Cell recognition based on atomic force microscopy and modified residual neural network.

Cell recognition methods are in high demand in cell biology and medicine, and the method based on atomic force microscopy (AFM) shows a great value in application. The difference in mechanical properties or morphology of cells has been frequently used to detect whether cells are cancerous, but this detection method cannot be a general means for cancer cell detection, and the traditional artificial feature extraction method also has its limitations. In this work, we proposed an analytic method based on the physical properties of cells and deep learning method for recognizing cell types. The residual neural network used for recognition was modified by multi-scale convolutional fusion, attention mechanism and depthwise separable convolution, so as to optimize feature extraction and reduce operation costs. In the method, the collected cells were imaged by AFM, and the processed images were analyzed by the optimized convolutional neural network. The recognition results of two groups of cells (HL-7702 and SMMC-7721, SGC-7901 and GES-1) by this method show that the recognition rate of dataset with the combination of cell surface morphology, adhesion and Young's modulus is higher, and the recognition rate of the dataset with optimal resolution is higher. Our study indicated that the recognition of physical properties of cells using deep learning technology can serve as a universal and effective method for the automated analysis of cell information.

Large-scale fabrication of an ultrathin broadband absorber using quasi-random dielectric Mie resonators.

Ultrathin broadband absorber maintaining a near-uniform low reflectivity over a broadband wavelength is essential for many optical applications, such as light harvesting and nanoscale imaging. Recently, there has been considerable interest in employing arrays of high-index dielectric Mie resonators on surfaces to trap light and reduce the reflectivity. For such Mie-resonant metasurfaces, however, antireflection properties featuring both a flat low reflectance curve and a wide bandwidth are hard to be satisfied simultaneously, and an efficient large-scale nanofabrication technique rarely exists. Here, we present a high-throughput laser interference induced quasi-random patterning (LIIQP) technique to fabricate quasi-random Mie resonators in large scale. Mie resonators with feature sizes down to sub-100 nm have been fabricated using a 1064 nm laser source. Each Mie resonator concentrates light at its shape-dependent resonant frequency, and all such resonators are arranged quasi-randomly to provide both rich (with broadband Fourier components) and strong (with large intensities) Fourier spectra. Specifically, a near-uniform broadband reflectivity over 400-1100 nm spectrum region has been confined below 3% by fabricating a large-scale ultrathin (around 400 nm) absorber. Our concept and high-throughput fabrication technique allows the rapid production of quasi-random dielectric Mie-resonant metasurfaces in a controllable way, which can be used in various promising applications including thin-film solar cells, display, and imaging.

Electrical characterization of tumor-derived exosomes by conductive atomic force microscopy.

The physical properties of tumor-derived exosomes have gained much attention because they are helpful to better understand the exosomes in biomedicine. In this study, the conductive atomic force microscopy (C-AFM) was employed to perform the electrical characterizations of exosomes, and it obtained the topography and current images of samples simultaneously. The exosomes were absorbed onto the mica substrates coated with a gold film of 20 nm thick for obtaining the current images of samples by C-AFM in air. The results showed that the single exosomes had the weak conductivity. Furthermore, the currents on exosomes were measured at different bias voltages and pH conditions. It illustrated that the conductivity of exosomes was affected by external factors such as bias voltages and solutions with different pH values. In addition, the electrical responses of low and high metastatic potential cell-derived exosomes were also compared under different voltages and pH conditions. This work is important for better understanding the physical properties of tumor-derived exosomes and promoting the clinical applications of tumor-derived exosomes.

Electrical conductivity measurement ofλDNA molecules by conductive atomic force microscopy.

Conductive atomic force microscopy (C-AFM) is a powerful tool used in the microelectronics analysis by applying a certain bias voltage between the conducting probe and the sample and obtaining the electrical information of sample. In this work, the surface morphological information and current images of the lambda DNA (λDNA) molecules with different distributions were obtained by C-AFM. The 1 and 10 ngµl-1DNA solutions were dripped onto mica sheets for making randomly distributed DNA and DNA network samples, and another 1 ngµl-1DNA sample was placed in a DC electric field with a voltage of 2 V before being dried for stretching the DNA sample. The results show that the current flowing through DNA networks was significantly higher than the stretched and random distribution of DNA in the experiment. TheI-Vcurve of DNA networks was obtained by changing the bias voltage of C-AFM from -9 to 9 V. The currents flowing through stretched DNA at different pH values were studied. When the pH was 7, the current was the smallest, and the current was gradually increased as the solution became acidic or alkaline.

Self-Assembly of DNA molecules in magnetic Fields.

In this work, a rich variety of self-assembled DNA patterns were obtained in the magnetic field. Herein, atomic force microscopy (AFM) was utilized to investigate the effects of the concentration of DNA solution, intensity and direction of magnetic field and modification of mica surface by different cations on the self-assembly of DNA molecules. It was found that owning to the change of the DNA concentration, even under the same magnetic field, the DNA self-assembly results were different. Thein situtest results showed that the DNA self-assembly in an magnetic field was more likely to occur in liquid phase than in gas phase. In addition, whether in a horizontal or vertical magnetic field, a single stretched dsDNA was obtained in a certain DNA concentration and magnetic field intensity. Besides, the modification of cations on the mica surface significantly increased the force between the DNA molecules and mica surface, and further changed the self-assembly of DNA molecules under the action of magnetic field.

Imaging the Substructures of Individual IgE Antibodies with Atomic Force Microscopy.

The interactions between antibodies and substrates directly affect their conformations and thus their immune functions. Therefore, it is desirable to study the structures of antibodies at the single molecule level. Herein, the substructures of Immunoglobulin E (IgE) on solid surfaces were investigated. For this purpose, tapping-mode atomic force microscopy (AFM) was applied to observe individual IgE substructures adsorbed onto Mg2+ and Na+ modified mica substrates in air. As expected, the AFM images revealed that the IgE antibodies exhibited different conformations on the surface of mica substrate consisting of the four basic orientations: three domain, two equivalent domain, two unequal domain, and single domain morphologies. Moreover, the differences in the different orientations in single IgE antibodies were also identified clearly.

Highly Active PdNi/RGO/Polyoxometalate Nanocomposite Electrocatalyst for Alcohol Oxidation.

A PdNi/RGO/polyoxometalate nanocomposite has been successfully synthesized by a simple wet-chemical method. Characterizations such as transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction analysis, and X-ray photoelectron spectroscopy are employed to verify the morphology, structure, and elemental composition of the as-prepared nanocomposite. Inspired by the fast-developing fuel cells, the electrochemical catalytic performance of the nanocomposite toward methanol and ethanol oxidation in alkaline media is further tested. Notably, the nanocomposite exhibits excellent catalytic activity and long-term stability toward alcohol electrooxidation compared with the PdNi/RGO and commercial Pd/C catalyst. Furthermore, the electrochemical results reveal that the prepared nanocomposite is attractive as a promising electrocatalyst for direct alcohol fuel cells, in which the phosphotungstic acid plays a crucial role in enhancing the electrocatalytic activities of the catalyst.

Novel α, ß-Unsaturated Sophoridinic Derivatives: Design, Synthesis, Molecular Docking and Anti-Cancer Activities.

Using sophoridine 1 and chalcone 3 as the lead compounds, a series of novel α, ß-unsaturated sophoridinic derivatives were designed, synthesized, and evaluated for their in vitro cytotoxicity. Structure-activity relationship (SAR) analysis indicated that introduction of α, ß-unsaturated ketone moiety and heterocyclic group might significantly enhance anticancer activity. Among the compounds, 2f and 2m exhibited potential effects against HepG-2 and CNE-2 human cancer cell lines. Furthermore, molecular docking studies were performed to understand possible docking sites of the molecules on the target proteins and the mode of binding. This work provides a theoretical basis for structural optimizations and exploring anticancer pathways of this kind of compound.

Effect of resveratrol on SH-SY5Y cells studied by atomic force microscopy.

In this paper, the effects of resveratrol on the viability, morphology, biomechanics and bioelectricity of SH-SY5Y cells were studied by atomic force microscopy. MTT assay showed that resveratrol had a dose effect on SH-SY5Y cells, and its activity was related to drug concentration and drug action time. With the increase of resveratrol concentration or the extension of action time, the activity of SH-SY5Y cells decreased obviously. Atomic force microscope (AFM) was employed to quantitatively analyze the physical changes of cells. AFM study shows that resveratrol can transform SH-SY5Y cells from spindle to sphere, and increase the cell height and decrease the cell adhesion. Also, the elastic modulus increases under the action of low concentration of resveratrol decreases under the action of high concentration of resveratrol, and the electric signal decreases. This study reveals the impact of resveratrol on SH-SY5Y cells from the biological and biophysical perspectives, which is helpful for a more comprehensive understanding of the interaction mechanism between resveratrol and SH-SY5Y cells. These techniques have potential applications in evaluating the effects of chemical substances on cells and screening targeted drugs.

pH-induced changes in IgE molecules measured by atomic force microscopy.

The environment surrounding proteins is tightly linked to its dynamics, which can significantly influence the conformation of proteins. This study focused on the effect of pH conditions on the ultrastructure of Immunoglobulin E (IgE) molecules. Herein, the morphology, height, and area of IgE molecules incubated at different pH were imaged by atomic force microscopy (AFM), and the law of IgE changes induced by pH value was explored. The experiment results indicated that the morphology, height and area of IgE molecules are pH dependent and highly sensitive. In particular, IgE molecules were more likely to present small-sized ellipsoids under acidic conditions, while IgE molecules tend to aggregate into large-sized flower-like structures under alkaline conditions. In addition, it was found that the height of IgE first decreased and then increased with the increase of pH, while the area of IgE increased with the increase of pH. This work provides valuable information for further study of IgE, and the methodological approach used in this study is expected to developed into AFM to investigate the changes of IgE molecules mediated by other physical and chemical factors. RESEARCH HIGHLIGHTS: The ultrastructure of IgE molecules is pH dependent and highly sensitive. IgE molecules were tend to present small-sized ellipsoids under acidic pH. Alkaline pH drives IgE self-assembly into flower-like aggregates.

Two mutations on S2 subunit were critical for Vero cell tropism expansion of infectious bronchitis virus HV80.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR690/S motif at the S2' cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR690/S motif at the S2' cleavage site induced low-level cell-cell fusion, while H855Q substitution hindered cell-cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines.

Review on the applications of atomic force microscopy imaging in proteins.

Protein is an important component of human tissues and cells, which is closely related to various life activities. Therefore, studying the conformation of proteins can not only provide better analytic insight into its interactions with biomacromolecules, but also contribute to the biological and medical research. In recent years, the atomic force microscopy with excellent capacity shines brilliantly in obtaining information of biological samples, especially in the research of proteins. Herein, we highlight the advances and main contributions in the understanding of proteins conformation and its interactions with biomacromolecules by atomic force microscopy imaging technology. In addition, we have found that the relevant information of a single investigated object could be directly measured and obtained by atomic force microscopy, which avoids the indirectness and complexity of data analysis. Therefore, it is reasonable to believe that this work will be conducive to promoting the development of atomic force microscopy in the research field of protein imaging, and promoting its further applications in immunology, medicine and biology.

Direct imaging of antigen-antibody binding by atomic force microscopy.

Direct observation of antigen-antibody binding at the nanoscale has always been a considerable challenging problem, and researchers have made tremendous efforts on it. In this study, the morphology of biotinylated antibody-specific Immunoglobulin E (IgE) immune complexes has been successfully imaged by atomic force microscopy (AFM) in the tapping-mode. The AFM images indicated that the individual immune complex was composed of an IgE and a biotinylated antibody. Excitingly, it is the first time that we have actually seen the IgE binding to biotinylated antibody. Alternatively, information on the length of IgE, biotinylated antibodies and biotinylated antibody-specific IgE immune complexes were also obtained, respectively. These results indicate the versatility of AFM technology in the identification of antigen-antibody binding. This work not only lays the basis for the direct imaging of the biotinylated antibody-IgE by AFM, but also offers valuable information for studying the targeted therapy and vaccine development in the future.

Recombinant infectious bronchitis coronavirus H120 with the spike protein S1 gene of the nephropathogenic IBYZ strain remains attenuated but induces protective immunity.

Infectious bronchitis (IB) is a highly infectious viral disease responsible for major economic losses in the poultry industry. A reverse genetic vaccine is a safe, rapid, and effective method of achieving IB prevention and control. In this study, we constructed the recombinant strain, rH120-S1/YZ, using a reverse genetic system, based on the backbone of the H120 vaccine strain, with the S1 gene replaced with that of the QX-like nephropathogenic strain, ck/CH/IBYZ/2011, isolated in China. The results of dwarf chicken embryos, growth kinetics, and viral titration in the embryos demonstrated that the biological characteristics of the recombinant virus remained unchanged. Like the rH120-infected group and in contrast to the rIBYZ-infected group, no mortality, clinical signs, or lesions were observed in the lungs or kidneys of young chickens inoculated with rH120-S1/YZ. The viral loads in various tissues, cloacal, and oral swabs was lower in most types of samples, indicating that the rH120-S1/YZ strain was highly safe in chicks. Compared to rH120 vaccination group, when the efficacy of this strain was evaluated against the QX-like IBV strain, better protection, with 100% survival rate and no disease symptom or gross lesion was observed in the chickens vaccinated with rH120-S1/YZ. Increased levels of IBV-specific antibodies were detected in the serum of the rH120-S1/YZ-vaccinated animals 14 days post-vaccination. Collectively, our results suggest that the recombinant strain, rH120-S1/YZ, may represent a promising vaccine candidate against QX-like IBVs.

The V617I Substitution in Avian Coronavirus IBV Spike Protein Plays a Crucial Role in Adaptation to Primary Chicken Kidney Cells.

The naturally isolated avian coronavirus infectious bronchitis virus (IBV) generally cannot replicate in chicken kidney (CK) cells. To explore the molecular mechanism of IBV adapting to CK cells, a series of recombinant viruses were constructed by chimerizing the S genes of CK cell-adapted strain H120 and non-adapted strain IBYZ. The results showed that the S2 subunit determines the difference in cell tropism of the two strains. After comparing the amino acid sequences of S protein of CK cell-adapted strain YZ120, with its parental strain IBYZ, three amino acid substitutions, A138V, L581F, and V617I, were identified. Using YZ120 as the backbone, one or more of the above-mentioned substitutions were eliminated to verify the correlation between these sites and CK cell tropism. The results showed that the CK cell tropism of the YZ120 strain depends on the V617I substitution, the change of L581F promoted the adaptation in CK cells, and the change at 138 position was not directly related to the CK cell tropism. Further validation experiments also showed that V617I had a decisive role in the adaptation of IBV to CK cells, but other areas of the virus genome also affected the replication efficiency of the virus in CK cells.

Anti-degranulation response of herbal formula in RBL-2H3 cells.

Allergic diseases not only bring serious economic burden to the patients, but also consume a lot of substantial resources of social medical systems. Thus, the prevention and treatment of allergic diseases are imperative. In this study, the anti-degranulation activity of herbal formula was evaluated using the rat basophil leukemia cells (RBL-2H3) as in vitro model. The morphological and biophysical properties of RBL-2H3 cells before and after treatment with herbal formula were also determined. Notably, the herbal formula exhibits clearly inhibited degranulation by RBL-2H3 cells in a concentration-dependent manner without cytotoxic effect. Therefore, this herbal formula can be used as an alternative and promising therapeutic agent to ameliorate allergic diseases.

The change in sex hormone binding globulin and the influence by gestational diabetes mellitus in fetal period.

OBJECTIVE: To investigate the influence of gestational diabetes mellitus (GDM) on the change of SHBG in fetus. METHOD: Forty-eight pregnant women with GDM and 86 women with normal pregnancy were included in the study. The following were measured in the serums of pregnant women, amniotic fluids, and umbilical cord serums: glucose, insulin, peptide-C, SHBG, and sex hormones. RESULTS: SHBGs in pregnant women's serums were, when compared with the control group: in male fetuses 308.06 +/- 55.64 vs. 445.21 +/- 50.07 (p < 0.01) and in female fetuses 312.38 +/- 56.61 vs. 451.05 +/- 52.87 (p < 0.01). When comparing the levels of SHBGs in amniotic fluids, inclusive of the control group, the following were in male fetuses 8.35 +/- 1.07 vs. 8.41 +/- 1.09 (p = NS) and in female fetuses 8.31 +/- 0.97 vs. 8.39 +/- 0.94 (p = NS). For the levels of SHBGs in umbilical cord serums and comparison to the control group were: in male fetuses 41.44 +/- 8.83 vs. 40.24 +/- 7.50 (p = NS) and in female fetuses 39.93 +/- 7.04 vs. 39.69 +/- 7.16 (p = NS). The concentration of SHBG in amniotic fluid had no significant relationship to glucose, dehydroepiandrosterone (DHEAS), estradiol, and total and free testosterone, but had an extremely negatively correlated to insulin and peptide-C (p < 0.01) in GDM group. CONCLUSION: Although the concentration of SHBG does not change in fetus when pregnant woman is complicated with GDM, it is already influenced by the fetal regulation on hyperinsulinemia.

Attention-Based Multi-NMF Deep Neural Network with Multimodality Data for Breast Cancer Prognosis Model.

Today, it has become a hot issue in cancer research to make precise prognostic prediction for breast cancer patients, which can not only effectively avoid overtreatment and medical resources waste, but also provide scientific basis to help medical staff and patients family members to make right medical decisions. As well known, cancer is a partly inherited disease with various important biological markers, especially the gene expression profile data and clinical data. Therefore, the accuracy of prediction model can be improved by integrating gene expression profile data and clinical data. In this paper, we proposed an end-to-end model, Attention-based Multi-NMF DNN (AMND), which combines clinical data and gene expression data extracted by Multiple Nonnegative Matrix Factorization algorithms (Multi-NMF) for the prognostic prediction of breast cancer. The innovation of this method is highlighted through using clinical data and combining multiple feature selection methods with the help of Attention mechanism. The results of comprehensive performance evaluation show that the proposed model reports better predictive performances than either models only using data of single modality, e.g., gene or clinical, or models based on any single NMF improved methods which only use one of the NMF algorithms to extract features. The performance of our model is competitive or even better than other previously reported models. Meanwhile, AMND can be extended to the survival prediction of other cancer diseases, providing a new strategy for breast cancer prognostic prediction.

Pathogenicity of a QX-like strain of infectious bronchitis virus and effects of accessory proteins 3a and 3b in chickens.

QX-like genotype infectious bronchitis virus (IBV) has become prevalent in recent years. Few studies have reported the effects of accessory proteins 3a and 3b on pathogenicity in vivo. We developed a reverse genetics system to manipulate the genome of a QX-like IBV strain IBYZ. Recombinant viruses rIBYZ-ScAUG3a, rIBYZ-ScAUG3b and rIBYZ-ScAUG3ab were generated. These viruses do not express the accessory proteins 3a, 3b, or 3ab due to a mutation in the AUG start codons. In SPF embryonated eggs, the recombinant viruses grew to the same viral load as parental strain rIBYZ. The pathogenicity of rIBYZ and recombinant viruses was examined in 1-day-old SPF chickens. In SPF chickens, rIBYZ-ScAUG3a had a lower mortality than rIBYZ. The clinical signs, gross lesions and histopathological changes of rIBYZ-ScAUG3a group were comparable to those of rIBYZ group. However, viral distribution and viral shedding showed that the viral loads of rIBYZ-ScAUG3a were lower than those of rIBYZ in tissue samples and swab specimens. The rIBYZ-ScAUG3b and rIBYZ-ScAUG3ab strains showed attenuated pathogenicity compared to rIBYZ, as no chickens died and all the parameters tested were considerably low. This study indicates that the absence of accessory proteins 3a and 3b in IBV lead to attenuated pathogenicity in chickens. Protein 3b has a greater effect on pathogenicity than protein 3a. These findings may be used in vaccination trials for the development of a new live-attenuated vaccine.

Effect of extract from ginseng rust rot on the inhibition of human hepatocellular carcinoma cells in vitro.

Hepatocellular carcinoma (HCC) is one of major leading causes of cancer death worldwide. As a traditional medicine, the anti-cancer function of ginseng is being growingly recognized and investigated. However, the effect of ginseng rust rot on human HCC is unknown yet. In this study, the HCC cells were treated with different parts of mountain cultivated ginseng rust rot and compared with human normal liver cells. The morphology, survival rate and ß-actin expression of the cells were changed by introducing the ginseng epidermis during the incubation process. Notably, the results reveal that the ginseng epidermis can induce apoptosis by altering the morphologies of cells, indicating the practical implication for the HCC treatment and drug development.