Cyclopropenone, Cyclopropeniminium Ion, and Cyclopropenethione as Novel Electrophilic Warheads for Potential Target Discovery of Triple-Negative Breast Cancer.

Because very few targets are currently available for drug development, triple-negative breast cancer (TNBC) has been defined as one of the most difficult diseases for chemotherapy. Herein, we describe a suite of novel electrophilic warheads, which we have used in chemical proteomics studies in a search for potential targets for TNBC. Binding site analysis revealed that these warheads can modify not only highly nucleophilic residues, including cysteine and lysine, but also weakly nucleophilic residues. Cys12 of Kirsten rat sarcoma (KRASG12C) was successfully labeled by cyclopropenone and the cyclopropeniminium ions. Moderate inhibitory activity against TNBC cells was achieved with these novel electrophile-based probes. Activity-based protein profiling reveals that these electrophiles can covalently label a series of essential protein targets, including ALDH2, LRPPRC, and FABP5 from MDA-MB-231 cells. Further functional validation experiments demonstrated that FABP5 might be a potential target for TNBC.

Numerical Simulation of the Shear Capacity of a GFRP-Strengthened Natural Bamboo-Bolt Composite Joint.

As an ecological green building material, natural bamboo has many advantages such as a light weight, high strength, and short growth cycle. Natural bamboo is widely used in landscape architecture and fabricated structures. However, in bamboo building structures, the most common bolted joints often appear cleaved along the grain. In this paper, glass fiber-reinforced polymer (GFRP) is designed to wrap and improve the shear capacity of natural bamboo-bolt composite joints. According to the corresponding material parameters, the finite element model of composite joints is established, and the key influencing variables of the bearing capacity, namely the bolt diameter, bamboo tube outer diameter, and screw end distance, are analyzed. In addition, according to the European analytical yield model of bolted connections, the analytical calculation method of the bearing capacity is proposed and compared with the experimental and simulated values. The results showed that the numerical model and the modified analytical model can suitably describe the bearing capacity of composite joints, and a higher bolt diameter, along with the bamboo outer diameter, will lead to a higher ultimate load of the composite joints. Moreover, the bearing capacity of composite joints has no obvious relationship with the end spacing.

Development and application of novel electrophilic warheads in target identification and drug discovery.

Nucleophilic amino acids play important roles in maintenance of protein structure and function, covalent modification of such amino acid residues by therapeutic agents is an efficient way to treat human diseases. Most of current clinical drugs are structurally limited to α,ß-unsaturated amide as an electrophilic warhead. To alleviate this issue, many novel electrophiles have been developed in recent years that can covalently bind to different amino acid residues and provides a unique way to interrogate proteins, including "undruggable" targets. With an activity-based protein profiling (ABPP) approach, the activity and functionality of a protein and its binding sites can be assessed. This facilitates an understanding of protein function, and contributes to the discovery of new druggable targets and lead compounds. Meanwhile, many novel inhibitors bearing new reactive warhead were developed and displayed remarkable pharmaceutical properties. In this perspective, we have reviewed the recent remarkable progress of novel electrophiles and their applications in target identification and drug discovery.

Salidroside regulates inflammatory pathway of alveolar macrophages by influencing the secretion of miRNA-146a exosomes by lung epithelial cells.

The purpose of this study was to explore the investigative mechanism of salidroside (SAL) on LPS-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The exosomes from RLE-6TN are extracted and identified by transmission electron microscopy, particle size analysis and protein marker detection, and co-cultured with NR8383 cells. The ALI/ARDS model of SD rats was established by LPS (10 mg/kg) intratracheal instillation. Following a four-hour intratracheal instillation of LPS, 50 µl of RLE-6TN exosomes were injected through the tail vein. After that, SAL and miR-146a antagomir were injected into the tail vein for 72 h, respectively. As the changes of HE stain, body weight and ALI score are observed. The expression of miR-146a, TLR4, NF-kB, IRAK1, TRAF6 and their related proteins were detected by RT-PCR and Western blot, respectively. TNF-α, IL-6, IL-8 and IL-1 ß inflammatory factors were detected by ELISA. The expression of miR-146a, NF-kB, IRAK, TRAF6 and related inflammatory factors in LPS-induced NR8383 was significantly higher than that in the control group, while SAL has greatly reduced the expression of TLR4 mediated NF-kB inflammatory pathway and related inflammatory factors. SAL can significantly improve the LPS-induced lung morphological abnormalities, slowed down the rate of weight loss in rats, and reducing the ALI score. The expression trend of NF-kB, IRAK, TRAF6 and related inflammatory factors in rats' lung tissues was consistent with that in NR8383 cells. SAL has a protective effect on ALI/ARDS caused by sepsis, which is likely to be developed to a potential treatment for the disease. To sum up, this study provides a new theoretical basis for the treatment of ALI/ARDS with SAL.

Thymosin­ß 4 induces angiogenesis in critical limb ischemia mice via regulating Notch/NF­κB pathway.

Thymosin­ß 4 (Tß4) has been reported to exert a pro­angogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tß4 on critical limb ischemia (CLI). The present study aimed therefore to investigate the mechanisms and pro­angiogenic effects of Tß4 in CLI mice. Tß4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NF­κB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesis­related factors angiopoietin­2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and α­smooth muscle actin (α­SMA) and the Notch/NF­κB pathways­related factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NF­κB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tß4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NF­κB, and phosphorylated (p)­p65 in HUVEC. In addition, Tß4 promoted the expression of CD31, α­SMA Ang2, tie2, VEGFA, N1ICD and p­p65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tß4, whereas Tß4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tß4 may promote angiogenesis in CLI mice via regulation of Notch/NF­κB pathways.

Investigating the Accuracy of Ultrasound-Ultrasound Fusion Imaging for Evaluating the Ablation Effect via Special Phantom-Simulated Liver Tumors.

The goal of this study was to investigate the accuracy of ultrasound-ultrasound (US-US) fusion imaging for evaluating the ablation effect via phantom-simulated liver tumors. Twenty special phantom models were established, ablated and divided into a complete ablation group (n = 10) and an incomplete ablation group (n = 10). US-US fusion imaging was performed to evaluate the ablation effect. Gross specimens were observed as a standard reference. In this US-US fusion imaging study, the registration success rate was 100% (20/20), and the assessment time was 3.8 ± 0.9 min. The accuracy rate of the evaluation was 100% (20/20). There was no significant difference in the residual pseudo-tumoral area between the evaluation with US-US fusion imaging and gross specimen observation (p = 0.811), and the measurement error was 1.1 ± 0.6 mm. In conclusion, the feasibility and accuracy of US-US fusion imaging when evaluating the ablation effect can be investigated with this phantom-simulated liver tumor ablation model in an ideal state.

[TCP wear particles causes injury of periprosthetic osteocytes in the mouse calvaria].

OBJECTIVE: To study whether tricalcium phosphate(TCP) wear particles cause injuries of periprosthetic osteocytes in the mouse calvaria, and to explain its molecular mechanism. METHODS: Thirty six-week(ICR)male mice were randomly divided into sham group, model (TCP) group and 3-methyladenine (3-MA) group. A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation over the periosteum around the middle suture of calvaria in mice. On the second postoperative day, the autophagy specific inhibitor 3-MA (1.0 mg/kg) was subcutaneously injected to the calvaria in the 3-MA-treated mice every other day. After 2 weeks, blood and the calvaria were obtained. Micro-CT was used to detect bone mineral density(BMD), bone volume fraction (BVF) and porosity number. HE staining and flow cytometry were performed to analyze the viability and apoptosis of periprosthetic osteocytes. The serum levels of dentin matrix protein 1(DMP-1) and sclerostin (SOST) were determined by ELISA. The proteins expressions of DMP-1, SOST, Beclin-1 and microtuble-associated protein 1 light chain 3 (LC-3) were detected by Western blot in the calvaria osteocytes. RESULTS: Compared with the sham group, the mice in the TCP group showed that a significant decrease in the viability of periprosthetic osteocytes, but obvious increases in number of osteocytes death and osteocytes apoptosis (P<0.05), and in serum level and protein expression of SOST; significant decreases in serum level and protein expression of DMP-1 (P<0.05), and remarkable up-regulation of autophagy-related factors beclin-1 and the conversion of LC3-Ⅱ from LC3-I in the calvaria osteocytes. Compared with TCP group, the mice in the 3-MA group showed that injuries of calvaria osteocytes were obviously aggravated, and osteocytes apoptosis was significantly increased (P<0.05). CONCLUSIONS: TCP wear particles can cause injuries of periprosthetic osteocytes via activation of apoptosis and autophagy, which promotes osteolysis around the prosthesis osteolysis and joint aseptic loosening.

Immediate evaluation and guidance of liver cancer thermal ablation by three-dimensional ultrasound/contrast-enhanced ultrasound fusion imaging.

PURPOSE: To investigate the feasibility and value of three-dimensional ultrasound/contrast-enhanced ultrasound (3D US-CEUS) fusion imaging for the immediate evaluation of technical success and the guidance of supplementary ablation during the liver cancer thermal ablation procedure. MATERIALS AND METHODS: Patients diagnosed with malignant liver cancer intending to receive thermal ablation including radiofrequency ablation (RFA) or microwave ablation (MWA) were enrolled. 3D US-CEUS fusion imaging was used to immediately assess the technical success and guide supplementary ablation. Contrast-enhanced computed tomography/magnetic resonance imaging (CECT/CEMRI) was performed one month after ablation to assess the technique effectiveness of the ablation. The registration success rate, duration time of 3D US-CEUS fusion imaging, technique effectiveness rate and major complications were recorded. RESULTS: In total, 76 patients with 95 tumours who underwent RFA or MWA and assessed by 3D US-CEUS fusion imaging were enrolled. The registration success rate of 3D US-CEUS fusion imaging was 93.7% (89/95), and the duration time was 4.0 ± 1.1 min. Thirty lesions received supplementary ablation immediately during the procedure. The technique effectiveness rate of the ablation was 98.8% (81/82). There were no major complications related to ablation. CONCLUSIONS: 3D US-CEUS fusion imaging is a feasible and valuable technique for the immediate evaluation and guidance of supplementary ablation during the liver cancer thermal ablation procedure.

Inhibition of osteolysis after local administration of osthole in a TCP particles-induced osteolysis model.

PURPOSE: Wear debris-induced osteolysis and aseptic loosening are the most frequent late complications of total joint arthroplasty leading to revision of the prosthesis. However, no effective measures for the prevention and treatment of particles-induced osteolysis currently exist. Here, we investigated the efficacy of local administration of osthole on tricalcium phosphate (TCP) particles-induced osteolysis in a murine calvarial model. METHODS: TCP particles were implanted over the calvaria of ICR mice, and established TCP particles-induced osteolysis model. On days one, four, seven, ten and thirteen post-surgery, osthole (10 mg/kg) or phosphate buffer saline (PBS) were subcutaneously injected into the calvaria of TCP particles-implanted or sham-operated mice. Two weeks later, blood, the periosteum and the calvaria were collected and processed for bone turnover markers, pro-inflammatory cytokine, histomorphometric and molecular analysis. RESULTS: Osthole (10 mg/kg) markedly prevented TCP particles-induced osteoclastogenesis and bone resorption in a mouse calvarial model. Osthole also inhibited the decrease of serum osteocalcin level and calvarial alkaline phosphatase (ALP) activity, and prevented the increase in the activity of tartrate resistant acid phosphatase (TRAP) and cathepsin K in the mouse calvaria. Furthermore, osthole obviously reduced the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) into the periosteum. Western blotting demonstrated TCP particles caused a remarkable endoplasmic reticulum (ER) stress response in the mouse calvaria, which was obviously blocked by osthole treatment. CONCLUSION: These results suggest that local administration of osthole inhibits TCP particles-induced osteolysis in the mouse calvarial in vivo, which may be mediated by inhibition of the ER stress signaling pathway, and it will be developed as a new drug in the prevention and treatment of destructive diseases caused by prosthetic wear particles.

Comparative proteomics and expression analysis of five genes in Epicauta chinensis larvae from the first to fifth instar.

Blister beetle is an important insect model for both medicinal and pure research. Previous research has mainly focused on its biology and biochemistry, but very little data is yet available in the molecular biology. This study uses differential proteomics technology to analyze the soluble proteins extracted from each of the 5 instars larvae of Epicauta chinensis. 42 of the differentially-expressed proteins were identified successfully by MALDI-TOF/TOF-MS. Some of these proteins' function and their expression profiles are analyzed. Our analysis revealed dynamics regulation of the following proteins: Axin-like protein pry-1 (APR-1), dihydrolipoyl dehydrogenase (DLD), vitellogenin (Vg) and lysozyme C (Lmz-S). APR-1 negatively regulates the Wnt signaling pathway. Its overexpression could result in embryo, leg, eye and ovary ectopica or malformation. DLD catalyzes the pyruvate into acetyl-CoA, the latter is the starting material of juvenile hormone (JH) and ipsdienol biosynthesis through the MVA pathway in insects. While Vg synthesis can be regulated by JH and stimulated by food factors. So DLD may affect the synthesis of JH, ipsdienol and Vg indirectly. The activity of lysozyme is an indicator of the immunity. Nutrition/food should be taken into account for its potential role during the development of larva in the future. Among the five genes and their corresponding proteins' expression, only hsc70 gene showed a good correspondence with the protein level. This reflects the fluctuating relationship between mRNA and protein levels.

Thymosin beta4 induces angiogenesis through Notch signaling in endothelial cells.

Thymosin beta4 (Tß4) has multi-functional roles in angiogenesis and arteriogenesis, but little is known about its mechanism. The Notch signaling pathway is important in regulation of angiogenic behavior of endothelial cells, in addition to vascular endothelial growth factor (VEGF). Whether, Tß4 regulates angiogenesis through Notch signaling pathway is not clear. In this article, we evaluated the effect of Notch signaling in Tß4-induced angiogenesis in human umbilical vein endothelial cell (HUVEC). Our results revealed that Tß4 increased Notch1 and Notch4 expression in a dose and time-dependent manner. The inhibition of Notch1 or Notch4 with siRNA or the Notch receptor inhibitor DAPT significantly prevented Tß4-induced HUVEC tube formation and lymphocyte transendothelial migration. The inhibition of Notch1 or Notch4 also blocked Tß4-induced VEGF and HIF-1α expression. VE-cadherin is the major endothelial adhesion molecule in the control of angiogenesis. Tß4 significantly reduced VE-cadherin expression levels in HUVEC, while the inhibition of Notch signaling prevented Tß4-induced VE-cadherin down-regulation. The results of this study suggest that Tß4 induces HUVEC angiogenesis through Notch signaling pathway.

Relationship between serum thymosin ß4 levels and coronary collateral development.

BACKGROUND: Thymosin ß4 was recently demonstrated to be a potential stimulator of angiogenesis and arteriogenesis in both in-vivo and in-vitro studies. Little is known about the relationship between serum thymosin ß4 levels and the extent of coronary collaterals in patients with coronary artery disease. HYPOTHESIS: Serum thymosin ß4 independently predicts the formation of coronary collaterals in patients with coronary artery disease. MATERIALS AND METHODS: A total of 190 consecutive patients who underwent coronary angiography were divided into two groups according to coronary angiograms and collateral formation: the poor collateral group consisting of 103 patients with at least more than or equal to one coronary stenosis of greater than or equal to 75% without visible collateral circulation, and the good collateral group consisting of 87 patients with at least more than or equal to one coronary stenosis of greater than or equal to 75% with well-developed collateral circulation. Collateral score was analyzed using the Cohen-Rentrop classification. RESULTS: Serum thymosin ß4 levels were 1024.50 ± 516.92 ng/ml and 1373.00 ± 1082.88 ng/ml for patients in the poor collateral group and the good collateral group, respectively. Serum thymosin ß4 levels in the good collateral group were significantly higher than those in the poor collateral group (P<0.01). After adjustment in the multiple ordinal logistic regression model, serum thymosin ß4 levels showed a strong independent association with the collateral Cohen-Rentrop score (χ(2)=4.45, odds ratio=1.00, 95% confidence interval: 1.000-1.002, P=0.035). CONCLUSION: Elevated serum thymosin ß4 level is independently associated with a significant enhancement in coronary collateralization and patients in the good collateral tend to have a higher thymosin ß4 level.

An elevated level of BNP in plasma is related to the development of good collateral circulation in coronary artery disease.

OBJECTIVE: B-type natriuretic peptide (BNP) was recently demonstrated to be a potential stimulator of angiogenesis and arteriogenesis. The correlation between BNP level and collateral formation in patients with coronary artery disease (CAD) has not been reported. METHODS AND RESULTS: The study included 311 consecutive patients who underwent coronary angiography were divided into three groups according to coronary angiography and collateral formation: normal group (100 patients with normal coronary angiographic findings); poor collateral group (116 patients with at least one coronary stenosis of ≥75% without visible collateral circulation); and good collateral group (95 patients with at least one coronary stenosis of ≥75% with well-developed collateral circulation). Collateral score was analyzed using the Cohen-Rentrop classification. Plasma BNP levels were 45.77 ± 4.66 pg/ml, 116.40 ± 28.15 pg/ml, and 254.20 ± 42.85 pg/ml for patients in normal, poor collateral, and good collateral groups, respectively. Plasma BNP levels in the latter were significantly higher than in the normal group (p < 0.01) and poor collateral group (p < 0.05). There were no significant differences between the good collateral group and poor collateral group when compared with left ventricular ejection fraction (LVEF), left ventricular dimensions at end diastole (LVEDd), age, severity of angiographic disease, and other cardiovascular risk factors. After adjustment in the multiple ordinal logistic regression model, plasma BNP levels showed a strong independent association with collateral Cohen-Rentrop score (χ(2 )= 5.636, OR = 1.002, 95% CI 1.000-1.004, p = 0.018). CONCLUSIONS: An elevated level of BNP in plasma is independently associated with collateral development; patients with good collaterals tend to have a higher BNP level.

Plasma hyaluronan and collateral development in patients with coronary artery disease.

OBJECTIVE: In-vivo studies have shown that hyaluronan (HA) can promote angiogenesis and arteriogenesis, which results in accelerated collateral development. This study is aimed at investigating the association between plasma HA levels and the extent of coronary collaterals, in patients with coronary artery disease. METHODS: A total of 253 consecutive patients, who underwent coronary angiography, were divided into three groups according to coronary angiograms: normal group, 81 patients with normal coronary angiographic findings; poor collateral group, 98 patients with at least one coronary stenosis of at least 75%, but without visible collateral circulation; good collateral group, 74 patients with at least one coronary stenosis of at least 75% with well-developed collateral circulation. Plasma HA levels were measured by radioimmunoassay. The correlation between HA levels and the extent of coronary collaterals according to the Cohen-Rentrop classification was calculated by cumulative logits models. RESULTS: Plasma HA levels were 43.71+/-2.91, 61.77+/-4.10, and 131.97+/-11.76 ng/ml, for patients in the normal, poor collateral, and good collateral groups, respectively. The good collateral group had significantly higher plasma HA levels than the poor collateral (P<0.001) and normal group (P<0.001), whereas there was no significant difference between the normal and poor collateral group. HA levels elevated with increasing Rentrop score, and the cumulative logits model showed a strong graded association between plasma HA levels and the collateral Cohen-Rentrop score (odds ratio=1.021, chi2=17.811, 95% confidence interval: 1.011-1.031, P=0.000). CONCLUSION: This study suggests that elevated plasma HA levels are associated with a significant enhancement in coronary collateralization. HA may serve as a novel potential biomarker for collateral formation in patients with coronary artery disease.

A study on the correlation between IL1RAPL1 and human cognitive ability.

This study aimed to investigate the effects of IL1RAPL1 on the human cognitive ability. Four genetic marker sites, i.e., DXS1218, DXS9896, rs6526806 and rs12847959 on IL1RAPL1 were genotyped in 332 Qinba Mountain Area children. Meanwhile, a cognition test with a C-WISC scale was performed to study the relationship of genotype with cognition test scores. Results indicated that genotypes of DXS1218, DXS9896 and rs12847959 were associated with memory/concentration factor intelligence quotient (IQ) (P=0.027, 0.042, 0.029, respectively). DXS1218 also associated with full IQ, verbal IQ, and performance IQ (P=0.006, 0.014, 0.006, respectively). rs12847959 were related to verbal comprehension factor and perceptual organization factor IQ (P=0.021, 0.043, respectively). Further study on rat brain revealed that Il1rapl was mainly expressed in memory/concentration-associated encephalic regions, such as hippocampus, dentate fascia, osmesis perithelium, and piriform cortex. mRNA expression levels of Il1rapl in brains of rats with different learning and memory abilities showed significant difference. Combined data suggested that IL1RAPL1 affected human cognitive ability to some extent, especially the memory and concentration capability.