C. elegans vulva is an excellent model for the study of developmental biology and cell–cell signaling. The developmental induction of vulval precursor cells (VPCs) to assume the 3°-3°-2°-1°-2°-3° patterning of cell fates occurs with 99.8% accuracy. During C. elegans vulval development, an EGF signal from the anchor cell initiates the activation of RasLET-60 > RafLIN-45 > MEKMEK-2 > ERKMPK-1 signaling cascade to induce the 1° cell. The presumptive 1° cell signals its two neighboring cells via NotchLIN-12 to develop 2° cells. In addition, RasLET-60 switches effectors to RalGEFRGL-1 > RalRAL-1 to promote 2° fate. Shin et al. (2019) showed that RalGEFRGL-1 is a dual-function protein in VPCs fate patterning. RalGEFRGL-1 functions as a scaffold for PDKPDK-1 > AktAKT-1/2 modulatory signaling to promote 1° fate in addition to propagating the RasLET-60 modulatory signal through RalRAL-1 to promote 2° fate. The deletion of RalGEFRGL-1 increases the frequency of VPC patterning errors 15-fold compared to the wild-type control. We speculate that RalGEFRGL-1 represents an “insulated switch”, whereby the promotion of one signaling activity curtails the promotion of the opposing activity. This property might increase the impact of the switch on fidelity more than two separately encoded proteins could. Understanding how developmental fidelity is controlled will help us to better understand the origins of cancer and birth defects, which occur in part due to the misspecification of cell fates.The  C. elegans vulva is an excellent model in which to study cell–cell signaling. Six specialized equipotent epithelial cells, the vulval precursor cells (VPCs), are arranged along the ventral midline. During the L3 stage, the VPCs are induced to form the pattern of 3°-3°-2°-1°-2°-3° cell fates. The 1° and 2° cells go through three rounds of division and proceed to form the vulva; an epithelial tube connects the uterus with the external environment. The 3° cells divide once and then fuse with the surrounding epithelium. This pattern occurs with 99.8% accuracy [RGL-1, that encodes dual functions [RGL-1 orchestrates opposing 1°- and 2°-promoting modulatory cascades to help promote developmental fidelity. However, the study hints at a far more important interpretation of bifunctional RalGEFRGL-1.The C. elegans VPC fate patterning: (1) the sequential induction model, (2) the graded signal model, and (3) the mutual antagonism model. In the sequential induction model, induction occurs in a stepwise fashion: the 1° fate is induced first, then the 2° fate. The EGFLIN-3 ligand, produced by the anchor cell (AC) in the ventral gonad, activates EGFRLET-23 in the nearest VPC, which, in turn, activates RasLET-60 to trigger activation of the RafLIN-45 > MEKMEK-2 > ERKMPK-1 canonical MAP kinase cascade to induce 1° fate [LIN-12 to assume 2° fate [LIN-12 is necessary and sufficient to induce 2° fate.Three models are used to frame our understanding of del, and  the mutu 2° fate ,8. This LIN-3 gradient can dictate their ultimate fate [LIN-3 or the activation of EGFRLET-23 modulates VPC fate selection [LIN-3. However, two lines of evidence suggest that sequential induction—1°, then 2°—takes precedence over the graded signal. First, mutations disrupting either the well-known EGFLIN-3 > RasLET-60 > MAP kinase or Notch cascades result in absent 1° or 2° cells, respectively. Second, while disruption of the EGFLIN-3 > RasLET-60 > MAP kinase results in a vulvaless phenotype, i.e., no induction of any vulval fates [LIN-12 results in missing 2° cells but the continued presence of 1° cells [LIN-12 but induction of 1° fate is not compromised. To restate, 1° fate can be induced in the absence of 2°-promoting signal, but 2° cells cannot be induced in the absence of 1°-promoting signal. The sequential induction and graded signal models were not reconciled for a long time [The graded signal model is based on the observation that the position of isolated VPCs within the EGFate fate ,10. Furtelection ,12. Takeal fates , the dis1° cells : 2° cellong time .LET-60  and NotchLIN-12 (2°-promoting) cascades provide the skeleton of VPC patterning, while the graded signaling is overlaid to improve the fidelity of this process [RAL-1 propagates this 2°-promoting signal by activation of a MAP4K > p38 MAP kinase signaling cascade [LET-60 switches between utilization of RafLIN-45 and RalGEFRGL-1 effectors remains unknown. Mirroring the 2°-promoting RasLET-60 > RalGEFRGL-1 > RalRAL-1 modulatory signal in support of NotchLIN-12, an PI3KAGE-1 > PDKPDK-1 > AktAKT-1/2 modulatory signal promotes 1° fate in support of the core RasLET-60 > RafLIN-45 1°-promoting signal [AGE-1 is unknown: it could be another RasLET-60 input, or it could be EGFR or InsRDAF-2. To summarize, to properly pattern the VPCs at high fidelity, each vulval contributing cell fate type—1° and 2°—requires a core, essential signaling cascade and a modulatory, inessential signaling cascade.We were able to reconcile these two models by discovering the mechanism by which the graded signal is interpreted: effector switching by RasLET-60 . Oncogen 2° fate . RasLET- process . RalRAL- cascade . The mecg signal . ActivatLIN-12 receptor is internalized and degraded, thus preventing conflicting 2°-promoting signals in a VPC that is assuming 1° fate [LIP-1 phosphatase .RGL-1 is a dual-function protein in VPC fate patterning, ii) that the two functions are genetically separable, iii) that RalGEFRGL-1 likely functions as a scaffold for PDKPDK-1 > AktAKT-1/2 signaling in addition to propagating the RasLET-60 signal through RalRAL-1, and iv) that deletion of RalGEFRGL-1 does not alter the relative balance of 1° and 2° fates in VPC patterning but v) increases the frequency of patterning errors 15-fold about the wild-type control [RGL-1 orchestrates the two opposing modulatory signals in VPC fate patterning: Akt promotion of 1° fate and Ral promotion of 2° fate. That both activities are embodied in the same signaling molecule raises thought-provoking questions about the functional significance of this concurrence. We speculate that both signals embodied in the same protein represent an “insulated switch,” whereby the promotion of one signaling activity curtails the promotion of the opposing activity and vice-versa, perhaps thus increasing the impact of the switch on fidelity more than could two separately encoded proteins. This model also casts the frequently described phenomenon of “pathway cross-talk” in an interesting new light.Here, we reflect on a study that suggests an additional, fourth model that increases developmental fidelity, which we term “insulated switches” in signaling. Specifically, Shin et al. 2019) show that i) RalGEF019 show RGL-1 was uncovered serendipitously: the deletion of RalRAL-1 reduced 2°-promoting signaling while the deletion of RalGEFRGL-1 caused no effect on patterning [RGL-1 functions as an intermediary between RasLET-60 and RalRAL-1 [RGL-1 or RalRAL-1 to cause the same signaling defect. A natural hypothesis, eventually confirmed, was that RalGEFRGL-1 plays opposing and genetically separable roles in VPC fate patterning. Answering this question was technically demanding. Since the VPCs induced at the mid-L3 stage comprise approximately one-ten thousandth of the volume of the animal, relatively minute and transient changes in conventional signaling biomarkers like phospho-ERK or phospho-Akt are not detectable. Furthermore, no transcriptional reporters have been described as readouts of the modulatory cascades of RasLET-60 > RalGEFRGL-1 > RalRAL-1 or PI3KAGE-1 > PDKPDK-1 > AktAKT-1/2 during patterning of VPC fates, precluding a quantitative readout of fluorescent reporters of activity of those cascades. Consequently, the authors used phenotypic analysis to dissect these signals, by counting VPCs whose fate were transformed from 3° to 1° or 3° to 2° [The dual function of RalGEFRalRAL-1 , we expe3° to 2° .RGL-1 or RalRAL-1 conferred no obvious VPC patterning defects in an otherwise wild-type animal, consistent with a modulatory signal rather than the necessary and sufficient core signals [LET-60, let-60(n1046gf), which harbors a moderately activating G13E mutation, to provide a “sensitized” genetic background that responds to genetic perturbations of modulatory proteins. let-60(n1046gf) confers 3°-to-1° cell fate transformations, resulting in ectopic 1° lineages . Mutations that reduce the function of genes that act downstream of RasLET-60 suppress the induction of let-60(n1046gf)-dependent ectopic 1° cells -dependent ectopic 1° cells, while a reduction in this cascade function reduces induction of ectopic 1° cells [let-60(n1046gf) 1°-promoting activity partially enhance or suppress the induction of ectopic 1° cells, as assayed by counting induced cells [To understand these approaches, we need to understand the use of sensitized genetic backgrounds to reveal pathway activity through the use of genetic principles of parallelism and epistasis ,27,28. S signals ,19. Shins e.g., ,30). Mut. MutRGL-1° cells . In cont1° cells . Such aned cells ,19.RGL-1 > RalRAL-1 without altering the other properties of these proteins. The R139H missense mutation in RalRAL-1 and R361Q putative GEF dead mutation in RalGEFRGL-1 selectively disrupt 2° signaling in the VPCs and enhance 1° induction in the let-60(n1046gf) background [RGL-1 promotes 2° fate via the canonical GEF-dependent activation of RalRAL-1. In an experiment that bypassed the GEF requirement of RalGEFRGL-1 with activated Ral, the effect of deletion of RalGEFRGL-1 on the 1° promoting function could be detected. Therefore, RalGEFRGL-1 performs an additional GEF-independent function that antagonizes its canonical function.Shin et al. 2019) were able to unravel this signaling puzzle using these concepts of parallelism and epistasis. First, the authors defined genetic tools to selectively ablate the 2°-promoting signal of RalGEF019 were RGL-1 in promoting 1° fate, epistasis experiments were performed that point to the deletion of RalGEFRGL-1 suppressing the effect of deletion PTENDAF-18 and constitutively activated PDKPDK-1, and partially suppressing the effect of constitutively activated AktAKT-1. These results are consistent with the model that RalGEFRGL-1 functions as a scaffold for PDKPDK-1 and AKTAKT-1 in the modulatory 1°-promoting PI3KAGE-1 cascade. This observation is consistent with mammalian studies showing that mammalian RalGDS physically interacts with PDK and Akt as a scaffold [RGL-1 functioning in parallel to PI3KAGE-1 > PDKPDK-1 > AktAKT-1. Thus, RalGEFRGL-1 likely balances the functions of two modulatory cascades: canonical GEF-dependent activation of RalRAL-1 and promotion of 2° fate, and non-canonical GEF-independent scaffolding of PDKPDK-1 and AktAKT-1 to promote 1° fate.To further investigate the non-canonical GEF-independent activity of RalGEFscaffold ,32. HoweRGL-1, which requires the scoring of many more animals than used in sensitized backgrounds (1200 per genotype). Strikingly, the baseline error rate in VPC patterning was 15-fold higher in the two deletion mutants than in the wild type (P < 0.0001). Notably, environmental perturbations had no effect on different mutant backgrounds [RGL-1 function is not to defend against environmental variability but to decrease the variability in the system, i.e., fluctuations in signaling output or “noise”.To circumvent the requirement for a sensitized background, the authors counted patterning errors in the wild type vs. two strains with deleted RalGEFRGL-1 functions. Most animals still patterned the VPCs correctly using the core signaling cascades of RasLET-60 > RalRAL-1 > MAP kinase and NotchLIN-12. However, this 15-fold change in rate of developmental error reveals the function performed by modulatory cascades in patterning VPCs fates. These cascades likely reinforce initial commitment to fates analogously to the mechanisms of mutual antagonism discussed above. If both activities were encoded separately, a GEF intermediate between RasLET-60 and RalRAL-1 on the one hand and a scaffold for PDKPDK-1 on the other, we would never take note of the phenomenon. It is the dual function of RalGEFRGL-1 in the process that draws our attention as unusual. Does the bifunctionality of RalGEFRGL-1 have special mechanistic significance? We next discuss the possible meanings of this phenomenon.The rate of patterning errors changed from 0.2% in the wild type to 3.0% upon loss of both RalGEFRGL-1 is an “insulated switch”: both activities in the same protein are important to reduce signaling noise  cell biological or (2) steric hindrance. In a cell biological mechanism, the putative RalGEFRGL-1 as only a GEF of RalRAL-1 and a scaffold is only a scaffold. Yet many larger proteins perform multiple functions. They bind to targets other than those involved in their canonical function. Canonical functions are usually identified accidentally: what did we find first? It is hard to break out of that chronological bias, which leads to a conceptual bias.In molecular pathways we tend to think linearly; linearity is built into the word “pathway.” For instance, we tend to think about RalGEFRGL-1 itself: had it been originally discovered as a scaffold for PDKPDK-1 and AktAKT-1 signaling, its name would be very different than “RalGEFRGL-1”. If we think about these signaling systems as signaling networks instead of pathways, then these dual-functioning proteins are not unusual, but instead a key feature of signaling. Mechanistically, this dual-functional property of proteins at the network level could encode far more richness into signaling networks. Combinatorial functionality produces far more complexity. How do we generate, or build, one of the most complicated animals on the planet with the same number of genes as we use to generate one of the simplest animals? The answer probably is combinatorial functions of proteins: dual functionality (or more than two: many). Importantly, we also can get increased developmental fidelity with this special feature of the dual-functioning protein.These ideas lead us to the concept of “cross talk” in signal transduction. Researchers frequently describe one signaling axis communicating with another signaling axis, and this phenomenon is frequently referred to as “cross talk.” However, many such events are orphans, or stand-alone observations that are not followed up on to investigate the biological consequences of the activity. However, this phenomenon is frequently described in the context of the “canonical pathway” vs the novel cross-talk function, which represents an intrinsic bias. We see this in the naming of RalGEFRGL-1 protein that can function to promote both a primary and secondary fate. How would we test this? Each activity would need to be separately mutated by creating two different genes in the animal so that they are no longer linked. One is only a scaffold, one only a signaler, but both are expressed at same levels and in same cells. This approach might break the insulated signal in RalGEFRGL-1, so the signaling activities are still encoded but the switch no longer exists. Since we do not know where the scaffolding activity is encoded in RalGEFRGL-1, this approach might not be technically feasible.We speculate about the role of the insulated switch: can a dual-functional protein reduce noise more than two single-function proteins, i.e., a signaler and a scaffold? Can the same protein simultaneously promote one activity while also working to reduce the opposing activity? We further speculate that proteins in the signaling world that can be identified as obviously multifunctional could be insulated switches. One such protein is PLC-epsilon, an isozyme of the phospholipase C (PLC) family that functions both in the transduction of signal and as a shared effector protein in Ras-, Rho-, and heterotrimeric G protein -mediated signaling. Thus, crosstalk between heterotrimeric and small GTPase signaling pathways is sensed and mediated by multifunctional protein PLC-epsilon (reviewed in ). AnotheC. elegans occurs with 99.8% accuracy. Many layers of signaling network orchestrate together to generate high fidelity by minimizing mis-specified or ambiguous fates. This accuracy is generated by the combination of (1) sequential induction, (2) graded signal, (3) mutual antagonism, and (4) the orchestration of two modulatory cascades promoting 1° and 2° signaling by the dual functioning protein RalGEFRGL-1. The study by Shin et al. (2019) revealed this new, latter mechanism by using genetic manipulations, the combined principles of epistasis and parallelism. Cancer often develops from increased noise and error rate, misspecification or variable specification of cells. Birth defects may also be initiated from increased noise during development. Therefore, the signaling networks that reinforce the fidelity of cell fate patterning will provide us with important clues to understand the disease’s origins. For organisms with thirty trillion cells, more noise reduction becomes increasingly important. In mammals, there are four RalGEF/RalGDS-encoding genes. Would these operate as insulated switches with four-fold redundancy? There might be more combinatorial properties, such as scaffold specificity for different PDK and Akt isoforms or splice variants. We conclude that insulated switches could provide additional layers of fidelity and buffering of signaling networks against stochasticity during development.The patterning of the VPC fate in


Porcine growth hormone (pGH) is most important hormone which is involved in the growth and development of pig. However, a series of studies have indicated that neonatal pig is insensitive to pGH; the reason for this phenomenon is still not fully understood. In this work, we try to investigate this issue from the angle of intracellular signaling induced by pGH. In the present study, porcine hepatocytes from neonatal pig were used as a model, and confocal laser scanning microscopy (CLSM), Western blot, co-immunoprecipitation and colocalization assay were used to study pGH’s signaling properties in hepatocytes of neonatal pig and explore the possible mechanism(s) for why intracellular signaling is insensitive to pGH. The results indicated that Janus kinase 2 and signal transducers and activators of transcription 5/3/1 (JAK2-STATs) signaling are not activated. We further investigated the possible mechanism(s) by which JAK2-STATs’ signaling is not activated by pGH and growth hormone receptor (GHR) and found that the negative regulatory molecules of JAK2-STATs signaling may be associated with this phenomenon in the hepatocytes of neonatal pig. In addition, we also explored pGH’s biology in hepatocytes from neonatal pig, it can be found that pGH/GHR could translocate into the cell nucleus, which implies that pGH/GHR may exhibit physiological roles based on their nuclear localization. We found that pGH could not trigger intracellular signaling in the hepatocytes of neonatal pigs, but not young pigs, which provides an important explanation for why the growth of neonatal pig is GH independent. Growth hormone (GH) plays important roles in the regulation of growth and development in mammals  increases growth rate, improves feed efficiency, protein synthesis and increases muscle growth markedly  to explore intracellular signaling induced by pGH in the hepatocytes of neonatal pig; (2) to find a possible answer for why pGH is not sensitive in neonatal pig from the angle of pGH-induced intracellular signaling. Porcine hepatocyte is an important target cell of pGH and also is an ideal somatic cell model to study pGH-induced intracellular signaling  were purchased from Sigma . Phospho-JAK2 and JAK2 were from Cell Signaling Technology . Phospho-STAT5/3/1 and total STAT5/3/1 antibodies were obtained from Santa Cruz . PVDF membranes, ECL and BSA were from Millipore. Porcine GHR, β-actin and normal mouse/rabbit lgG were obtained from Abcam . Cell culture plates  were purchased from Corning Costar . Fetal calf serum (FCS) was obtained from Invitrogen . Lysis buffer was purchased from Beyotime Biotechnology . Collagenase was obtained from Hua Cheng Biological Inc . All other reagents were purchased from Sigma .Porcine hepatocytes were isolated according to our previous methods  for pGH internalization analysis, the cells were washed with PBS, the FITC-pGH were then added into the plates and incubated for different durations. The porcine hepatocytes were then washed for three times with PBS and fixed with 4% paraformaldehyde at 37°C for 20 min. After washing, the cell nuclei were stained with DAPI. After washing for three times with PBS, the cells were observed using confocal laser scanning microscopy (Olympus FV3000); (2) for colocalization analysis, the cells were fixed and blocked, the cells were then treated with the indicated antibodies. After washing, the cells were incubated with second antibodies labeled with Alexa Fluor 488 (green) and Alexa Fluor 555 (red). After washing for three times, the cells were observed using CLSM.p-value <0.05 was considered statistically significant.The data are presented as the mean ± standard error (S.E.). The results were analyzed by one-way analysis of variance using Statistical Analysis System (SAS) software . A We first preliminarily evaluate hepatic cell surface GHR expression in the hepatocytes of 1–7 days old and 100 days old pig by CLSM, as indicated in We first check the expression level of signaling molecules  in the hepatocytes of neonatal pigs. As shown in Previous studies have reported that the hepatocyte of growing young pig (∼60 kg) is very sensitive to pGH  are very sensitive to pGH, which exhibits sensitive STATs’ signaling response in time-course experiments. Based on this, we investigated STAT5/3/1 activation under pGH stimulation in neonatal hepatocytes under the same culture conditions. As shown in We first checked the interactions between pGH and GHR by CLSM; we can see that pGH and GHR could interact with each other on the cells, which indicated that pGH could interact normally with GHR  ). Therefore, we checked the expression of these negative regulatory molecules, as shown in Since pGH/GHR fails to activate the intracellular signaling pathway in the hepatocytes of neonatal pig, does it mean that pGH/GHR has no effect in neonatal pig? Traditional view believes that pGH/GHR exhibits its physiological functions in cell membrane; however, recent studies also show that GH/GHR can transport into cell nuclei, where they also can exert important roles (such as proliferation)  (Sjögren et al. Recently, a series of studies have shown that pGH not only exhibits its physiological roles in cell membrane but also exerts its biological activities by its nuclear translocation (Brooks et al. In summary, in the current work, we found that JAK2-STATs signaling is not sensitive to pGH in newborn pigs, which provides a possible explanation for why pGH is unresponsive in newborn pigs. Based on this, we propose a new paradigm for pGH’s functions in neonatal pig.


Oryza sativa L.), severe seed sterility has been considered a barrier preventing cultivation of autotetraploids since the 1930s. Recently, a series of studies identified two fertile autotetraploids, identified herein as the PMeS (Polyploid Meiosis Stability) and Neo-Tetraploid lines. Here, we summarize their characteristics, focusing on the recovery of seed fertility, and discuss potential future directions of study in this area, providing a comprehensive understanding of current progress in the study of fertile tetraploid rice, a classical, but promising, concept for rice breeding.Ploidy manipulation is an efficient technique for the development of novel phenotypes in plant breeding. However, in rice ( The phenomenon of polyploidy, which refers to the multiplication of chromosome sets within cells, often doubling a normal (diploid) set into a quadruple (tetraploid) set, is a widespread and distinctive feature of the higher plants . Polyplohttp://ricecrp.org/), as a means of providing new genetic materials for future crop improvement. In this review, we focus on autotetraploid rice, especially its fertility, because low seed fertility has been regarded as a barrier obstructing its use. In the course of summarizing two pioneering studies on autotetraploid rice with high seed fertility, we will discuss potential future directions for ploidy manipulation in rice, a classical but promising concept.In crop species, it has been frequently observed that polyploidization causes plants to grow larger, more quickly, and with higher yields, compared to their diploid relatives , althougOryza sativa L.) is a diploid species with two sets of 12 chromosomes (2n = 24). [Although Asian cultivated rice may have experienced whole genome duplication more than 96 million years ago  has significant effects on the pollen fertility of autotetraploid hybrids, and that pollen fertility further decreased with increasing allelic interaction. They also found abnormal chromosomal behavior in the hybrids with low pollen fertility and suggested that the gene interactions of hybrid sterility loci tend to increase the chromosomal abnormalities that cause the partial abortion of male gametes, leading to the decline in seed set of the autotetraploid rice hybrids (San and Sbn) at loci Sa and Sb, respectively  derived from different autotetraploid parents. If so, a QTL analysis of high seed fertility could be performed by developing recombinant inbred lines derived from crosses between autotetraploids. The Neo-Tetraploid lines show different expression of some important meiosis stage specific- and meiosis-related genes  responsible for high fertility in these tetraploid rice varieties will be necessary. Both the PMeS and Neo-Tetraploid rice lines have been developed from the progenies of crosses between the ed genes . Genes/Qed genes . These roid line  may suppoid line  may causAlthough their origin is unclear, identifying the genes responsible for high seed fertility in the PMeS and Neo-Tetraploid lines will enable the transfer of this technique to other autotetraploid rice varieties through marker-assisted selection or genome editing. A diverse set of fertile autotetraploid rice varieties will greatly increase their potential usefulness in future agriculture. Ploidy manipulation, a classical concept, will revive in the field of rice breeding in the near future.YKo, DK, and YKi contributed to conceptualization of this review. YKo wrote original draft. DK and YKi reviewed and edited the original manuscript.YKo and YKi are funded by JSPS KAKENHI Grant Numbers 18K05565 and 19H00937, respectively.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.


Interest has emerged in biased agonists at the mu opioid receptor (MOR) as a possible means for maintaining potent analgesis with reduced side effect profiles. While approaches measuring in vitro biased agonism are used in the development of these compounds, their therapeutic utility will ultimately be determined by in vivo functional effects. Nonhuman primates (NHPs) are the most translational model for evaluating the behavioral effects of candidate medications, but biased signaling of these drugs at NHP MOR receptors has been unstudied. The goal of the current work was to characterize MOR ligand bias in rhesus macaques, focusing on agonists that have previously been reported to show different patterns of biased agonism in rodents and humans. Downstream signaling pathways that responded to MOR activation were identified using a luciferase reporter array. Concentration-response curves for specific pathways  were generated using six agonists previously reported to differ in terms of signaling bias at rodent and human MORs. Using DAMGO as a reference ligand, relative cAMP, NF-ĸB and MAPK/JNK signaling by morphine, endomorphin-1, and TRV130 were found to be comparable between species. Further, the bias patterns of across ligands for NF-ĸB and MAPK/JNK were largely similar between species. There was a high degree of concordance between rhesus macaque and human MOR receptor signaling bias for all agonists tested, further demonstrating their utility for future translational behavioral studies. Recent advances in the study of G-protein coupled receptor (GPCR) pharmacology have demonstrated that various intracellular pathways can be differentially activated in a ligand-specific manner, a phenomenon referred to as biased agonism . AccordiSeveral promising studies ,12,13,14Due to their genetic and brain morphology similarities to humans, nonhuman primates (NHPs) are the most translational preclinical model for predicting the therapeutic and toxicological effects of candidate medications, including opioid analgesics ,31,32,33Part of the reason why the majority of in vitro assays investigating signaling bias are done largely (or solely) in humans is because of the widespread use of assays for measuring β-arrestin recruitment based on enzyme fragment complementation technology. The commercially available tools for this approach, which requires the construction of modified fusion proteins for either β-arrestin or both GPCR and β-arrestin , only exMacaca mulatta) and human MORs, focusing specifically on downstream signaling pathways as a downstream proxy of G-protein and β-arrestin recruitment. The overall hypothesis was that there would be a high degree of concordance in the bias factors for each of the compounds between rhesus macaque and human MORs.The overall goal of this study was to compare relative potencies and efficacies of a series of MOR agonists that have been reported to vary in terms of signaling bias in rhesus macaque . Although this study is agnostic to the mechanisms through which the signaling pathways are ultimately activated or inhibited in an effort to capture the presumed bias present in the agonists, we focused on these specific pathways to represent downstream results of both G-protein and β-arrestin response. We transduced the luciferase reporter constructs from each of the three chosen signaling pathways into HEK293 cells and created the stable cell lines through puromycin selection. To verify the selection results and confirm reporter response, we used GPCR independent substrates to stimulate each signaling pathway. Treated cells showed significantly higher luciferase readings than saline-treated cells , demonst50, Emax of each curve and the species comparisons are shown in Human and rhesus macaque MORs were then transfected into the cell lines containing the signaling reporters. These cell lines were expanded and treated with six MOR ligands: morphine, DAMGO, met-enkephalin, β-endorphin, endomorphin-1, and TRV130. The generated concentration-response curves are shown in For the cAMP pathway, all ligands demonstrated a concentration-dependent downregulation as expected in both human and rhesus macaque MORs a,d. No smax except TRV130 was significantly higher than multiple ligands in both species.At the NF-ĸB pathway, all ligands produced a concentration-dependent stimulation in both species b,e, agaimax, again, showed no significant differences between species  .In the human MOR, a consistent pattern emerges for morphine a, met-enIn the rhesus macaque MOR, morphine f was staNext ∆∆log  following the manufacturer’s protocol, and bacteria were shaken at 37 °C at 225 rpm overnight in LB medium (Invitrogen). Plasmids were purified using the Plasmid Plus Maxi kit , confirmed by Sanger sequencing, and stored at −20 °C until use.pcDNA3.1/V5-His-TOPO vectors  containing either human (NM_000914) or rhesus macaque (NM_001032824)  methods . Cloned 2 culture dishes  at 37 °C with 5% CO2. They were stably transfected with either human or rhesus macaque OPRM1 plasmids using Lipofectamine 3000 reagent (Invitrogen) followed by selection using geneticin (Gibco) for two weeks. PCR was used to confirm gene expression.HEK293 cell lines were purchased  and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin  in 100 mmSeparately, HEK293 cell lines expressing luciferase reporters for second messenger signaling pathways were generated using stable transduction of Cignal Lenti-Reporter assays (Qiagen). These assays make use of lentiviral particles carrying luciferase reporter genes under the transcriptional control of specific second messenger signaling pathway transcriptional response elements. Here, cell lines were generated for reporting activation of the cAMP, NF-ĸB and MAPK/JNK pathways using lentiviral particles with a multiplicity of infection equal to 2. After transduction over three days, puromycin selection was performed, and cells were grown to confluence as above. Confirmation of activity was accomplished with GPCR independent substrates .OPRM1 using Attractene (Qiagen). The cells were then treated with either saline or the MOR agonist DAMGO (10 μM) for 6 h. Because MOR is known to be Gi-linked -enkephalin; Bachem, Torrance, CA, USA), met-enkephalin (Sigma–Aldrich), β-endorphin , endomorphin-1 , and TRV130 . All ligands except TRV130, which was first dissolved in DMSO (20%), were dissolved in water, then prepared as serial dilutions to concentrations between 10−11 to 10−5 M. GPCR independent substrates including forskolin, TNF-α and PMA (Sigma–Aldrich) were used to activate cAMP, NF-ĸB and MAPK/JNK respectively. All GPCR independent substrates were diluted in DMSO to final concentrations of 10 μM for forskolin, 50 ng/mL for TNF-α, and 10 ng/mL for PMA.We chose six MOR agonists that include endogenous and exogenous ligands and that vary in terms of G-protein bias: morphine , DAMGO  overnight to 60–70% confluence. We then transiently transfected the plasmids containing either the rhesus macaque or human p value less than 0.05 was considered statistically significant following Bonferroni correction for multiple testing.To verify that the luciferase reporter genes were working in our generated stable cell lines, we treated these cells with saline and substrates that stimulate the signaling pathways independent of GPCR activation  for 20 h. We then lysed the cells and measured luciferase activities. An unpaired t-test (two-tailed) was used to compare between groups, 50 (potency) and Emax (efficacy) for the signaling pathways between species and between different ligands within species. Comparisons with p value less than 0.05 were considered to be significantly different, again using a Bonferroni correction. The Emax values used in this study were normalized as follows: For the cAMP pathway, cells treated with 1 μM forskolin defined 100%, and cells treated with 1 μM forskolin and 1 μM DAMGO defined 0%. For the NF-ĸB and MAPK/JNK pathways, data from saline-treated cells defined 0% and 1 μM DAMGO-treated cells defined 100%. The EC50 for each ligand was defined by the molar concentration required to generate a half-maximal response. To calculate bias factors, curves were fit with an operational model [mE was the possible maximum response, n was the transducer slope; both parameters were equal for each calculation. A was the molar concentration of the ligand, KA was the equilibrium dissociation constant of ligand A and A were determined in the operational model, and log . An extra sum-of-squares F test was used to compare the differences of Log ECal model :ResponseUsing DAMGO as the reference ligand, normalized transduction coefficients ∆log (Using cAMP as a reference signaling pathway to generate the double normalized transduction coefficients ∆∆log (Finally, the bias factor for each condition was calculated using the following equation:∆log (t-test, p value less than 0.05 was considered statistically significant.For the comparisons between these parameters, we used a two-way unpaired Student


Rapid eye movement (REM) sleep behavior disorder (RBD) is a risk factor for developing Parkinson’s disease (PD) and may represent its prodromal state. We compared neuropsychological and neuropsychiatric phenotypes of idiopathic (i) RBD, PD and healthy comparators (HC) in order to identify iRBD specific characteristics. Thirty-eight patients with iRBD, 38 PD patients with RBD (PD + RBD), 38 PD patients without RBD (PD-RBD) and 38 HC underwent a comprehensive neurological, neuropsychological and neuropsychiatric evaluation. iRBD, PD + RBD and PD-RBD performed worse than HC in short-term verbal memory, praxia, language and executive functions. iRBD had higher levels of anxiety, depression, apathy and alexithymia than HC. iRBD had higher levels of apathy than PD + RBD. Both PD groups had higher levels of anxiety and depression than HC. Surprisingly, iRBD performed better than all groups in long-term verbal memory. Patients diagnosed with iRBD are characterized by poor global cognitive performance, but better long-term memory and higher levels of depression, anxiety, alexithymia and apathy. Alexithymia and apathy in patients diagnosed with iRBD may be the expression of precocious derangement of emotional regulation, subsequently observed also in PD. Cognitive and neuropsychiatric symptoms of iRBD are early clinical manifestations of widespread neurodegeneration. Rapid Eye Movement (REM) sleep Behavior Disorder (RBD) is a parasomnia characterized by loss of normal skeletal muscle atonia during REM sleep, such that patients “act out” dreams, often violently, which is potentially harmful for themselves and their bedpartner . The idiPrevious studies identified in patients with iRBD ,9,10,11,The aim of the present study is to identify specific neuropsychological and neuropsychiatric features of patients diagnosed with iRBD and to compare their symptoms with those found in HC, PD + RBD and PD without RBD (PD-RBD). Considering iRBD as the prodromal state of synucleinopathy, we anticipated iRBD had a cognitive and neuropsychiatric profile similar to PD + RBD and PD-RBD, different from healthy comparators (HC). In particular, we predicted that patients with iRBD experience executive dysfunctions and motivational and emotional dysregulation.The study was carried out on 38 patients diagnosed with iRBD, 38 with PD + RBD and 38 with PD-RBD, according to international guidelines ,29. All Common inclusion criteria for all groups were: (1) age between 55 and 85 years; and (2) vision and hearing sufficient for compliance with testing procedures.Specific inclusion criteria in iRBD were: (1) iRBD diagnosis made by video-polysomnography (v-PSG) according to International Classification of Sleep Disorders-3rd Edition (ICSD-3) criteria ; (2) no Specific inclusion criteria in patients with PD were: (1) PD diagnosis made in according to the UK Parkinson’s Disease Society Brain Bank diagnostic criteria ; (2) MinCommon exclusion criteria for all participants enrolled were the following: (1) presence of major medical illnesses ; (2) known or suspected history of alcoholism, drug dependence and abuse, head trauma and major psychiatric disorders (apart from mood or anxiety disorders) according to the DSM-V criteria ; (3) anyThe study was approved by the Ethical Committee of Fondazione Santa Lucia IRCCS and, in accordance with the Helsinki Declaration, each subject signed an informed consent form prior to enrollment.The sociodemographic and neurological features of patients were collected at enrollment by neurologists with expertise on parkinsonism and sleep disorders. The severity of parkinsonian symptoms was measured by the Unified Parkinson’s Disease Rating Scale—part III (UPDRS-III), and PD severity was staged according to the modified Hoehn and Yahr (H&Y) scale . DopaminWithin 2 weeks from enrollment, all participants underwent a structured psychiatric interview Structured Clinical Interview for DSM-5 Disorders—Clinician Version (SCID-5-CV), SCID-5 Research Version and SCID-5-Personality Disorders, for the identification of mental disorders . All psyAll patients were submitted to a detailed neuropsychological evaluation , includiThe severities of symptoms of anxiety, alexithymia, apathy, anhedonia and depression were assessed in all participants . SpecifiHedonic tone was measured by the Snaith-Hamilton Pleasure Scale (SHAPS). Severity of depressive symptoms was investigated by the Beck Depression Inventory  . Cognitit-test. Levene’s test was used to test for equality variances.Differences in sociodemographic and clinical characteristics among groups were assessed by Chi-square test and univariate analysis of variance (ANOVA) followed by Fisher’s Protected Least Significant Difference post-hoc tests, where appropriate. Post-hoc tests were repeated by Tukey’s Honestly Significant Difference test. Differences in clinical characteristics between PD + RBD and PD-RBD were analyzed using paired p < 0.05. In the case of significant effect, we conducted a series of one-way ANOVAs followed by Fisher’s PLSD test post-hoc comparisons, when appropriate. The Bonferroni’s correction was applied before interpreting the significance of ANOVAs and of post-hoc tests, to control the risk of type I error. Post-hoc tests were repeated by Tukey’s HSD test. Cohen’s f was calculated to estimate effect size.To investigate differences in cognitive performances among diagnostic groups, we conducted a one-way multivariate analysis of variance (MANOVA) with a single independent variable  with four levels and 10 dependent variables . The omnibus level of significance for MANOVA was set at p < 0.05. In the case of significant effects, we conducted a series of one-way ANOVAs followed by Fisher’s PLSD test post-hoc comparisons, when appropriate. The Bonferroni’s correction was applied before interpreting the significance of ANOVAs and of post-hoc tests, to control the risk of type I error. Post-hoc tests were repeated by Tukey’s HSD test. Cohen’s f was calculated to estimate effect size.To investigate differences in severity of neuropsychiatric symptoms among groups, we conducted a MANOVA with a single independent variable  with four levels and five dependent variables . The omnibus level of significance for MANOVA was set at As expected, we found a significantly higher score in the UPDRS-III in the two groups of patients diagnosed with PD compared to iRBD. No significant differences were found in illness duration, daily levodopa equivalent dosage and H&Y stage between the two groups diagnosed with PD . 3.30 = 8.337; p < 0.0001; and η2p = 0.246) in neuropsychological scores among groups.MANOVA model indicated global difference . As to neuropsychiatric characteristics, the MANOVA model showed global difference among groups  at the first visit was not performed here, because we used a continuous value approach on cognitive dimensions/symptoms. However, the presence of MCI is going to be considered as a possible early indicator of conversion from iRBD to a neurodegenerative disease ,52. ThusIn conclusion, patients diagnosed with iRBD have specific cognitive and neuropsychiatric phenotypes characterized by poor global cognitive performance, but better long-term memory, and higher level of depression, anxiety, alexithymia and apathy. In particular, phenomenology of alexithymia and apathy in iRBD indicates precocious derangement of emotional regulation, subsequently observed also in PD. Although iRBD is a well-known predictive clinical manifestation of neurodegenerative disease onset, our results highlight that peculiar symptoms could be accepted as early clinical markers in development of PD or other types of neurodegenerative diseases, and should be routinely evaluated in clinical setting.


WNT ligands interact with distinct families of cell surface receptors to initiate signaling in response to the various environmental cues that orchestrate cell physiology and tissue homeostasis. For example, WNT signaling directs cytoskeletal modulations, organelle dynamics and specific transcriptional programs that regulate cell growth, differentiation, and migration. While WNT signaling was initially best studied in embryogenesis and carcinogenesis, quite naturally, this important aspect of life was also discovered to orchestrate interactions between the immune system and invading pathogens and tumors, as well as the microbiota that co-exist with the host. Many gaps remain in our understanding of how WNT signaling is involved in immune defense, host cell interactions with tumors and phylogenetically diverse microbes. This Research Topic comprises articles that provide a comprehensive picture of the current understanding of WNT signaling in response to pathogens, commensals, and tumors. With a view toward exploiting these novel insights for therapeutic applications, considerations of targeting specific WNTs and WNT signaling intermediates in the various scenarios are discussed.Mukherjee et al., Ljungberg et al., Silva-Garcia et al., Jati et al., Rogan et al., Li et al., Cosin-Roger et al., Patel et al., and  Martin-Orozco et al..WNTs comprise a family of secreted glycol-lipo-protein ligands that bind to cell surface receptors including Frizzled G protein-coupled receptors, as well as ROR and Ryk tyrosine kinases to elicit signal transduction. Although WNT signaling is broadly categorized into 2 principal types, β-catenin-dependent and β-catenin independent, considerable crosstalk exists among the signaling intermediates of WNT signaling pathways in complex modes that are mostly context dependent. The intricate features of the diverse WNT signaling cascades under physiological states and different pathophysiological settings have been covered categorically in the articles by Salmonella Typhimurium depresses WNT signaling in the endothelium by virtue of its T3SS effector system and utilizes the vascular leakiness to enhance its dissemination . Some pathogens, for example S. flexneri on the other hand, take advantage of the inflammatory component of WNT signaling for propagation of infection . Along the same lines, Silva-Garcia et al. have outlined the role of various bacterial virulence factors in the regulation of the WNT-β-catenin pathway during disease pathogenesis. Ljungberg et al. provide an overview of bacteria-induced WNT responses in experimental systems and patient samples, and the functional consequences in the context of immune responses. Some examples include impact on immune cell differentiation, antimicrobial defense, inflammation, and the cross-talk between innate and adaptive immunity. Adding another twist to this topic, Jati et al. have described how actin cytoskeleton organization by WNT5A signaling dictates the killing of some pathogens by utilizing the host autophagy machinery. Jati et al. also raise how WNT signaling might facilitate differentiating between pathogenic and non-pathogenic microbes, given the plethora of beneficial commensals that have evolved with our system.Being an important component of cell and tissue homeostasis, WNT signaling is often exploited by pathogens for their own survival. For example, In view of the ongoing tug of war between the host and pathogens for WNT signaling components during the onset and progression of infections, it would be fair to state that appropriate and targeted inhibition or activation of WNT signaling may be beneficial for the host's ability to control pathogens. Such operations, however, would naturally vary case to case.Cosin-Roger et al., evidence of the association of WNT signaling with tumorigenesis was first reported in 1991, when mutations in the Adenomatous Polyposis Coli (APC) gene were linked with the development of colorectal cancer. Anomalous expression and function of the APC gene product, which regulates β-catenin functions as a transcriptional co-activator, is now known to be associated with uncontrolled cell proliferation in cancer. Cosin-Roger et al. and Li et al. project WNT-β-catenin signaling as a high priority target for therapeutic intervention in the treatment of cancers. In their review, Patel et al. describe the role of tumor infiltrating macrophages in colorectal cancer, commenting on the high level of expression of WNT2 and WNT5A in the macrophages and its correlation with cancer progression. They further describe how WNT-β-catenin signaling is involved in the functional polarization of tumor-associated macrophages that facilitates tumor growth. Martin-Orozco et al. discuss the role of WNT-β-catenin signaling in augmenting drug resistance of tumors through expression of drug export pumps. Importantly, reports of the role of WNT signaling in tumor regression also exist. For example, evidence has been presented that WNT5A acts as a tumor suppressor in various cancers. In fact Foxy-5 is a hexapeptide mimic of WNT5A, which is currently being studied in clinical trials as a tumor suppressor .Consistent with an important role in directing transcription, cytoskeletal motility and cell proliferation and differentiation, dysregulated WNT signaling is associated with the development of tumors. With the discovery that WNT signaling also shapes immune cell functions, it is increasingly appreciated that the immune response to tumors is shaped by WNT signaling in both tumor and immune cells. As explained by As dysregulated WNT signaling is explored as a therapeutic target in cancer, it is important to consider that the applied interventions will define and re-shape the associated immune response. With pro- and anti-tumorigenic functions of WNT signaling in specific tumor settings it will be important to evaluate the therapeutic benefits and potential risks of WNT-targeted interventions in different cancers.Activation of WNT signaling is an integral part of host responses to microbial encounter and tumorigenesis. Given the complexity of WNT signaling, special emphasis should be placed on the nature of the WNT response, as well as the understanding of functional impact of individual WNT ligands and their concerted action in response to infection and tumorigenesis. This will be essential to evaluate strategies for WNT-directed therapeutic interventions that might prove particularly valuable considering the significant challenges posed by drug resistance in pathogens as well as tumors.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The primary cilium, a microtubule based organelle protruding from the cell surface and acting as an antenna in multiple signaling pathways, takes center stage in the formation of the cerebral cortex, the part of the brain that performs highly complex neural tasks and confers humans with their unique cognitive capabilities. These activities require dozens of different types of neurons that are interconnected in complex ways. Due to this complexity, corticogenesis has been regarded as one of the most complex developmental processes and cortical malformations underlie a number of neurodevelopmental disorders such as intellectual disability, autism spectrum disorders, and epilepsy. Cortical development involves several steps controlled by cell–cell signaling. In fact, recent findings have implicated cilia in diverse processes such as neurogenesis, neuronal migration, axon pathfinding, and circuit formation in the developing cortex. Here, we will review recent advances on the multiple roles of cilia during cortex formation and will discuss the implications for a better understanding of the disease mechanisms underlying neurodevelopmental disorders. The cerebral cortex contains dozens of different types of neurons . This diPrimary cilia are small protrusions from the cell surface and act as signaling hubs in neural development. Harboring the cell surface receptors for multiple signaling pathways, this tiny organelle receives the signals controlling corticogenesis and converts them into cellular responses, for example into changes in gene expression and/or alterations in the migratory behavior of cells and axons. Astonishingly, the cilium can even influence axon pathfinding decisions by the axonal growth cone which is separated from the cilium by large distances, in case of the corticospinal motor neurons by several centimeters . MoreovePrimary cilia are microtubule based cell organelles that function as sensors for chemical and mechanical cues from the cellular environment. Nine outer microtubule doublets emanate from the basal body at the ciliary base to form the ciliary axoneme which protrudes from the cell surface  Fisch a. PrimaryGli2 mutant mice while Gli3 null mutants present with severe morphological and molecular defects in the dorsal telencephalon   . These feriments . In cont mutants . InteresGpr161 encodes an orphan G protein coupled receptor (GPCR) that acts as a negative regulator of Shh signaling by controlling cAMP levels and thereby Gli3 processing and repressor formation  cause dwarfism, microcephaly and intellectual disability  and Filamin A genes  abolished hotspot formation and resulted in defective centrosome dynamics and altered nucleokinesis. Interfering with the centrosomal localization of PKA phenocopied these migratory defects strongly suggesting a direct link between the primary cilium and the centrosome through cAMP signaling. Interestingly, the authors found a similar cAMP hotspot in migrating cortical projection neurons. While this raises the interesting possibility that cAMP signaling is similarly involved in somal translocation of cortical projection neurons, it remains to be seen whether other signaling pathways and in particular Reelin signaling which is critical for correct cortical layering  and epilepsy . A majoral axons . Arrivinal axons . In addial axons . These fircuitry .Arl13b conditional inactivation in interneurons caused alterations in interneuron morphology and electrophysiological properties with reductions in synaptic buttons density and synaptic size (In models, single neurons are often depicted with their primary cilium protruding from the surface of the neuronal soma at a distance from dendrites and synapses. However, projection neurons and interneurons are very densely packed in the developing and adult cortex and as such cilia will be in close contact with other neurons, their dendrites and axons. This raises the interesting possibility that due to their position cilia represent excellent candidates as modifiers of neuronal circuitry. This certainly is an area much to learn about in the developing cerebral cortex but we are starting to gain insights into ciliary roles in other forebrain neurons in shaping neuronal connectivity. In addition to cortical interneurons, the medial ganglionic eminence generates striatal interneurons that regulate the activity of medium spiny neurons in the striatum and thereby striatal output . It was tic size . Moreovetic size  accumulatic size .In this review, we have summarized our current knowledge on the role of primary cilia during corticogenesis. This tiny organelle plays multiple and remarkably varied roles at various steps of cortical development. A major focus in the future will be to elucidate how a combination of defects in ciliary controlled signaling pathways contribute to this variability in phenotypes. Throughout this review, we have emphasized the importance of ciliary mediated processing of Gli3 for cortical patterning and stem cell development reflecting a strong requirement to suppress Hedgehog signaling in these early steps of corticogenesis. In contrast, Hedgehog signaling becomes increasingly more active at later stages to control the formation of olfactory bulb interneurons and of glial cells  and estaKH-T and TT conceived the idea and wrote the manuscript. Both 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.


The Coronavirus Disease 2019 (COVID-19) pandemic has infected over 10 million people globally with a relatively high mortality rate. There are many therapeutics undergoing clinical trials, but there is no effective vaccine or therapy for treatment thus far. After affected by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), molecular signaling pathways of host cells play critical roles during the life cycle of SARS-CoV-2. Thus, it is significant to identify the involved molecular signaling pathways within the host cells. Drugs targeting these molecular signaling pathways could be potentially effective for COVID-19 treatment.In this study, we developed a novel integrative analysis approach to identify the related molecular signaling pathways within host cells, and repurposed drugs as potentially effective treatments for COVID-19, based on the transcriptional response of host cells.dexamethasone was top-ranked in the prediction, which was the first reported drug to be able to significantly reduce the death rate of COVID-19 patients receiving respiratory support.We identified activated signaling pathways associated with the infection caused SARS-CoV-2 in human lung epithelial cells through integrative analysis. Then, the activated gene ontologies (GOs) and super GOs were identified. Signaling pathways and GOs such as MAPK, JNK, STAT, ERK, JAK-STAT, IRF7-NFkB signaling, and MYD88/CXCR6 immune signaling were particularly activated. Based on the identified signaling pathways and GOs, a set of potentially effective drugs were repurposed by integrating the drug-target and reverse gene expression data resources. In addition to many drugs being evaluated in clinical trials, the The integrative genomics data analysis and results can be helpful to understand the associated molecular signaling pathways within host cells, and facilitate the discovery of effective drugs for COVID-19 treatment. Coronavirus Disease 2019 (COVID-19) in the U.S. and over 10 million cases  globally, based on the COVID-19 Dashboard For the signaling network analysis, the 307 KEGG (Kyoto Encyclopedia of Genes and Genomes) –12 signaFor the gene ontology (GO)  analysisThe KEGG signaling pathway analysis was conducted to identify the potentially activated signaling pathways within the lung host cells after SARS-CoV-2 infection see Fig. -Upper. Ahttps://www.wired.com/story/ai-uncovers-potential-treatment-covid-19-patients/, and https://www.clinicaltrialsarena.com/news/eli-lilly-to-study-baricitinib-for-covid-19-treatment/), was reported in the Lancet [Based on the drug-target interaction data derived from DrugBank and the identified signaling network, 220 drugs were identified to inhibit 97 target genes on the signaling network  , and compared with the drugs identified from the signaling pathway analysis. Based on the dashboard of clinical trials for COVID-19 treatment, 114 FDA approved drugs were reported from 1,132 clinical trials globally . Drug combinations (drug cocktails) targeting on different targets can be potentially synergistic for COVID-19 treatment.Moreover, the identified drugs inhibiting the predicted signaling pathways were compared with drugs used in the clinical trials for COVID-19 treatment. Specifically, drugs that have been tested or are currently being tested in clinical trials globally were identified from the covid-trials dashboard  and the number of genes in GOs between 10 and 1000. Then the common activated GOs between NHBE and A549_ACE2 cells and between NHBE and CALU-3 cells were unified. After empirically removing the unrelated and general GOs, 73 GOs were kept. The full list of enriched GOs was provided in the supplementary file (Cell-line-GO.txt). Then, the clustering analysis was employed to divide the activated GOs into 5 sub-groups (named super-GOs). Among the 73 GOs, 212 up-regulated genes were kept. Figure The gene signatures in each of 5 super-GO clusters were used as the query input of the CMAP database to identify drugs capable of inhibiting the associated genetic pathways. Specifically, 258 drugs appeared in the top 100 drugs in at least one of the 5 super-GOs. There were 26 common drugs among the total drugs derived from aforementioned signaling network analysis and the super-GO analysis  samples  of COVIDTo identify drugs using updated CMAP database, the 51 GO terms were clustered into five super-GOs. Then genes associated with individual super-GOs were used as the signatures to identify the associated drugs that can potentially inhibit the individual GOs. In total, 120 drugs were identified that appeared at least two times in the top 100 drugs associated with the five super-GOs. Importantly, there were 49 overlapping drugs between the drug lists identified the super-GO gene signatures of cell lines infected by SARS-CoV-2 and human tissue samples of COVID-19 respectively . Also, the top 180 common up- and 183 common down-regulated genes between NHBE and CALU-3 cell lines were selected to identify potentially effective drugs in the CMAP database. Specifically, 30 out of 180 up-regulated genes, and 39 out of 183 down-regulated genes were not available in the CMAP database. Thus 150 up-regulated and 144 down-regulated genes were used. Among the top 100 FDA drugs, there were 13 drugs also appeared in the clinical trials, i.e., sildenafil, lenalidomide, dexamethasone, sitagliptin, simvastatin, azithromycin, formoterol, thalidomide, fluoxetine, lopinavir, valsartan, verapamil, chloroquine. The best rank of dexamethasone was 6. The prediction is similar to the prediction using GO term analysis. Therefore, the prediction can be used as additional evidence of how these drugs can potentially reverse the differentially expressed genes.For the human samples, there were 156 common up-regulated genes and 162 common down-regulated genes between the human lung tissues and BALF samples. respectively. The common up- and down-regulated genes were used to identify potentially effective drugs in the CMAP database. Specifically, 31 out of 156 up-regulated genes, and 15 out of 162 down-regulated genes were not available in the CMAP database. Thus 125 up-regulated and 147 down-regulated genes were used. Among the top 100 FDA drugs, there were 5 drugs also appeared in the clinical trials, i.e., valsartan, doxycycline, isotretinoin, metformin, progesterone. The best rank of dexamethasone was 330 (not top-ranked). The results are not consistent with the GO-term based analysis. One possible reason is that much noisy genes were identified in the human tissue and BALF samples as signatures to identify the related drugs in the CMAP database. Therefore, GO term analysis is helpful to identify biologically meaningful gene sets as gene set signatures to identify drugs in CMAP database.Currently, there is no effective new drugs or vaccine approved for the treatment of COVID-19, though rapid developments are occurring and being translated into clinical use. As the disease continues to spread, it is becoming increasingly important to develop a treatment modality in the most time-efficient manner. One such approach is to use genetic information to inform the repurposing of available medications. This preliminary and exploratory analysis uses transcriptional response (gene expression) profiles from human host cells before and after the infection with the SARS-CoV-2. In the analysis results, some potentially important targets, signaling pathways, and a set of GOs activated within host cells after viral infection, were identified. Moreover, a set of drugs registered for COVID-19 treatment globally were also identified in the analysis. These discoveries can be helpful to facilitate the design of future clinical trials for the COVID-19 treatment. epithelial, which may be further modified on a person-to-person basis. Second, the signaling network analysis is a long-standing challenge, and the models could be improved by integrating the KEGG signaling pathways with the GOs (to include more genes) and also incorporating transcriptomic response data of cells and human tissue samples of COVID-19 patients to uncover the core signaling networks involved in the life cycle of SARS-CoV-2 within host cells. Third, the unbiased list of medications generated and presented was not filtered by route of administration or clinical applicability. For instance, the immune dampening chemotherapeutic agent docetaxel was identified, but would likely not be administered to an infected patient due to concern for augmenting viral replication. Therefore, further pipelines and additional information are needed to analyze the potential effects of these medications, and to continue developing this computational approach to medication repurposing. In addition, we will investigate both up-regulated and down-regulated genes, which could be helpful to understand the potential mechanism of viral-host signaling interactions. Network analysis and its applications for drug and drug combination prediction are challenging problems [This exploratory computational study still has some limitations that can be further improved in the future work. First, the genetic data are derived from an in vitro analysis and will inevitably have a gene expression profile different from an in vivoproblems –38. It cAdditional file 1. Table S1: Drugs inhibiting targets on the signaling network.


Antidepressants are prescribed for the treatment of a number of psychiatric disorders in children and adolescents, however there is still controversy about whether they should be used in this population. This meta-review aimed to assess the effects of antidepressants for the acute treatment of attention-deficit/hyperactivity disorder (ADHD), anxiety disorders (ADs), autistic spectrum disorder (ASD), enuresis, major depressive disorder (MDD), obsessive-compulsive disorder (OCD), and posttraumatic stress disorder (PTSD) in children and adolescents. Efficacy was measured as response to treatment  and tolerability was measured as the proportion of patients discontinuing treatment due to adverse events. Suicidality was measured as suicidal ideation, behavior (including suicide attempts) and completed suicide. PubMed, EMBASE, and Web of Science were systematically searched (until 31 October 2019) for existing systematic reviews and/or meta-analyses of double-blind randomized controlled trials. The quality of the included reviews was appraised using AMSTAR-2. Our meta-review included nine systematic reviews/meta-analyses . In terms of efficacy this review found that, compared to placebo: fluoxetine was more efficacious in the treatment of MDD, fluvoxamine and paroxetine were better in the treatment of AD; fluoxetine and sertraline were more efficacious in the treatment of OCD; bupropion and desipramine improved clinician and teacher-rated ADHD symptoms; clomipramine and tianeptine were superior on some of the core symptoms of ASD; and no antidepressant was more efficacious for PTSD and enuresis. With regard to tolerability: imipramine, venlafaxine, and duloxetine were less well tolerated in MDD; no differences were found for any of the antidepressants in the treatment of anxiety disorders (ADs), ADHD, and PTSD; tianeptine and citalopram, but not clomipramine, were less well tolerated in children and adolescents with ASD. For suicidal behavior/ideation, venlafaxine (in MDD) and paroxetine (in AD) were associated with a significantly increased risk; by contrast, sertraline (in AD) was associated with a reduced risk. The majority of included systematic reviews/meta-analyses were rated as being of high or moderate in quality by the AMSTAR-2 critical appraisal tool . One included study was of low quality and two were of critically low quality. Compared to placebo, selected antidepressants can be efficacious in the acute treatment of some common psychiatric disorders, although statistically significant differences do not always translate into clinically significant results. Little information was available about tolerability of antidepressants in RCTs of OCD and in the treatment of ADHD, ASD, MDD, and PTSD. There is a paucity of data on suicidal ideation/behavior, but paroxetine may increase the risk of suicidality in the treatment of AD and venlafaxine for MDD. Findings from this review must be considered in light of potential limitations, such as the lack of comparative information about many antidepressants, the short-term outcomes and the quality of the available evidence. There are many classes of antidepressants, which include selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs), noradrenaline and specific serotonergic antidepressants (NASSAs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors (MAOIs). Antidepressants are one of the possible intervention strategies for a number of mental health conditions in adults as well as in young people. Indeed, several antidepressants are currently licensed for the treatment of child and adolescent psychiatric disorders, with specific indications varying across countries. For instance, in the USA, fluoxetine and escitalopram are Food and Drug Administration (FDA)-approved for major depressive disorder (MDD), fluoxetine, sertraline, fluvoxamine, and clomipramine for obsessive-compulsive disorder (OCD), duloxetine for generalized anxiety disorder (GAD) and the combination of olanzapine and fluoxetine for bipolar depression . In the Over the past decade, the use of antidepressants in children and adolescents has increased in many Western countries. From 2005 to 2012, the prevalence of antidepressant use has increased from 1.3% to 1.6% in the USA, from 0.7% to 1.1% in the UK, from 0.6% to 1.0% in Denmark, from 0.5% to 0.6% in Netherlands, and from 0.3% to 0.5% in Germany .We conducted a meta-review of the existing literature on RCTs in children and adolescents across a number of disorders (see list below). We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) approach . The prost October 2019 for systematic reviews and meta-analyses of double-blind RCTs on the use of orally-administered antidepressants in the treatment of children and adolescents (aged 18 or below) with a diagnosis of anxiety disorder (AD), ADHD, autistic spectrum disorder (ASD), Enuresis, MDD, OCD, and posttraumatic stress disorder (PTSD) according to standard operationalized criteria such as Diagnostic and Statistical Manual of Mental Disorders (DSM)-III, DSM-III-R, DSM-IV(TR), DSM-5, International Classification of Diseases (ICD)-10 or Research Diagnostic Criteria. We searched a long list of antidepressants .Finally, in terms of suicidal behavior/ideation venlafaxine in children/adolescents with depression and paroxetine in those with ADs, respectively, were associated with a significantly increased risk, sertraline was associated with a reduced risk in youth with anxiety, and no cases of completed suicides were reported in studies of OCD and PTSD.Overall, the evidence from our meta-review is only partially in line with the current license status of antidepressants in children and adolescents. In fact, our findings support: 1) the current license of fluoxetine for MDD; 2) the approval of fluoxetine and sertraline for OCD [e.g., USA  the guidelines from the National Institute of Clinical Care and Excellence  on the uOur findings, which point to the possible efficacy of at least some of the so-called “antidepressants” for the treatment of a number of psychiatric disorders, lend support to the Neuroscience-based Nomenclature (NbN) initiative  which prvia the AMSTAR-2 tool   anxiety  on AD).  anxiety .It should also be noted that a high placebo response may impact the estimated efficacy of antidepressants . Indeed,In terms of tolerability, the evidence we gathered showed that, whilst some antidepressants  were less well tolerated than placebo in the treatment of acute major depression, their tolerability was not statistically different from that of placebo in the treatment of ADs, ADHD, and PTSD. These findings could suggest a less good tolerability of antidepressants in young people with depression as compared to other disorders. However, this conclusion should be taken very cautiously, as the inclusion criteria for the participants, and hence their clinical characteristics that may impact on tolerability, varied across the included meta-analyses.Our meta-review also focused on the risk of suicidal behavior and ideation, a very relevant and controversial topic . Whilst In addition to the methodological issues in accurately estimating the prevalence and magnitude of the problem due to the fact that many trials  only relied on spontaneous reports of suicidal behavior and/or ideation, we highlight here that, since the warning, there has been a decline in the prescription of antidepressants in young people, alongside an increase in the rate of suicidal events among patients with severe depression . These fIn addition to the strengths of this meta-review, which was based on a comprehensive search of the literature and an assessment of the quality of the systematic reviews/meta-analyses, a number of limitations should be noted. First, we did not include all available systematic reviews and meta-analyses; rather, we retained only the most comprehensive/recent, so we may have missed some relevant data. It is worth noting that the latest guidance from the Cochrane handbook does not indicate a specific way to select systematic reviews/meta-analyses to be included in meta-reviews; rather, it offers a number of options, including our approach .It is also possible that the retained meta-analyses did not include RCTs that would provide relevant information for the present meta-review, owing to the specific inclusion criteria of each meta-analysis. However, checking the references of the included systematic reviews/meta-analyses, we found only two small studies  that would not change the results materially . We also restricted the search to limited number of antidepressants (see Method section for our protocol) and to articles in English only. It is likely that other pharmacological interventions are also used for the disorders under investigation in this article, however we decided to focus on the antidepressants which are more frequently prescribed in real world practice .Second, the systematic reviews/meta-analyses were focused on RCTs only, which have well-known limitations in terms of duration , reporting of adverse events and selection of participants, which limits their external validity in terms of efficacy and tolerability. Even if we searched for unpublished data, it is known that the published literature is also biased substantially as a result of selective outcome reporting , not onlResults from our analysis should be taken with caution and our meta-review is not informative on the long-term effects of antidepressants in children. Whilst conducting RCTs in the long-term is challenging from a practical and ethical standpoint, the use of discontinuation trials , which aThird, our meta-review does not allow for the comparison of the efficacy and tolerability of antidepressants across a number of disorders. Therefore, based on our findings it would be misleading to conclude that, for instance, venlafaxine is associated with increased risk of suicidal behavior/ideation in major depression but not in AD, as data for major depression and anxiety, respectively, were derived from two different network meta-analyses with different inclusion/exclusion criteria.Fourth, as per review protocol, we limited the analysis of adverse events to those that resulted in discontinuation. The number of people dropping out from treatment because of side effects can be considered a pragmatic measure of severity of symptoms, however, this information contributes only to part of the full clinical picture. Adverse events reduce quality of life and therefore may reduce the benefits of antidepressants, despite not resulting in discontinuation. Moreover, specific adverse events—no matter how severe they are—are important for patients and for the shared decision-making process . TolerabIn conclusion, the results from our analysis of aggregate data should be contextualized, incorporating patient’s values and preferences . TreatmeThe datasets generated for this study are available on request to the corresponding author.Protocol was designed by KB, AT, SC, and AC. Search was performed by KB and reference screening undertaken by KB, AT, and SC. Data extraction was performed by KB and double-checked by AT or SC. The manuscript was written by KB, AT, and SC and reviewed by AC. All authors contributed to the article and approved the submitted version.AC is supported by the National Institute for Health Research (NIHR) Oxford cognitive health Clinical Research Facility, by an NIHR Research Professorship (grant RP-2017-08-ST2-006), by the NIHR Oxford and Thames Valley Applied Research Collaboration and by the NIHR Oxford Health Biomedical Research Centre (grant BRC-1215-20005). The views expressed are those of the authors and not necessarily those of the UK National Health Service, the NIHR, or the UK Department of Health.Two authors (AC and SC) are authors on two of the systematic reviews included in our meta-review [Cipriani et al. ; CorteseThe remaining 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.


Cryptosporidium spp. and Giardia duodenalis are common gastrointestinal parasites with a broad range of hosts, including humans, livestock, and wildlife. To examine the infection status and assess the zoonotic potential of Cryptosporidium spp. and G. duodenalis in dairy cattle in Gansu, China, a total of 1414 fecal samples were collected from the rectum, with one sample collected from each individual animal. All the samples were tested using nested PCR based on the small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp. and G. duodenalis. The overall infection rates of Cryptosporidium spp. and Giardia duodenalis were 4.2% (n = 59) and 1.0% (n = 14), respectively. Four Cryptosporidium species were identified: C. andersoni (n = 42), C. parvum (n = 12), C. bovis (n = 5), and C. ryanae (n = 1). In further analyses of subtypes of C. parvum isolates based on the 60 kDa glycoprotein (gp60) gene, five were successfully subtyped as IIdA19G1 (n = 4) and IIdA15G1 (n = 1). All 14 G. duodenalis isolates were identified as assemblage E using the triosephosphate isomerase (tpi) gene. The relatively low positive rates of Cryptosporidium spp. and G. duodenalis detected here and the predominance of non-human pathogenic species/assemblages of these parasites indicated their unique transmission dynamics in this area and the low level of threat posed to public health. However, continuous monitoring and further studies of these parasites should be conducted for the prevention and control of these pathogens. Cryptosporidium spp. and Giardia duodenalis are common gastrointestinal parasites with a broad range of hosts, including humans, livestock, and wildlife [wildlife . These pwildlife . When inwildlife .Cryptosporidium species and more than 70 genotypes have been described using molecular diagnostic tools based on the small subunit ribosomal RNA (SSU rRNA) gene [Cryptosporidium spp. have been identified in dairy cattle, with C. parvum, C. bovis, C. andersoni and C. ryanae being the most common species [C. scrofarum, C. felis, C. suis, and C. hominis also have been detected in dairy cattle [Cryptosporidium parvum has been classified into at least 19 subtype families: IIa to IIi and IIk to IIt, by means of sequencing analysis of the gp60 gene [At least 44 NA) gene , 38. Pre species . Additioy cattle . Cryptosp60 gene , 17. In p60 gene . In othep60 gene .SSU rRNA, G. duodenalis was classified into eight distinct assemblages (A–H), of which assemblages A and B can infect humans and various animals [According to molecular characterization based on sequence analyses of  animals . Assembl animals . However animals . In dair animals , 35.Cryptosporidium spp. and G. duodenalis in pre-weaned dairy calves varies among different areas in China. The predominant species of Cryptosporidium spp. in pre-weaned calves in Ningxia is C. parvum, whereas in Henan and Shandong, it is C. bovis, and C. andersoni is the primary species in Shaanxi Province [G. duodenalis assemblage E has been detected in Beijing [G. duodenalis assemblage E  and assemblage E mixed with A  have been found in Hebei and Tianjin [Cryptosporidium spp. and G. duodenalis infections in dairy cattle could not only be potential sources for humans acquiring cryptosporidiosis and giardiasis but also could cause great economic losses [In China, dairy cattle have been regarded as a significant reservoir for human acquisition of cryptosporidiosis and giardiasis . The disProvince , 22, 24. Beijing , while G Tianjin ; assembl Tianjin . Cryptosc losses , 31.Cryptosporidium spp. and G. duodenalis in dairy cattle in Gansu province.Gansu Province, located on the northwestern plateau of China, is one of the key zones of both the ancient Silk Road and the New Silk Road Economic Belt. The main climatic feature of Gansu is arid conditions with scarce precipitation, making it one of the driest places in the world. According to official statistics, there are approximately 25.6 million people and 4.4 million dairy cattle in Gansu Province. To date, however, there have been few epidemiological investigations, and little is known about the prevalence and molecular characterization of parasites in dairy cattle in Gansu province. The purpose of the present study was to assess the prevalence and determine the species and genotypes of This study was conducted in accordance with the Chinese Laboratory Animal Administration Act of 1988. The research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University. The farmers gave permission to collect fecal samples, and no animals were injured during the collection.Fresh fecal samples were collected directly from the rectum using disposable gloves, and the fecal samples were placed in containers with ice packs and quickly transported to the laboratory. All fecal samples were placed in the refrigerator at 4 °C until DNA extraction. A total of 1414 samples were collected between April 2015 and July 2015 from nine dairy cattle farms in Gansu , and oneWe used an E.Z.N.A.™ Stool DNA Kit  to extract the genomic DNA, of which the first step is adding 200 mg X glass beads and 540 SLX-Mlus buffer to stool samples, then vortex at maximum speed for 10 min. The extracted DNA was stored at −20 °C before PCR amplification.Cryptosporidium spp. were identified based on the small subunit (SSU) rRNA gene by nested PCR [C. parvum was subtyped with nested PCR targeting the gp60 gene [Giardia duodenalis was identified based on the SSU rRNA [tpi) genes by nested PCR [Cryptosporidium spp. SSU rRNA gene and C. parvum gp60 gene were performed in 25 μL volume, including 1 μL template DNA or primary PCR product, 2.5 μL 10× KOD-Plus PCR buffer, 2.5 μL dNTPs (2 mM), 1.5 μL MgSO4 (25 mM), 0.5 μL of each primer (25 μM), 16 μL double distilled water, and 0.5 μL KOD-Plus amplification enzyme (1 unit/μL) . A total of 35 cycles were carried out; each of these consisted of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min. There was also an initial hot start at 94 °C for 3 min and a final extension at 72 °C for 7 min. The secondary cycling conditions were identical to those used in the primary PCR. The PCR reaction mixtures for the SSU rRNA and tpi loci of G. duodenalis were conducted: 2.5 μL 10× PCR buffer, 2 μL dNTPs (1.25 mM), 0.3 μL each primer (25 μM), 0.2 μL rTaq DNA polymerase (1 unit/μL) , 2 μL DNA sample, 16.7 μL double distilled water, and 1 μL of bovine serum albumin (10 mg/mL). Thirty-five PCR cycles  with an initial hot start (96 °C for 4 min) and final extension (72 °C for 4 min) were carried out. The secondary PCR cycle conditions were identical to the primary round except that the annealing temperature was increased to 59 °C.sted PCR , and C. p60 gene . GiardiaSSU rRNA  and triosted PCR . The prihttp://blast.ncbi.nlm.nih.gov) using Clustal X 2.10 .The positive secondary PCR products were sequenced bidirectionally by SinoGenoMax Biotechnology Co., Ltd . To confirm different species or genotypes, sequences obtained in this study were aligned with reference sequences downloaded from GenBank (http://www.megasoftware.net) based on evolutionary distances calculated with the Kimura 2-parameter model. The reliability of these trees was assessed with a bootstrap analysis of 1000 replicates.To infer the phylogenetic relationships of the detected samples, neighbor-joining (NJ) trees were constructed with the MEGA X program , was calculated. The chi-square test was used to compare differences in infection rates between different age groups and clinical symptoms. A two-tailed MT820515–;MT820519, ;MT821528, and ;MT821529.The representative nucleotide sequences obtained from this study were submitted to GenBank under the following accession numbers: ;Cryptosporidium spp., and the overall infection rate was 4.2%. The highest infection rate  was detected on a farm in Hezheng county, followed by Minle county  and Ganzhou city . Equal infection rates (3.2%) were detected in Shandan (n = 4) and Jinta counties (n = 3), and the infection rates in Gaotai, Linze, and Sunan counties, and Yuzhong district were 2.9% (n = 4), 2.4% (n = 3), 2.3% (n = 9), and 2.2% (n = 5), respectively. Fourteen out of 1414 fecal samples were positive for G. duodenalis, with an overall infection rate of 1.0%. The highest infection rate  of G. duodenalis was observed on a farm in Gaotai, followed by Linze  and Jinta . Equal infection rates  were detected in Ganzhou and Minle, and the infection rates in Shandan and Sunan were 0.8% (n = 1) and 0.5% (n = 2), respectively. There was no G. duodenalis detected in the Yuzhong or Hezheng , the predominant species, accounted for 71.2% of all Cryptosporidium spp. infections and was detected on six farms, while C. parvum (n = 12), C. bovis (n = 5), and C. ryanae (n = 1) were detected on six, three, and one farms, respectively. In the further analyses of subtypes of the 12 C. parvum isolates, five isolates were successfully subtyped, including subtype IIdA15G1 (n = 1) and subtype IIdA19G1 (n = 4)  .G. duodenalis SSU rRNA gene showed that all 14 positive samples belonged to assemblage E. Two distinct genotypes from 12 assemblage E isolates were observed including E1 (n = 10) and E2 (n = 2) by amplification and sequencing of the tpi gene.Sequencing analyses of the C. andersoni was found in all age groups except the <3-month group, while C. parvum was only found in <3-month-old dairy cattle. Cryptosporidium bovis and C. ryanae were only detected in 3–11-month-old dairy cattle. With the exception of >24-month-old dairy cattle, G. duodenalis assemblage E was detected in all age groups, including nine in <3-month-old dairy cattle, three in 3–11-month-old dairy cattle, and two in 12–24-month-old dairy cattle  compared to the reference sequence ;MK796098. The sequences belonging to C. parvum, C. bovis, and C. ryanae had 100% homology to the reference sequences ;MT002720, ;MH028031 and ;MK982468, respectively. Of the G. duodenalis tpi gene, E1 and E2 exhibited 100% homology to the reference sequences ;KY769101 and ;KY432851, respectively. To estimate phylogenetic inferences among the identified positive samples, NJ trees were inferred in MEGA X  was observed in the <3-month-old group, followed by the 3–11-month-old group , the 12–24-month-old group , and the >24-month-old group . Compared with the <3-month-old group, an OR of 0.47 , an OR of 0.38 , and an OR of 0.27  were associated with the 3–11-month-old group , 12–24-month-old group, and >24-month-old group, respectively. The infection rates of G. duodenalis were 7.5% (n = 9), 1.8% (n = 3), and 0.4% (n = 2) in <3-month-old, 3–11-month-old, and 12–24-month-old animals, respectively. There was a significant negative correlation between the G. duodenalis infection rate and age in dairy cattle, as an OR of 0.23  was associated with the 3–11-month-old group, and 0.05  was associated with the 12–24-month-old group  compared to calves with diarrhea , with an OR of 0.16   and without diarrhea  were not statistically significant (p = 0.312)  . However= 0.312) .Cryptosporidium spp. was 4.2%. Similar or slightly lower infection rates have been reported in previous studies of Cryptosporidium spp. in dairy cattle in other provinces such as NXHAR  [G. duodenalis was 1.0%, which was consistent with the infection rates reported in NXHAR  [Cryptosporidium spp. and G. duodenalis in dairy cattle may vary according to geographical location. Moreover, water-borne transmission is an important route of Cryptosporidium spp. and G. duodenalis cross-species transmission [In the present study, the overall infection rate of 23/1366) , Qinghai23/1366) , Shaanxi23/1366) , and Gan23/1366) . The inf23/1366) , 29, Anh23/1366) , 40, and29/1366) , and was29/1366) , Heilong29/1366) , and Jia29/1366) . These dCryptosporidium species in <3-month-old, 3–11-month-old, 12–24-month-old and > 24-month-old groups were C. parvum, C. bovis, C. andersoni and C. andersoni, respectively. Remarkably, the distribution of the four most common Cryptosporidium species is related to the age of dairy cattle. Cryptosporidium parvum is often detected in pre-weaned calves, which currently result in pre-weaned calf diarrhea. Cryptosporidium bovis and C. ryanae are commonly seen in post-weaned calves and yearlings, and C. bovis is more common than C. ryanae [C. andersoni is more likely to infect adult dairy cattle, which normally leads to lower milk production and weight loss [The dominant . ryanae . Howeverght loss .C. parvum isolates, 5 were identified as subtype IIdA19G1 (n = 4) or subtype IIdA15G1 (n = 1) based on sequencing analysis of the gp60 gene, and these 2 subtypes have also frequently been found in bovines [Of the 12  bovines . In addi bovines , 19. Mea bovines , 30. ThuG. duodenalis assemblage E was the dominant assemblage, consistent with the results reported worldwide [G. duodenalis transmission for the protection of animals and breeders in the area, although the zoonotic risk of G. duodenalis was limited in this study.In this study, orldwide . These rorldwide , 39. TheCryptosporidium spp. and G. duodenalis were relatively low in this area. Moreover, the study revealed the presence of C. parvum subtypes IIdA19G1 and IIdA15G1, and only G. duodenalis assemblage E was identified in dairy cattle in Gansu province, indicating that zoonotic transmission of Cryptosporidium spp. and G. duodenalis is possible but unlikely. Therefore, it is necessary to adopt effective strategies to prevent and control the transmission of Cryptosporidium spp. and G. duodenalis among dairy cattle and humans in Gansu province.In conclusion, the infection rates of The authors declare that they have no conflicts of interest.