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.

The P5-type ATPase Spf1 is required for development and virulence of the rice blast fungus Pyricularia oryzae.

Pyricularia oryzae (synonym Magnaporthe oryzae) is a plant pathogen causing major yield losses in cultivated rice and wheat. The P-type ATPases play important roles in cellular processes of fungi, plants, and animals via transporting specific substrates through ATP hydrolysis. Here, we characterized the roles of a P5-ATPase, Spf1, in the development and virulence of P. oryzae. Deletion of SPF1 led to decreased hyphal growth and conidiation, delayed spore germination and appressorium formation, reduced penetration and invasive hyphal extension, and attenuated virulence. Appressorium turgor, however, was not affected by deletion of SPF1. The co-localization of Spf1-GFP and an endoplasmic reticulum (ER) marker protein, Lhs1-DsRed2, indicated that Spf1 is an ER-localized P5-ATPase. An ER stress factor, 0.5 µg/ml tunicamycin (TUNI), inhibited the growth of ∆spf1, but another ER stress factor, 5 mM dithiothreitol (DTT), promoted the growth of ∆spf1. Treatment with chemicals for oxidative stress (5 mM H2O2 and 0.8 mM paraquat) also promoted the growth of ∆spf1. Gene expression assays showed that unfolded protein response (UPR) components KAR2, OST1, PMT1, ERV29, PDI1, SCJ1, SEC61, a Ca2+ channel-related P-type ATPase gene PMR1, and a calcineurin-dependent transcription factor CRZ1 were significantly up-regulated in ∆spf1, suggesting activation of UPR in the mutant. These lines of experimental evidence indicate that SPF1 is involved in some basal ER mechanisms of P. oryzae including UPR pathway and responses to ER related stresses, therefore, affecting fungal development and virulence. However, the detailed mechanism between Spf1 and virulence still awaits future researches.

A disulfiram-loaded electrospun poly(vinylidene fluoride) nanofibrous scaffold for cancer treatment.

Disulfiram (DSF), an FDA approved drug for the treatment of alcoholism, has shown its effectiveness against diverse cancer types. Thus, we developed a disulfiram-loaded scaffold using the electrospinning method to enhance the stability of DSF and to facilitate its appropriate distribution to tumor tissues. The drug release profile of the disulfiram-loaded scaffold was examined by high-performance liquid chromatography. We obtained mechanical and morphological characterizations of A549 cells treated with different scaffolds by various techniques to evaluate its antitumor properties. This work revealed that the cells after the treatment with the disulfiram-loaded scaffold exhibited a lower height and a larger elastic modulus compared with the untreated cells and those treated with the neat electrospun fibers. The changes were the indicators of cell apoptosis. Taken collectively, the results indicate that DSF was successfully incorporated into the electrospun fibers, and the disulfiram-loaded scaffold has great potential for inhibiting the regional recurrence of cancer.

Mechanical properties study of SW480 cells based on AFM.

Since the invention of the atomic force microscope (AFM), it has been widely applied in biomedicine. One of the most important applications is used as an indenter tool to do the indentation experiment in order to get the mechanical properties of cells. In this paper, SW480 cells were used as the test subjects. Through the analysis of the contact and indentation, Young's modulus (E), which is an important parameter of cancer cells, has been estimated. Experimental results show that different mechanical models should be chosen to calculate the E in different indentation depths. Here, the E of SW480 cells was (2.5 ± 0.8) KPa at the indentation depth of 99 nm.

A kelch domain cell end protein, PoTea1, mediates cell polarization during appressorium morphogenesis in Pyricularia oryzae.

The rice blast fungus Pyricularia oryzae differentiates into an infection structure, called an appressorium, for plant penetration. The process of appressorium formation requires the transformation of polarized growth to isotropic growth, while penetration requires the opposite growth transformation from isotropic to polarized. Polarized growth requires coordinated organization of cytoskeletal elements, such as microtubule and actin. We identified PoTea1, a homolog of Tea1 from Schizosaccharomyces pombe, and characterized its roles in P. oryzae. After PoTEA1 deletion, ∆Potea1 displayed slowed hyphal growth, decreased sporulation, increased hyphal branches, abnormal two-celled spores, and reduced plant penetration and virulence. During appressorium formation, ∆Potea1 developed a long germ tube with a small appressorium, leading to delayed appressorium differentiation and reduced glycogen and lipid droplet degradation. ∆Potea1 is defective in cAMP-PKA and Pmk1 MAPK pathways. PoTea1 localized at hyphal tips and appressoria as bright dots and was highly dynamic during appressorium formation. PoTea1 formed a complex with itself by self-assembly that was highly dependent on its kelch motif. The coiled-coil motif C2 of PoTea1 is involved in self polymerization and appressorium formation. Benomyl and latrunculin A, two cytoskeleton inhibitors, disturbed the stable localization of PoTea1 at vegetative hyphal tips. We speculate that PoTea1 functions in appressorium formation and virulence by mediating cell polarity in P. oryzae.

An appressorium membrane protein, Pams1, controls infection structure maturation and virulence via maintaining endosomal stability in the rice blast fungus.

The rice blast fungus Magnaporthe oryzae spores differentiate and mature into functional appressoria by sensing the host surface signals. Environmental stimuli are transduced into cells through internalization during appressorium formation, such as in the cAMP-PKA pathway. Here, we describe a novel contribution to how appressoria mature on the surface of a leaf, and its connection to endosomes and the cAMP-PKA pathway. An appressorium membrane-specific protein, Pams1, is required for maintaining endosomal structure, appressorium maturation, and virulence in M. oryzae. During appressorium development, Pams1 was translocated from the cell membrane to the endosomal membrane. Deletion of PAMS1 led to the formation of two types of abnormal appressoria after 8 h post inoculation (hpi): melanized type I had a reduced virulence, while pale type II was dead. Before 8 hpi, Δpams1 formed appressoria that were similar to those of the wild type. After 8 hpi, the appressoria of Δpams1 was differentiated into two types: (1) the cell walls of type I appressoria were melanized, endosomes were larger, and had a different distribution from the wild type and (2) Type II appressoria gradually stopped melanization and began to die. The organelles, including the nucleus, endosomes, mitochondria, and endoplasmic reticula, were degraded, leaving only autophagic body-like vesicles in type II appressoria. The addition of exogenous cAMP to Δpams1 led to the formation of a greater proportion of type I appressoria and a smaller proportion of type II appressoria. Thus, defects in endosomal structure and the cAMP-PKA pathway are among the causes of the defective appressorium maturation and virulence of Δpams1.

PoRal2 Is Involved in Appressorium Formation and Virulence via Pmk1 MAPK Pathways in the Rice Blast Fungus Pyricularia oryzae.

Pyricularia oryzae is an important plant pathogenic fungus that can severely damage rice and wheat crops, leading to significant reductions in crop productivity. To penetrate into and invade tissues of its plant host, this fungus relies on an invasive structure known as an appressorium. Appressorium formation is rigorously regulated by the cAMP-PKA and Pmk1 MAPK pathways. Here, we identified PoRal2, a homologous protein of Schizosaccharomyces pombe Ral2, and characterized its roles in fungal development and virulence in P. oryzae. PoRal2 contains N-terminal kelch repeats and C-terminal BTB domains. PoRal2 is involved in sporulation, aerial hypha and conidiophore differentiation, appressorium formation, plant penetration, and virulence. During appressorium formation, ∆Poral2 mutants generate appressoria with long germ tubes on hydrophobic surfaces. ∆Poral2 mutants exhibited a defective response to exogenous cAMP and the activated RAS2 G18V on a hydrophilic surface, indicating impairment in the cAMP-PKA or Pmk1 MAPK signaling pathways. Deletion of PoRAL2 leads to lowered Pmk1 phosphorylation level in the mutant. Moreover, PoRal2 is found to interact with Scd1, Smo1, and Mst50, which are involved in activation of Pmk1. In addition, the expression levels of MPG1, WISH, and PDEH in the cAMP-PKA pathway, RAS2 in both the cAMP-PKA and Pmk1 MAPK pathways, and melanin biosynthesis genes (ALB1, BUF1, and RSY1) were significantly down-regulated in the ∆Poral2. Therefore, PoRal2 is involved in fungal development and virulence by its crosstalk in the cAMP-PKA and Pmk1 MAPK signaling pathways.

Role refinement of melanin synthesis genes by gene knockout reveals their functional diversity in Pyricularia oryzae strains.

Pyricularia oryzae is a plant pathogenic fungus that severely affects rice production. Past studies, primarily using mutants generated by spontaneous mutations or artificial physical and chemical mutagenesis, have determined that melanin is required for appressorium turgor, penetration, and virulence of P. oryzae. However, these roles need to be verified by gene knockout and/or overexpression in different strains considering the potential differences in the level of virulence. Here, we confirmed the indispensable roles of melanin in the development and virulence of P. oryzae by knocking out and over-expressing three melanin synthesis genes (ALB1, RSY1, and BUF1) in two wild-type strains (Guy11 and 70-15). Deletion of ALB1, RSY1, or BUF1 led to loss of melanin and virulence in both strains. ALB1, RSY1 and BUF1 in Guy11, and BUF1 in 70-15 were required for conidiation, respectively. ALB1, RSY1, and BUF1 were required for conidial resistance to environmental stresses (UV exposure, oxidization, and freezing damage) in both strains. Guy11 cells had greater amounts of melanin and more transcripts of melanin synthesis genes than 70-15 cells. Paired culture experiments between the deletion or over-expression mutants of melanin synthesis genes suggested that the reaction catalyzed by Buf1, but not Alb1 and Rsy1, was likely a rate-limiting step in melanin biosynthesis in 70-15. These results expand our understanding on melanin and its synthesis genes in P. oryzae as well as its responses to biotic and abiotic environments.

A VASt-domain protein regulates autophagy, membrane tension, and sterol homeostasis in rice blast fungus.

Sterols are a class of lipids critical for fundamental biological processes and membrane dynamics. These molecules are synthesized in the endoplasmic reticulum (ER) and are transported bi-directionally between the ER and plasma membrane (PM). However, the trafficking mechanism of sterols and their relationship with macroautophagy/autophagy are still poorly understood in the rice blast fungus Magnaporthe oryzae. Here, we identified the VAD1 Analog of StAR-related lipid transfer (VASt) domain-containing protein MoVast1 via co-immunoprecipitation in M. oryzae. Loss of MoVAST1 resulted in conidial defects, impaired appressorium development, and reduced pathogenicity. The MoTor (target of rapamycin in M. oryzae) activity is inhibited because MoVast1 deletion leads to high levels of sterol accumulation in the PM. Site-directed mutagenesis showed that the 902 T site is essential for localization and function of MoVast1. Through filipin or Flipper-TR staining, autophagic flux detection, MoAtg8 lipidation, and drug sensitivity assays, we uncovered that MoVast1 acts as a novel autophagy inhibition factor that monitors tension in the PM by regulating the sterol content, which in turn modulates the activity of MoTor. Lipidomics and transcriptomics analyses further confirmed that MoVast1 is an important regulator of lipid metabolism and the autophagy pathway. Our results revealed and characterized a novel sterol transfer protein important for M. oryzae pathogenicity.Abbreviations: AmB: amphotericin B; ATMT: Agrobacterium tumefaciens-mediated transformation; CM: complete medium; dpi: days post-inoculation; ER: endoplasmic reticulum; Flipper-TR: fluorescent lipid tension reporter; GO: Gene ontology; hpi: hours post-inoculation; IH: invasive hyphae; KEGG: kyoto encyclopedia of genes and genomes; MoTor: target of rapamycin in Magnaporthe oryzae; PalmC: palmitoylcarnitine; PM: plasma membrane; SD-N: synthetic defined medium without amino acids and ammonium sulfate; TOR: target of rapamycin; VASt: VAD1 Analog of StAR-related lipid transfer; YFP, yellow fluorescent protein.

Single-cell patterning technology for biological applications.

Single-cell patterning technology has revealed significant contributions of single cells to conduct basic and applied biological studies in vitro such as the understanding of basic cell functions, neuronal network formation, and drug screening. Unlike traditional population-based cell patterning approaches, single-cell patterning is an effective technology of fully understanding cell heterogeneity by precisely controlling the positions of individual cells. Therefore, much attention is currently being paid to this technology, leading to the development of various micro-nanofabrication methodologies that have been applied to locate cells at the single-cell level. In recent years, various methods have been continuously improved and innovated on the basis of existing ones, overcoming the deficiencies and promoting the progress in biomedicine. In particular, microfluidics with the advantages of high throughput, small sample volume, and the ability to combine with other technologies has a wide range of applications in single-cell analysis. Here, we present an overview of the recent advances in single-cell patterning technology, with a special focus on current physical and physicochemical methods including stencil patterning, trap- and droplet-based microfluidics, and chemical modification on surfaces via photolithography, microcontact printing, and scanning probe lithography. Meanwhile, the methods applied to biological studies and the development trends of single-cell patterning technology in biological applications are also described.

Influence of magnetic field on morphological structures and physiological characteristics of bEnd.3 cells cultured on polypyrrole substrates.

This paper employs a spin-coated method to construct conductive polypyrrole (PPy) substrates which present superior properties for controlling the morphological structures and functions of bEnd.3 cells. The PPy substrates with a homogeneous particle size, uniform distribution and proper roughness show enhanced hydrophilic characteristics and improve cell adhesion to the substrates. The changes in the mechanical properties of cells and the responses to the designed substrates and magnetic field are also explored. Due to the synergistic effect between the magnetic field and the conductive PPy substrate, the cells cultured in such an environment exhibit applanate shapes with more branches and enhanced cell viability. In addition, the cells preferentially extend along the magnetic field direction. The mechanical characteristics of cells change significantly under varying magnetic intensity stimulations (5-16 mT). The satisfying effect on cells' morphology and outgrowth is acquired at the magnetic intensities of 9-10 mT and duration of 20 min, compared with other stimulated groups, while retaining cell viability. Moreover, the cells express higher adhesion up to 5.2 nN. The results suggest that the application of the PPy substrates and magnetic field is a promising candidate for the protection of neurovascular units and treatment of neurological diseases.

AFM-detected apoptosis of hepatocellular carcinoma cells induced by American ginseng root water extract.

American ginseng as a common and traditional herbal medicine has been used in cancer treatment for many years. However, the effect of American ginseng on the cancer cell response (i.e. apoptosis) has not been fully understood yet. Previous studies demonstrated that cellular apoptosis was associated with the changes of mechanical and morphological properties. Therefore, in this study, mechanical and morphological characterizations were carried out by both atomic force microscope (AFM) and inverted optical microscope to investigate the apoptosis of hepatocellular carcinoma (SMMC-7721) cells affected by American ginseng root water extract (AGRWE). The results showed that the cells treated with AGRWE exhibited significantly larger surface roughness, height and elastic modulus values than control group. Moreover, those parameters were upregulated under the higher concentration of AGRWE and longer culture time. Consequently, it indicates that the mechanical and morphological properties can be used as the apoptotic characteristics of SMMC-7721 cells. Also, the increased surface roughness and elastic modulus of cells under the AGRWE treatment have shown that the apoptosis of SMMC-7721 cells can be enhanced by AGRWE. This will provide an important implication for hepatocelluar carcinoma treatment and drug development.

Study of morphological and mechanical features of multinuclear and mononuclear SW480 cells by atomic force microscopy.

This article studies the morphological and mechanical features of multinuclear and mononuclear SW480 colon cancer cells by atomic force microscopy to understand their drug-resistance. The SW480 cells were incubated with the fullerenol concentrations of 1 mg/ml and 2 mg/ml. Morphological and mechanical features including the height, length, width, roughness, adhesion force and Young's modulus of three multinuclear cell groups and three mononuclear cell groups were imaged and analyzed. It was observed that the features of multinuclear cancer cells and mononuclear cancer cells were significantly different after the treatment with fullerenol. The experiment results indicated that the mononuclear SW480 cells were more sensitive to fullerenol than the multinuclear SW480 cells, and the multinuclear SW480 cells exhibited a stronger drug-resistance than the mononuclear SW480 cells. This work provides a guideline for the treatments of multinuclear and mononuclear cancer cells with drugs.

Compensation of the magnetic force imaging by scanning directions.

It was found that the results of magnetic force microscope (MFM) imaging were different with the probe scanning directions. This paper studied the effect of scanning directions on the MFM imaging, and a method for the distortion compensation was proposed to reduce the errors. In the study, three different scanning directions with the angles of 0°, 45° and 90° were used to measure the magnetic domain structures distributions of magnetic sample. The experimental results have shown that the scanning direction parallel to the magnetic domain structure will cause a minimum phase shift difference and lead to a structure distortion. A method for compensating the distortions was proposed. With this method, the distorted structures were corrected and the effect of scanning directions on the MFM imaging was significantly reduced. This work provides a way for the acquisition of the correct images of magnetic structures using an MFM and the improvement of imaging quality in a wide range of MFM applications.