Optimization of Protein-Protein Interaction Measurements for Drug Discovery Using AFM Force Spectroscopy

Increasingly targeted in drug discovery, protein-protein interactions challenge current high throughput screening technologies in the pharmaceutical industry. Developing an effective and efficient method for screening small molecules or compounds is critical to accelerate the discovery of ligands for enzymes, receptors and other pharmaceutical targets. Here, we report developments of methods to increase the signal-to-noise ratio (SNR) for screening protein-protein interactions using atomic force microscopy (AFM) force spectroscopy. We have demonstrated the effectiveness of these developments on detecting the binding process between focal adhesion kinases (FAK) with protein kinase B (Akt1), which is a target for potential cancer drugs. These developments include optimized probe and substrate functionalization processes and redesigned probe-substrate contact regimes. Furthermore, a statistical-based data processing method was developed to enhance the contrast of the experimental data. Collectively, these results demonstrate the potential of the AFM force spectroscopy in automating drug screening with high throughput

Electronic properties and 4f→ 5d transitions in Ce-doped Lu2SiO5: a theoretical investigation

This is an electronic version of an article published in Journal of Materials Chemistry. Ning, L., Lin, L., Li, L., Wu, C., Duan, C., Zhang, Y. and Luis Seijo. "Electronic properties and 4f 5d transitions in Ce-doped Lu2SiO5: a theoretical investigation". Journal of Materials Chemistry 22 (2012): 13723-1373

Theoretical study on structural properties and 4f 5d transitions of locally charge-compensated Ce3+ in CaF2

Reprinted with permission from Ning, L., Wu, C.,Li, L., Lin,L., Duan, C., Zhang, Y. and Luis Seijo. "Theoretical study on structural properties and 4f 5d transitions of locally charge-compensated Ce3+ in CaF2". The Journal of Physical Chemistry C 116.34 (2012): 18419-18426.The structural properties and 4f → 5d transitions of Ce3+ in CaF2, with local charge compensation by an interstitial fluoride (Fi ′) or an oxygen substitution for fluoride (OF′), have been studied using the density functional theory (DFT) within the supercell model and the wave function-based embedded cluster calculations, respectively. The DFT results indicate that the incorporation of locally charge-compensated Ce3+ in CaF2 induces an anisotropic distortion of the structure around the dopant site. On the basis of the DFToptimized structures, the Ce-centered embedded clusters are constructed, on which the wave function-based CASSCF/CASPT2/ RASSI−SO calculations at the spin−orbit level are performed to obtain the Ce3+ 4f1 and 5d1 level energies. The calculated 4f−5d transition energies and relative intensities are in good agreement with available experimental results. From the present calculations, we conclude that the 5d1 level missing in the low-temperature absorption spectrum of the tetragonal Ce center with Fi ′ compensation is the second-lowest one, and the absorption at this level is overshadowed by an adjacent cluster band usually assigned to Ce clusters and thus was not observed in experiments. We also assign the two closely spaced absorption lines around 3118.5 Å observed in experiments to the lowest two quasi-degenerated 4f → 5d transitions of the monoclinic center with Fi ′ compensation rather than those of the trigonal center as proposed earlier. Finally, we analyze the structural and electronic reasons for the large reduction (∼2000 cm−1 ) of the lowest 4f → 5d transition energy from a Fi ′ to a nearest-neighbor OF′ compensation, in terms of the changes in the centroid energy difference and crystal- field splittingThis work was supported by the NSFC (Grants 11174005, 11074315, 90922022, and 10804001) and the Program for Innovative Research Teams in Anhui Normal University of China. L.S. acknowledges support from MEC-Spain (Grant MAT2011-24586

Autophagy protects against palmitate-induced apoptosis in hepatocytes

BACKGROUND: Non-alcoholic fatty liver disease, one of the most common liver diseases, has obtained increasing attention. Palmitate (PA)-induced liver injury is considered a risk factor for the development of non-alcoholic fatty liver disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism in response to various stresses. In this study, we investigated whether autophagy plays a protective role in the progression of PA-induced hepatocytes injury. RESULTS: Annexin V-FITC/PI staining by FCM analysis, TUNEL assay and the detection of PARP and cleaved caspase3 expression levels demonstrated that PA treatment prominently induced the apoptosis of hepatocytes. Meanwhile, treatment of PA strongly induced the formation of GFP-LC3 dots, the conversion from LC3I to LC3II, the decrease of p62 protein levels and the increase of autophagosomes. These results indicated that PA also induced autophagy activation. Autophagy inhibition through chloroquine pretreatment or Atg5shRNA infection led to the increase of cell apoptosis after PA treatment. Moreover, induction of autophagy by pretreatment with rapamycin resulted in distinct decrease of PA-induced apoptosis. Therefore, autophagy can prevent hepatocytes from PA-induced apoptosis. In the further study, we explored pathway of autophagy activation in PA-treated hepatocytes. We found that PA activated PKCα in hepatocytes, and had no influence on mammalian target of rapamycin and endoplasmic reticulum stress pathways. CONCLUSIONS: These results demonstrated that autophagy plays a protective role in PA-induced hepatocytes apoptosis. And PA might induce autophagy through activating PKCα pathway in hepatocytes

Quantifying the controls on evapotranspiration partitioning in the highest alpine meadow ecosystem

Quantifying the transpiration fraction of evapotranspiration (T/ET) is crucial for understanding plant functionality in ecosystem water cycles, land‐atmosphere interactions, and the global water budget. However, the controls and mechanisms underlying the temporal change of T/ET remain poorly understood in arid and semiarid areas, especially for remote regions with sparse observations such as the Tibetan Plateau (TP). In this study, we used combined high‐frequency laser spectroscopy and chamber methods to constrain estimates of T/ET for an alpine meadow ecosystem in the central TP. The three isotopic end members in ET (δET), soil evaporation (δE), and plant transpiration (δT) were directly determined by three newly customized chambers. Results showed that the seasonal variations of δET, δE, and δT were strongly affected by the precipitation isotope (R2 = 0.53). The δ18O‐based T/ET agreed with that of δ2H. Isotope‐based T/ET ranged from 0.15 to 0.73 during the periods of observation, with an average of 0.43. This mean result was supported by T/ET derived from a two‐source model and eddy covariance observations. Our overarching finding is that at the seasonal timescale, surface soil water content (θ) dominated the change of T/ET, with leaf area index playing only a secondary role. Our study confirms the critical impact of soil water on the temporal change of T/ET in water‐limited regions such as the TP. This knowledge sheds light on diverse land‐surface processes, global hydrological cycles, and their modeling

Oral Human Papillomavirus Infection and Its Risk Factors among 5,410 Healthy Adults in China, 2009–2011

Abstract Background: Despite the established link between oral human papillomavirus (HPV) infection and a subset of oropharyngeal squamous cell carcinoma (OSCC), little is known about the epidemiology of oral HPV infection among healthy adults in China. Methods: Oral swab specimens and questionnaires were collected from 5,410 individuals (ages 25–65 years). HPV DNA in oral exfoliated cells was tested by general primer-mediated (SPF1/GP6+) PCR and sequencing. Univariate and multivariate analyses were performed to assess the associations between exposure factors and oral infection. Results: Alpha mucosal HPV types were detected in 0.67% [95% confidence interval (CI), 0.47%–0.93%] of 5,351 β-globin–positive specimens, and cutaneous HPV in 5.46% (95% CI, 4.86%–6.10%). HPV 16 and 3 were the most prevalent types of α mucosal (0.43%; 95% CI, 0.27%–0.64%) and cutaneous HPV (4.17%; 95% CI, 3.65%–4.74%), respectively. The prevalence of α mucosal HPV decreased with increasing age (25–65 years) from 0.93% to 0.36% (Ptrend = 0.033), and was associated with self-reported history of oral disease [adjusted odds ratio (OR), 4.78; 95% CI, 1.07–21.41]. In 1,614 heterosexual couples, cutaneous HPV in one partner was found to increase the other partner's risk of cutaneous HPV infection (adjusted OR, 2.33; 95% CI, 1.22–4.48). Conclusions: Oral HPV infection, particularly with α mucosal types, is rare among healthy adults in China. A younger age and a history of oral disease imply higher risk of α mucosal HPV infection. Impact: This study addresses the paucity of epidemiological data on oral HPV infection among healthy population in China. Cancer Epidemiol Biomarkers Prev; 23(10); 2101–10. ©2014 AACR.</jats:p

Quantitative Analysis and Comparison Study of [18F]AlF-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 Using a Reference Tissue Model

With favorable pharmacokinetics and binding affinity for αvβ3 integrin, 18F-labeled dimeric cyclic RGD peptide ([18F]FPPRGD2) has been intensively used as a PET imaging probe for lesion detection and therapy response monitoring. A recently introduced kit formulation method, which uses an 18F-fluoride-aluminum complex labeled RGD tracer ([18F]AlF-NOTA-PRGD2), provides a strategy for simplifying the labeling procedure to facilitate clinical translation. Meanwhile, an easy-to-prepare 68Ga-labeled NOTA-PRGD2 has also been reported to have promising properties for imaging integrin αvβ3. The purpose of this study is to quantitatively compare the pharmacokinetic parameters of [18F]FPPRGD2, [18F]AlF-NOTA-PRGD2, and [68Ga]Ga-NOTA-PRGD2. U87MG tumor-bearing mice underwent 60-min dynamic PET scans following the injection of three tracers. Kinetic parameters were calculated using Logan graphical analysis with reference tissue. Parametric maps were generated using voxel-level modeling. All three compounds showed high binding potential (BpND = k3/k4) in tumor voxels. [18F]AlF-NOTA-PRGD2 showed comparable BpND value (3.75±0.65) with those of [18F]FPPRGD2 (3.39±0.84) and [68Ga]Ga-NOTA-PRGD2 (3.09±0.21) (p>0.05). Little difference was found in volume of distribution (VT) among these three RGD tracers in tumor, liver and muscle. Parametric maps showed similar kinetic parameters for all three tracers. We also demonstrated that the impact of non-specific binding could be eliminated in the kinetic analysis. Consequently, kinetic parameter estimation showed more comparable results among groups than static image analysis. In conclusion, [18F]AlF-NOTA-PRGD2 and [68Ga]Ga-NOTA-PRGD2 have comparable pharmacokinetics and quantitative parameters compared to those of [18F]FPPRGD2. Despite the apparent difference in tumor uptake (%ID/g determined from static images) and clearance pattern, the actual specific binding component extrapolated from kinetic modeling appears to be comparable for all three dimeric RGD tracers