Novel GPIb-independent platelet aggregation induced by botrocetin: Implications for diagnosis and antithrombotic therapy.

BACKGROUND: Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand diseases and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. OBJECTIVE: To explore the alternative receptor(s)/mechanisms participate in botrocetin-induced platelet aggregation. METHODS: The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic and αIIbß3-deficient mice, Bernard-Soulier syndrome (BSS) and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing ELISA. Botrocetin-αIIbß3 and botrocetin-GPIbα interactions were measured using ELISA and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. RESULTS: Botrocetin could induce aggregation of platelets from a BSS patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbß3 and facilitated αIIbß3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbß3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbß3, and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbß3 and GPIb-mediated platelet aggregation, spreading and thrombus formation. CONCLUSION: Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbß3 and GPIbα.

INPP4B ensures that ILC1s and NK cells set up a productive home office.

In this issue of JEM, Peng et al. (https://doi.org/10.1084/jem.20230124) identify inositol polyphosphate 4-phosphatase type II (encoded by Inpp4b) as an important enzyme for tissue-resident ILC1 and NK cell survival, signal transduction, and anti-tumor immunity.

Direct Access to α,ß-Unsaturated Ketones via Rh/MgCl2-Mediated Acylation of Vinylsilanes.

We report herein the facile and practical construction of α,ß-unsaturated ketones via rhodium-catalyzed direct acylation of vinylsilanes with readily available and abundant carboxylic acids. This protocol features access to a diverse array of synthetically useful functionalities with moderate to excellent yields. More importantly, the late-stage functionalization of pharmaceuticals was also realized with synthetically useful yield.

Viper venoms drive the macrophages and hepatocytes to sequester and clear platelets: novel mechanism and therapeutic strategy for venom-induced thrombocytopenia.

Venomous snakebites cause clinical manifestations that range from local to systemic and are considered a significant global health challenge. Persistent or refractory thrombocytopenia has been frequently reported in snakebite patients, especially in cases caused by viperidae snakes. Viper envenomation-induced thrombocytopenia may persist in the absence of significant consumption coagulopathy even after the antivenom treatment, yet the mechanism remains largely unknown. Our study aims to investigate the mechanism and discover novel therapeutic targets for coagulopathy-independent thrombocytopenia caused by viper envenomation. Here we found that patients bitten by Protobothrops mucrosquamatus and Trimeresurus stejnegeri, rather than Naja. atra may develop antivenom-resistant and coagulopathy-independent thrombocytopenia. Crude venoms and the derived C-type lectin-like proteins from these vipers significantly increased platelet surface expression of neuraminidase and platelet desialylation, therefore led to platelet ingestion by both macrophages and hepatocytes in vitro, and drastically decreased peripheral platelet counts in vivo. Our study is the first to demonstrate that desialylation-mediated platelet clearance is a novel mechanism of viper envenomation-induced refractory thrombocytopenia and C-type lectin-like proteins derived from the viper venoms contribute to snake venom-induced thrombocytopenia. The results of this study suggest the inhibition of platelet desialylation as a novel therapeutic strategy against viper venom-induced refractory thrombocytopenia.

Diffuse reduction of spleen density is a novel prognostic marker for intrahepatic cholangiocarcinoma after curative resection.

BACKGROUND: Diffuse reduction of spleen density (DROSD) is related to cancer prognosis; however, its role in intrahepatic cholangiocarcinoma (ICC) remains unclear. AIM: To assess the predictive value of DROSD in the prognosis of ICC after curative resection. METHODS: In this multicenter retrospective cohort study, we enrolled patients with ICC who underwent curative hepatectomy between 2012 and 2019. Preoperative spleen density was measured using computed tomography. Overall survival (OS) and recurrence-free survival (RFS) rates were calculated and compared utilizing the Kaplan-Meier method. Univariable and multivariable Cox regression analyses were applied to identify independent factors for OS and RFS. A nomogram was created with independent risk factors to predict prognosis of patients with ICC. RESULTS: One hundred and sixty-seven ICC patients were enrolled. Based on the diagnostic cut-off values (spleen density ≤ 45.5 Hounsfield units), 55 (32.9%) patients had DROSD. Kaplan-Meier analysis indicated that patients with DROSD had worse OS and RFS than those without DROSD (P < 0.05). Cox regression analysis revealed that DROSD, carcinoembryonic antigen level, carbohydrate antigen 19-9 level, length of hospital stay, lymph node metastasis, and postoperative complications were independent predictors for OS (P < 0.05). The nomogram created with these factors was able to predict the prognosis of patients with ICC with good reliability (OS C-index = 0.733). The area under the curve for OS was 0.79. CONCLUSION: ICC patients with DROSD have worse OS and RFS. The nomogram is a simple and practical method to identify high-risk ICC patients with poor prognosis.

A positive feedback loop between Periostin and TGFß1 induces and maintains the stemness of hepatocellular carcinoma cells via AP-2α activation.

BACKGROUND: Liver cancer stem cells (LCSCs) play key roles in the metastasis, recurrence, and chemotherapeutic resistance of hepatocellular carcinoma (HCC). Our previous research showed that the POSTN gene is closely related to the malignant progression and poor prognosis of HCC. This study aimed to elucidate the role of POSTN in generating LCSCs and maintaining their stemness as well as the underlying mechanisms. METHODS: Human HCC tissues and matched adjacent normal tissues were obtained from 110 patients. Immunohistochemistry, western blotting (WB), and RT-PCR were performed to detect the expression of POSTN and stemness factors. The roles of transforming growth factor (TGF)-ß1 and AP-2α in the POSTN-induced stemness transformation of HCC cells were explored in vitro and in vivo using LCSCs obtained by CD133+ cell sorting. RESULTS: The high expression of POSTN was correlated with the expression of various stemness factors, particularly CD133, in our HCC patient cohort and in TCGA and ICGC datasets. Knockdown of POSTN expression decreased the abilities of HCC cell lines to form tumours in xenograft mouse models. Knockdown of POSTN expression also suppressed cell viability and clone formation, invasion, and sphere formation abilities in vitro. Knockdown of AP-2α attenuated the generation of CD133+ LCSCs and their malignant behaviours, indicating that AP-2α was a critical factor that mediated the POSTN-induced stemness transformation and maintenance of HCC cells. The role of AP-2α was verified by using a specific αvß3 antagonist, cilengitide, in vitro and in vivo. Activation of POSTN could release TGFß1 from the extracellular matrix and initiated POSTN/TGFß1 positive feedback signalling. Furthermore, we found that the combined use of cilengitide and lenvatinib suppressed the growth of HCC cells with high POSTN expression more effectively than the use of lenvatinib alone in the patient-derived xenograft (PDX) mouse model. CONCLUSIONS: The POSTN/TGFß1 positive feedback pathway regulates the expression of stemness factors and the malignant progression of HCC cells by regulating the transcriptional activation of AP-2α. This pathway may serve as a new target for targeted gene therapy in HCC.

C-Gait for Detecting Freezing of Gait in the Early to Middle Stages of Parkinson's Disease: A Model Prediction Study.

OBJECTIVE: Efficient methods for assessing walking adaptability in individuals with Parkinson's disease (PD) are urgently needed. Therefore, this study aimed to assess C-Gait for detecting freezing of gait (FOG) in patients with early- to middle-stage PD. METHOD: People with PD (PWP) diagnosis (Hoehn and Yahr stages 1-3) were recruited from April 2019 to November 2019 in Beijing Rehabilitation Hospital. The participants performed six items of walking adaptability on an instrumented treadmill augmented with visual targets and obstacles (C-Mill). The patient's walking adaptability was evaluated by C-Gait assessment and traditional walking tests, and FOG-related indexes were collected as outcome measures. Two discriminant models were established by stepwise discriminant analysis; area under the receiver operating characteristic (ROC) curve (AUC) was used to validate the models. RESULT: In total, 53 patients were included in this study. Most C-Gait assessment items had no or low correlations with traditional walking tests. The obstacle avoidance (r = -0.639, P = 0.003) and speed of adaptation (r = -0.486, P = 0.035) items could lead to FOG with high sensitivity. In addition, the C-Gait assessment model (AUC = 0.755) had slightly better discrimination of freezers from non-freezers compared with traditional walking test models (AUC = 0.672); specifically, obstacle avoidance and speed of adaptation have uniquely discriminant potential. CONCLUSION: C-gait assessment could provide additional value to the traditional walking tests for PD. Gait adaptability assessment, as measured by C-Gait, may be able to help identify freezers in a PD population.

Copper-Catalyzed Preparation of Benzo[3,4]indolo[1,2-b]isoquinoline-8-ones and Photoluminescence Exploration.

A facile synthesis of benzo[3,4]indolo[1,2-b]isoquinolin-8-ones is described. Under copper catalysis, the reaction proceeds with a high efficiency and a broad reaction scope. A deuteration experiment shows that the KIE value is 2.85. From the results on mechanism studies, copper-catalyzed C-H activation, intramolecular cis-addition of alkynes, and reductive elimination are involved. Moreover, this skeleton is indeed a new fluorophore, and its photophysical properties are also investigated.

WISP2 promotes cell proliferation via targeting ERK and YAP in ovarian cancer cells.

BACKGROUND: Wnt-inducible signaling pathway protein 2 (WISP2) is a wnt1-induced signaling pathway protein 2. Although studies indicate that WISP2 may promote the development of various tumors, its role in ovarian cancer remains unclear. The objective of the current study was to analyze the effects of WISP2 on the proliferation and migration of ovarian cancer cells in vitro and in vivo. RESULTS: Immunohistochemistry and western blotting indicated that WISP2 was highly expressed in various ovarian cancer tissues and cell lines, but weakly expressed in normal ovary tissue. WISP2 deletion inhibited cell growth, clone formation, and migration of ovarian cancer cells while promoting cell apoptosis and affecting the cell cycle. This growth inhibitory effect caused by WISP2 loss is due to the inhibition of phosphorylated extracellular signal-related kinase (p-ERK)1/2, as well as CCAAT/enhancer-binding protein α (CEBPα) and CEPBß. In addition, WISP2 deletion also activated the Yes-associated protein (YAP). CONCLUSION: WISP2 deletion inhibits ovarian cancer cell proliferation by affecting ERK signaling pathways.

Organoids of liver diseases: From bench to bedside.

Understanding the occurrence, development, and treatment of liver diseases is the main goal of hepatopathology research. Liver diseases are not only diverse but also highly heterogeneous among individuals. At present, research on liver diseases is conducted mainly through cell culture, animal models, pathological specimens, etc. However, these methods cannot fully reveal the pathogenic mechanism and therapeutic characteristics of individualized liver diseases. Recent advances in three-dimensional cell culture technology (organoid culture techniques) include pluripotent stem cells and adult stem cells that are cultured in vitro to form self-organizing properties, making it possible to achieve individualized liver disease research. This review provides a comprehensive overview of the development of liver organoids, the existing and potential applications of liver regenerative medicine, the pathogenesis of liver disease heterogeneity, and drug screening.

Integrated physiological and proteomic analysis reveals underlying response and defense mechanisms of Brachypodium distachyon seedling leaves under osmotic stress, cadmium and their combined stresses.

Drought stress, a major abiotic stress, commonly occurs in metal-contaminated environments and affects crop growth and yield. In this study, we performed the first integrated phenotypic, physiological, and proteomic analysis of Brachypodium distachyon L. seedling leaves under polyethylene glycol (PEG) mock osmotic stress, cadmium (Cd2+), and their combined stresses. Combined osmotic and Cd2+ stress had more significant effects than each individual stress on seedling growth, and the physiological traits and ultrastructures of leaves. Totally 117 differentially accumulated protein (DAP) spots detected by two-dimensional difference gel electrophoresis (2D-DIGE) were identified, and representing 89 unique proteins under individual and combined stresses. These DAPs were involved in photosynthesis/respiration (34%), energy and carbon metabolism (21%), stress/defense/detoxification (13%), protein folding and degradation (12%), and amino acid metabolism (7%). Principal component analysis (PCA) revealed that DAPs from the Cd2+ and combined stresses grouped much closer than those from osmotic stress, indicating Cd2+ and combined stresses resulted in more changes to the leaf proteome than osmotic stress alone. Protein-protein interaction analyses showed that a 14-3-3 centered sub-network could play important roles in responses to abiotic stresses. An overview pathway of proteome metabolic changes in Bd21 seedling leaves under combined stresses is proposed, representing a synergistic responsive network and underlying response and defense mechanisms. SIGNIFICANCE: Drought stress is one of the major abiotic stresses, which commonly occurs in metal-contaminated environments, and affects crop growth and yield performance. We performed the first integrated phenotypic, physiological and proteomic analysis of Brachypodium distachyon L. seedling leaves under drought (PEG), cadmium (Cd2+) and their combined stresses.

Association between Pax8-PPARandgamma;1 Rearrangement and Follicular Thyroid Cancer: a Meta-Analysis.

BACKGROUND: Pax8 and peroxisome proliferator-activated receptor gamma 1 gene (Pax8-PPARγ1) are important factors in tumors. Several studies have suggested that follicular thyroid cancer may arise from Pax8- PPArγ1 rearrangement. In order to have a better understanding of the association between Pax8-PPARγ1 rearrangement and follicular thyroid cancer, we conducted the presenmt meta-analysis. MATERIALS AND METHODS: The information was extracted from PubMed, EMBASE and Web of Science. Statistic analysis was performed with Stata12.0 software. Odds ratios (ORs) were calculated using a fixed-effects model. We also performed heterogeneity and publication bias analyses. RESULTS: Nine studies including 198 follicular thyroid cancer patients and 268 controls were considered eligible. The frequency of Pax8-PPARγ1 rearrangement was significantly higher in the follicular thyroid cancer group than in the control group, with a pooled OR of 6.63 (95%CI=3.50-12.7). In addition, through subgroup analysis, the OR between Pax8-PPARγ1 rearrangement and follicular thyroid cancer was 6.04 (95%CI = 3.18-11.5) when using benign tumor tissues as controls. The OR for the method subgroup was 9.99 (95% CI =4.86-20.5) in the RT-PCR. CONCLUSIONS: The final results demonstrated that Pax8-PPARγ1 rearrangement has significant association with follicular thyroid cancer.

Insertion of an intracisternal A particle retrotransposon element in plasma membrane calcium ATPase 2 gene attenuates its expression and produces an ataxic phenotype in joggle mutant mice.

Using forward genetic analysis, we identified the insertion of an intracisternal A particle (IAP) retrotransposon element in the plasma membrane calcium ATPase 2 gene (Pmca2/Atp2b2) in the joggle mouse, a novel mutant that displays ataxic gait by postnatal day 12. Expression of Pmca2 mRNA in the joggle mouse is only 5% of that in the wild type mouse. The insertion is located 15 bp downstream of the donor splice site of the exon containing the initiation codon. Chimeric mRNA composed of the 5'-region of Pmca2 and the IAP element were detected, indicating that some of the primary transcripts are terminated by polyadenylation signals in long terminal repeats of the IAP element. We also identified cryptic splice sites in the IAP element that are likely involved in aberrant splicing of the Pmca2 primary transcripts that leads to rapid degradation of mRNA through nonsense mediated mRNA decay. Ataxia was observed in compound heterozygous mice carrying the joggle mutation and the wriggle mutation, a previously reported missense Pmca2 mutant. Thus, we attributed ataxia in joggle mice to reduced expression of Pmca2, resulting from insertion of the IAP element.

Mapping of jog locus to the region between D6Mit104 and D6Mit336 on mouse chromosome 6.

The joggle mouse is a recessive ataxic mutant carrying an unknown mutation in a C3H/He (C3H)-derived chromosomal segment. Taking advantage of the mouse genome database, we selected 127 DNA microsatellite markers showing heterozygosity between C3H and C57BL/6J (B6) and a first round of screening for the joggle mutation was performed on B6-jog/+ partial congenic mice (N4). We identified 4 chromosomal regions in which 13 microsatellite markers show heterozygosity between C3H and B6. Then, we analyzed the genotype of these 4 chromosomal regions in mice that showed the joggle phenotype and mapped the jog locus between markers D6Mit104 (111.4 Mb) and D6Mit336 (125.1 Mb) (an interval of 13.7 Mb) on chromosome 6. By using a partial congenic strain together with the mouse genome database, we successfully mapped the chromosomal localization of the jog locus much more efficiently than by conventional linkage analysis.