Abstract Recent studies have demonstrated that one‐carbon metabolism plays a significant role in cancer development. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme of one‐carbon metabolism, has been reported to be dysregulated in many cancers. However, the specific role and mechanism of MTHFD2 in lung adenocarcinoma (LUAD) still remains unclear. In this study, we evaluated the clinicopathological and prognostic values of MTHFD2 in LUAD patients. We conducted a series of functional experiments in vivo and in vitro to explore novel mechanism of MTHFD2 in LUAD. The results showed that MTHFD2 was significantly up‐regulated in LUAD tissues and predicted poor prognosis of LUAD patients. Knockdown of MTHFD2 dramatically inhibited cell proliferation and migration by blocking the cell cycle and inducing the epithelial‐mesenchymal transition (EMT). In addition, MTHFD2 knockdown suppressed LUAD growth and metastasis in cell‐derived xenografts. Mechanically, we found that MTHFD2 promoted LUAD cell growth and metastasis via AKT/GSK‐3β/β‐catenin signalling. Finally, we identified miR‐30a‐3p as a novel regulator of MTHFD2 in LUAD. Collectively, MTHFD2 plays an oncogenic role in LUAD progression and is a promising target for LUAD diagnosis and therapy.
Thrombin is a central enzyme in haemostasis and thrombosis, and a proven target for anticoagulant therapies. Different classes of thrombin inhibitors, while exerting therapeutic benefits in most clinical trials, have different indications, dosing regimens, and bleeding complications. To gain more insight into the underlying mechanisms for their differential clinical profiles, we compared four marketed and representative agents, including dabigatran, hirudin, bivalirudin (direct thrombin inhibitors, DTIs), and heparin (an indirect thrombin inhibitor), in two in vitro spike-in assays with concentration titrations covering their therapeutic ranges. The two assays were the Thrombinoscope TGA with plasma, triggered by low tissue factor (1 nM TF), and TEG with whole blood, triggered by 1:8000 Recombiplastin (equivalent to low TF), with or without a threshold level of tPA to induce fibrinolysis. In TGA, the largest effect was prolongation of lag time, with the potency of the three DTIs rank-ordered as hirudin>dabigatran>bivalirudin; regarding peak, slope, and ETP, while complete inhibition was achieved with 1-2 μM dabigatran or hirudin, bivalirudin had no effect even at 4 μM, possibly due to its short half life in plasma. In TEG, the three DTIs prolonged clotting time (R) in the same rank order as TGA; for clot strength (MA), while all four agents reduced MA in synergy with tPA, only hirudin reduced MA without tPA, likely due to its highest potency. With tPA-induced fibrinolytic activity (Ly30), dabigatran and bivalirudin enhanced Ly30 (dabigatran>bivalirudin), but hirudin and heparin did not. This contrast might involve differential access to clot-bound thrombin. Heparin had a steep dose-response curve for both lag time in TGA and R in TEG, which is in line with its very narrow therapeutic index. All three DTIs, but not heparin, displayed the previously reported paradoxical increase in peak and slope in TGA in the low concentration range, suggesting this is indeed a class effect of DTI. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. These results in combination with known clinical properties are informative on efforts to define the optimal profiles of new anticoagulants.
Coagulation Factor XII (FXII) is an attractive novel target for antithrombotic therapies as studies with FXIIa inhibitors and FXII knockout mouse suggest FXII is critically involved in pathological thrombus formation but is dispensable for hemostasis. To further assess the role of FXII in thrombosis and hemostasis, FXII knockout or knockdown models in rat were established and characterized. FXII knockout in rat was generated via the Zinc Finger nuclease (ZFN) technology and confirmed by lack of detectable zymogen in circulation. Titratable FXII knockdown in rat was achieved with a siRNA reagent that is potent on FXII mRNA in liver and selective against all other coagulation factors. FXII knockout in rats produced marked antithrombotic efficacy in both the arteriovenous shunt model (~100% clot weight reduction), and the 10% FeCl 3 -induced carotid artery thrombosis model (no reduction in blood flow post injury), without any increase in the cuticle bleeding time compared to the WT control rats. Ex vivo aPTT and ellagic acid-triggered thrombin generation assay (TGA) exhibited corresponding changes whereas ex vivo PT or TF (20 pM)-triggered TGA was indistinguishable from WT rats. Rats that received a single dose of FXII siRNA (at 0 (non-targeting siRNA control), or 0.01, or 0.03, or 0.1, or 0.3 mg/kg) exhibited dose-dependent knockdown in FXII mRNA in the liver and FXII zymogen in plasma (90% and 97%, respectively, for the 0.3 mg/kg dose) at day 7 post dosing. FXII knockdown was associated with dose-dependent antithrombotic efficacy (with the 0.3 mg/kg dose delivering near maximal efficacy in both models) and minimal increase in cuticle bleeding times (<50% across all doses). Ex vivo pharmacodynamic markers (aPTT and TGA) tracked with the knockdown levels and efficacy. In summary, our findings in both the knockout model and the knockdown model in rats confirmed and extended literature observations with regard to the highly desirable benefit-to-risk profile of targeting FXII in preclinical species; the siRNA tool will allow detailed studies on level of target engagement needed for translatable efficacy; the KO rat will be valuable for addressing any mechanism-based safety concerns as well as off-target effects of putative FXIIa inhibitors.
Objective To explo re the influence of diabetes on the macrophages and interdigitating cells in rat thymus and the roles of them in the thymus involution.?Methods Using the rat model of alloxan-diabetes and the enzyme-histochenmistry, immuno histochemistry and electron microscopy, qualitative and semi-quantitative analys es and morphologic observation were made on the macrophages and interdigitating cells in thymus.?Results In diabetic rat thymus, the m acrophages were strongly positive for ACP, NSE, Ly and AACT,the number of them increased evidently, groups of the macrophages gathered in the cortex and cortic omedullary zone, especially in the cortex. The interdigitating cells, strongly p ositive for S-100 protein, were evidently reduced in number and mainly distribut ed in the medulla and corticomedullary zone. Electron microscopy revealed apopto tic cells and bodies engulfed by the macrophages in the cortex, but cystic vacuo les and granules engulfed by the macrophages in the corticomedullary zone.?Conclusion In diabetic rat thymus, the macrophages are eviden tly increased in number and enzyme activities, and show heterogenous changes in enzyme activity, morphology and function. The interdigitating cells are evidentl y decreased in number. These cells both may play important roles in the thymus i nvolution.
Thrombin is a central enzyme in hemostasis and thrombosis, and a proven target for anticoagulant therapies. We compared four marketed and representative thrombin inhibitors, heparin, hirudin, bivalirudin, and dabigatran, in in-vitro spike-in assays that covered their therapeutic ranges. The assays employed were low tissue factor (1 pmol/l)-triggered thrombin generation assay (TGA) with plasma and 1 : 8000 Recombiplastin-triggered thromboelastography (TEG) with whole blood, with or without tissue plasminogen activator (tPA)-induced fibrinolysis. The three direct thrombin inhibitors (DTIs) prolonged TGA lag time and TEG clotting time (R) with a potency stack-ranking of hirudin > dabigatran ≅ bivalirudin. Heparin had the most steep concentration–response curve for both parameters. In TGA, 1–2 μmol/l dabigatran or hirudin resulted in complete inhibition on peak, slope, and endogenous thrombin potential, whereas bivalirudin had no effect on these parameters up to 10 μmol/l. All three DTIs, but not heparin, displayed a paradoxical increase in peak and slope in the low concentration range. In TEG, whereas all four agents reduced clot strength (maximal amplitude) in synergy with tPA, hirudin was the only DTI that reduced maximal amplitude appreciably without tPA. Dabigatran had the strongest potentiating effect on tPA-induced fibrinolytic activity (Ly30). With regard to the effects on coagulation and clot strength (lag time, R, and maximal amplitude) in the respective therapeutic range, dabigatran elicited the most modest changes. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. The contrasts between the agents are consistent with their known properties and are informative on efforts to define the optimal profiles of new anticoagulants.
Hypoxanthine−guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325−334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to kcat, phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s-1) and reverse (9 vs 0.17 s-1) directions. In the forward direction, product pyrophosphate dissociates rapidly (>12 s-1) followed by release of IMP (6.0 s-1). In the reverse direction, Hx dissociates rapidly (9.5 s-1) and PRPP dissociates slowly (0.24 s-1). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq ≈ 1.6 × 105), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase·IMP complex has been solved to 2.4 Å resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.
Multiple coagulation factors in the intrinsic cascade are emerging targets for new anticoagulant therapies. Rat and rabbit are two commonly utilized preclinical species for studying thrombosis and hemostasis. The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target coagulation factors in rat and rabbit. Plasma kallikrein was selected for rat studies as plasma kallikrein knockout mouse had been described before in thrombosis and hemostasis models. Factor X (FX) was selected for rabbit studies as a rich dataset of FXa inhibitors in rabbits exists in literature. siRNAs that produced over 90% knockdown of rat plasma prekallikrein mRNA and rabbit FX mRNA in vitro were identified from cellular screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day seven post dosing in both species. Both prekallikrein siRNA and FX siRNA resulted in dose-dependent and selective knockdown of target gene mRNA in the liver of respective species with maximum reduction of over 90% on day 7 following a single dose of the siRNA-LNP. Plasma prekallikrein siRNA at the highest dose (0.5 mg/kg) produced 92% knockdown on the circulating zymogen in the rats, associated with modest (~45%) clot weight reduction in the arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. These observations are consistent with the modest antithrombotic phenotype and absence of bleeding diathesis reported for the knockout mouse. FX siRNA at the highest dose (1.0 mg/kg) produced 98% knockdown on the circulating zymogen in the rabbits, accompanied with significant prolongation in ex vivo aPTT (103%) and PT (202%) measurements, consistent with the literature findings on pharmacodynamic effects of FXa inhibitors in rabbit. Results thus fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat and rabbit, via systemic delivery of siRNA. The titratable knockdown platform will not only strengthen target validation in thrombosis research but also facilitate studies on hemophilia diseases and hemostatic agents thereof.
Abstract Recent developments of deep learning methods have demonstrated their feasibility in liver malignancy diagnosis using ultrasound (US) images. However, most of these methods require manual selection and annotation of US images by radiologists, which limit their practical application. On the other hand, US videos provide more comprehensive morphological information about liver masses and their relationships with surrounding structures than US images, potentially leading to a more accurate diagnosis. Here, we developed a fully automated artificial intelligence (AI) pipeline to imitate the workflow of radiologists for detecting liver masses and diagnosing liver malignancy. In this pipeline, we designed an automated mass-guided strategy that used segmentation information to direct diagnostic models to focus on liver masses, thus increasing diagnostic accuracy. The diagnostic models based on US videos utilized bi-directional convolutional long short-term memory modules with an attention-boosted module to learn and fuse spatiotemporal information from consecutive video frames. Using a large-scale dataset of 50 063 US images and video frames from 11 468 patients, we developed and tested the AI pipeline and investigated its applications. A dataset of annotated US images is available at https://doi.org/10.5281/zenodo.7272660.
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