Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells.

Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique-topography and recognition (TREC) imaging-to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells.

Tyrosine phosphorylation of Dbl regulates GTPase signaling.

Rho GTPases are molecular "switches" that cycle between "on" (GTP-bound) and "off" (GDP-bound) states and regulate numerous cellular activities such as gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses. Dysregulation of GTPases is a key feature of many diseases, especially cancers. Guanine nucleotide exchange factors (GEFs) of the Dbl family are activated by mitogenic cell surface receptors and activate the Rho family GTPases Cdc42, Rac1, and RhoA. The molecular mechanisms that regulate GEFs from the Dbl family are poorly understood. Our studies reveal that Dbl is phosphorylated on tyrosine residues upon stimulation by growth factors and that this event is critical for the regulated activation of the GEF. These findings uncover a novel layer of complexity in the physiological regulation of this protein.

Radiopharmaceutical study on Iodine-131-labelled hypericin in a canine model of hepatic RFA-induced coagulative necrosis.

PURPOSE: Hypericin (HYP) has been found avid to necrosis in small animal studies. We sought to evaluate the tissue distribution of (131)I-HYP in a large animal model and to explore the theranostic utilities of (131)I-HYP after radiofrequency ablation (RFA). MATERIALS AND METHODS: This animal experiment was approved by the institutional ethics committee. Twenty-five male dogs were enrolled and subjected to transabdominal hepatic RFA. (131)I-HYP was prepared by an electrophilic substitution method and intravenously administered at 0.5 mCi/kg. Systemic and regional distributions of (131)I-HYP were monitored dynamically by single-photon emission computed tomography/computed tomography (SPECT-CT), gamma counting, autoradiography, and fluorescent and light microscopy at different time points up to 14 days. Experimental data were quantified and statistically analysed. RESULTS: Most of the tissues and organs retained (131)I-HYP only transiently. (131)I-HYP was mainly metabolised in the liver and excreted into the bile. (131)I-HYP gradually accumulated in the RFA-induced necrosis with a peak concentration occurring within 2 days and lasting over 2 weeks as visualised by in vivo SPECT-CT and ex vivo autoradiography and fluorescent microscopy, and quantified by radioactivity and fluorescence measurements. Accumulation of (131)I-HYP was low in both the necrosis centre and normal liver tissue. CONCLUSION: (131)I-HYP showed persistent high affinity to hepatic thermo-coagulative necrosis, but only a transient uptake by normal liver in dogs. Necrosis caused by RFA could be indicated by (131)I-HYP on nuclear imaging, which suggests a supplementary measure for tumour detection and therapy.

Subcellular spectroscopic markers, topography and nanomechanics of human lung cancer and breast cancer cells examined by combined confocal Raman microspectroscopy and atomic force microscopy.

The nanostructures and hydrophobic properties of cancer cell membranes are important for membrane fusion and cell adhesion. They are directly related to cancer cell biophysical properties, including aggressive growth and migration. Additionally, chemical component analysis of the cancer cell membrane could potentially be applied in clinical diagnosis of cancer by identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined Raman microspectroscopy (RM) and atomic force microscopy (AFM) technique was applied to detect the difference in membrane chemical components and nanomechanics of three cancer cell lines: human lung adenocarcinoma epithelial cells (A549), and human breast cancer cells (MDA-MB-435 with and without BRMS1 metastasis suppressor). Raman spectral analysis indicated similar bands between the A549, 435 and 435/BRMS1 including ~720 cm(-1) (guanine band of DNA), 940 cm(-1) (skeletal mode polysaccharide), 1006 cm(-1) (symmetric ring breathing phenylalanine), and 1451 cm(-1) (CH deformation). The membrane surface adhesion forces for these cancer cells were measured by AFM in culture medium: 0.478 ± 0.091 nN for A549 cells, 0.253 ± 0.070 nN for 435 cells, and 1.114 ± 0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62 ± 0.682 mN m(-1) for A549 cells, 2.105 ± 0.691 mN m(-1) for 435 cells, and 5.448 ± 1.081 mN m(-1) for 435/BRMS1 cells.

Lactobacillus plantarum LP45 inhibits the RANKL/OPG signaling pathway and prevents glucocorticoid-induced osteoporosis.

Objective: To examine the potential effect of the probiotic strain Lactobacillus plantarum LP45 on osteoporosis and to explore the involved molecular mechanisms. Methods: A rat model of glucocorticoid-induced osteoporosis (GIO) was established, which was also orally administered with increasing doses of LP45 for 8 weeks. After the termination of the 8-week treatment, the tibia and femur bones of rats were analyzed for bone histomorphometry, bone mineral content (BMC), and bone mineral density (BMD). Femoral biomechanics were assessed. In addition, levels of osteocalcin, tartrate-resistant acid phosphatase 5 (TRAP5), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) in the serum and bone marrow were also measured using ELISA, Western blot, and real time-polymerase chain reaction. Results: GIO caused obvious defects in tibia and femur bone structures, in terms of tissue/bone volume, trabecular separation, trabecular thickness, and trabecular number, which could be rescued by LP45 dose dependently. The GIO-induced reductions in BMC, BMD, osteoblast surfaces per bone surface (BS), as well as elevated osteoclast surface per BS were largely restored by LP45 administration dose-dependently. LP45 also increased femoral biomechanics of GIO rats. Importantly, LP45 dose-dependently restored the changes of osteocalcin, TRAP5, OPG, and RANKL in the serum as well as bone marrow of GIO rats. Conclusion: Oral LP45 administration could significantly prevent bone defects in GIO rats, suggesting its potential as a dietary supplement with beneficial effects against osteoporosis, which might involve the RANKL/OPG signaling pathway.

Distribution patterns and driving mechanism of soil protozoan community at the different depths of Larix principis-chinensis forest in the Luya Mountain, China.

To reveal the assembly mechanisms of soil protozoan community in subalpine forest ecosystems, we analyzed the composition and diversity of protozoan communities and their drivers at the six strata (the litter profile, humus profile, 0-10 cm, 10-20 cm, 20-40 cm and 40-80 cm) of soil profiles in subalpine Larix principis-rupprechtii forest in Luya Mountain using Illumina Miseq high-throughput sequencing technology. The results showed that protozoa in the soil profiles belonged to 335 genera, 206 families, 114 orders, 57 classes, 21 phyla, and 8 kingdoms. There were five dominant phyla (relative abundance >1%) and 10 dominant families (relative abundance >5%). The α diversity decreased significantly with increasing soil depth. Results of PCoA analysis showed that the spatial composition and structure of protozoan community differed significantly across soil depths. The results of RDA analysis showed that soil pH and soil water content were important factors driving protozoan community structure across soil profile. Null model analysis suggested that the heterogeneous selection dominated the processes of protozoan community assemblage. Molecular ecological network analysis revealed that the complexity of soil proto-zoan communities decreased continuously with increasing depth. These results elucidate the assembly mechanism of soil microbial community in subalpine forest ecosystem.

NGMMs: Neutrosophic Gaussian Mixture Models for Breast Ultrasound Image Classification.

Ultrasound imaging is commonly used for diagnosing breast cancers since it is non-invasive and inexpensive. Breast ultrasound (BUS) image classification is still a challenging task due to the poor image quality and lack of public datasets. In this paper, we propose novel Neutrosophic Gaussian Mixture Models (NGMMs) to more accurately classify BUS images. Specifically, we first employ a Deep Neural Network (DNN) to extract features from BUS images and apply principal component analysis to condense extracted features. We then adopt neutrosophic logic to compute three probability functions to estimate the truth, indeterminacy, and falsity of an image and design a new likelihood function by using the neutrosophic logic components. Finally, we propose an improved Expectation Maximization (EM) algorithm to incorporate neutrosophic logic to reduce the weights of images with high indeterminacy and falsity when estimating parameters of each NGMM to better fit these images to Gaussian distributions. We compare the performance of the proposed NGMMs, its two peer GMMs, and three DNN-based methods in terms of six metrics on a new dataset combining two public datasets. Our experimental results show that NGMMs achieve the highest classification results for all metrics.

TGF beta-mediated BIM expression and apoptosis are regulated through SMAD3-dependent expression of the MAPK phosphatase MKP2.

Transforming growth factor-beta (TGFbeta) induces the expression of the pro-apoptotic protein BIM, and mediates apoptosis in hepatocytes and B lymphocytes. BIM is regulated through a post-translational mechanism involving ERK-dependent phosphorylation and ubiquitin-mediated proteasomal degradation. Here, we show that TGFbeta induces BIM through its rapid inhibition of ERK, thereby preventing the phosphorylation and degradation of BIM. TGFbeta, through a SMAD3-dependent mechanism, transcriptionally induces the mitogen-activated protein kinase (MAPK) phosphatase MKP2, encoded by an immediate early gene, to attenuate ERK and promote the accumulation of BIM protein. Overexpression of MKP2 in hepatocytes modulates ERK-mediated phosphorylation of BIM and apoptosis in the absence of TGFbeta, whereas its ablation in pro-B cells, derived from MKP2-deficient mice, protects cells from TGFbeta-mediated apoptosis, and blocks TGFbeta-induced ERK inhibition and BIM induction. Furthermore, in pro-B cells derived from SMAD3-deficient mice, induction of MKP2 by TGFbeta, inhibition of ERK, induction of BIM and apoptosis do not occur. Our results indicate that MKP2 mediates TGFbeta-dependent apoptosis by linking SMAD3 to the modulation of ERK activity and mitochondrial-mediated pro-apoptotic events.

Appearance variation adaptation tracker using adversarial network.

Visual trackers using deep neural networks have demonstrated favorable performance in object tracking. However, training a deep classification network using overlapped initial target regions may lead an overfitted model. To increase the model generalization, we propose an appearance variation adaptation (AVA) tracker that aligns the feature distributions of target regions over time by learning an adaptation mask in an adversarial network. The proposed adversarial network consists of a generator and a discriminator network that compete with each other over optimizing a discriminator loss in a mini-max optimization problem. Specifically, the discriminator network aims to distinguish recent target regions from earlier ones by minimizing the discriminator loss, while the generator network aims to produce an adaptation mask to maximize the discriminator loss. We incorporate a gradient reverse layer in the adversarial network to solve the aforementioned mini-max optimization in an end-to-end manner. We compare the performance of the proposed AVA tracker with the most recent state-of-the-art trackers by doing extensive experiments on OTB50, OTB100, and VOT2016 tracking benchmarks. Among the compared methods, AVA yields the highest area under curve (AUC) score of 0.712 and the highest average precision score of 0.951 on the OTB50 tracking benchmark. It achieves the second best AUC score of 0.688 and the best precision score of 0.924 on the OTB100 tracking benchmark. AVA also achieves the second best expected average overlap (EAO) score of 0.366, the best failure rate of 0.68, and the second best accuracy of 0.53 on the VOT2016 tracking benchmark.

Label-free discrimination and quantitative analysis of oxidative stress induced cytotoxicity and potential protection of antioxidants using Raman micro-spectroscopy and machine learning.

Diesel exhaust particles (DEPs) are major constituents of air pollution and associated with numerous oxidative stress-induced human diseases. In vitro toxicity studies are useful for developing a better understanding of species-specific in vivo conditions. Conventional in vitro assessments based on oxidative biomarkers are destructive and inefficient. In this study, Raman spectroscopy, as a non-invasive imaging tool, was used to capture the molecular fingerprints of overall cellular component responses (nucleic acid, lipids, proteins, carbohydrates) to DEP damage and antioxidant protection. We apply a novel data visualization algorithm called PHATE, which preserves both global and local structure, to display the progression of cell damage over DEP exposure time. Meanwhile, a mutual information (MI) estimator was used to identify the most informative Raman peaks associated with cytotoxicity. A health index was defined to quantitatively assess the protective effects of two antioxidants (resveratrol and mesobiliverdin IXα) against DEP induced cytotoxicity. In addition, a number of machine learning classifiers were applied to successfully discriminate different treatment groups with high accuracy. Correlations between Raman spectra and immunomodulatory cytokine and chemokine levels were evaluated. In conclusion, the combination of label-free, non-disruptive Raman micro-spectroscopy and machine learning analysis is demonstrated as a useful tool in quantitative analysis of oxidative stress induced cytotoxicity and for effectively assessing various antioxidant treatments, suggesting that this framework can serve as a high throughput platform for screening various potential antioxidants based on their effectiveness at battling the effects of air pollution on human health.

Transparent and strong polymer nanocomposites generated from Pickering emulsion gels stabilized by cellulose nanofibrils.

We report here the development of transparent and strong polymer composites reinforced by unmodified cellulose nanofibrils (CNFs) with a Pickering emulsion gelation strategy. The CNFs entangle and firmly stabilize on the surface of emulsion droplets containing polymethyl methacrylate (PMMA) solution, leading to the gelation of the emulsions. CNFs/PMMA composites were generated via vacuum filtration and solvent washing of the gel and a subsequent two-step hot pressing. The composites contained a unique self-assembled two-tier hierarchy of CNFs networks and demonstrate promising transparency, tensile strength, flexibility, and an extremely low thermal expansion. Remarkably, these properties are highly tunable with varying the concentration of CNFs and the volume ratio of the water to oil phase. This work offers a facile route to realize the well dispersion of unmodified CNFs in hydrophobic polymer matrix and achieve high performance of polymeric materials reinforced by CNFs.

Microfluidic chip for non-invasive analysis of tumor cells interaction with anti-cancer drug doxorubicin by AFM and Raman spectroscopy.

Raman spectroscopy has been playing an increasingly significant role for cell classification. Here, we introduce a novel microfluidic chip for non-invasive Raman cell natural fingerprint collection. Traditional Raman spectroscopy measurement of the cells grown in a Polydimethylsiloxane (PDMS) based microfluidic device suffers from the background noise from the substrate materials of PDMS when intended to apply as an in vitro cell assay. To overcome this disadvantage, the current device is designed with a middle layer of PDMS layer sandwiched by two MgF2 slides which minimize the PDMS background signal in Raman measurement. Three cancer cell lines, including a human lung cancer cell A549, and human breast cancer cell lines MDA-MB-231 and MDA-MB-231/BRMS1, were cultured in this microdevice separately for a period of three days to evaluate the biocompatibility of the microfluidic system. In addition, atomic force microscopy (AFM) was used to measure the Young's modulus and adhesion force of cancer cells at single cell level. The AFM results indicated that our microchannel environment did not seem to alter the cell biomechanical properties. The biochemical responses of cancer cells exposed to anti-cancer drug doxorubicin (DOX) up to 24 h were assessed by Raman spectroscopy. Principal component analysis over the Raman spectra indicated that cancer cells untreated and treated with DOX can be distinguished. This PDMS microfluidic device offers a non-invasive and reusable tool for in vitro Raman measurement of living cells, and can be potentially applied for anti-cancer drug screening.

miR-22 promotes apoptosis of osteosarcoma cells via inducing cell cycle arrest.

To study the effects of miR-22 on the proliferation and the apoptosis of osteosarcoma MG-63 cell line and to explore the potential molecular mechanism that miR-22 regulates this biological process. Quantitive real-time polymerase chain reaction (RT-qPCR) was performed to explore the miRNA level of miR-22. The MG-63 cell line was infected with miR-22 mimics for establishment of miR-22 overexpression. Non-infected cells were in blank group and cells infected with empty vector were served as negative control (NC group). MTT assay was conducted to measure cell viability. The cell cycle and apoptosis were explored using flow cytometry and the apoptosis-related markers were detected by western blotting. RT-qPCR results revealed that the miR-22 miRNA level in the MG-63 cells was significantly lower than that in osteoblasts (P<0.05). MTT assay showed that the MG-63 cells infected with miR-22 mimics exhibited markedly decreased proliferation ability compared with blank and empty vector (NC) groups. Next, we found that overexpression of miR-22 remarkably increased the apoptosis of the MG-63 cells, evidenced from the flow cytometry results and elevated Bax and reduced Bcl-2. Furthermore, results revealed that percentage of the cells at G0/G1 phase in miR-22 mimic group (66.75±3.67%) was significantly higher than blank (52.9±2.58%) and NC (50.5±2.45%) groups. miR-22 attenuated the proliferation and induced the apoptosis of the MG-63 cells via promoting G0/G1 cell cycle arrest. Thus, miR-22 may have the potential to be a novel therapeutic in treatment of osteosarcoma.

Nanomedicine-based combination of gambogic acid and retinoic acid chlorochalcone for enhanced anticancer efficacy in osteosarcoma.

In this study, gambogic acid (GA) and retinoic acid chlorochalcone (RACC) co-loaded glycol chitosan nanoparticle was successfully developed and studied for its therapeutic efficacy against osteosarcoma cancer cells. The GA/RACC loaded glycol chitosan nanoparticles (RGNP) was nanosized and exhibited a controlled release of drug in either pH 7.4 and pH 5.0. Owing to the strong positive charge on the RGNP surface, efficiency cellular uptake was observed in cancer cells. Moreover, a synergistic combination of GA and RACC were effectively suppressed the tumor growth progression. The half maximal inhibitory concentration (IC50) values in MG63 cells were 0.89µg/ml and 0.35µg/ml for GA and RGNP after 24h. The results clearly suggest the synergist effect of GA and RACC in effectively inhibiting the cancer cell proliferation. The RGNP as expected induced a remarkably higher apoptosis of cancer cells with ∼28%. Overall, combination of GA and RACC encapsulated in a nanocarrier could be an effective strategy to treat osteosarcoma. Future studies will focus on the in vivo evaluation of GA/RACC-loaded polymeric nanoparticles.

Effect of variation of FGF2 genotypes on the risk of osteosarcoma susceptibly: a case control study.

OBJECTIVE: Genetic factors play an important role in osteosarcoma (OS) etiology and fibroblast growth factor 2 (FGF2) gene single polymorphisms may be involved. The aim of this study was to test whether FGF2 variants are associated with susceptibility to OS in a Chinese population. METHODS: A total of 151 subjects who were diagnosed as OS and 225 healthy age-matched controls were enrolled in the present study. Thers11737764 C/T SNP in FGF2 gene was genotyped in all the subjects. The SPSS software was used to investigate the association between the rs11737764 genotypes and OS susceptibility or severity. RESULTS: The genotype frequencies of the FGF2 rs11737764 C/T polymorphism were 44.4% (CC), 50.3% (CT) and 5.3% (TT) in OS patients, and 55.6% (CC), 43.1% (CT) and 1.3% (TT) in controls. Rs11737764 C/T was found to be significantly associated with increased risk and OS no matter what genetic model was used. CONCLUSION: In conclusion, our data demonstrated the FGF2 SNP rs11737764 was significantly associated with increased osteosarcoma susceptibility in Chinese Han Population.

Label-free and non-invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum-free media.

Traditional approaches to characterize stem cell differentiation are time-consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) - both considered as non-invasive techniques - are applied to detect the biochemical and biophysical properties of trophoblast derived stem-like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed. Monitoring trophoblast cells differentiation.

A shortest-path graph kernel for estimating gene product semantic similarity.

BACKGROUND: Existing methods for calculating semantic similarity between gene products using the Gene Ontology (GO) often rely on external resources, which are not part of the ontology. Consequently, changes in these external resources like biased term distribution caused by shifting of hot research topics, will affect the calculation of semantic similarity. One way to avoid this problem is to use semantic methods that are "intrinsic" to the ontology, i.e. independent of external knowledge. RESULTS: We present a shortest-path graph kernel (spgk) method that relies exclusively on the GO and its structure. In spgk, a gene product is represented by an induced subgraph of the GO, which consists of all the GO terms annotating it. Then a shortest-path graph kernel is used to compute the similarity between two graphs. In a comprehensive evaluation using a benchmark dataset, spgk compares favorably with other methods that depend on external resources. Compared with simUI, a method that is also intrinsic to GO, spgk achieves slightly better results on the benchmark dataset. Statistical tests show that the improvement is significant when the resolution and EC similarity correlation coefficient are used to measure the performance, but is insignificant when the Pfam similarity correlation coefficient is used. CONCLUSIONS: Spgk uses a graph kernel method in polynomial time to exploit the structure of the GO to calculate semantic similarity between gene products. It provides an alternative to both methods that use external resources and "intrinsic" methods with comparable performance.

Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function.

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.