While C-terminal truncations are known to accelerate α-synuclein aggregation, the understanding of how N-terminal truncations influence fibril formation lags behind. Here, we show that the removal of N-terminal residues outside the canonical amyloid core weakens the fibril seeding efficiency and hinders propagation. Remarkably, a unique, asymmetric amyloid core is revealed by cryogenic electron microscopy in the N-terminally truncated 41–140 variant, highlighting the strong influence of the N terminus in dictating fibril structure. This work offers insights into the interplay of truncations and full-length proteins; thus, changes in their relative levels could prove to be beneficial for molecular intervention. The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson’s disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41–140). This behavior can be explained by the unique fibril structure that is adopted by 41–140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent β-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended β-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations. Amyloid formation of α-synuclein (α-syn) is a pathological feature of Parkinson’s disease (PD), multiple-system atrophy (MSA), and dementia with Lewy bodies , 2. An a410α-Syn truncations originate from incomplete degradation, which has been attributed to various cytosolic –15 and l1518SI Appendix, Fig. S1). Generally, residues between 37 and 97 constitute the fibril core with a few exceptions that involve additional residues in the N terminus, which include phosphorylated Y39 fibrils with an extended core of 1–100  has revealed fibril structures for full-length α-syn 1–140) , 30–32, 30 , 30–32,30of 1–100  and two 2) stabilized by an intermolecular salt bridge between K80 and E83. Collectively, these results establish the important role of N-terminal residues in fibril formation and structure.Here, we sought to understand the role of the N terminus in α-syn fibril formation by removing different N-terminal residues and evaluating their effects on aggregation kinetics, fibril structure, and propagation. Three ∆N-terminal constructs  have been examined, in which the first 13-, 35-, and 40-residues in the N terminus were deleted A. We spet1/2) of 44 ± 4 h, which was reduced to 33 ± 4 h at 70 µM . Specifically, t1/2 values were 25 ± 3, 20 ± 2, and 19 ± 2 h for 70 µM 14‒140, 36‒140, and 41‒140, respectively.To assess the role that the N terminus plays in fibril assembly, three ∆N-α-syn proteins were generated: 14–140, 36–140, and 41–140 A. Their at 70 µM C. While SI Appendix, Fig. S2). Densitometric analysis of the aggregation reactions revealed that only for 14‒140 does the lower final ThT signal correlate with a reduced amount of aggregated proteins (50% compared to 85% full-length). In contrast, at least 90% of both 36‒140 and 41‒140 are in the pelleted fraction, which is comparable to 1‒140, suggestive of ThT sensitivity to fibril polymorphism.Interestingly, lower final ThT intensities were consistently observed for the ΔN-α-syn variants. To determine whether the observed ThT intensity differences are due to relative amounts of aggregated proteins, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed after ultracentrifugation . The twisted fibrils have varying helical pitches ranging from ∼54 to 300 nm. For 41‒140, the main species are twisted fibrils with a helical pitch ∼53 nm with a minor population of fibril bundles . These twisted fibrils are not seen for 1‒140.Negative-stain TEM images taken postaggregation show fibril differences at the ultrastructural level D–G. As pSI Appendix, Fig. S6). In contrast, 41‒140 was more protease sensitive, especially at the higher PK concentrations. Liquid chromatography–mass spectrometry analysis  indicated that there is a consistent C-terminal cutting site at residues Y125/E126 (in which / denotes cut site) for all constructs. For 36‒140 and 41‒140, another cut site at L113/E114 is also revealed. In the N terminus, 1‒140 revealed cleavages at A18/A19, A19/E20, and A30/G31 with the smallest PK-resistant core being residues 31‒125. For 14‒140, 36‒140, and 41‒140, their smallest cores are 36‒125, 36‒113, and 41‒113, respectively. An additional cleavage site at H50/G51 was also observed for 41‒140, resulting in a 51‒125 fragment. It is interesting to note that cut sites E35/G36, H50/G51, and L113/E114 are not observed in the full-length protein, suggesting that the structures of full-length and ΔN-α-syn fibrils are distinct.To evaluate the protease-resistant structured cores of the ΔN-α-syn fibrils, limited proteolysis was performed using a broad-spectrum protease, proteinase K (PK). SDS-PAGE analysis revealed greater resistance for 14‒140 and 36‒140 compared to degradation profiles of 1‒140 ; however, the uncertainty in our measurements precluded the evaluation whether there were significant differences among the ΔN-variants and the full-length fibrils for either cell line. Based on these results, these ΔN-fibrils have comparable cytotoxicity to cells under the conditions examined.The cytotoxicity of 1‒140, 14‒140, 36‒140, and 41‒140 α-syn fibrils were examined in two different cell lines: human SH-SY5Y neuroblastoma and rat dopaminergic N27 cells. Cell viability was assessed by mitochondrial activity after 48 h treatment with fibrils (1 μM). Fibrils were prepared by ultracentrifugation, and the pelleted fibrils were quantified prior to treatment. Using a standard 3--2,5-diphenyltetrazolium bromide assay, impaired mitochondrial activity was consistently observed from all fibril treatments compared to the buffer control . As a reference, self-seeding by preformed 1‒140 fibrils (5%) accelerated the aggregation of the full-length protein by abrogating the lag phase and enhancing the final ThT signal. This result is recapitulated at a higher seed concentration . By comparison, only the addition of 14‒140 fibrils stimulated 1‒140 aggregation by shortening the lag time and increasing the final ThT signal, whereas in the presence of either 36‒140 or 41‒140 fibrils, 1‒140 exhibited indistinguishable kinetics to that of itself alone. SDS-PAGE analysis indicated less pelleted material after ultracentrifugation for 14‒140 seeded reactions, supporting that 14‒140 fibrils are inferior seeds for 1‒140 . Postaggregation SDS-PAGE analysis revealed that while 14‒140 has a higher level of aggregated materials compared to the unseeded control, a similar amount of pelleted protein was obtained for 36‒140, cautioning that higher ThT signals do not necessarily correlate to fibril amounts.Having established that 36‒140 and 41‒140 fibrils cannot cross-seed the full-length soluble protein, we investigated whether the reverse reaction would also be prohibitive, which would further substantiate the importance of N-terminal residues in fibril propagation. Efficient seeding was observed in the presence of full-length fibrils for both 14‒140 and 36‒140, with lag phases abolished and higher final ThT signals A and B. Right), which are not characterized from those of the unseeded reaction . Interestingly, while the fibril morphology of 36‒140 remained heterogenous (Right), the observed helical pitches of the twisted fibrils were ∼90 and 220 nm, which again are different from those of the unseeded reaction , indicating an effect of the full-length seeds on the growth of 36‒140 fibrils. It is evident that 1‒140 was not able to promote 41‒140 fibril growth since the fibrils are highly reminiscent (Right) to those formed from the 41‒140 control .This cross-seeding by full-length fibrils of 14‒140 was corroborated by TEM images taken postaggregation, which revealed more homogeneous fibrils with a half pitch of ∼80 nm A, Right,rogenous B, Right,iniscent C, Right Collectively, these cross-seeding experiments suggest that different mechanisms are at play depending on the fibril seeds and soluble proteins used. Since 14‒140, but neither 36‒140 or 41‒140 fibrils, could enhance 1‒140 aggregation, residues between 14 and 35 could be important in the productive recruitment of full-length protein. Alternatively, the removal of the N-terminal 35 residues could completely change the fibril structure that is favored only by ΔN-variants. However, when using seeds with an intact N terminus, only 41‒140 failed to template, which would indicate that residues 36 through 40 are more important in either scenario, in the replication of the full-length structure and the formation of a different fibril structure.SI Appendix, Table S3). The twisted fibrils seen in negative-stain TEM . A cross-sectional density map of the fibril is also shown in Bottom Left.To gain an understanding to our cross-seeding observations, we sought to obtain fibril structural information by cryo-EM. Out of the three ∆N-constructs studied, 41‒140 fibrils had the most homogenous population, and thus, the fibril structure of 41‒140 was successfully determined by cryo-EM at near-atomic resolution , with clear side-chain density that permitted unambiguous de novo atomic model building. A different number of residues was resolved for the two asymmetric protofilaments, comprising residues E46‒K96 and E61‒D98 for chains A and B, respectively. Chain A folds into a β-strand–rich architecture including the β-arch kernel (E57–T72) that is present in other full-length α-syn structures . While specific residue assignment was not possible, it likely represents residues between G41 and K60. This extra density also results in compaction of residues E46–H50 in chain A that is otherwise more extended in the main polymorph. Similar to published structures . Additional hydrogen-bonding is found between the two Q62 residues. This interface . We note that K80 from chain B is found buried at the interface, which was also found in the hydrophobic core of the E46K fibril structure, in which it is interacting with a resolved water molecule  . In the Ac1–140 structure, β7 is folded back and packs against β6.A comparison of chains A and B of 41‒140 to the acetylated (Ac) 1‒140 protomer (Protein Data Bank [PDB] code 6OSJ) is shown in Fig. S1) , 31–33. The overlay of chain B with Ac1‒140 shows striking conformational differences. There are only 13 overlapping residues, A76 to A89, which span strands β6 and β7. This extended hairpin conformation composed of residues G68 to V95 appears unique to this structure thus far. Collectively, these results support the idea that N-terminal residues modulate the mechanism of amyloid assembly and contribute to the molecular interactions that define fibril structure.SI Appendix, Figs. S3–S5). Structure determination of 41‒140 by cryo-EM revealed a fibril conformation in which two asymmetric protofilaments with different lengths of ordered residues interwind to form a twisted fibril .Fibril polymorphism is a defining feature of pathological amyloids, yet mechanistic insight into how multiple structures are generated from a given polypeptide chain is still an open question. In this work, we explored the impact of the N terminus on α-syn fibril formation by using a series of N-terminal truncations. The removal of 13-, 35-, and 40-residues from the N terminus–influenced aggregation behaviors , and alld fibril . A larged fibril . An inted fibril A. IncludSince the N-terminal region (residues 1 through 36) is unresolved and thus presumed to be largely disordered in most full-length α-syn fibril structures, the impact of its removal on the 41‒140 structure was initially surprising. However, recent work has demonstrated the participation of N-terminal residues in two 1‒140 polymorphs in which residues V16 through E20 form a hydrophobic steric zipper with residues S87 through A91 . Also, fUsing cross-fibril propagation experiments, we conclusively show the importance of the N terminus in α-syn fibril formation. As the N terminus is removed, ΔN-α-syn fibrils were shown to be poor seeds for soluble full-length α-syn . This is37VLYV40, in facilitating the formation of the full-length structure. This postulate is supported by recent work on a Δ36–42 α-syn mutant (From a mechanistic standpoint, two recent studies also have highlighted the involvement of N-terminal residues in fibril formation. Specifically, residues 1–11  and 1–12n mutant , which sWith ∆N-α-syn modulating aggregation kinetics as well as fibril structure and propagation, a central question is whether these species are beneficial or detrimental in a cellular environment. It is known that both N- and C-terminal truncations are prevalent in both PD and healthy individuals , and somSI Appendix.Details regarding reagents, molecular cloning, protein expression and purification, aggregation kinetics, TEM, liquid chromatography–mass spectrometry, cell culture and cell viability assay, cryo-EM sample preparation, and data collection and processing as well as model and refinement are provided in Supplementary File


Thymic stromal lymphopoietin (TSLP), a distant paralog of the cytokine IL-7, has been shown to be associated with atherosclerosis. However, the effect of plasma TSLP level after acute myocardial infarction (AMI) remains largely unclear. Thus, we aimed to assess the relationship between the concentration of TSLP at admission and the risk of major adverse cardiovascular events (MACE) in AMI patients.A total of 175 patients with AMI and 145 unstable angina (UA) controls were recruited in the present study. The clinical characteristics were collected, and MACE was recorded during hospitalization and the follow-up period after discharge.p < 0.001, respectively]. Multivariate linear regression analysis revealed that Troponin-I  was an independent factor for TSLP. According to the median of TSLP concentrations, all the AMI patients were divided into the high-level group  and the low-level group . In a receiver operating characteristic curve analysis, the area under the curve for TSLP as a predictor of AMI was 0.674 with a cut-off value of 9.235 pg/ml. After a median follow-up of 14 months, Kaplan-Meier survival analysis showed no significant difference in MACE-free survival between the two groups (p = 0.648). Finally, the multivariate logistic regression analyses demonstrated that TSLP was a negative predictor of MACE in AMI patients .The median value  of TSLP concentrations in the AMI group was higher than that in the UA group [11.18 (8.14–15.22) vs. 8.56 (5.26–11.94) pg/ml, Plasma TSLP levels were elevated in patients with AMI than those in UA. The lower TSLP concentration was associated with MACE after AMI. Coronary artery disease (CAD) has emerged as a common cause of death over the past few decades. Acute myocardial infarction (AMI) is one of the most serious manifestations of CAD, with a high fatality rate and poor prognosis . TherefoThymic stromal lymphopoietin (TSLP) is a distant analog of the cytokine interleukin-7 (IL-7) and a member of the four-helix-bundle cytokine family . TSLP isThe most common cause of death in patients with AMI is a malignant arrhythmia or heart failure, which may be closely related to inflammatory responses and is one of the causes of long-term mortality after AMI. However, there are very few studies on plasma TSLP in patients with AMI. Hence, this study aimed to investigate the correlation between plasma TSLP level with AMI and to evaluate the correlation between plasma TSLP level and major adverse cardiovascular events (MACE) after AMI.A cross-sectional observational trial was conducted in a single center. All subjects were recruited in the Heart Center of Beijing Chaoyang Hospital, Capital Medical University between May 2019 and November 2019. A total of 175 patients were diagnosed with AMI, and 145 unstable angina (UA) patients as controls were included in the present study. Criteria for AMI diagnosis were as follows: clinical symptoms, typical electrocardiogram changes, elevated cardiac biomarkers (cardiac troponin-I and creatine kinase MB), and coronary angiography. The exclusion criteria were: (1) asthma, (2) autoimmune diseases, (3) pregnancy, (4) neoplasm, (5) severe liver or kidney disease, and (6) severe inflammatory or infectious diseases. The clinical characteristics, including demographic data, history, and echocardiography were collected at the time of enrollment. The study protocol was in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Beijing Chao-Yang Hospital of Capital Medical University. All patients provided written informed consent for participation in the trial.All the blood samples were collected on the first morning after admission under a fasting state from the peripheral vein. Plasma was obtained from the blood samples by centrifugation at 3,000 rpm for 15 min and stored at −80°C, without repeated freeze-thaw cycles until analysis. Plasma concentrations of TSLP were measured using ELISA . The operation procedures followed the instructions of the ELISA kit. The intra-assay and inter-assay variations were <5%. The laboratory results, including type b natriuretic peptide (BNP), leukocytes, platelets, troponin I, creatine kinase-MB (CK-MB), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), D-dimer, and low-density lipoprotein cholesterol (LDL-c), were determined at the clinical laboratory center using standard protocols. The Synergy between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery (SYNTAX) score was calculated to assess the severity of coronary lesions according to the data of coronary angiogram. The Global Registry of Acute Coronary Events (GRACE) risk score is a practical risk assessment tool for in-hospital prognosis.SD or median and interquartile range (25th and 75th percentiles). Categorical variables were performed using the chi-square test or Fisher's exact test. The two-sample t-test and the Mann–Whitney U-test were performed for comparisons of continuous variables. The Kolmogorov-Smirnov test was performed to check normality.Categorical variables were presented as frequencies (percentage) and continuous variables as mean ± p < 0.1 indicated the inclusions in the multivariate model. The Variance Inflation Factor (VIF) test was performed to collinearity diagnosis and logarithmic transformation is used for data conversion of non-normal distribution variables. Clinical events of the all patients were systematically followed-up. The median follow-up time of the present study was 14  were performed to identify the variables most strongly associated with TSLP levels in all patients. The univariable with y was 14 , 14 montp < 0.001, respectively] (p = 0.248). According to the median value of TSLP concentrations, all patients with AMI were divided into the high-level group (TSLP level ≥ 11.18 pg/mL) (N = 91) and the low-level group (TSLP <11.18 pg/mL) (N = 84), and the clinical parameters between the two groups were compared. There was no statistical difference in the demographic characteristics between the two groups. The higher levels of TSLP were associated with Hemoglobin (p = 0.01). To evaluate cardiac function by echocardiography, plasma TSLP concentration had no significant correlation with left ventricular ejection fraction (LVEF) or baseline left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), or left ventricular end-systolic diameter (LVESD) (p = 0.072).The median value (25th and 75th percentiles) of TSLP concentrations was higher in the AMI group than in the UA group  . The non (LVESD) . We furtr = 0.13, p = 0.03), white blood cell count , total cholesterol , LDL-C , Troponin-I , CK-MB , D-dimer  and BNP . Additionally, plasma TSLP level was also correlated with GRACE score  and LVESD . TSLP level was negatively correlated with lymphocytes  and LVEF   .p = 0.004) was an independent determinant of TSLP.n = 3) in the high-TSLP group and 13 patients  in the low-TSLP group experienced MACE. The Kaplan-Meier survival analysis showed that there was no significant difference between the two groups  (p = 0.024). This association remained still significant in the multivariate analysis, which showed that TSLP levels were the independent determinants with OR of 0.778  (The ROC curve for TSLP as a predictor of AMI showed an area under the curve of 0.674 (95% CI:0.615–0.733). A cutoff point of 9.235 pg/ml for TSLP had a sensitivity of 65.7% and a specificity of 62.1% in predicting AMI. Among the 175 AMI patients, those with TSLP > 9.235 pg/ml were more likely to smoke (= 0.047) . Further= 0.032) .In the current study, we, for the first time, evaluate the relationship between plasma TSLP levels and AMI, and its clinical outcomes. The data revealed that TSLP plasma levels were significantly higher in AMI patients than in UA patients. Moreover, TSLP plasma levels were associated with inflammatory markers, myocardial injury markers, and BNP in the CAD patients. Based on the previous findings, we speculated that circulating TSLP concentrations might be associated with the MACE in the AMI population.Thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, was originally found in the culture supernatant of a mouse thymic stromal cell line that exerts its biological function through the TSLPR . This cyAtherosclerosis is a chronic inflammatory disorder characterized by complex immunological interactions between residential vascular cells and professional immunocytes, including T-cells, macrophages, and smooth muscle cells . MortaliHeart failure (HF) is a major factor affecting the prognosis of AMI, with a significant risk of morbidity and mortality. Macrophages are the major responder cells of the immune system after AMI . There aThis study had several limitations. First, plasma TSLP level and MACE are not causally associated. The long-term prognostic value of TSLP for patients with AMI and the underlying mechanisms need to be studied in the future. Second, this was a cross-sectional study conducted in a single-center, with a low evidence-based level. Third, this study had a small sample size; so, a large sample size study is needed to verify our findings. Finally, we collected blood samples at admission and did not have serial plasma TSLP data at follow-up.This was the first study to demonstrate that the plasma TSLP concentration was increased in patients with AMI compared with the UA controls. The lower TSLP level is an independent predictor of MACE in AMI patients. Understanding the correlation between TSLP concentration and other established prognostic factors may facilitate the prognosis and management of AMI.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing Chao-Yang Hospital of Capital Medical University. The patients/participants provided their written informed consent to participate in this study.XY and LZ: designed the study. YHZ, YPZ, and CH: collected the data and performed the statistical analysis. YHZ, ZG, and ZM: wrote the manuscript. YL, XY, and LZ: revised 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Xenopus and zebrafish, highlighting the similarities and differences of the regulatory networks between species. We also review recent advances regarding the molecular nature of DV patterning, including the initiation of the DV axis, the formation of the BMP gradient, and the regulatory molecular mechanisms behind BMP signaling during the establishment of the DV axis. Collectively, this review will help clarify our current understanding of the molecular nature of DV axis formation.One of the most significant events during early embryonic development is the establishment of a basic embryonic body plan, which is defined by anteroposterior, dorsoventral (DV), and left-right axes. It is well-known that the morphogen gradient created by BMP signaling activity is crucial for DV axis patterning across a diverse set of vertebrates. The regulation of BMP signaling during DV patterning has been strongly conserved across evolution. This is a remarkable regulatory and evolutionary feat, as the BMP gradient has been maintained despite the tremendous variation in embryonic size and shape across species. Interestingly, the embryonic DV axis exhibits robust stability, even in face of variations in BMP signaling. Multiple lines of genetic, molecular, and embryological evidence have suggested that numerous BMP signaling components and their attendant regulators act in concert to shape the developing DV axis. In this review, we summarize the current knowledge of the function and regulation of BMP signaling in DV patterning. Throughout, we focus specifically on popular model animals, such as  The embryonic body plan includes the anteroposterior, dorsoventral (DV), and left-right axes, all of which are established according to cell movements and rearrangements during vertebrate embryonic development. These patterning events ultimately form tissues and organs, which were originally induced from three germ layers located along the body axis . In partAround the early 1920s, the embryologist Hans Spemann and his colleagues transplanted the dorsal lip of a blastopore to the ventral side of a host embryo during amphibian gastrulation. They found that the transplanted cells induced the ventral cells of the host embryo to form a duplicated body axis as development progressed, which included the central nervous system . Given tDrosophila, the DV axis is mainly formed by reciprocal interaction between maternal and zygotic genes. The maternal gene dorsal is ubiquitously expressed in the oocyte, as is the Dorsal protein  to direct the dorsal-related tissues . ConsequIn amphibian embryos, the establishment of the DV axis relies on the redistribution of localized maternal determinants in yolk cells during embryo fertilization. Under the influence of gravity, the cortex in oocytes transports to the animal pole. This is due to the influence of sperm entry in a process called cortical rotation. Maternal β-catenin then begins to accumulate in the future dorsal area, which is exactly opposite to the point of sperm entry. The dorsal determinants stabilize β-catenin in the future dorsal area, resulting in pigment differences that form a gray crescent . Then, tPrevious studies have shown that the transportation of zebrafish dorsal determinants is associated with DV patterning. For instance, partial removal of the vegetal yolk causes a severely ventralized phenotype with no dorsal structures like nerve tissues and dorsal mesoderm . This inXenopus embryos. This caused developmental defects in the DV axis  to knock down wnt11 function in  DV axis . 2 yearse embryo .Maternal β-catenin plays an important role in the formation of the shield and DV axis in zebrafish. For instance, the mutant ichabod embryo has a mutation in the region of the β-catenin2 promoter. This mutation causes decreased β-catenin2 expression and results in serious defects in the establishment of the DV axis . MaternaIn 2011, a team led by Bernard Thisse discovered that the potential maternal dorsal determinant in zebrafish was not Wnt11, but rather Wnt8a. Secretion of the Wnt8a protein resulted in a dorsal-high/ventral-low Wnt activity gradient, which was restricted by two Wnt inhibitors, Sfrp1a and Frzb. These proteins were essential to trigger the canonical β-catenin pathway . HoweverEstablishing the dorsal organizer of the embryo relies on both maternal factors and zygotic genes. Zygotic BMP genes are required for patterning of the DV axis in both vertebrates and invertebrates. Upon the formation of the dorsal organizer, a BMP signaling gradient is formed along the DV axis. This gradient is critical, with different signaling levels specifying different tissue types. More specifically, high levels of BMP signaling are required for a ventral fate while low levels are required for a dorsal one.BMPs belong to the transforming growth factor-β (TGF-β) superfamily, which was originally identified by their ability to induce ectopic bone formation. They are involved in massive developmental programs and cellular behaviors, including embryo morphogenesis, mesoderm formation, DV patterning, cell proliferation, and tissue homeostasis . So far,The signal transduction for the BMP pathway is highly conserved across species. As shown in BMP signaling plays a crucial role in the establishment of the DV axis. From an evolutionary perspective, the generation of the BMP signaling gradient during DV patterning is remarkably conserved . A betteThere is no consensus on the process of the formation of BMP gradient. It is generally thought that the mature oocyte is symmetric about the dorsal-ventral axis prior to fertilization. After fertilization, the maternal factors activate the ubiquitous expression of zygotic BMP at first . For insThe binding of BMP ligands to their receptors is regulated by two kind of factors with opposite functions. One type of factors are secreted antagonists that bind to BMP ligands and block signal transduction; the second promote ligand-receptor binding as membrane-anchored co-receptors . GeneralThe molecular network that generates the BMP gradient is conserved across evolution despite of the large differences in shape and size .Xenopus orthologues, forming a BMP gradient to pattern the DV axis , and secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. In planarians, the expression of BMPs and their putative antagonists are spatially opposed. BMPs and their antagonists form a regulatory circuit to regulate the BMP gradient and establish DV patterning  and BMP antagonists Sizzled, along with the organizer-secreted extracellular BMP antagonists Chordin, Noggin, Follistatin like 1b (Fstl1b), and Cerberus .Drosophila, Tolloid acts as a zinc metalloproteinase that cleaves Sog to release BMPs  domains to enhance its cleavage activity  and Crossveinless 2 (CV2) have dual activities in regulating BMP signaling. In ignaling . Tsg strhibition  , expressed on the ventral side as part of the BMP synexpression group, is a pseudoreceptor that shapes the BMP signaling gradient by negatively modulating BMP signaling  Mullins. Bambi s process .Smad1/5/8 act as major transducers downstream of BMP receptors. The deletion of maternal smad5 leads to a severe dorsalized phenotype, similar to that observed in the bmp2b/bmp7 mutant; comparatively, lack of zygotic smad5 causes weak dorsalization, suggesting maternal Smad5 plays a primary role during DV patterning . In zebrXenopus embryos. This suggests a critical role for tightly controlled protein stability during early embryogenesis /Cadherin-dependent cell-cell adhesiveness .Xenopus Spemann organizer or shield of the zebrafish embryo to the ventral-most part of a host embryo, they induce a secondary body axis at the site of the graft , the molecular nature of the generation and maintenance of the BMP signaling gradient during DV patterning has come to light. Although our knowledge of the regulatory network behind the embryonic BMP gradient has seen tremendous advancement, we are far from a complete understanding regarding the interplay between the BMP signaling gradient and embryo patterning. For example, the formation of the BMP signaling gradient relies on a restricted distribution of BMP ligands and their antagonists. However, the dynamic diffusion, interaction, and degradation of these extracellular proteins have so far not been thoroughly investigated using long-term observation and quantitative analyses. Furthermore, the identification of direct target genes of the BMP gradient is urgently needed, particularly those that are essential for DV axis patterning and ventral tissue specification through genome-wide analyses. In addition, mammals—including human beings—have different degrees of robustness for their body axial patterning. However, no expressed sequence homologous to pinhead has yet been found in bird and mammalian species. At present, little is known about the molecular mechanisms underlying the substantial DV polarity in these higher organisms. The answers to these important scientific questions will greatly promote our understanding of the molecular mechanisms in embryonic DV patterning.


The content of free SH increased significantly (p < 0.05) at 100 MPa. FTIR spectrum and fluorescence analysis confirmed the changes in the secondary and tertiary structures. The experimental results indicated that the structural and functional properties of HHP-pretreated RBPH improved.Rice bran protein (RBP) hydrolysis was conducted after high hydrostatic pressure (HHP) pretreatment. The structural and functional properties of HHP-pretreated rice bran protein hydrolysates (RBPH) were investigated. HHP pretreatments were conducted at 100, 200, and 300 MPa; then, enzymatic hydrolysis at atmospheric pressure was performed using trypsin. An RBPH sample that had not been pretreated by HHP was used as a control. Free sulfhydryl (SH) content, SDS-PAGE profiles, high-performance size exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectrum, scanning electron microscopy (SEM), intrinsic fluorescence spectrum, solubility, and emulsifying and foaming properties were evaluated. Changes in particle size and ζ-potential were monitored. Compared with the control, the results of solubility, the emulsifying activity index (EAI) and the emulsifying stability index (ESI) increased significantly ( Rice bran is a low-priced, underutilized major by-product of rice processing . Rice brPhysical, chemical, and enzymatic modification are common modification methods used at present . High hyThe objective of the current work was to investigate the influence of HHP pretreatment on the structural and functional properties of RBPH, including its solubility, emulsifying activity index (EAI), emulsifying stability index (ESI), foaming capacity (FC), and foaming stability (FS). Besides, the mechanism of HHP pretreatment on RBPH was studied by observing the particle size and ζ-potential, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), scanning electron microscopy (SEM), high-performance size exclusion chromatography (HPSEC) and Fourier transform infrared (FTIR) spectrum. The study will provide a basis for further elucidating the mechanisms of the functional properties of RBP modified by HHP pretreatment.4 U/g) was obtained from Sigma-Aldrich Co. . Deionized water was used. Chemicals and reagents were of analytical grade. Fresh rice bran was purchased from Heilongjiang Great Northern Wilderness Agribusiness Group Co., Ltd. . RBP was obtained in laboratory condition. Trypsin .RBP was extracted in the laboratory according to Zang et al. . Rice brw/v). RBP solutions (200 mL in each bag) were packed in polyethylene plastic bags, sealed after exhausting the air, placed in HHP equipment. Samples were treated by HHP at 100, 200, and 300 MPa for 30 min. The indicated pressure was reached in 1 to 2 min; this pressure was kept for 30 min and released to normal pressure in 1 to 2 min. Each HHP treatment was conducted three times.HHP pretreatment was carried out at 25 °C in HHP equipment , with water used as hydrostatic fluid. RBP was dispersed in a 0.01 M phosphate buffer ). Hydrolysis was performed in triplicate, and the reaction was stopped by heating the hydrolysates for 10 min at 90 °C. Next, the pH of the solution was maintained at 7 with 0.5 M NaOH, and the supernatant was obtained by centrifugation at 6000× g for 15 min at 4 °C. The supernatant was then stored at −20 °C until analysis.The hydrolysis experiment was carried out after HHP treatment. Hydrolysis of RBP by trypsin was performed at atmospheric pressure (0.1 MPa) for 60 min according to Zang et al. , with sow/v), and the pH was adjusted to 7.0. The dispersion was then centrifuged at 10,000× g for 20 min. The protein content in the supernatant was measured by Lowry’s method using bovine serum albumin (BSA) as the standard. Equation (1) was used to calculate the solubility.Solubility was determined according to Zang et al.  with sliw/v), then 0.5% (w/v) of an RBPH (12 mL) solution was mixed with soybean oil (4 mL) to prepare the emulsion by homogenizing for 1 min at 10,000 rpm. Next, 50 μL of the microemulsion was removed from the bottom of the emulsion and immediately diluted with 5 mL of 0.1% sodium dodecyl sulfate (SDS) solution and vortexed. After that, the absorbance of the mixtures was measured at 500 nm for 0 min and 10 min; 0.5% (w/v) of the RBPH solution was used as a control. Equations (2) and (3) were used to calculate EAI and ESI, respectively:0 and A10 are the absorbance determined at 0 min and 10 min, respectively. T0 is 0 min, and T10 is 10 min.The procedures of Li et al.  were usew/v), and then 20 mL of 1% (w/v) of the RBPH sample solution diluted with deionized water was homogenized at 10,000 rpm for 1 min. The homogenized sample was left to stand for 30 min at room temperature to calculate the FS. FC and FS were calculated by Equations (4) and (5):1 (mL) is volume before whipping, V2 (mL) is volume after whipping, V3 (mL) is volume after standing.FC and FS were measured by the method of Zhang et al. . Brieflyw/v). The particle size and polydispersity index (PDI) analyses were performed with a particle size distribution instrument . The particle size was characterized by the d4,3 (volume average particle size).The samples were diluted with a phosphate buffer  to a protein concentration of 1% (w/v). ζ-potential was determined by the methods of Tang et al. [The samples were diluted with a phosphate buffer  to a protein concentration of 1%  and boiled in boiling water for 5 min. 6 μL solution of each sample was loaded into proper well separately after cooling to room temperature. The stacking gel and the separating gel experiments were carried out at 90 V and 120 V, respectively. The gels were stained by coomassie Brilliant Blue R-250.SDS-PAGE analysis was done by the method of Laemmli . The RBPw/v) was solubilized in 5 mL of a Tris-Gly buffer solution . This solution was added to a 2-nitrobenzoic acid (DTNB) (20 μL) reagent. The absorbance of the mixture was measured at 412 nm. The solution without DTNB was used as the blank.SH content was determined by the method of Shimada & Cheftel , with mi412 was absorbance at 412 nm, C was the solids content in protein solution (mg/mL), and D was the dilution factor. The -SH content was calculated as Equation (6): w/v) protein concentration was obtained with a phosphate buffer . A 0.45 μm cellulose acetate membrane was used to remove impurities. The sample was injected onto a Shodex protein KW-804 column , with a 0.01 M phosphate buffer (pH 7.0) and 0.3 M NaCl used as the mobile phase. The flow rate was set at 1 mL/min. The eluent was monitored at 280 nm.HPSEC experiments were carried out using an Agilent 1100 liquid chromatograph equipped with a photodiode array detector according to Guan et al. , with soThe surface morphology of each sample was analyzed by Scanning electron microscopy . The samples were coated with Au using an ion sputter to a thickness of 15 nm and observed at an accelerating voltage of 5.0 kV.−1 with a resolution of 4 cm−1 and 64 scans. The structure of freeze-dried RBPH samples were measured by an FTIR spectrometer . The samples were pressed into pellets with KBr powder at a proportion of 1:100. The scanning range was set to 500–4000 cmw/v). Emission wavelengths were collected between 300 and 450 nm with a constant slit of 2.5 nm.Fluorescence spectrometry measurements were carried out by excitation at 290 nm by the method of Liu et al.  with slip < 0.05, and analysis of variance (ANOVA) and Duncan’s test were used. All experiments were conducted in triplicate and SPSS 22.0 software was used to express the results as the mean values ± standard deviations.Statistical comparisons were made at the significance level of p < 0.05). This may be the reason why the structure of the protein became loose through the HHP pretreatment, especially the exposure of hydrophilic and hydrophobic groups. Enzyme action sites in protein that are easily enzymatically digested were exposed, and the exposure of hydrophilic groups can make protein disperse or hydrate more readily [Solubility is crucial for protein in food applications . Figure  readily . HHP pre readily . p < 0.05) higher. When the pressure increased, EAI first increased and then decreased, and the maximal value was at 200 MPa. The present results share similarity to those of Wang et al. [p < 0.05) compared with the ESI of the control group, whereas ESI decreased slightly at 300 MPa. The balance between hydrophilicity and lipophilicity determines the emulsifying properties of proteins [The effects on the EAI and ESI of RBPH produced by HHP pretreatment are shown in g et al. . These rproteins . The strproteins . The decp < 0.05). This trend is in parallel to the results of Maria et al. [The FC and FS of RBPH pretreated by HHP are shown in a et al. .Compared with the control group, the FC of RBPH increased because of the HHP pretreatment, which increased the solubility of RBPH, unfolded part of the RBP structure, exposed buried hydrophobic groups, and increased the molecules’ flexibility, consequently promoting protein adsorption in the foaming process and reducing interfacial tension between air and water. Li et al.  stated tIn this study, the trend of the FC data was in accordance with the trends of solubility, EAI and ESI .p < 0.05) compared with the control. PDI also exhibited the same tendency as particle size. As reported, high pressure made the particle size smaller [ smaller . The treThe data showed the average particle size of the control group was the largest out of all four samples. With the increase in pressure, the mean particle size of RBPH first decreased to 278 nm at 200 MPa, then increased to 433 nm at 300 MPa. The PDI showed the same trend. This result may be caused by the three-dimensional structure of RBP molecules becoming loose through HHP, as the RBP molecules had a certain degree of dissociation and extension [p < 0.05) changes in the ζ-potential of the control and the RBPH samples pretreated by HHP of –2.81 mV (control group), –5.1 mV (100 MPa), –3.68 mV (200 MPa) and –3.25 mV (300 MPa), respectively.The existence of protein on the surface of the droplet may generate an electric charge . ζ-potenCompared with the control group, the absolute value of RBPH’s ζ-potential in an aqueous dispersion was relatively larger. Theoretically, the absolute value of ζ-potential is associated with the colloidal stability of protein dispersion. In general, the absolute value of ζ-potential is positively correlated with intermolecular electrostatic repulsion. The greater the intermolecular repulsion, the better the stability of the colloid . TherefoThe electrophoretic bands of the control group and RBPH treated at 100–300 MPa are shown in Current studies on protein hydrolysis have indicated that HHP treatment can destroy the disulfide bond of protein , and thaAs reported, a protein’s structure can be changed by HHP, such as by unfolding, so that protease can enter the binding site for hydrolysis more effectively, increasing the sensitivity of protein to the enzyme . With thFree SH is a vital chemical bond that stabilizes the conformation of proteins . It is up < 0.05); this result may be the reason why breaking of the noncovalent bonds induced unfolding of protein [p < 0.05) at 100 MPa. However, when the pressure was higher than 100 MPa, the SH content of all RBPH samples gradually decreased as the pressure increased (p < 0.05). The result was similar to that of the SH content change trend found in a previous study [ protein . The SH us study . The autus study . At 100 In this study, HPSEC was used to characterize the molecular weight distribution of RBPH pretreated by HHP. In the HPSEC profile of RBPH, there were four major elution peaks . These pWith the increase in pressure, the amount of RBPH with a molecular weight of 22–27 kDa decreased gradually, while the proportions of RBPH with a weight of 18–22 kDa and below 14 kDa increased. A small proportion of high-molecular-weight RBPH began to appear at 200 MPa. The amount of hydrolysate with a molecular weight above 27 kDa was greatest at 300 MPa out of all the samples. This phenomenon indicated that the hydrolysis and aggregation of protein occurred interactively. HHP pretreatment could unfold the protein structure to facilitate hydrolysis, and excessive pressure (300 MPa) pretreatment can lead to the aggregation of RBPH. This was the cause of the amount of low-molecular-weight RBPH  reducing further at 300 MPa. The experiment data of this work  revealedThe surface morphology of RBP and RBPH is exhibited in −1 to 4000 cm−1. Changes in a protein’s secondary structure are generally evaluated by Amide I stretching  and the Amide II shift  [−1. When the pressure was 100 MPa, it shifted to 3409 cm−1. When the pressure increased, the characteristic peak continued to shift. The characteristic peak shifted to 3413 cm−1 at 300 MPa. The results showed that the characteristic absorption peak of -OH stretching vibration was a blue shift after HHP pretreatment, which may be due to the effect of the HHP pretreatment on the hydrogen bonds. The absorption peak at 2924 cm−1 represents the stretching vibration of -CH. It shifted to 2926 cm−1 after HHP pretreatment. And the stretching vibration peak of C=O shifted from 1654 cm−1 in the control group to 1667 cm−1 in the HHP pretreatment samples, and the characteristic peak of the stretching vibration of C-N shifted from 1545 cm−1 to 1547 cm−1. Generally speaking, a protein’s secondary structure is associated with the hydrogen bonds [In this experiment, the secondary structure of RBPH was studied using FTIR spectroscopy. As shown in etching) ,53,54,55en bonds . These rmax) and fluorescence intensity change at the same time can the spatial structure of protein be characterized [Generally, the environmental polarity of tryptophan (Trp) affects the emission fluorescence spectra of protein. Trp is a sensitive means of characterizing the conformation of proteins and is considered as a measure for estimating the changes in a protein’s tertiary structure . A previcterized .The intrinsic fluorescence spectrum of RBPH is depicted in Overall, this work studied the structural and functional properties of RBPH pretreated by HHP. The current work indicated that RBPH exhibited significant improvements over the control in terms of the solubility, emulsifying properties, and foaming properties. The findings may indicate that HHP changed the spatial structure of RBP, thus enhancing the efficiency and effect of hydrolysis. The SDS-PAGE and free SH content results indicated that the primary structure of RBP altered after HHP pretreatment. The FTIR and fluorescence spectrum results showed that HHP changed the spatial structure of RBP. With the increase in pressure, the volume of the average particle size first decreased and then increased. The absolute value of the ζ-potential of the HHP-pretreated RBPH in an aqueous dispersion was larger than that of the control. Thus, the data obtained from the experiment indicated that HHP pretreatment could effectively improve the functionality of RBPH. This processing technology could broaden the potential application of RBP in food products. Further studies should be implemented to investigate the proper hydrolysis degree of RBP achieved through HHP for application in various food products.


Xenopus laevis oocyte expression system of CB1 and CB2 receptors, interacting with heteromeric GIRK1/2 channels and a regulator of G protein signaling, RGS4. This ex vivo system enables the discovery of a wide range of ligands interacting orthosterically or allosterically with CB1 and/or CB2 receptors. WIN55,212-2, a non-selective agonist of CB1 and CB2, was used to explore the CB1- or CB2-GIRK1/2-RGS4 signaling cascade. We show that WIN55,212-2 activates CB1 and CB2 at low concentrations whereas at higher concentrations it exerts a direct block of GIRK1/2. This illustrates a dual modulatory function, a feature not described before, which helps to explain the adverse effects induced by WIN55,212-2 in vivo. When comparing the effects with other typical cannabinoids such as Δ9-THC, CBD, CP55,940, and rimonabant, only WIN55,212-2 can significantly block GIRK1/2. Interestingly, the inward rectifier potassium channel, IRK1, a non-G protein-coupled potassium channel important for setting the resting membrane voltage and highly similar to GIRK1 and GIRK2, is not sensitive to WIN55,212-2, Δ9-THC, CBD, CP55,940, or rimonabant. From this, it is concluded that WIN55,212-2 selectively blocks GIRK1/2.The coupling of cannabinoid receptors, CB1 and CB2, to G protein-coupled inward rectifier potassium channels, GIRK1 and GIRK2, modulates neuronal excitability in the human brain. The present study established and validated the functional expression in a  Cannabichannels . The GIRchannels ,15. GIRKchannels . Notablychannels ,18,19.Heterotetramers of GIRK subunits exist as primary functional GIRK channels that regulate neuronal excitability in the brain ,16. The in vivo [Xenopus oocyte system co-expressing CB1 and GIRK1/4, WIN55,212-2 enhanced currents carried by GIRK1/4 through activating CB1 [−/− mice [The binding of several cannabinoids to CB1 and/or CB2 activates the G-proteins-mediated signaling cascade and then produces coordinated changes in several cellular effector systems including triggering GIRK channels ,22. For in vivo , recogniin vivo . Neverthin vivo . Additioin vivo . In a Xeting CB1 . CB2, hoting CB1 . Further−/− mice . Based oXenopus oocyte system efficiently co-expressing CB1 or CB2, GIRK1/2, and a regulator of the G protein signaling protein 4 (RGS4). Furthermore, we characterized the effects of WIN55,212-2 on CB1-GIRK1/2-RGS4, CB2-GIRK1/2-RGS4, and GIRK1/2 systems, and compared the effects of several typical cannabinoids. In consideration of the high similarity of inward rectifier K+ (IRK) channels to GIRK channels and wide expression as well as vital functions of IRK1 (Kir2.1) in the human body, the effects of WIN55,212-2 were also tested on an IRK1-expression Xenopus oocyte system. This assay provides a straightforward and efficient methodology for examining cannabinoid-stimulated effects. Moreover, the results may partly explain the contradictory effects produced by WIN55,212-2 in vivo.Thus, this study aims to further explore the interactions between WIN55,212-2 and cannabinoid receptors coupling to GIRK channels, particularly the GIRK1/2 expressed in the human brain. First of all, we built and validated a reliable Xenopus laevis frogs  were approved by the Animal Ethics Committee of the KU Leuven (Project No. P186/2019) following regulations of the European Union (EU) concerning the welfare of laboratory animals as declared in Directive 2010/63/EU. Oocytes were isolated from ovarian tissue surgically removed during hypothermia and 0.1% tricaine  induced anesthesia. After recovery from anesthesia, frogs were returned to their tanks in the Aquatic Facility of KU Leuven and monitored daily.All procedures for the use and handling of adult female 2+-free ND96 solution. Isolated stage V-VI oocytes were then maintained in ND96 solution containing Theofylline and Gentamicin at 16 °C. The ND96 solution was composed of 96 mM NaCl, 2 mM MgCl2, 2 mM KCl, 5 mM HEPES, and 1.8 mM CaCl2, with a final pH of 7.5.The oocytes were enzymatically defolliculated by collagenase (3 mg/mL)  digestion at 16 ℃ on a rocker platform in a Cain vitro using the appropriate RNA polymerase . Among these cDNAs, CB1, CB2, IRK1, and RGS4 cDNAs contain pGEMHE vectors which were constructed as described before [Xenopus β-globin gene that flanks the cloned cDNA and enhances RNA stability in Xenopus oocytes [+ currents on day 2 or 3 for GIRK1/2 and IRK1, and day 4 or 5 for CB-GIRK1/2-RGS4 coupling.Linearized cDNAs of human CB1, CB2, GIRK1, GIRK2, IRK1, and RGS4 were used to transcribe cRNAs d before , contain oocytes . The con+ currents through GIRK1/2 were measured using a two-electrode voltage-clamp amplifier . Electrodes were fabricated from borosilicate glass tubes  by a programmable microelectrode puller . Electrodes were filled with 3 M KCl and had tip resistances of 0.8 to 1.5 MΩ. Membrane currents from voltage-clamped oocytes were digitized using a Digidata 1550 low-noise data acquisition system  and a Dell PC running pCLAMP 10.1 software .Macroscopic K+ (HK) solution composed of 96 mM KCl, 2 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES with a final pH of 7.5. The resulting increase in inward K+ current represents a ‘basal’ K+ current  that is following primarily receptor-independent GIRK channel activity. Rapid application and washout of CB agonist WIN 55,212-2  in HK produced the reversible receptor-dependent K+ current  and was performed with a controlled perfusion system that rapidly switched the perfusion of two syringe reservoirs (syringe A and syringe B) connected to syringe needles, two-way stopcocks, and tubing. The syringe reservoirs contained HK (syringe A) and HK plus WIN55,212-2 (syringe B). The perfusion system was located next to the oocyte. In addition to the abovementioned −90 mV voltage protocol, a 1-s voltage ramp protocol was also applied from −150 to +60 mV from a holding potential of −20 mV during perfusion.Oocytes were placed in a 0.2 mL recording chamber continuously perfused with ND96 solution. After electrode impalement and clamping the membrane potential to −90 mV, the perfusion solution was changed to a high Kv/v), which did not cause changes of currents in non-injected oocytes, or oocytes expressing IRK1, GIRK1/2, and CB-GIRK1/2-RGS4. The 50 μM and 100 μM concentrations of WIN55,212-2 almost reached saturation of WIN55,212-2 in HK, indicated as ≈50 μM and ≈100 μM. Flow, through the perfusion system, was gravity-driven. All recordings were performed at room temperature (21 to 23 °C).For experiments to determine the concentration-current response relationship, a range of WIN 55,212-2 concentrations (in HK) were tested for each oocyte via a manifold that connected multiple syringe reservoirs containing different concentrations of WIN55,212-2. DMSO in each WIN55,212-2 solution was ≤1%  were calculated using the Prism 8.0 program . Statistical comparisons between the various experimental groups were performed by one-way ANOVA where p < 0.05 was considered significant. Experiments were replicated for three to six oocytes (n = 3–6).The EC+ currents, which are known to depend on free G protein βγ-subunits present in the oocytes because of the inherent activity of G proteins [K,basal). In the presence of HK, K+ current enhancement was immediately evoked on the application of 1 μM WIN55,212-2  and was reversible via washing out WIN55,212-2 by HK. The receptor-dependent K+ currents represent that GIRK1/2 were activated by CB1 or CB2 coupled specifically to GIRK1/2. As a control, K+ currents carried by GIRK1/2 were not significantly enhanced upon the application of 1 μM WIN55,212-2 in oocytes injected with only GIRK1/2 .First of all, we aim to assess the properties of receptor-dependent GIRK1/2 activation elicited by cannabinoid receptors CB1 and CB2. In addition to cannabinoid receptors and GIRK1/2 in this system, RGS4 was also transfected to the oocytes. RGS proteins have been previously reported to maintain the robustness of GPCR-GIRK coupling . Moreoveproteins , were obK,intrinsic) that was responsible for IK,basal, non-injected oocytes were tested for sensitivity to HK. As a result, IK,intrinsic, which was 0.158±0.0595 μA (n = 5), constituted a negligible proportion of the total IK,basal. Exchanging HK to HK plus 1 μM WIN55,212-2 did not change IK,intrinsic (n = 5), suggesting no effect of WIN55,212-2 on intrinsic oocyte receptors or other endogenous ion channels.To determine the intrinsic current  was applied to independent oocytes (three oocytes per concentration). Note the progressively higher response of IK,WIN with higher WIN55,212-2 concentrations . + currents produced by ≈100 μM WIN55,212-2 in the CB-GIRK1/2-RGS4 coupling expression system.We investigated the concentration-response relations of the effects of WIN55,212-2 on the CB-GIRK1/2-RGS4 coupling expressed in trations . However55,212-2 . For thecurrents . SubsequK,WIN/IK,basal × 100) as a function of the WIN55,212-2 concentration reveals a concentration-dependent increase and subsequent decrease in the response of IK,WIN  for activating CB1  for activating CB2 . Nonetheless, as shown in + currents in a concentration-dependent manner, which indicates high concentrations of WIN55,212-2 blocked GIRK1/2. Therefore, the reduction of inward K+ currents produced by WIN55,212-2 observed in the results of “3.2” was caused by the blockage of GIRK1/2 in the CB-GIRK1/2-RGS4 coupling expression system. In other words, these results suggest that high concentrations of WIN55,212-2 can block the effect of GIRK1/2 activated by CB1 and CB2, presumably following direct interaction with the GIRK channels.WIN55,212-2 is a well-characterized agonist of CB1 and CB2; thus, it can exert activity by activating cannabinoid receptors. Moreover, low concentrations of WIN 55,212-2 (≤1 μM) can activate receptor-dependent inward K+ currents, n = 3) when compared with heterotetramer GIRK1/2 .GIRK2 can also form homomultimers in the human body ,33 in adCannabis sativa, commonly known as marijuana. They are phytocannabinoids that have been extensively investigated and are classified as classical cannabinoids at the structural level [+ currents were also observed when the oocytes expressing GIRK1/2 were perfused with HK. Interestingly, the addition of 100 μM Δ9-THC to HK slightly enhanced the response of inward K+ currents , cannabidiol , CP55,940 (a non-selective agonist of CB1 and CB2), and rimonabant (a selective antagonist of CB1) also interact with GIRK1/2. Among these cannabinoids, Δ9-THC and CBD are derived from the plant al level   [Xenopus oocytes [Xenopus oocyte expression system, compared with the previously built system [Xenopus oocyte expression system is that different combinations of membrane-bound proteins can be freely expressed: for example, co-inject mRNA encoding CB1 and TRPV1 , or CB2 and μ-opioid receptors, to examine the possible promiscuity of ligands, or CB1 with different types of voltage-gated ion channels . Besides, structure-function research, based on the expression of mutant CB1/CB2, can also be conducted using this system. As such, this functional bioassay can unravel hitherto unknown coupling(s) of signal-transmission pathways, e.g., the cannabinoid and opioid pathways, allowing us to better understand the properties of some cannabinoids [Cannabinoid receptors have been expectantly viewed as hopeful targets for the treatment of diverse diseases. Therefore, many expression systems of CB1 and CB2 have emerged in response to promptly discovering drug candidates by screening potential ligands  spleen) . The CB- spleen) . Among R oocytes . This st oocytes . Further oocytes . Thus, Ct system . Another+ currents by activating CB1 or CB2. However, we further found that high concentrations of WIN55,212-2  in HK blocked GIRK1/2 activated by CB1 or CB2. Concentration-response curves of WIN55,212-2 are shown in + currents. In contrast, in the CB2-GIRK1/2-RGS4 coupling and GIRK1/2, 10 μM WIN55,212-2 in HK slightly reduced inward K+ currents. Thus, we consider that the affinity of 10 μM WIN55,212-2 for CB1 may be stronger than that for CB2 and GIRK1/2. Moreover, ≈50 μM WIN55,212-2 probably shared a similar affinity for both CB1 and GIRK1/2, while the affinity of ≈100 μM WIN55,212-2 for GIRK1/2 may become stronger than that for CB1 in the CB1-GIRK1/2-RGS4 coupling (i (the binding constant) values for CB1 ranged from 1.89 to 123 nM, and the Ki values for CB2 varied from 0.280 to 16.2 nM, depending on various CB1 and CB2 expression systems [Interestingly, dual modulation of CB-GIRK1/2-RGS4 coupling was elicited by WIN55,212-2 in the oocyte expression system. As a non-selective agonist of CB1 and CB2, WIN55,212-2 in HK (≤1 μM) undoubtedly augmented inward Kcoupling . Neverth systems . TherefoFunctionally, WIN55,212-2 under high concentrations significantly blocked GIRK1/2 activated by CB1 or CB2, and thus inhibited the CB-GIRK1/2 signaling. Previous studies have shown that after chronic administration of WIN55,212-2 to adult male rats, CB1 binding and mRNA levels were reduced in the substantia nigra and the striatum . Moreovein vivo. Similarly, the concentrations of Δ9-THC in vivo are, as far as we know, also unknown after chronic exposure of single Δ9-THC, though many studies have revealed the plasma concentration of Δ9-THC in humans after chronic treatment of cannabis. Meanwhile, cannabis/cannabinoid-based medicines are increasingly being used to treat several diseases such as epilepsy, chronic pain, multiple sclerosis, and neurodegenerative diseases [Heavy or regular cannabis/cannabinoid abuse, generally defined as daily or almost daily use over a prolonged period, has been linked to cognitive dysfunction and increased risk of developing psychiatric symptoms, including schizophrenia spectrum disorders, acute psychosis, mania, and a motivational syndrome . Neverthdiseases ,53,54,55diseases ,57.+ currents under the high concentration. This result may be attributed to the amino-alkylindole-type structure of WIN55,212-2 which is distinct from the structures of other tested cannabinoids. On the other hand, human IRK1 was not sensitive to high concentrations of WIN55,212-2, Δ9-THC, CBD, CP55,940, and rimonabant. The core cytoplasmic domain elements of GIRK1 or GIRK2 channels are very similar to that of IRK1 [Finally, we compared the effects of ≈100 μM WIN55,212-2 with the effects of 100 μM of other typical cannabinoids Δ9-THC, CBD, CP55,940, and rimonabant on GIRK1/2. As shown in  of IRK1 . The bin of IRK1 . The str of IRK1 . We hypoXenopus oocytes. The CB2-GIRK1/2-RGS4 coupling system is the first successful heterologous expression system of CB2. This research firstly reveals the dual modulation of cannabinoid receptors and brain GIRK1/2 coupling by WIN55,212-2 in the oocyte expression system. Although WIN55,212-2 is an agonist of CB1 and CB2 and can activate CB-GIRK signaling in the CB-GIRK1/2-RGS4 coupling, it can also block GIRK1/2 activated by CB1 and CB2 under high concentrations. Moreover, WIN55,212-2 also can directly block GIRK1/2 in a concentration-dependent manner. Comparing the effects of WIN55,212-2 with the effects of Δ9-THC, CBD, CP55,940, and rimonabant which are also typical cannabinoids on GIRK1/2 shows that only WIN55,212-2 can significantly block GIRK1/2 at the high concentration. However, the high concentration of WIN55,212-2, Δ9-THC, CBD, CP55,940, and rimonabant did not affect IRK1, which is highly similar to GIRK channels, representing the selective blockage of WIN55,212-2 on GIRK1/2. The blockage of GIRK1/2 in CB-GIRK signaling provides new insights on the cannabinoid receptors-mediated side effects induced by the chronic accumulation of WIN55,212-2 in vivo. This research also provides a straightforward methodology to rapidly test the effects of cannabinoids/potential cannabinoids on CB1 and CB2 ex vivo.The present research built and validated a reliable and efficient CB1 and CB2 expression system coupled to GIRK1/2 and RGS4 in


Mycobacterium tuberculosis (MTb) and Mycobacterium leprae (M. leprae) is observed in PLHTLV. Reports of opportunistic infections and high frequency of crusted scabies in patients with HTLV-1 points to immune impairment in those individuals. Human T lymphotropic virus type 1 may influence the persistence of pathogens, exemplified by the higher rates of Schistosoma mansoni and Strongyloides stercoralis  treatment failure observed in PLHTLV. This retrovirus is also associated with increased tuberculosis (TB) severity with some evidence pointing to a deleterious impact on leprosy outcome as well. These findings are supported by immune alterations observed in those co-infected individuals. Although the role of HTLV-1 in HCV outcome is debatable, most data indicate that HTLV may negatively impact the clinical course of hepatitis C. Co-infections may also influence the risk of developing HTLV-1 associated disease, but data are still limited. The impact of HTLV-1 on the response to more common infections, might contribute to the increased mortality rate of HTLV-1. Large scale prospective controlled studies on the prevalence and impact of HTLV-1 in co-infections and vice-versa are needed. Human T lymphotropic virus type 1 impact in public health is broad. Measures to increase awareness and to prevent new infections are needed.Human T lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes lifelong T-cell infection in humans, impacting the host immune response. This virus causes a range of clinical manifestations, from inflammatory conditions, including neuronal damage  to life-threatening leukemia . Human T lymphotropic virus type 1 is also associated with increased risk of all-cause mortality, but the mechanisms remain unclear. As a blood-borne and sexually transmitted infection (STI), HTLV-1 shares transmission routes to many other pathogens and although it has worldwide distribution, it affects mainly those in low- and middle-income tropical areas, where the prevalence of other infectious agents is high. These factors contribute to a high incidence of co-infections in people living with HTLV-1 (PLHTLV). This comprehensive review addresses the impact of HTLV-1 on several co-infections and vice-versa. There is evidence of higher rates of HTLV-1 infection in association with other blood borne  and sexually transmitted  infections but whether this represents increased susceptibility or opportunity is unclear. Higher frequency of  The immune imbalance that occurs in HTLV-1 infection is linked to the pathogenesis of HTLV-1 associated diseases. The neurological disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), that greatly impacts patient‘s quality of life and can result in wheelchair dependency, and other conditions, such as uveitis and thyroiditis are examples of the many inflammatory manifestations associated with HTLV-1. Adult T-cell leukemia/lymphoma (ATL), the usually fatal neoplasm caused by HTLV-1 is also linked to immunological impairment as well as monoclonal expansion and transformation of an infected T-cell. There are several evidences that patients with ATL have severe immunosuppression which is associated with opportunistic infections and other malignancies , hepatitis B virus (HBV), human papillomavirus (HPV), and herpes simplex virus (HSV). Moreover, the distribution of HTLV-1, with high prevalence observed mainly in low- and middle-income countries (LMIC) , Mycobacterium tuberculosis (MTb), and Mycobacterium leprae (M. leprae), is high. Thus, HTLV-1 infected individuals can be co-infected with a range of pathogens. Indeed, the prevalence of co-infection is high in some settings. The interactions between coexisting pathogens are complex and may affect the natural course of both infections. While an anti-viral immune response involves a dominant Th1 type response, the ideal response to some parasites, e.g., helminths, usually involves a dominant Type-2 response. Therefore, the response to co-infections can be conflicting from an immunological perspective (Human T lymphotropic virus type 1 (HTLV-1) causes lifelong infection mainly in CD4gnancies , 5. Unprs (LMIC) , overlapspective . Howeverp < 0.01] but confirmatory tests for HTLV-1 infection were not performed  vs. 2.6% , OR = 1.18 (95% CI = 0.48–2.89), p = 0.71]  vs. 11.9% , erformed . Similarerformed . In the erformed . In Biss = 0.71] . The aut = 0.71] . There w [3.7% 6/2 vs. 2.6 = 0.71]  19)..p < 0.01p < 0.05) (p < 0.001). In a multivariate analysis HTLV-1 infection was associated with TB history in Peru   of HTLV-1 patients had a history of TB compared with 2.9%  of uninfected patients  . When an < 0.05) . No diff1.6–3.9) . This wa1.6–3.9) .M. leprae from Yemen, Senegal, Ethiopia, and a non-endemic area for HTLV-1 from Brazil (Curitiba). However, the sample size in each setting was small  suggesting an increased susceptibility to develop TB if HTLV-1 infected or vice versa, the first option being most biologically plausible  (MTb.The data indicate that HTLV-1 is more common in patients with lausible . While p23–9.77) , which wp = 0.02, in Baltimore) and positive syphilis serology  were associated with confirmed HTLV-1/2 infection  (p = 0.22] (p = 0.03) and the prevalence of HTLV-1 infection was higher in patients seropositive for syphilis than in seronegative patients   ] and males [OR = 3.56 (1.24–10.22)] and hypothesized that although the efficiency of HTLV-1 sexual transmission is lower from female-to-male, it can be increased by the presence of a penile ulcer or concurrent syphilis . In the nfection . This asnfection . The fre62–12.9) . An asso62–12.9)  and Braz62–12.9) . In Japa62–12.9) . In Hait = 0.22] , while i = 0.22] . In Peru 0.0001) . Another 0.0001) . In a ST.7–52.3) . In Zair.7–52.3) . Two stuion test , nor in  > 0.05] . Meta-an > 0.05] . WhetherChlamydia trachomatis (C. trachomatis) amongst Peruvian female sex workers  (C. trachomatis was higher in cases [63.2% (60/102)] than controls   . As the authors highlighted, the limited number of co-infected individuals may have influenced the results  . This asdom use)  and in a > 0.05) . When tht HTLV-1 . Zunt et results .Human T lymphotropic virus type 1 infection was associated with HSV-2 seropositivity in pregnant women from Haiti   and femap = 0.03)  than uninfected women (mean age 36 years) attending a gynecology clinic  . Human T = 0.03) . In HTLV = 0.03) . However = 0.03) . Figure There is a consistency in the literature to indicate a relationship between HTLV-1 and coinfection with other sexually transmitted infections (STIs) . However, causation has not been demonstrated. Prospective studies are required and would be helpful to distinguish between HTLV-1 infection acquired sexually and vertically.Several studies report a high frequency of HCV and HBV infection in PLHTLV. Unfortunately, most of them do not include a control group. Studies from China, Brazil, and Sweden confirmed a higher prevalence of HTLV-1 infection among patients that are seropositive for HCV –54. PoolHuman T lymphotropic virus type 1 can cause pulmonary disease that may result in important parenchymal damage and bronchiectasis . HoweverSt. stercoralis co-infection. Patients with HTLV-1 have more frequently positive stool test for Ss and treatment failure (persistent parasite excretion despite treatment). This topic is addressed in another study.Human T lymphotropic virus type 1 infection may also influence the ability of other parasites to persist within the co-infected host. This was described in HTLV-1 and Schistosoma mansoni infection among HTLV-1 infected individuals from Brazil  has been reported (p < 0.001), but both were carried out in Brazil by the same group, and probably have an overlap of patients (S. mansoni eggs in stool samples 2 months after therapy with praziquantel was more frequent in HTLV-1 co-infected patients than in patients with schistosomiasis only . Re-exposure to parasite was excluded in all patients. One co-infected patient was not able to eradicate the helminthic infection even after eight courses of praziquantel (Schistosoma mansoni in people living with HTLV-1 (PLHTLV) but this is better characterized for Strongyloides infection.Similarly, high frequency of reported . Two avapatients . Treatmeiquantel . AlthougSchistosoma sp infection elicits a Th-2 type response. Although it seems to be important for parasite clearing, it can be associated with the disease‘s pathophysiology   , 58. HowRegarding TB severity in HTLV-1 infected individuals, the findings are contradictory. In Peru, HTLV-1 patients co-infected with TB reported more frequently a history of relatives‘ deaths due to TB infection . Human TM. leprae/HTLV-1 co-infected patients: Mortality rates 5.5/100 vs. 3.6/100 person-years of observation, resulting in a risk ratio of 1.4 (95% CI 1.04–1.89) for those infected with HTLV-1  and may be considered a marker of HTLV-1 infection (p < 0.01) (p < 0.02)  is an infrequent, albeit severe and highly contagious infection caused by massive infestation of the mite, nfection . In Bahi < 0.01) . History < 0.02) . In Peru < 0.02) , 82, whi < 0.02)  and all  < 0.02) . Among H < 0.02) . In the < 0.001] . Crusted< 0.001] , 85–87, < 0.001] , 82, 88 < 0.001] , 89, 90.< 0.001] . Therefop = 0.033]  vs. 13.8% (49/356),  vs. 13.8ividuals . Dissemiaribbean . Pulmonaaribbean . Cryptocaribbean –96.Histoplasma capsulatum is a ubiquitous fungus that usually causes asymptomatic infection. Patients co-infected with HIV, those with primary immunodeficiencies or under immunosuppressive treatments are at risk of developing progressive disseminated histoplasmosis (PDH), a clinical illness with extra-pulmonary involvement. The mortality of PDH can be as high as 85% in untreated patients, and 25% despite a standard “adequate” therapeutic regimen. Severe gastrointestinal histoplasmosis in Peru  (togenes) .S mansoni but this needs to be substantiated. Whilst it is plausible that HTLV-1 infection may impact the course of the many mycotic infections that co-exist in HTLV-1 endemic areas the need for better studies to determine the epidemiological and biological associations between HTLV-1 and infections such as histoplasmosis, paracoccidioidomycosis, and cryptococcosis is clear (The strongest evidence points to increased pathology due to mycobacterium in the presence of HTLV-1. There is a suggestion that HTLV-1 infection may favorably modulate the impact on is clear .Schistosoma antigens downregulate in vitro pro-inflammatory cytokine response (IFN-γ and CXCL9) by peripheral blood lymphocytes from HTLV-1 infected individuals  was observed among asymptomatic carriers when compared to individuals with HAM   in a retrospective association  (During the follow-up (mean 6.75 years) of 36 HTLV-1 infected patients with ID from Bahia, 47.2% developed HAM and one individual developed ATL . In a mo = 0.02) . Moreove = 0.02) , 115. In = 0.02) . A numbeData regarding the impact of co-infections on HTLV-1 outcome is extremely limited and more studies are needed.There is evidence of higher rates of HTLV-1 infection in association with other blood borne and STIs but whether this represents increased susceptibility or opportunity is unclear. The data suggest that HTLV-1 increases susceptibility to a range of infection related disease resulting in either higher risk of disease if exposed, longer persistence or greater severity. This indicates that PLHTLV may have a degree of immunosuppression regardless of symptomatic or asymptomatic HTLV-1 clinical status. Co-infections may also impact the risk of developing HTLV-1 associated disease, but data are still extremely limited.Human T lymphotropic virus type 1 infection is a neglected infection that is responsible not only for diverse and severe inflammatory diseases, such as HAM, uveitis, pulmonary diseases, Sjogren syndrome, and arthritis, and the malignant disease ATL, but also a degree of immunosuppression that can increase susceptibility to and/or severity of co-infections, many of which are also neglected tropical diseases. More subtle impact of HTLV-1 on the response to more common infections, might contribute to the increased mortality rate of HTLV-1, but this will only be determined by large scale prospective controlled studies. Identification of HTLV-1 status should therefore be included in population-based studies. Human T lymphotropic virus type 1 should be considered a global health concern as its impact in public health is evident. Measures to improve the global awareness about HTLV-1 and to prevent its transmission are necessary.GT designed the study, supervised the project, and revised the manuscript. CR performed data compilation, analysis, and wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.GT was supported by NIHR Imperial College Trust.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.