PKCζ phosphorylates VASP to mediate chemotaxis in breast cancer cells.

Breast carcinoma (BC) is one of the most common malignant cancers in females, and metastasis remains the leading cause of death in these patients. Chemotaxis plays an important role in cancer cell metastasis and the mechanism of breast cancer chemotaxis has become a central issue in contemporary research. PKCζ, a member of the atypical PKC family, has been reported to be an essential component of the EGF-stimulated chemotactic signaling pathway. However, the molecular mechanism through which PKCζ regulates chemotaxis remains unclear. Here, we used a proteomic approach to identify PKCζ-interacting proteins in breast cancer cells and identified VASP as a potential binding partner. Intriguingly, stimulation with EGF enhanced this interaction and induced the translocalization of PKCζ and VASP to the cell membrane. Further experiments showed that PKCζ catalyzes the phosphorylation of VASP at Ser157, which is critical for the biological function of VASP in regulating chemotaxis and actin polymerization in breast cancer cells. Furthermore, in PKCζ knockdown BC cells, the enrichment of VASP at the leading edge was reduced, and its interaction with profilin1 was attenuated, thereby reducing the chemotaxis and overall motility of breast cancer cells after EGF treatment. In functional assays, PKCζ promoted chemotaxis and motility of BC cells through VASP. Our findings demonstrate that PKCζ, a new kinase of VASP, plays an important role in promoting breast cancer metastasis and provides a theoretical basis for expanding new approaches to tumor biotherapy.

Hybridization Chain Reaction-Based Electrochemical Biosensors by Integrating the Advantages of Homogeneous Reaction and Heterogeneous Detection.

The conventional hybridization chain reaction (HCR)-based electrochemical biosensors usually require the immobilization of probes on the electrode surface. This will limit the applications of biosensors due to the shortcomings of complex immobilization processes and low HCR efficiency. In this work, we proposed astrategy for the design of HCR-based electrochemical biosensors by integrating the advantages of homogeneous reaction and heterogeneous detection. Specifically, the targets triggered the autonomous cross-opening and hybridization oftwobiotin-labeled hairpin probes to form long-nicked dsDNA polymers. The HCR products with many biotin tags were then captured by a streptavidin-covered electrode, thus allowing for the attachment of streptavidin-conjugated signal reporters through streptavidin-biotin interactions. By employing DNA and microRNA-21 as the model targets and glucose oxidase as the signal reporter, the analytical performances of the HCR-based electrochemical biosensors were investigated. The detection limits of this method were found to be 0.6 fM and 1 fM for DNA and microRNA-21, respectively. The proposed strategy exhibited good reliability for target analysis in serum and cellular lysates. The strategy can be used to develop various HCR-based biosensors for a wide range of applications because sequence-specific oligonucleotides exhibit high binding affinity to a series of targets. In light of the high stability and commercial availability of streptavidin-modified materials, the strategy can be used for the design of different biosensors by changing the signal reporter and/or the sequence of hairpin probes.

Isolation of BVDV-1a, 1m, and 1v strains from diarrheal calf in china and identification of its genome sequence and cattle virulence.

Bovine viral diarrhea virus (BVDV) is an important livestock viral pathogen responsible for causing significant economic losses. The emerging and novel BVDV isolates are clinically and biologically important, as there are highly antigenic diverse and pathogenic differences among BVDV genotypes. However, no study has yet compared the virulence of predominant genotype isolates (BVDV-1a, 1b, and 1m) in China and the emerging genotype isolate BVDV-1v. The serological relationship among these genotypes has not yet been described. In this study, we isolated three BVDV isolates from calves with severe diarrhea, characterized as BVDV-1a, 1m, and novel 1v, based on multiple genomic regions [including 5-untranslated region (5'-UTR), Npro, and E2] and the phylogenetic analysis of nearly complete genomes. For the novel genotype, genetic variation analysis of the E2 protein of the BVDV-1v HB-03 strain indicates multiple amino acid mutation sites, including potential host cell-binding sites and neutralizing epitopes. Recombination analysis of the BVDV-1v HB-03 strain hinted at the possible occurrence of cross-genotypes (among 1m, 1o, and 1q) and cross-geographical region transmission events. To compare the pathogenic characters and virulence among these BVDV-1 genotypes, newborn calves uninfected with common pathogens were infected intranasally with BVDV isolates. The calves infected with the three genotype isolates show different symptom severities (diarrhea, fever, slowing weight gain, virus shedding, leukopenia, viremia, and immune-related tissue damage). In addition, these infected calves also showed bovine respiratory disease complexes (BRDCs), such as nasal discharge, coughing, abnormal breathing, and lung damage. Based on assessing different parameters, BVDV-1m HB-01 is identified as a highly virulent strain, and BVDV-1a HN-03 and BVDV-1v HB-03 are both identified as moderately virulent strains. Furthermore, the cross-neutralization test demonstrated the antigenic diversity among these Chinese genotypes (1a, 1m, and 1v). Our findings illustrated the genetic evolution characteristics of the emerging genotype and the pathogenic mechanism and antigenic diversity of different genotype strains, These findings also provided an excellent vaccine candidate strain and a suitable BVDV challenge strain for the comprehensive prevention and control of BVDV.

In Situ S-Doping Strategy of Promoting Iron Coordinated by Nitrogen-Doped Carbon Nanosheets for Efficient Oxygen Reduction Reaction.

Improving transition metal-nitrogen-carbon (M-N-C) as a noble-metal-free catalyst for the oxygen reduction reaction (ORR) is critical to achieve low-cost electrochemical energy conversion. Herein, an in situ S doping strategy of enhancing Fe-N-C activity for ORR was developed by newly designed Fe(II) ion coordinated S-containing bis(imino)-pyridine-based polymers as precursors, which were synthesized through copolymerizing three monomers of 2, 6-diacetylpyridine (DAP), triamterene (TIT), and 2,5-dithiobiurea (DTB) as both N and S sources. All samples derived from various molar ratios of the three monomers possess a self-supporting structure of nanosheets. Additionally, incorporating DTB into the copolymer can not only strongly affect the derived coordinative species of N dopants to Fe atom but also effectively induce the synergistic effect between S dopants and FeNx moieties, resulting a significant improvement for ORR. The S-doped Fe-N-C nansheets with Fe coordinated by 4 pyrrolic N dopants exhibit the highest ORR activity and stability in alkaline media with a higher power output of Zn-air battery than that of the same loading of Pt/C. Theoretical calculation identifies that the thiophenic S dopant adjacent to Fe-pyrrolic N moiety can decrease the d band center of Fe atom, greatly weakening the energy profiles of oxygenated intermediates and thus enhancing ORR. In addition, because of the designability of transition metal coordinated S-containing bis(imino)-pyridine based polymers in the work, therefore, it is believable that this strategy would open a wide space to explore the structural relationship between precursors and MNx active sites with S dopants for the purpose of achieving highly efficient and robust M-N-C catalysts for energy-related electrocatalysis.

[Effect of Long-term Straw Returning on the Mineralization and Priming Effect of Rice Root-carbon].

Long-term straw returning to the field changes the environmental conditions of rice paddy soil, which affects the mineralization and priming effect of residual rice roots in the soil, but the direction and intensity of its influence is not clear. Therefore, based on a long-term fertilization field experiment, 13C-CO2 isotopic labeling technology and laboratorial incubation were used to analyze the characteristics of mineralization of rice roots and native soil organic carbon, the intensity and direction of the priming effect, and the source partitioning of CO2 emissions in three treatments, consisting of no fertilization (CK), chemical fertilizer (CF), and straw returning with chemical fertilizer (CFS). The results showed that after 120 days of flooding incubation, the root residue (R) increased the cumulative CO2 emissions by 617.41-726.27 mg·kg-1. The cumulative CO2 emissions from roots and root mineralized proportions in the CFS+R and CF+R treatments were 470.82 and 444.04 mg·kg-1, respectively, and 18.8% and 17.8%, respectively. These were significantly higher than those in the CK+R treatment (384.19 mg·kg-1, 15.4%). There was no significant difference in the cumulative CO2 emissions from native soil organic carbon among the three treatments. However, the mineralized proportion of native soil organic carbon in the CFS+R treatment (4.2%) was significantly lower than that in the CF+R and CK+R treatments (5.4% and 5.8%). The priming effect in the CFS+R treatment was 29.6%, which was significantly lower than that in the CK+R treatment (42.5%) and higher than that in the CF+R treatment (14.4%). A total of 23.47% to 27.59% of the cumulative CO2 emission of the flooded paddy soil was from the roots, and the remainder was from the soil. In addition, the proportion of CO2 emission caused by the priming effect was smaller in the CFS+R treatment than that in the CK+R treatment and larger than that in the CF+R treatment. In summary, the long-term straw returning in the flooded paddy soil will increase the mineralization potential of rice roots, but it is more conducive to the stability of the native soil organic carbon.

AGR2-induced cholesterol synthesis drives lovastatin resistance that is overcome by combination therapy with allicin.

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a multifunctional protein under physiological and pathological conditions. In this study we investigated the roles of AGR2 in regulating cholesterol biogenesis, lipid-lowering efficiency of lovastatin as well as in protection against hypercholesterolemia/statin-induced liver injury. We showed that AGR2 knockout significantly decreased hepatic and serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in mice with whole-body or hepatocyte-specific Agr2-null mutant, compared with the levels in their wild-type littermates fed a normal chow diet (NCD) or high-fat diet (HFD). In contrast, mice with AGR2 overexpression (Agr2/Tg) exhibited an increased cholesterol level. Mechanistic studies revealed that AGR2 affected cholesterol biogenesis via activation of AKT/sterol regulatory element-binding protein-2 (SREBP2), to some extent, in a PDI motif-dependent manner. Moreover, elevated AGR2 led to a significant decrease in the lipid-lowering efficacy of lovastatin (10 mg· kg-1· d-1, ip, for 2 weeks) in mice with hypercholesterolemia (hyperCho), which was validated by results obtained from clinical samples in statin-treated patients. We showed that lovastatin had limited effect on AGR2 expression, but AGR2 was inducible in Agr2/Tg mice fed a HFD. Further investigations demonstrated that drug-induced liver toxicity and inflammatory reactions were alleviated in hypercholesterolemic Agr2/Tg mice, suggesting the dual functions of AGR2 in lipid management and hyperCho/statin-induced liver injury. Importantly, the AGR2-reduced lipid-lowering efficacy of lovastatin was attenuated, at least partially, by co-administration of a sulfhydryl-reactive compound allicin (20 mg· kg-1· d-1, ip, for 2 weeks). These results demonstrate a novel role of AGR2 in cholesterol metabolism, drug resistance and liver protection, suggesting AGR2 as a potential predictor for selection of lipid-lowering drugs in clinic.

Acidic/Alkaline Stress Mediates Responses to Azole Drugs and Oxidative Stress in Aspergillus fumigatus.

A human host exploits stresses such as acidic/alkaline pH, antifungal drugs, and reactive oxygen species to kill microbial pathogens such as the fungus Aspergillus fumigatus. However, A. fumigatus is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of the human host? In this observation, we show that simultaneous exposure to acidic pH and oxidative stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills A. fumigatus synergistically in vitro. Interestingly, A. fumigatus is resistant to the combination of alkaline pH and oxidative stress. Quantitative real-time PCR analyses showed that acidic/alkaline pH stress can mediate oxidative stress responses in A. fumigatus by regulating the expression of catalase-encoding genes. We further show that A. fumigatus is sensitive to the combination of acidic/alkaline stress and azole drug stress. Transcriptome analysis revealed that the sensitivity of A. fumigatus to azole drugs under acidic/alkaline conditions may be related to changes in genetic stability, sphingolipid metabolism, lipid metabolism, and amino acid metabolism. Collectively, our findings suggest that combinatorial stress represents a powerful fungicidal mechanism employed by hosts against pathogens, which suggests novel approaches to potentiate antifungal therapy. IMPORTANCE The human host combats fungal infections via phagocytic cells that recognize and kill fungal pathogens. Immune cells combat Aspergillus fumigatus infections with a potent mixture of chemicals, including reactive oxygen species, acidic/alkaline stress, and antifungal drugs. However, A. fumigatus is relatively resistant to these stresses in vitro. In this observation, we show that it is the combination of acidic/alkaline pH and oxidative or azole stress that kills A. fumigatus so effectively, and we define the molecular mechanisms that underlie this potency. Our findings suggest that combinatorial stress is a powerful fungicidal mechanism employed by hosts, which suggests novel approaches to potentiate antifungal therapy. This study provides a platform for future studies that will address the combinatorial impacts of various environmental stresses on A. fumigatus and other pathogenic microbes.

Anterior gradient 2 increases long-chain fatty acid uptake via stabilizing FABP1 and facilitates lipid accumulation.

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a well-established oncogene. Here, we found that Agr2-/- mice had a decreased fat mass and hepatic and serum lipid levels compared with their wild-type littermates after fasting, and exhibited reduced high-fat diet (HFD)-induced fat accumulation. Transgenic mice overexpressing AGR2 (Agr2/Tg) readily gained fat weight on a HFD but not a normal diet. Proteomic analysis of hepatic samples from Agr2-/- mice revealed that depletion of AGR2 impaired long-chain fatty acid uptake and activation but did not affect de novo hepatic lipogenesis. Further investigations led to the identification of several effector substrates, particularly fatty acid binding protein-1 (FABP1) as essential for the AGR2-mediated effects. AGR2 was coexpressed with FABP1, and knockdown of AGR2 resulted in a reduction in FABP1 stability. Physical interactions of AGR2 and FABP1 depended on the PDI motif in AGR2 and the formation of a disulfide bond between these two proteins. Overexpression of AGR2 but not a mutant AGR2 protein lacking PDI activity suppressed lipid accumulation in cells lacking FABP1. Moreover, AGR2 deficiency significantly reduced fatty acid absorption in the intestine, which might be resulted from decreased fatty acid transporter CD36 in mice. These findings demonstrated a novel role of AGR2 in fatty-acid uptake and activation in both the liver and intestine, which contributed to the AGR2-mediated lipid accumulation, suggesting that AGR2 is an important regulator of whole-body lipid metabolism and down-regulation of AGR2 may antagonize the development of obesity.

Oxidative stress induced autophagy in cancer associated fibroblast enhances proliferation and metabolism of colorectal cancer cells.

Tumors are comprised of malignant cancer cells and stromal cells which constitute the tumor microenvironment (TME). Previous studies have shown that cancer associated fibroblast (CAF) in TME is an important promoter of tumor initiation and progression. However, the underlying molecular mechanisms by which CAFs influence the growth of colorectal cancer cells (CRCs) have not been clearly elucidated. In this study, by using a non-contact co-culture system between human colorectal fibroblasts (CCD-18-co) and CRCs (LoVo, SW480, and SW620), we found that fibroblasts existing in tumor microenvironment positively influenced the metabolism of colorectal cancer cells, through its autophagy and oxidative stress pathway which were initially induced by neighboring tumor cells. Therefore, our data provided a novel possibility to develop fibroblasts as a potential target to treat CRC.

Mitigating effects of ex situ application of rice straw on CH4 and N2O emissions from paddy-upland coexisting system.

The in situ application of rice straw enhances CH4 emissions by a large margin. The ex situ application of rice straw in uplands, however, may mitigate total global warming potential (GWP) of CH4 and N2O emissions from paddy-upland coexisting systems. To evaluate the efficiency of this practice, two field trials were conducted in rice-rice-fallow and maize-rape cropping systems, respectively. Year-round measurements of CH4 and N2O emissions were conducted to evaluate the system-scaled GWP. The results showed that CH4 accounted for more than 98% of GWP in paddy. Straw removal from paddy decreased 44.7% (302.1 kg ha-1 yr-1) of CH4 emissions and 51.2% (0.31 kg ha-1 yr-1) of N2O emissions, thus decreased 44.8% (7693 kg CO2-eqv ha-1 yr-1) of annual GWP. N2O accounted for almost 100% of GWP in upland. Straw application in upland had insignificant effects on CH4 and N2O emissions, which increased GWP only by 91 kg CO2-eqv ha-1 yr-1. So, the transfer of straw from paddy to upland could decrease GWP by 7602 kg CO2-eqv ha-1 yr-1. Moreover, straw retention during late rice season contributed to 88.2% of annual GWP increment. It is recommended to transfer early rice straw to upland considering GWP mitigation, nutrient recycling and labor cost.

Testing and optimization of a semiautomatic prostate boundary segmentation algorithm using virtual operators.

Image analysis tasks such as size measurement and landmark-based registration require the user to select control points in an image. The output of such algorithms depends on the choice of control points. Since the choice of points varies from one user to the next, the requirement for user input introduces variability into the output of the algorithm. In order to test and/or optimize such algorithms, it is necessary to assess the multiplicity of outputs generated by the algorithm in response to a large set of inputs; however, the input of data requires substantial time and effort from multiple users. In this paper we describe a method to automate the testing and optimization of algorithms using "virtual operators," which consist of a set of spatial distributions describing how actual users select control points in an image. In order to construct the virtual operator, multiple users must repeatedly select control points in the image on which testing is to be performed. Once virtual operators are generated, control points for initializing the algorithm can be generated from them using a random number generator. Although an initial investment of time is required from the users in order to construct the virtual operator, testing and optimization of the algorithm can be done without further user interaction. We illustrate the construction and use of virtual operators by testing and optimizing our prostate boundary segmentation algorithm. The algorithm requires the user to select four control points on the prostate as input.

Semiautomatic three-dimensional segmentation of the prostate using two-dimensional ultrasound images.

In this paper, we report on two methods for semiautomatic three-dimensional (3-D) prostate boundary segmentation using 2-D ultrasound images. For each method, a 3-D ultrasound prostate image was sliced into the series of contiguous 2-D images, either in a parallel manner, with a uniform slice spacing of 1 mm, or in a rotational manner, about an axis approximately through the center of the prostate, with a uniform angular spacing of 5 degrees. The segmentation process was initiated by manually placing four points on the boundary of a selected slice, from which an initial prostate boundary was determined. This initial boundary was refined using the Discrete Dynamic Contour until it fit the actual prostate boundary. The remaining slices were then segmented by iteratively propagating this result to an adjacent slice and repeating the refinement, pausing the process when necessary to manually edit the boundary. The two methods were tested with six 3-D prostate images. The results showed that the parallel and rotational methods had mean editing rates of 20% and 14%, and mean (mean absolute) volume errors of -5.4% (6.5%) and -1.7% (3.1%), respectively. Based on these results, as well as the relative difficulty in editing, we conclude that the rotational segmentation method is superior.