling inhibition/promotion in many cancers are recruiting patients. There is still a lot to do in the field of GBM immunotherapy. Various toll-like-receptor (TLR) family members are detected in the central nervous system (CNS). They are mainly expressed in neurons and glial structures, where they play a role in recognizing unfamiliar molecules, some postapoptotic antigens consequently control repair processes and modulate inflammatory action .Furthermore, TLR occurrence was noticed and widely described in glioblastoma-multiforme (GBM) mature cell fractions . StudiesThe TLR family is a group of ten receptors (TLR-1–TLR-10) characterized by the detection of a particular pattern of micro-organisms (pathogen-associated molecular patterns (PAMPs)), which are invariable for most pathogens and not present in mammalian organisms. TLR receptors are also sensitive to particles secreted during necrosis and cell death called danger-associated molecular patterns (DAMPs) ,21,22,23Toll receptors (TRs) were detected in drosophila embryos by Hashimoto et al. (1988) ,36. HoffTo bind lipopolysaccharides, TLR-4 requires co-operating molecule myeloid differentiation 2 molecule (MD-2), extracellularly stabilizing the ligand during activation. After antigen ligation, the receptor dimerizes and activates TIR domains; to properly signal, TLRs require MyD88, an essential adaptor activated during the first stages after pattern ligation to promote an immune response. Activation of the transmembrane receptoral structure of TLR-4 transfers signaling in two major ways, the TIRAP–MyD88-dependent pathway controlling primary nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promotion, and the combined production of inflammatory cytokines. Activation of MyD88 results in IL-1R-associated kinase 1/TNFR-associated factor 6 (IRAK-1/TRAF-6) induction with collateral ubiquitination, and TRAF-6 promotes Transforming growth factor-beta-activated kinase 1 (TAK-1). Activation of TAK-1 results in the formation and promotion of the IKK/NF-κB complex. Mitogen-activated protein kinases-c-Jun. N-terminal kinase (MAPK-JNK) and extracellular signaling-regulated kinase (ERK1/2) kinases are also activated, which indicates activated protein-1 (Ap-1). Downsignaling promotes transcription factors, such as activated Ap1, stabilizing many genes with regulatory properties during inflammation ,46,47.The second toll-like receptor adaptor molecule-1-translocating chain-associating membrane (TRIF-TRAM) signaling pathway (MyD88-independent) activates interferon regulatory factor-3 (IRF-3). TRAM signaling, through TRIF activation, results in TRAF-3 and TRAF-6 promotion ; RIP is Numerous signaling pathways induce carcinogenesis, oncogenic transformation, and invasiveness of cancer cells. Interestingly, some of them promote NF-κB expression. NF-κB, as an important transcription factor, is involved in various cellular processes, including migration, proliferation, and survival. Inflammatory cytokines, PAMP and DAMP, could trigger further pro- NF-κB signaling throughout the activation of TNFRs and TLRs ,54,55,56Developments made in neuroimmunology and tumor biology revealed interesting properties of TLR. Activation and TLR-4 downsignaling seem to play an interesting role in the promotion of glioma growth and invasion. On the other hand, some studies discovered that, in some special conditions, they could take part in the anticancer response. The release of HMGB1 from GBM dead cells during radio and chemotherapy stimulates the TLR4 pathway. It leads directly to Dendritic Cell maturation and efficient tumor antigen presentation leading to GBM regression in animal models ,75,76,77miRNAs represent a group of short noncoding strands of RNA with length oscillating 20–22 nucleotides each. Around 30% of all genes are controlled and regulated by miRNAs. Molecules bind to the targeted mRNA with the 3′ region called the untranslated (UTR) monitoring the expression of particular genes through degradation or translation breakdown. As a result, miRNAs are engaged in various biological processes, such as homeostasis, growth, differentiation, immune activation, apoptosis, and stress response. Otherwise, they modulate the immune response to pathogen invasion throughout the TLR signaling pathway . Additionally, miRNA activity is similarly detected in normal healthy tissue in chronic diseases and cancers ,85,86,87Levels of miRNAs are significantly higher in GBM tissue and GBM cell lines ,90,91,92Immune checkpoints (ICs) are molecules with the ability to modulate T cells. Coinhibitory and costimulatory properties ensure a balanced immune response. ICs protect autoimmunity and optimize the adequate response of the immune system. When carcinogenesis occurs, IC signaling becomes a route of immune escape, leading to aggressive tumor invasion. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) was the first well-described IC molecule expressed on CD-8+ and CD-4+ T cells. CTLA-4 binds to the corresponding B7-1 (CD80) ligand on antigen-presenting cells (APCs). Interaction between both results in the inhibition of T cells. In GBM, CTLA-4 expression on CD8+ and CD4+ has a significant correlation with poor outcome and invasion of brain tissue. TLR signaling promotes the overexpression of CD80, enhancing the presentation of antigens. It strengthens CTLR-4-induced T-cell anergy and GBM expansion ,113,114.The PD-1/PD-1L axis is another IC pathway. PD-1 and PD-1L are the most potent immunomodulatory proteins. PD-1L was described in 1999 by the Dong study team as a B7-H1 molecule , which rA study performed by Beswick et al.  discusseGlioblastoma, as a highly aggressive neoplasm, activates or modulates many pathways promoting gliomagenesis. One of those downsignaling instances concerns wingless signaling (Wnt\claudines axis) occurring as a crucial controller of cell–cell interplay, and as a regulator of migration. Any abnormalities in Wnt lead to evolutive effects. Aberrations in Wnt balance between stimulation and inhibition usually promote carcinogenesis. The Wnt complex represents an extended network consisting of various components, crosstalk interactions, and multiple regulatory stages. Wnt usually starts with receptor and coreceptor stimulation. Receptors, such as low-density lipoprotein receptor-related protein 6 (LRP6), protein Tyr kinase 7 receptor (PTK7), Tyr kinase-like orphan receptor (ROR), skeletal muscle Tyr kinase receptor (MUSK), and Tyr kinase receptor (RYK), are treated as Wnt-related. The main regulation of receptors is intracellularly processed by phosphorylating kinases. Extracellular regulation takes place through active agonists, such as norrins, the R-spondin family, and antagonists WNT inhibitory factor (WIF), secreted frizzled-related protein (SFRP), sclerostin, and Dickkopf-related protein 1 (DKK1) ,136,137.Wnt pathway activation is connected with other molecules called claudins. Claudins are a family of proteins that determine tight junctions (TJs) . Claudin-5 is mainly expressed on the lung epithelium and brain tissue. Claudin-5, built-in TJs, bands in CNS endothelial cells. TJs create direct intercellular adhesion reducing cell-to-cell distance. In effect, they form the real connection between particular endothelial cells, strengthen the integrity of vessels, and control the diffusion of different ions and solutions. CNS embryonal development changes the permeability ratio. An immature form of the CNS vasculature, leaky blood vessels with characteristic fenestrations, are replaced by more integrating TJs. Claudin-5 contributes to the function of the blood barrier ,141,142.Dickkopf-related protein-3 (DKK-3), as was mentioned above, represents a group of proteins with strong suppressive features able to halt Wnt signaling. Moreover, DKK-3 seems to be an effective inhibitor of tumor-cell growth molecules. Casili et al. performed a study revealing DKK-3 to have a regulating influence on the Wnt pathway, resulting in increased caspase-3-dependent apoptosis .Caspase-3, a representative of the caspase family, is described as an effector caspase. Its properties, such as activation by many apoptotic inducers, result in rife activity leading to complete apoptosis in many cell lines. Overexpression of caspase-3 results in an apoptotic GBM phenotype, confirmed in patient samples. Studies suggested that a higher apoptotic ratio is associated with longer PFS in treating for GBM. Sensitivity to temozolomide (TMZ) treatment is also correlated with caspase-3 expression, and higher levels of caspase-3 promote TMZ efficiency. Interestingly, caspase-9, a less apoptotic molecule, is downexpressed after Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK-3) upregulation, usually increasing after TLR-4 stimulation. Highly invasive cancer-cell lines express caspase-9 more intensively ,150,151.Apoptosis, as a canonical biological interaction, regulates the subsistence of organisms and limits the expansion of GBM. A study connected the expression of DKK-3 and claudine-5 in healthy brain tissue, compared with the downexpression of both in GBM patients. Significantly lower levels of DKK-3 were observed in many types of neoplasms concerning prostate cancer. The absence of TLR-4 inhibits the growth of glioblastoma lines. The expression of TLR-4 interferes with Wnt downsignaling, promoting carcinogenesis. Additionally, the lack of TLR-4 maintains claudin-5 and DKK-3 proapoptotic properties, resulting in the limitation of tumor expansion. To sum up, the downexpression of TLR-4 promotes Wnt/DKK-3/claudin-5 signaling, which limits GBM invasiveness ,152,153.A study performed by Alvarado et al. revealed a lack of TLR-4 occurrence on cancer stem cells (CSCs), the most invasive and most aggressive subpopulations of GBM. In opposition to CSC mature GBM cells and non-CSCs, it demonstrated TLR-4 expression and a response to agonist ligation. CSCs are responsible for therapeutic ineffectiveness and tumor-mass progression. External stimulation, such as necrosis, hypoxia, uncontrolled proliferation, and acidic stress, characteristics of a tumor microenvironment, generate unfavorable conditions. Thanks to developed adaptive mechanisms, CSCs maintain the balance between self-restoration and differentiation in hostile habitats. Owing to limited TLR-4 expression, CSCs can survive in a malevolent environment. Experiments conducted by a study team explained the processes of suppressing the CSC subpopulation, in which TLR-4 signaling played a key role. The main discovery in those papers indicated intensive TLR-4 downsignaling, halting CSC expansion by reducing the activity of retinoblastoma binding protein 5 (RBBP5), which is naturally increased in this subpopulation. Another important thing to understand in the underlying processes in CSCs is that RBBP-5 promotes cardinal stem-cell transcription initiators, essential to achieve invasiveness and self-restoration. TLR-4 expression and downsignaling inhibit RBBP-5 by the phosphorylation of TBK-1. TLR signaling borders proliferative trends in CSC and GBM ,159,160.Recent studies concerning bladder tumors, prostate cancer, and colorectal cancer pointed at TLR signaling as a tumorigenic and tumor-progression factor ,163,164.Exposition of TLR-4 on LPS in in vitro studies resulted in tumor-cell-line progression. The promotion and proliferation of the U87 and U118 lines were detected, and an increase in invasion was realized in U87 . StimulaProsaposin (PSAP) conserved glycoprotein acts as a neurotropic factor in the glioblastoma environment. High levels of PSAP were detected in glioblastoma patients and were associated with unfavorable outcome ,170,171.LPS was examined as affecting GSCs, and mature glioma cells in the study performed by Han et al. rat model has proven that LPS stimulation prolonged survival time significantly in the observed glioma-cohort . InteresTLR-4 is expressed on the surface of the cell membrane and occurs on endosomes. Moreover, the receptor has the ability to dependently signal to MyD88 and TRIF complexes. Those characteristic features are the example of evolutionary controlled carefulness in precise antigen-detection and downsignaling-promotion mechanisms. TLR-4 coreceptor myeloid differentiation factor 2 (MD-2) has an appropriate pocket that binds the corresponding ligands. Immunotherapy disrupting TLR-4 and MD-2 binding or blocking their interplay was explored ,179,180.https://clinicaltrials.gov/ct2/show/NCT03782415).The macrophage migration inhibitory factor (MIF) cytokine is treated as a potent cytokine initiating immunity and inflammation processes ,190,191.https://clinicaltrials.gov/ct2/results?cond=ibudilast&term=&cntry=&state=&city=&dist=).Ibudilast is an agent that is able to diffuse the blood–brain barrier (BBB), developing antitumor activity by inhibiting MIF and phosphodiesterase-4 (PDE-4), also reducing TLR-4 downsignaling with pro-oncogenic properties. Action leads to excessive apoptosis and the limitation of cell proliferation in GBM ,197,198.Another clinical application of TLR-4 signaling concerns the antiglioma HSP vaccination. HSP, as a part of the heat shock protein peptide complex (HSSPC) , is charhttps://www.clinicaltrials.gov)  .TLR-4 signaling pathways and their wide spectrum of interactions seem to be promising targets for immunotherapy. Multimodal interplay and a variety of downsignaling encourage TLR-4 to be further considered as a potential aim for immune agents. Clinical trials contesting TLR-4 agonists and inhibitors are recruiting patients with various cancers. TLR-4-related treatment is being gradually introduced for GBM patients. There is still much to do in the field of effective immunotherapy.


Here, we report that the imp1 mutant is defective in the BR-receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Our set of data reveals that BR signaling transcriptionally and post-translationally represses the accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that BR signaling controls PIN-LIKES (PILS)-dependent nuclear abundance of auxin. We performed a forward genetic screen for   •Impaired BR perception enhances PILS5 overexpression phenotypes•BR signaling increases PILS protein turnover•BR signaling defines PILS-dependent nuclear abundance and signaling of auxin•PILS-dependent BR-auxin crosstalk affects organ growth Sun et al. reveal that BR signaling limits the accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. This alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development. The phytohormone auxin is a key regulator of plant growth and development. Indole-3-acetic acid (IAA), the most abundant endogenous auxin, is perceived by the nuclear F-Box protein TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and its close homologs . Auxin fMost IAA is synthesized in a two-step biosynthetic route, providing auxin in various tissues , 6, 7. ABesides local auxin metabolism, intercellular auxin transport is crucial to define auxin signaling gradients and maxima within plant tissues , 15. Thein silico screen, we have previously identified the PIN-LIKES (PILS) protein family of auxin transport facilitators, which resembles the predicted topology of PIN proteins  644 to serine [S]) in the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1)  ) , 26. In henotype A, proposbri1imp1 mutant independently of PILS5OE, we outcrossed the bri1imp1 mutation to Col-0 wild-type twice and revealed that the bri1imp1 mutant showed a similar reduction in the dark-grown hypocotyl length as PILS5OE, confirming a strong additive effect in imp1;PILS5OE mutant combination   A–S1D. ThBRI1 enhances PILS5-related phenotypes, we expressed pBRI1::BRI1-GFP in the imp1;PILS5OE mutant background. BRI1-GFP expression indeed complemented the imp1;PILS5OE mutant, resembling PILS5OE phenotypes  and BRRE-element (BR-response element) . After spinning down for 45 min at 4°C with 35,000 x g, the protein concentration was assessed using the Bradford method. Membranes were probed with a 1:5,000 dilution of GFP antibody  or 1:1,000 dilution of RFP antibody . As loading control, membranes were probe with a 1:2,000 dilution of Actin antibody . Horseradish peroxidase couple goat anti-mouse  or anti-rabbit  were used as secondary antibodies. The signals were detected and quantified using a Fusion Solo S (Vilber). Samples were used for three independent technical replicates.Five-day-old seedlings were transferred for 5 h either on plates with DMSO or 50 nM BL. Root material was ground to fine powder in liquid nitrogen and solubilized with extraction buffer [25 mM Tris, pH 7.5, 10 mM MgCl35S::GFP-PILS2, 35S::GFP-PILS3, 35S::PILS5-GFP, 35S::PILS6-GFP, and pDR5::GFP/RFP seedlings in Col-0 or mutant backgrounds were imaged with a Leica SP5 (Leica). Fluorescence signals for GFP , mRFP1  and propidium iodide (PI) staining  were detected with 20 × (water immersion) or 63 × (water immersion) objective. To image DII-VENUS and mDII-VENUS, Leica TCS SP8 equipped with a white laser was used, allowing us to separate GFP and YFP fluorophores. The fluorescence signal intensity  of the presented markers was quantified on raw images using the Leica software.5- or 6-d-old https://www.mathworks.com/help/matlab/ref/dde23.html) was used to obtain direct solutions of DDEs. All simulations were performed until 300 steps to account for multiple oscillations. Overall, our model incorporates BR and auxin signaling pathways [The dynamics of all components built in the model was simulated using delayed differential equations (DDEs) implemented in MATLAB Inc. The MATLAB-derived dde23 solver  with delay τ,αBR is BR production rate and kBR is rate of repression mediated by BZRD and δBR is a BR turnover rate.BR synthesis is inhibited by its own signaling, which is implemented by BZR dimers  is restricted by PILS auxin transport facilitators , 24,. α denotes the ARF-dependent transcription rate times amount of ARF homodimers (ARFD) and δaux is an auxin-dependent degradation rate. kARF is promoter association constant of ARFD. Next, ARF monomers (ARF) are described by the following mathematical equation,The dynamics of auxin signaling repressors (AUX/IAA)  are modeαARF and δARF are basal production and degradation rates of ARF monomer and θ represents AUX/IAA-dependent ARF sequestering that leads to negative feedback on AUX/IAA levels. γdAF and γaAF stand for dissociation and association rates of ARF dimers (ARFD) that follow the formula,Finally, PILS protein levels are coupled to BR and auxin signaling pathway through transcription and degradation and follow this formula,bP and δPIL are basal production and degradation rates of PILS proteins, respectively. αpAF denotes the ARF-dependent transcription rate and δPBR is an BR-dependent degradation rate of PILS. kAF is association constant of ARFD to PILS promoter and kBZ is a rate of repression mediated by BZR dimers.where αPILS transcription, PILS protein levels as well as auxin response measurements . PILS protein reporter  and estimating basal production from experiments  was modeled by reducing AUX/IAA-dependent sequestering of ARFs in Equation 8 such that parameter θ was inversely scaled with BIN2 levels. The reference set of experimentally fitted model parameters is shown below:BL treatments were modeled by adding constant external source of BR to right hand side of Equation 1; Φ = 0.05 μM. Estimated parameters for  mutants I and 3K Finally, key model parameters were varied ± 25% from the estimated values to test model robustness against intrinsic and extrinsic noise B. We couD and BZRD, amplitudes and periods were calculated, using peak find function (MATLAB Inc.) and subtracted to estimate phase differences between two oscillators. The phase differences were plotted, using violin plot function in MATLAB  together with probability density distributions performed with histfit function (https://www.mathworks.com/help/stats/histfit.html). The large variation in phases (broader distribution) indicates that two oscillatory pathways are out-of-sync, whereas sharper distributions reflect near-perfect synchrony between two signaling pathways.For each time-dependent solution of ARFOne of key findings from our model predictions was that PILS auxin transporters mediate the coupling between BR and auxin signaling pathways, presumably by synchronizing coupled oscillators and maintaining near-constant phase (phase-locking) between oscillations . Next, whttp://rsb.info.nih.gov/ij/) software. The root meristem length, width, and the fluorescence signal intensity  of the presented markers was quantified on raw images using the Leica software (LAS AF Lite). The western blot signals were quantified using a Fusion Solo S (Vilber).Hypocotyl and root lengths and GUS intensity  were measured and quantified with the ImageJ (http://www.graphpad.com) software. The significance of the data was evaluated using the Student’s t test in the case of two columns comparisons. One-way ANOVA followed by Tukey’s test was performed in the case of the multiple columns’ comparisons procedure. Two-way ANOVA followed by Bonferroni post-tests was carried out to compare two different genotypes at different treatments.Means and standard errors were calculated and the statistical significance was evaluated using the Graph Pad Prism5 (Representative data are shown throughout the text. All experiments have been performed in at least three replications.This study did not generate/analyze datasets/code.


Plants growing in any particular geographical location are exposed to variable and diverse environmental conditions throughout their lifespan. The multifactorial environmental pressure resulted into evolution of plant adaptation and survival strategies requiring ability to integrate multiple signals that combine to yield specific responses. These adaptive responses enable plants to maintain their growth and development while acquiring tolerance to a variety of environmental conditions. An essential signaling cascade that incorporates a wide range of exogenous as well as endogenous stimuli is multistep phosphorelay (MSP). MSP mediates the signaling of essential plant hormones that balance growth, development, and environmental adaptation. Nevertheless, the mechanisms by which specific signals are recognized by a commonly-occurring pathway are not yet clearly understood. Here we summarize our knowledge on the latest model of multistep phosphorelay signaling in plants and the molecular mechanisms underlying the integration of multiple inputs including both hormonal  and environmental (light and temperature) signals into a common pathway. We provide an overview of abiotic stress responses mediated via MSP signaling that are both hormone-dependent and independent. We highlight the mutual interactions of key players such as sensor kinases of various substrate specificities including their downstream targets. These constitute a tightly interconnected signaling network, enabling timely adaptation by the plant to an ever-changing environment. Finally, we propose possible future directions in stress-oriented research on MSP signaling and highlight its potential importance for targeted crop breeding. Under natural conditions, plants are need to continuously maintain a balance between growth and defense or adaptive responses that are dictated by the severity, duration, and developmental timing at which any particular stress occurs . The essETHYLENE RESPONSE FACTOR 1 (ERF1) was gradually induced by drought stress treatment over 12 h (reaching a maximum after 1 h), and this plays a positive role in drought, salt, and heat stress tolerance via stress-specific gene regulation  action have revealed that cytokinin modulates levels of stress-related genes/proteins . On the gulation .Arabidopsis thaliana).All these findings (and many others described below) show how important plant hormone signaling (including the MSP) and metabolism are during early stress responses. In the next few sections, we attempt to summarize recent observations on the biological relevance of MSP-mediated stress adaptation strategies in plants (mostly in RESPONSE REGULATORS (A-ARRs), the cytokinin primary-response genes, which also function as negative feedback regulators of the signaling pathway (APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene family, mediate a large fraction of the transcriptional response to cytokinins, affecting a set of cytokinin-responsive genes that largely overlaps with targets of type-B ARRs , located both at the plasma membrane and the endoplasmic reticulum . Recentl pathway . In tande-B ARRs . For morcis-acting GCC box and DRE elements depending on whether the stress conditions are abiotic or biotic  and ETHYLENE RESPONSE SENSOR 1 (ERS1) with a functional HK domain, and the HK-like sensor Ser/Thr kinases ETR2, ERS2, and ETHYLENE INSENSITIVE 4 (EIN4), that make up subfamily 2 . Ethylenarr2 mutant is less sensitive to treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) probably due to a positive effect of ARR2 on the expression of ERF1, an ethylene-responsive member of the ERF/AP2 transcription factor family  and the UV-B RESISTANCE 8 (UVR8) family proteins affect plant behavior in response to both quantity and quality of light. The individual signaling branches mediating responses to a wide range of wavelengths as well as their interplay has been extensively studied e.g., . Since lCOP1–SPA E3 ubiquitin ligase  and far-red (FR) light. Phytochromes are synthesized as light-insensitive apoproteins and the functional, i.e., light photoswitchable holoproteins (phys) are assembled following binding with the light-sensitive, linear tetrapyrrole chromophore cofactor phytochromobilin, that is imported to the cytosol from plastids . Upon abn ligase . PIFs con ligase . COP1 inn ligase . More deOne of the first pieces of evidence for direct crosstalk between MSP and light signaling was presented by CKI1 was found to be under the control of phyA, probably via PHY-INTERACTING FACTOR 1 (PIF1) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). Plants defective in phyA and HEME OXYGENASE1 (HO1), whose product is involved in the production of phytochromobilin, show misregulation of CKI1 expression, associated with attenuation of MSP responsiveness to cytokinins and occurrence of cytokinin-related phenotypes  controls the activity of (MSP) signaling pathway by transcriptional regulation of the constitutively active HK, thus controlling MSP sensitivity to its primary signal, cytokinin  that do not mediate hormone responses  kinase . Under uproteins . The abiproteins .2+ levels  and cytoskeletal rearrangement  . SnRK2s pression .Since ABA interacts tightly with MSP signaling during the abiotic stress response, the mutual crosstalk might occur at several levels including regulation of phosphorylation of individual signaling components as well as regulation of dynamic chromatin state . These iAltogether, the MSP pathway is not just the cytokinin signaling transduction system, but it acts as a signaling hub, integrating both intrinsic  regulation with that from environmental signals, including light, osmoregulation, and temperature . The impStress, in general, affects the metabolism of cytokinins, ABA, and ethylene, which in turn interact with specific kinases to regulate many stress-related biological processes ranging from regulation of shoot growth under stress to stomata movement. Drought results in the reduction of cytokinin content by modulating cytokinin metabolism and stimulating ABA and ethylene contents in a wide range of terrestrial plants e.g., . Even th2 uptake and water loss by transpiration  which is responsible for removing sodium ions from root xylem. Cytokinins were also shown to regulate salt stress−induced expression of the type−A response regulator ARR5 predominantly via ARR1 and ARR12, revealing the action of particular MSP components in the roots to control sodium accumulation in the shoots  and the type-A ARR genes ARR5, ARR7, and ARR15. This ethylene-mediated repression was supposed to be mediated by direct binding of EIN3 to the promoters of type-A ARRs, thus potentially representing another mechanistic link between canonical ethylene signaling and MSP during plant desiccation , NAC DOMAIN CONTAINING PROTEIN (ANAC), and DRE-BINDING PROTEIN 2A (DREB2A) transcription factors and their downstream genes may explain the increased stress tolerance of AHK1 overexpressing plants  is an important physiological adaptation trait of plants to drought. The recent findings of Furthermore, various types of abiotic stresses including cold, osmotic stress, salt, and drought differentially regulate the expression of several individual MSP components . As mentetr1-6 and etr1-7 mutants have a higher germination rate than wild-type seeds in the dark or following FR illumination (that inhibits seed germination in the WT) indicating a negative role for ETR1 in the control of germination  that further activate downstream ABA-unresponsive and ABA-activated SnRK2s . Fast reTemperature is another fundamental environmental factor, with 10–30°C being optimum for the growth of most higher plants . TemperaArabidopsis -mediated cold acclimation pathway. Based on the hypersensitive response of ahk2, ahk3, and arr7 mutants, as well as the insensitivity of ARR7 overexpression line to ABA, the authors propose that the MSP-mediated cold response is mediated via negative regulation of ABA signaling by AHK/ARRs, thus following the general scheme of negative cytokinin/ABA interaction as in the drought response described above  regulatory mechanisms, mediating the AHK2/3-dependent cold response , , , 2017. Rresponse .Metaphorically, sensor kinases serve as guards at the gate to the cell or nucleus, enabling the perception of various environmental signals. Besides HKs, acting as an integral part of canonical MSP signaling, other sensor kinases integrated into MSP signaling are phytochromes  similar to phyB mutants, demonstrating that circadian light input is modulated by the interaction of phyB with ARR3 and ARR4. Considering the absence of any correlation between sensitivity to cytokinins in higher-order arr mutants and the period phenotype, as well as the distinct (concentration-dependent) effects of exogenous cytokinins, ARR3 and ARR4 seem to control both the length of the period (independent of phyB) and the phase of the period (through phyB) independently of cytokinins (ARRs expression responds to temperature fluctuations independently of cytokinin levels (see above), we may say that type-A ARRs could represent an intersection for light and temperature signaling to fine-tune photomorphogenesis under adverse environmental conditions  in the control over context-dependent chromatin accessibility , the welThe conclusions and implications we summarize above point toward a number of important questions. How is the combination of individual inputs translated into the final MSP signaling output? How is signaling fidelity maintained? In other words, how does the plant recognize and discern the particular origin of the signal, given that a number of sensory kinases recognizing the different type of signals result in phosphorylation of AHPs, and act as a signaling hub within the pathway? Is there any variability in the sensitivity of MSP signaling to individual types of MSP inputs associated with ecotype-specific adaptation to the site of its origin? And finally, what are the mechanisms acting downstream of MSP signaling in the stress response, either independently or in concert with other signaling pathways?Future studies aimed at potential modification of the MSP signaling output at the level of the regulated genes and characterization of the underlying molecular mechanisms will be necessary to answer some of these questions. Studying the existing pool of natural genetic variability in MSP signaling affecting the responsiveness of the pathway to various inputs might provide important evidence on the potential role of MSP signaling and its downstream targets. They would also go a long way in shedding light on the ability of plants to successfully face environmental stresses which are after all an everyday reality of plant life. In combination with tools for targeted genome editing of plant genomes that have become available recently , this tyJS, KN, and JH performed the literature search. KN and JS created the figures. JS, KN, RV, and JH wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Drosophila eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aβ42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aβ42-mediated neurodegeneration.Alzheimer's disease  is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a  Abnormal cleavage of the amyloid precursor protein (APP) results in 42 amino acid long polypeptides hereafter referred to as Aβ42 peptides  is a progressive neurodegenerative disorder that affects the aging population and is predicted to continually increase in prevalence and incidence in the United States  to TNF receptors, named Wengen and Grindelwald in flies , the Drosophila JNKK that phosphorylates basket (bsk), the Drosophila JNK . The transcription factor Jun translocates to the nucleus to induce target genes of the JNK pathway , which is a dual-specificity phosphatase that negatively regulates bsk and thereby forms a negative feedback loop  and head involution defective (hid), as well as caspase-independent mechanisms  signaling pathway is implicated in Aβ42-mediated neurodegeneration BSC782/+, which rescued the Aβ42-mediated neurodegeneration phenotype. This deficiency uncovers 10 genes including the hpo gene. Further testing with the candidate genes uncovered by Df(2R)BSC782 revealed hpo as the causal genetic modifier of the neurodegeneration phenotype of Aβ42 overexpression. This suggested that hpo and other components of the Hippo signaling pathway may impact the Aβ42-mediated neurodegeneration phenotype. Here we report that the Hippo pathway affects Aβ42-mediated neurodegeneration phenotypes as hyperactivation of Hippo signaling leads to enhancement of Aβ42 toxicity, whereas downregulation of Hippo signaling rescues Aβ42-mediated neurodegeneration phenotype. Previously, we had reported that Aβ42 induced neuronal apoptosis via activation of a JNK–caspase-dependent pathway. Recently, JNK and Hippo pathway were shown to interact in several contexts, which prompted us to study if JNK–Hippo interactions affected the Aβ42-mediated neurodegeneration phenotype. Here we report that misexpression of Aβ42 induces JNK signaling, which in turn, induces Hippo signaling by blocking Yki activation. Activation of Hippo signaling in Aβ42-mediated neurodegeneration activates puc-lacZ, a reporter of JNK signaling. Here we present evidences to support a role for a positive feedback loop between JNK and Hippo signaling pathways that promotes Aβ42-mediated neurodegeneration in the Drosophila eye.In a genetic modifier screen, we identified a deficiency, http://flybase.bio.indiana.edu). Fly stocks used in this study were: GMR-Gal4 , UAS-yki   BSC782/+, a deficiency, which is located on the right arm of the second chromosome, and uncovers βTub56D, par-1, CG16926, CG7744, CG15120, mei-W68, oseg6, TBCB, rep, and hpo genes (listed in Flybase). For wild-type control, we used the Canton-S stock of D. melanogaster in this study. Fly stocks were maintained at 25°C on the regular cornmeal, yeast, molasses food medium.All fly stocks used in this study are listed at FlyBase , fixed in 4% paraformaldehyde in PBS for 20 min, and washed in PBS. We stained the tissue with a combination of antibodies following a previously published protocol  assays using a cell death detection kit from Roche Diagnostics. TUNEL assays allow identification of the cells undergoing cell death where the cleavage of double- and single-stranded DNA is labeled by a fluorescent tag (TMR Red)  adult eyes from Canton-S (wild-type), GMR> Aβ42, GMR> Aβ42+hpo following standardized protocols , Lane 5-GMR> Aβ42+hpoRNAi (loss-of-function). We incubated the blots with the Phospho SAPK/JNK (81E11)  and after appropriate washes incubated with horseradish peroxidase conjugated goat anti–rabbit IgG  secondary antibody. Signal was detected using super signal chemiluminescence substrate (Pierce). We captured the images using the BioSpectrum® 500 Imaging System and analyzed the blot images and band intensity. We used Microsoft Excel 2017 software for statistical analysis. We calculated the p-values using the Student two-tailed t-test. The error bars represent standard deviation from means.Protein samples were prepared from  showed highly reduced adult eye phenotype, The wild-type eye imaginal disc  developsDf(2R)BSC782, which in transheterozygous combination (Df(2R)BSC782/+) rescues the GMR> Aβ42-mediated neurodegeneration phenotype both in the eye imaginal disc BSC782 deficiency is located on the right arm of the second chromosome and uncovers 10 genes including hippo (hpo), a member of highly conserved Hippo growth regulatory pathway (Df(2R)BSC782 (Drosophila eye. Misexpression of hpo (GMR>hpo) results in the loss of photoreceptor neurons on the posterior margin of the eye imaginal disc  enhances neuronal loss and results in a stronger neurodegeneration phenotype in the eye imaginal disc  that results in mild overgrowth both in the eye disc  shows a strong rescue of Aβ42-mediated neurodegeneration both in the eye imaginal disc BSC782  using ganal disc , resultinal disc . As compnal disc , misexprnal disc  and the eye disc  and in tnal disc  and the hpo can rescue Aβ42-mediated neurodegeneration, we therefore tested the effects of modulation of Hippo signaling on Aβ42 accumulation. We used the 6E10 antibody that specifically detects the Aβ42 polypeptide. We observed robust Aβ42 accumulation in the eye imaginal discs from GMR> Aβ42  results in a strong suppression of the diap1-4.3-GFP reporter expression in the GMR>Aβ42 eye imaginal disc  results in 2-fold increase in cell death  background, suggesting increased JNK activity  results in a strong upregulation of puc-lacZ reporter expression  results in downregulation of puc-lacZ reporter expression  shows robust induction of puc-lacZ reporter in GMR expression domain  did not induce high levels of puc-lacZ . This resulted in stronger neurodegeneration in the eye imaginal disc (hpo and dominant negative bsk (GMR> Aβ42+hpo+bskDN). This also resulted in strong neurodegeneration both in the eye imaginal disc , we activated JNK signaling and blocked Hpo signaling at the same time by misexpression of activated nal disc  and the nal disc  as companal disc  as well nal disc . These fnal disc  as well nal disc . These anal disc .AD, an age-related progressive neurodegenerative disorder, manifests by progressive neuronal loss, brain atrophy, and cognitive impairments   BSC782/+, which can rescue Aβ42-mediated neurodegeneration. This deficiency uncovers hpo and other genes   (Moran et al., vice versa, it does not rescue neurodegenerative phenotypes both in the third instar eye-antennal imaginal disc and the adult eye (We previously reported the neuroprotective effects of the apical basal polarity gene dult eye . This obdult eye , 6 that dult eye . Based odult eye . There idult eye . We repodult eye . Both ofA growing body of epidemiological evidence and molecular investigations has shown some interesting links between cancer and AD (Battaglia et al., All datasets generated for this study are included in the article/supplementary material.MI and MT performed the experiments and data analysis. AdS, OP, NG, MR, and PD performed the experiments. MK-S performed data analysis and manuscript writing. AS developed the concept, performed data analysis and manuscript writing.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.