Real-time monitoring of caspase-3/8 activity by self-assembling nanofiber probes in living cells.

Caspase-3/8 are key members of the cysteine-aspartyl protease family with pivotal roles in apoptosis. We have designed and synthesized self-assembling probes, Nap-GFFpYDEVD-AFC and Nap-GFFpYIETD-AFC, with fluorescence 'turn-on' properties for real-time monitoring of Caspase-3/8 activity in living cells.

Self-assembling nitrilotriacetic acid nanofibers for tracking and enriching His-tagged proteins in living cells.

Specific and expeditious identification and enrichment of target proteins in living cells is often a challenging task. The hexahistidine (6His) tag is frequently used to label artificially engineered proteins produced in prokaryotic or eukaryotic cells. Utilizing the interaction between 6His-tag and nitrilotriacetic acid (NTA) mediated by divalent metal ions (Ni2+, Cu2+, Zn2+ or Co2+), we designed and synthesized a series of Nap-G/Biotin/ANA-FFpYGK-NTA probes that, assisted by alkaline phosphatase (ALP), self-assemble into nanofibers. The probe consists of an NTA group that specifically binds to 6His-tag, an FFpY group that promotes self-assembly facilitated by ALP, and a hydrophobic (Nap-G/ANA/Biotin) capping group for various applications. We demonstrate that the ANA-FFpYGK-NTA(Ni2+) nanofibers are fit for real-time tracking of His-tagged protein in living cells, and the Biotin-FFpYGK-NTA(Ni2+) nanofibers are for isolating His-tagged proteins and other proteins that they interact with.

Self-assembling peptide-etoposide nanofibers for overcoming multidrug resistance.

We developed a new strategy to overcome the MDR of etoposide using self-assembling nanofibers. Compared with the original etoposide, the inhibitory activity of Nap-GFFpYK-etoposide1/2 against murine Lewis lung cancer or breast cancer cells was increased 10 times, and 20 times on these cells with artificially overexpressed MDR1. Our method to synthesize and separate etoposide isomers provides a new strategy for the modification of this drug.

Compounds targeting YadC of uropathogenic Escherichia coli and its host receptor annexin A2 decrease bacterial colonization in bladder.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), and fimbrial tip adhesins, play important roles in UPEC colonization. Few fimbrial tip adhesins and their receptors on host cells, which have the potential to be the therapeutic targets, have been identified. METHODS: the UPEC wild-type strain CFT073, ΔyadC and the complemented strain were used to perform assays in vitro and in vivo. The effects of D-xylose targeting YadC on UPEC colonization were evaluated. A YadC receptor was identified by far-western blotting, LC-MS/MS and co-immunoprecipitation. The effects of compounds targeting the receptor on UPEC colonization were tested. FINDINGS: YadC was investigated for its mediation of UPEC adhesion and invasion to bladder epithelial cells in vitro; and its promotion of UPEC colonization in bladder in vivo. D-xylose, targeting YadC, showed prophylactic and therapeutic effects on UPEC colonization. Annexin A2 (ANXA2) was identified as a YadC receptor, involved in UPEC infection. ANXA2 inhibitors attenuated UPEC infections. The yadC gene was widely present in UPEC clinical isolates and phylogenetic analysis of yadC was performed. INTERPRETATION: YadC and its receptor ANXA2 play important roles in UPEC colonization in bladder, leading to novel treatment strategies targeting YadC or ANXA2 for acute UTIs. FUND: This study was supported by grants from the National Natural Science Foundation of China (NSFC) Programs (31670071 and 31970133), the National Key Technologies R&D Program, Intergovernmental international innovation cooperation (2018YFE0102000), Tianjin Science and Technology Commissioner Project (18JCZDJC36000), the Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2017ZD12). The Science Foundation of Tianjin Medical University (2016KY2M08).

Human rhomboid family-1 modulates clathrin coated vesicle-dependent pro-transforming growth factor α membrane trafficking to promote breast cancer progression.

BACKGROUND: Epidermal growth factor receptor (EGFR) signalling is critical in epithelial cancer development. Human rhomboid family-1 (RHBDF1) facilitates the secretion of TGFα, an EGFR ligand, in breast cancer; however, the underlying mechanism remains unclear. We evaluated the role for RHBDF1 in clathrin-coated vesicle (CCV)-dependent pro-TGFα membrane trafficking in breast cancer cells upon stimulation by G-protein coupled receptor (GPCR) agonists. METHODS: RHBDF1 was silenced in various breast cancer cells using shRNA. TGFα levels, subcellular localization, and secretion were evaluated using ELISA, immunofluorescent staining, and coimmunoprecipitation. Phosphorylation and expression of relevant proteins were measured by western blotting. RHBDF1-dependent cell viability and invasion were measured. FINDINGS: RHBDF1 mediates GPCR agonist-induced EGFR phosphorylation by promoting TGFα secretion in various types of breast cancer cells. RHBDF1 not only mediates ADAM17-dependent shedding of TGFα, but is essential in membrane trafficking of pro-TGFα. RHBDF1 silencing results in blocking of clathrin uncoating from CCV, a crucial step for the plasma membrane release of pro-TGFα. Interaction of RHBDF1 with auxilin-2, a CCV protein, determines the recruitment of HSC70 to CCV to facilitate clathrin uncoating. RHBDF1 function is required for the proliferation and mobility of breast cancer cells upon stimulation by Sphingosine 1 Phosphate (S1P), a GPCR agonist. We demonstrate a significant correlation between RHBDF1 overexpression and EGFR activation in breast cancer tissues. INTERPRETATION: RHBDF1 is an indispensable component of the protein trafficking machinery involved in GPCR-mediated EGFR transactivation, and is an attractive therapeutic target for cancer. FUND: National Natural Science Foundation of China (81,672,740 to ZSZ, 81,272,356 and 81,330,029 to LYL).

A novel Anti-Cancer Stem Cells compound optimized from the natural symplostatin 4 scaffold inhibits Wnt/ß-catenin signaling pathway.

Cancer stem cells (CSCs) are responsible for carcinogenesis, cancer progression, relapse, metastasis and drug resistance. Therefore, the development of drug molecules targeting CSCs plays a vital role in medicinal researching field. However, there are extremely rare molecules that selectively ablate CSCs. The research and development of drugs targeting CSCs is limited due to a lack of anti-CSCs lead compounds. In this study, an anti-CSCs lead compound 35b was discovered, which was derived from the natural chemical scaffold of Symplostatin 4. This compound exhibited a significantly suppressive effect on tumor growth both in vitro and in vivo. Additionally, 35b could significantly reduce the number of melanoma tumor spheres and decrease the percentage of ALDH+ melanoma cells. Further mechanism study illustrated that compound 35b could eliminate the melanoma CSCs by efficiently blocking Wnt/ß-catenin signaling pathway. Collectively, our findings would provide a novel chemical scaffold and alternative idea of molecular design for development of anti-CSCs drugs.

Discovery of N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives as potential antiproliferative agents by inhibiting MEK.

Mitogen activated protein kinase (MAPK) signal transduction pathway has been proved to play an important role in tumorigenesis and cancer development. MEK inhibitor has been demonstrated significant clinical benefit for blocking MAPK pathway activation and possibly could block reactivation of the MAPK pathway at the time of BRAF inhibitor resistance. Twenty N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives have been designed and synthesized as MEK inhibitors, and their biological activities were evaluated. Among these compounds, compound 7b showed the most potent inhibitory activity with IC50 of 91nM for MEK1 and GI50 value of 0.26µM for A549 cells. The SAR analysis and docking simulation were performed to provide crucial pharmacophore clues that could be used in further structure optimization.

Push Force Analysis of Anchor Block of the Oil and Gas Pipeline in a Single-Slope Tunnel Based on the Energy Balance Method.

In this paper, a single-slope tunnel pipeline was analysed considering the effects of vertical earth pressure, horizontal soil pressure, inner pressure, thermal expansion force and pipeline-soil friction. The concept of stagnation point for the pipeline was proposed. Considering the deformation compatibility condition of the pipeline elbow, the push force of anchor blocks of a single-slope tunnel pipeline was derived based on an energy method. Then, the theoretical formula for this force is thus generated. Using the analytical equation, the push force of the anchor block of an X80 large-diameter pipeline from the West-East Gas Transmission Project was determined. Meanwhile, to verify the results of the analytical method, and the finite element method, four categories of finite element codes were introduced to calculate the push force, including CAESARII, ANSYS, AutoPIPE and ALGOR. The results show that the analytical results agree well with the numerical results, and the maximum relative error is only 4.1%. Therefore, the results obtained with the analytical method can satisfy engineering requirements.

Scaffold-based pan-agonist design for the PPARα, PPARß and PPARγ receptors.

As important members of nuclear receptor superfamily, Peroxisome proliferator-activated receptors (PPAR) play essential roles in regulating cellular differentiation, development, metabolism, and tumorigenesis of higher organisms. The PPAR receptors have 3 identified subtypes: PPARα, PPARß and PPARγ, all of which have been treated as attractive targets for developing drugs to treat type 2 diabetes. Due to the undesirable side-effects, many PPAR agonists including PPARα/γ and PPARß/γ dual agonists are stopped by US FDA in the clinical trials. An alternative strategy is to design novel pan-agonist that can simultaneously activate PPARα, PPARß and PPARγ. Under such an idea, in the current study we adopted the core hopping algorithm and glide docking procedure to generate 7 novel compounds based on a typical PPAR pan-agonist LY465608. It was observed by the docking procedures and molecular dynamics simulations that the compounds generated by the core hopping and glide docking not only possessed the similar functions as the original LY465608 compound to activate PPARα, PPARß and PPARγ receptors, but also had more favorable conformation for binding to the PPAR receptors. The additional absorption, distribution, metabolism and excretion (ADME) predictions showed that the 7 compounds (especially Cpd#1) hold high potential to be novel lead compounds for the PPAR pan-agonist. Our findings can provide a new strategy or useful insights for designing the effective pan-agonists against the type 2 diabetes.

[Compare of CT scan of the nasopharynx in patients with obstructive sleep apnea-hypopnea syndrome and health].

OBJECTIVE: To explore the impact of nasopharynx and palatum durum on the pathogenesis and severity of OSAHS patients via CT scan, and to establish a foundation to select the suitable surgical procedures. METHOD: Thirty nine OSAHS patients diagnosed by PSG and 30 normal adults were included in this study. Successive CT scanning were undertaken from roof of nasopharynx to glottis and then the CT scan slices were reconstructed in sagittal plane through Efilm software. The parameters were measured in the midline of the nasopharynx,the longest section of the hard palate and then were analyzed by t test. RESULT: The subspinale-posterior nasal spine (A-PNS) of OSAHS patients were longer than those of normal adults. The posterior nasal spine-culminating point of nasopharynx (PNS-R) and posterior space of hard palate (PNS-B) of OSAHS patients were less than those of normal adults. A 95% confidence interval were calculated from the parameters of 30 normal adults. Among 39 OSAHS patients, the A-PNS in 6 patients were longer than the top limit of normal,the PNS-R in 10 patients and the PNS-B in 16 patients were less than the low limit of normal adults, 2 patients were seen the differences of both A-PNS and PNS, 5 were seen the differences of both PNS-R and PNS-B, and 1 were seen the differences of A-PNS and PNS-B. The AHI of 24 OSAHS patients with smaller nasopharynx and longer hard palate were larger than that of the other 15 OSAHS patients, meanwhile the SaO2 of the former patients were lower than the latter. CONCLUSION: Though the narrow of the upper airway in OSAHS patients mainly attribute to the nasopharynx, the nasopharyngeal size and the length of hard palate are also involved in the pathogenesis and severity of OSAHS and should be considered when choosing the surgical procedures.

[Prevention of bone loss by estradiol valerate combined with medroxyprogesterone acetate among postmenopausal women with osteopenia].

OBJECTIVE: To observe the efficacy of estradiol valerate (E(2)V) combined with medroxyprogesterone acetate (MPA) in prevention of bone loss among postmenopausal women with osteopenia. METHODS: Ninety-two women 1 to 8 years after menopause with osteopenia were randomly divided into 4 groups: 1) control group: 23 cases, taking calcium carbonate (Ca) 400 mg/d+ vitamin D 200 IU/D; 2) treatment group 1: 24 cases, taking E(2)V 1.0 mg/d+ MPA 2 mg/d+ Ca 400 mg/d; 3) treatment group 2: taking E(2)V 1.0 mg/d+ MPA 2 mg/d+ Ca 400 mg/d+ vitamin D 200 IU/d; and 4) treatment group 3: taking E(2)V 1.5 mg/d+ MPA 2 mg/d+ Ca 400 mg/d+ vitamin D 200 IU/d. The bone mineral density (BMD) of L(2- 4) and neck of femur was measured by DEXA method, and the bone metabolic markers such as serum alkaline phosphatase (ALP) and urine Ca/Cr and Ntx/Cr were examined just before the drug administration and 6, 12, and 18 months after the beginning of experiment. Vaginal bleeding and breast tenderness were recorded by the subjects themselves and examined by investigators. RESULTS: Seventy-eight subjects (84.5%) were followed up for I year and 77 of them (83.7%) were followed up for 1.5 years. The fasting morning Ca/Cr remained almost unchanged in the control group, and decreased by 43.8%, 33.3%, and 33.3% by the third month respectively and then remained unchanged in the treatment groups 1, 2, and 3. The urine Ntx/Cr decreased mildly but insignificantly in the control group (P > 0.05), and decreased since the 3rd month by 42.2%, 53.2%, and 29.1% respectively in the treatment groups 1 approximately 3 and then decreased very slowly. The ALP remained almost unchanged in the control group, and decreased by 6.8%, 17.0%, and 16.4% by the 6th months respectively and by 14.2%, 17.9%, and 28.8% by the 12th month and then remained almost unchanged in the treatment groups 1 approximately 3. The BMD of L2-4 increased by 7.1% +/- 4.6%, 6.8% +/- 5.3%, and 9.0% +/- 4.0% in the treatment 1 approximately 3 by the 18th month. The BMD of neck of femur increased by 4.1% +/- 3.2%, 4.0% +/- 5.9%, and 5.9% +/- 4.8% in the treatment 1 approximately 3 by the 18th month. Fracture occurred in 2 cases of the control group and 2 cases of the treatment group because of accidents. The vaginal bleeding rate was the highest in the treatment group 3 (47.4%). The breast tenderness rates were 27.3% approximately 47.6% in the 3 treatment groups. CONCLUSION: Administration of E(2)V I mg/d combined with MPA 2 mg/d is an optional regimen for postmenopausal women with intact uterine.