Enhancing maize's nitrogen-fixing potential through ZmSBT3, a gene suppressing mucilage secretion.

Maize (Zea mays) requires substantial amounts of nitrogen, posing a challenge for its cultivation. Recent work discovered that some ancient Mexican maize landraces harbored diazotrophic bacteria in mucilage secreted by their aerial roots. To see if this trait is retained in modern maize, we conducted a field study of aerial root mucilage (ARM) in 258 inbred lines. We observed that ARM secretion is common in modern maize, but the amount significantly varies, and only a few lines have retained the nitrogen-fixing traits found in ancient landraces. The mucilage of the high-ARM inbred line HN5-724 had high nitrogen-fixing enzyme activity and abundant diazotrophic bacteria. Our genome-wide association study identified 17 candidate genes associated with ARM across three environments. Knockouts of one candidate gene, the subtilase family gene ZmSBT3, confirmed that it negatively regulates ARM secretion. Notably, the ZmSBT3 knockout lines had increased biomass and total nitrogen accumulation under nitrogen-free culture conditions. High ARM was associated with three ZmSBT3 haplotypes that were gradually lost during maize domestication, being retained in only a few modern inbred lines such as HN5-724. In summary, our results identify ZmSBT3 as a potential tool for enhancing ARM, and thus nitrogen fixation, in maize.

Blockade of IL-6 inhibits tumor immune evasion and improves anti-PD-1 immunotherapy.

Long-standing inflammatory bowel disease predisposes to the development of colorectal cancer (CRC). Interleukin (IL) -6, a pivotal link between chronic inflammation and tumor progression, has recently been recognized as a potential therapeutic target. The effect of IL-6 on proliferation and metastasis of CRC by activating the STAT3 pathway has been widely demonstrated in recent years, but few on mediating tumor immune evasion. In this study, we found that IL-6 was remarkably overexpressed in CRC and its elevation was associated with a poor prognosis. We studied CRC tumorigenesis in vivo by inoculating MC38 tumors and induced-CRC model via AOM/DSS (azoxymethane/dextransulfate sodium) in IL-6 deficient (IL-6-/-) and wild-type (WT) mice and found that IL-6-/- mice were less susceptible to develop tumors, compared to WT mice. We detected CD8+ T cells via immunofluorescence and found they exhibit high expression in tumor of IL-6-/- mice. High level of IL-6 was found in colitis model, with down-regulation of MHC-I molecules. In in vitro experiments, we found that IL-6 may act as a negative regulator in IFNγ-STAT1-MHC-I signaling. In addition, vivo trials also confirmed that MHC-I mRNA level was negatively related to the existence of IL-6. Furthermore, the blockade of IL-6 also activated CD8+T-cell accumulation and led to the high PD-L1 expression in CRC, which can sensitize animals to anti-PD-1 therapy. Our study provides a research basis for the significant role of IL-6 in tumor evasion and highlights a novel target to improve the efficacy of immunotherapy.

Subcellular locations of potential cell wall proteins in plants: predictors, databases and cross-referencing.

The cell wall is the most striking feature that distinguishes plant cells from animal cells. It plays an essential role in cell shape, stability, growth and protection. Despite being present in small amounts, cell wall proteins (CWPs) are crucial components of the cell wall. The cell wall proteome generally consists of sensu stricto CWPs, apoplast proteins and extracellular secreted proteins. Currently, there is a need for the bioinformatics analysis of a tremendous number of protein sequences that have been generated from genomic, transcriptomic and proteomics research. Compared with intracellular proteins, the location prediction of CWPs is challenging because many aspects of these proteins have not been experimentally characterized, and there are no CWP-trained, specific predictors available. By introducing the biological relevance (particularly molecular aspects) of the cell wall and CWPs, we critically evaluated the accuracy of 16 state-of-the-art predictors and classical predictors for the prediction of CWPs using an independent database of Arabidopsis and rice proteins. All experimentally verified CWPs and non-CWPs were retrieved from the UniProt Knowledgebase. Based on the evaluation, we currently recommend the predictors mGOASVM, HybridGO-Loc and FUEL-mLoc for CWPs. Furthermore, we outlined the public databases that can be used to cross-reference the subcellular location of CWPs. We illustrate a flowchart of the subcellular location prediction and a cross-reference of possible CWPs. Finally, we discuss challenges and perspectives in the bioinformatics analysis of CWPs. It is hoped that this article will provide practical guidance regarding CWPs for nonspecialists and provide insight for bioinformatics experts to develop computational tools for CWPs.

2-DE-based proteomic analysis of protein changes associated with etiolated mesocotyl growth in Zea mays.

BACKGROUND: The mesocotyl connects the coleoptilar node and the basal part of the seminal root of maize (Zea mays) seedling. The mesocotyl pushes the shoot of the seedling out of the soil during seed germination; thus, its growth is highly related to deep-sowing tolerance. Although many studies on the maize mesocotyl have been carried out at physiological and molecular levels, the proteomic changes associated with cellular and physiological activities during mesocotyl growth are still unknown. RESULTS: In the present study, the maize hybrid Zhengdan 958 was used to study mesocotyl growth and accompanying protein changes. The dark-grown etiolated mesocotyls exhibited a slow-fast-slow feature, with significant changes in the levels of indole-3-acetic acid (IAA) and cellulose and the activity of peroxidase (POD). In particular, POD activity increased with mesocotyl growth, showing higher activity at the mature (lower) end of the mesocotyl. For the proteomic analysis, soluble proteins were extracted from etiolated mesocotyls dark-grown for 48 h, 84 h, and 132 h, corresponding to the initial, rapid, and slow growth periods, respectively, and subjected to separation by two-dimensional gel electrophoresis (2-DE). As a result, 88 differentially abundant proteins (DAPs) were identified using MALDI-TOF-TOF analysis. At 48 h, most DAPs were stress proteins, heat shock proteins and storage proteins; at 84 h, oxidation/reduction proteins, carbohydrate biogenesis-related proteins and cytoskeleton-related proteins were highly accumulated; at 132 h, the most striking DAPs were those involved in the synthesis and modification of the cell wall and the biogenesis of carbohydrates. Gene ontology (GO) analysis showed that changes in the abundance and proportion of DAPs were consistent with cellular and physiological activities and biological processes during mesocotyl growth. The accumulation of nine DAPs of interest was verified by immunoblotting and RT-qPCR. CONCLUSIONS: The present study revealed that the protein patterns in 2-D gels differed greatly with mesocotyl growth. At different growth periods, a specific set of DAPs participate in various biological processes and underlie the cellular and physiological activities of the mesocotyl. These results contributed to the understanding of mesocotyl growth and the cultivation of maize lines with deep-sowing tolerance.

Predictors of Lymph Node Metastasis and Prognosis in pT1 Colorectal Cancer Patients with Signet-Ring Cell and Mucinous Adenocarcinomas.

BACKGROUND/AIMS: The local excision of early colorectal cancer is limited by the presence of lymph node metastasis (LNM). Signet-ring cell carcinomas (SRC) and mucinous adenocarcinomas (MAC) are two relatively infrequent histological subtypes. However, little is known about the predictors of LNM and prognosis to support the feasibility of local excision in early-stage SRC and MAC. METHODS: The Surveillance Epidemiology and End Results Database were used to identify all patients with pT1 adenocarcinomas, including conventional adenocarcinoma (AC), MAC, and SRC. The prevalence of LNM was assessed, and the long-term survival rate in the above three types of colorectal cancer was calculated. RESULTS: SRC accounted for 0.3% and MAC accounted for 4.4% of the entire cohort of colorectal adenocarcinomas. Compared to AC, MRC and SRC were more often located in the proximal colon, and exhibited a higher grade. The incidence of LNM in AC, MAC, and SRC was 10.6%, 17.2%, and 33.3% for colon cancers and 14.8%, 25.9%, and 46.2% for rectal cancers, respectively. In patients with lymph nodes resected no less than 12, incidence of LNM in AC, MRC, and SRC was 12%, 21%, and 44% for colon tumors and 17%, 30%, and 14% for rectal tumors, respectively. Although, colon patients MAC showed an entirely worse survival rate than AC, rectum patients MAC showed a similar prognosis to AC. We found that in patients with rectal tumors, SRC had a worse 3 and 5-year prognosis than AC. However, for colon cancers, the prognosis of SRC was similar to that of AC. Histology was not found to be an independent prognostic factor in multivariate survival analysis. CONCLUSIONS: MAC and SRC are two distinct subtypes of colorectal cancer that require special attention despite their relatively rare prevalence. pT1 patients with SRC of the rectum and patients with MAC of the colon have higher incidences of LNM, and with these adverse outcomes, local excision is not recommended. AlthoughMAC of the rectum and SRC of colon have a high rate of LNM, the prognosis of these types are similar to that of AC.

The study of the role of purified anti-mouse CD193 (CCR3) antibody in allergic rhinitis mouse animal models.

The pathogenesis of allergic asthma is similar to that of allergic rhinitis, with inflammation cells producing and releasing inflammatory mediators and cytokines closely related to CCR3.Based on the theory of "one airway, one disease", the use of CCR3 monoclonal antibody may have a similar effect on allergic rhinitis. However, there are few studies on CCR3 monoclonal antibody in allergic rhinitis. Therefore, the aim of this study was to investigate the effective concentration of CCR3 monoclonal antibody, to compare the effects of different methods of administration, and to examine the lung condition of allergic mice to investigate whether antibody treatment protects the lungs. In this study, we constructed a mouse model of allergic rhinitis and intraperitoneally injected different doses of CCR3 monoclonal antibody (5, 10, and 20 uL/mg) to observe its therapeutic effect: observing changes in tissue morphology of nasal mucosa, infiltration of inflammation, and using ELISA to detect changes in relevant inflammatory mediators and cytokines, studying the role of CCR3 mAb in inhibiting CCR3-related actions on the nasal mucosa of allergic rhinitis mice. Furthermore, In addition, the therapeutic effects of intraperitoneal injection (i.p.) and intranasal administration (i.n.) were studied on the basis of effective concentrations.

Centrifugal Spinning Enables the Formation of Silver Microfibers with Nanostructures.

Silver nanowires (AgNWs) have received much attention and application in transparent electrodes, wearable electronic devices, and sensors. The hope is for these nanowires to eventually replace the most commonly used transparent electrode material-indium tin oxide (ITO). However, electrospinning used for the preparation of AgNWs on a large scale is limited by its low productivity and high electric field, while the alcohol-thermal method is limited to mixing by-product silver nanoparticles in silver nanowires. We demonstrate a novel and simple centrifugal spinning approach in order to successfully fabricate ultra-long silver microfibers based on AgNO3 and polyvinyl pyrrolidone (PVP). The centrifugal-spun precursor fiber and silver fiber can be prepared to as thin as 390 and 310 nm, respectively. Annealed fibers show typical nanostructures with grains down to a minimum size of 51 nm. Combinations of different parameters, including concentrations of PVP, needle size, and annealing temperature are also investigated, in order to optimize the spinning process of ultra-long silver microfibers. The feasibility of preparing silver microfibers by centrifugal spinning is preliminarily verified, examining prospects for mass production. Furthermore, numerous strategies related to assisting the creation of silver nanofibers using centrifugal spinning are presented as possibilities in future development.

Multilocation proteins in organelle communication: Based on protein-protein interactions.

Protein-protein interaction (PPI) plays a crucial role in most biological processes, including signal transduction and cell apoptosis. Importantly, the knowledge of PPIs can be useful for identification of multimeric protein complexes and elucidation of uncharacterized protein functions. Arabidopsis thaliana, the best-characterized dicotyledonous plant, the steadily increasing amount of information on the levels of its proteome and signaling pathways is progressively enabling more researchers to construct models for cellular processes for the plant, which in turn encourages more experimental data to be generated. In this study, we performed an overview analysis of the 10 major organelles and their associated proteins of the dicotyledonous model plant Arabidopsis thaliana via PPI network, and found that PPI may play an important role in organelle communication. Further, multilocation proteins, especially phosphorylation-related multilocation proteins, can function as a "needle and thread" via PPIs and play an important role in organelle communication. Similar results were obtained in a monocotyledonous model crop, rice. Furthermore, we provide a research strategy for multilocation proteins by LOPIT technique, proteomics, and bioinformatics analysis and also describe their potential role in the field of plant science. The results provide a new view that the phosphorylation-related multilocation proteins play an important role in organelle communication and provide new insight into PPIs and novel directions for proteomic research. The research of phosphorylation-related multilocation proteins may promote the development of organelle communication and provide an important theoretical basis for plant responses to external stress.

STOML2 interacts with PHB through activating MAPK signaling pathway to promote colorectal Cancer proliferation.

BACKGROUND: Highly expressed STOML2 has been reported in a variety of cancers, yet few have detailed its function and regulatory mechanism. This research aims to reveal regulatory mechanism of STOML2 and to provide evidence for clinical therapeutics, via exploration of its role in colorectal cancer, and identification of its interacting protein. METHODS: Expression level of STOML2 in normal colon and CRC tissue from biobank in Nanfang Hospital was detected by pathologic methods. The malignant proliferation of CRC induced by STOML2 was validated via gain-of-function and loss-of-function experiments, with novel techniques applied, such as organoid culture, orthotopic model and endoscopy monitoring. Yeast two-hybrid assay screened interacting proteins of STOML2, followed by bioinformatics analysis to predict biological function and signaling pathway of candidate proteins. Target protein with most functional similarity to STOML2 was validated with co-immunoprecipitation, and immunofluorescence were conducted to co-localize STOML2 and PHB. Pathway regulated by STOML2 was detected with immunoblotting, and subsequent experimental therapy was conducted with RAF inhibitor Sorafenib. RESULTS: STOML2 was significantly overexpressed in colorectal cancer and its elevation was associated with unfavorable prognosis. Knockdown of STOML2 suppressed proliferation of colorectal cancer, thus attenuated subcutaneous and orthotopic tumor growth, while overexpressed STOML2 promoted proliferation in cell lines and organoids. A list of 13 interacting proteins was screened out by yeast two-hybrid assay. DTYMK and PHB were identified to be most similar to STOML2 according to bioinformatics in terms of biological process and signaling pathways; however, co-immunoprecipitation confirmed interaction between STOML2 and PHB, rather than DTYMK, despite its highest rank in previous analysis. Co-localization between STOML2 and PHB was confirmed in cell lines and tissue level. Furthermore, knockdown of STOML2 downregulated phosphorylation of RAF1, MEK1/2, and ERK1/2 on the MAPK signaling pathway, indicating common pathway activated by STOML2 and PHB in colorectal cancer proliferation. CONCLUSIONS: This study demonstrated that in colorectal cancer, STOML2 expression is elevated and interacts with PHB through activating MAPK signaling pathway, to promote proliferation both in vitro and in vivo. In addition, combination of screening assay and bioinformatics marks great significance in methodology to explore regulatory mechanism of protein of interest.

[Study on expression and mechanism of serum differential proteins after rush immunotherapy of allergic rhinitis].

Objective:To detect the expression of differentially expressed proteins in serum of patients with allergic rhinitis who were allergic to dust mites before and after 6-day rush immunotherapy. The three differentially expressed proteins, CRP, CTHRC1 and WDR89, were detected and identified. The immunoregulatory effects and significance of these three differentially expressed proteins in rush immunotherapy of allergic rhinitis were analyzed and discussed. Method:The serum samples of 15 patients with allergic rhinitis, 15 patients with rush immunotherapy and 10 patients with healthy control group were collected. The samples were studied by isobaric tags for relative and absolute quantitation（iTRAQ） technique. The related differential proteins were determined by two-dimensional gel electrophoresis and mass spectrometry, and the rationality of the screened differential proteins was tested and verified by Cluster3.0 software and Java TreeView software. Finally, the selected CRP, CTHRC1 and WDR89 proteins were identified by enzyme-linked immunosorbent assay（ELISA）. Result:In this study, 893 proteins were detected and 53 differential proteins were identified. Compared with healthy control group, 24 proteins which was statistically significant were found in allergic rhinitis group, which were closely related to the occurrence of allergic rhinitis, including 10 up-regulated proteins and 14 down-regulated proteins. Compared with the allergic rhinitis group, patients with allergic rhinitis underwent 6 days of rush immunotherapy. There were 29 proteins whose expression of proteins with a difference of P value of less than 0.05 and 1.2 times higher, which were related to the effect after the incremental phase of rush immunotherapy was completed, of which 12 were up-regulated and 17 were down-regulated. Compared with healthy control group, the expression of up-regulated of allergic rhinitis group and the expression of down-regulated protein after 6 days of rush immunotherapy were CTHRC1, WDR89; Compared with healthy control group, AR group was down-regulated and the expression of up-regulated protein after 6 days of rush immunotherapy was CRP. CRP, CTHRC1 and WDR89 proteins were identified by enzyme linked immunosorbent assay（ELISA）, and it was found that the differential expression of CTHRC1 and WDR89 in AR and RIT was statistically significant（P<0.05）, but the differential expression of serum CRP in AR and RIT was not statistically significant（P>0.05）. Conclusion:Serum protein CTHRC1 and WDR89 are closely related to the pathogenesis of allergic rhinitis, and played a role in the regulation of rush immunotherapy, while serum protein CRP has no significant effect on AR and RIT.

Effects of lentivirus-mediated CCR3 RNA interference on the function of mast cells of allergic rhinitis in mice.

The CC chemokine receptor 3 (CCR3) expressed by eosinophils, mast cells and Th2 cells is closely related to allergic diseases. The objective of this study was to explore whether silencing of CCR3 with short hairpin RNAs (shRNAs) delivered by a lentiviral vector could impact the function of mast cells in a murine model of allergic rhinitis (AR) in vivo. The murine model of allergic rhinitis (AR) inducing by ovalbumin (OVA) was constructed, and the BALB/c mice were divided into normal control group, AR group, controlshRNA treated group and lentiviral CCR3-shRNA treated group. The recombinant lentivirus vectors which express a short hairpin RNA (shRNA) targeting the CCR3 were dropped into the nasal cavity of OVA-sensitized mice before the challenges. Real-time fluorescence quantitative PCR and western blotting were performed to observe inhibitory effect of CCR3 gene. Nasal symptoms of mice and OVA-specific IgE in each group were assessed. Concentrations of histamine, tryptase and Prostaglandin D2 (PGD2) in bone marrow, peripheral blood and nasal mucosa were analyzed. Furthermore, histological analysis and electron microscopy analysis were applied to detect the histology changes of nasal mucosa and the infiltration of mast cells in nasal mucosa. The results showed that administration of CCR3shRNA could effectively inhibit the expression of the CCR3 gene in bone marrow, peripheral blood and nasal mucosa, which reduced the nasal symptoms, the level of OVA-specific IgE, the inflammatory cells and mast cells infiltration into nasal cavity, and relieved the histopathological changes of nasal mucosa. In addition, intervention of CCR3shRNA could reduce the levels of the histamine, tryptase and PGD2 in bone marrow, peripheral blood and nasal mucosa. These results suggest that inhibition of CCR3 gene expression by shRNAs lentiviral vectors can effectively attenuate migration, infiltration and degranulation of mast cells in local tissues and alleviate the inflammation of allergic rhinitis mice.

Direct Assembly of 3D-BCN Microspheres as a Microsupercapacitor Electrode for Wearable Energy Storage.

Scalable and cost-effective fabrication of three-dimensional (3D) boron carbon nitride (BCN) microspheres was first demonstrated by hydrothermal and annealing methods. In particular, the specific surface area of 3D-BCN-4 reached 1390.12 m2 g-1 and had a high hierarchical pore structure. An all-printed solid-state flexible microsupercapacitor (MSC) based on 3D-BCN-4 microspheres as an electrode material was fabricated for the first time by a screen printing method, which also provided efficacious properties. The single MSC areal capacitance reached 41.6 mF cm-2. Furthermore, the remarkable mechanical flexibility was also achieved for the device with evidence that no obvious capacitance loss occurred even upon bending to 180°, and the device had a 93.3% capacitance retention after 1000 cycles. In addition, the maximum energy density reached 0.00832 mW h cm-2, and the highest power density was 2 mW cm-2. These results show the synthesis of 3D-BCN by a facile and effective method with excellent electrochemical performance, which should provide a promising direction to wearable energy storage devices.

Correction: Modified TCA/acetone precipitation of plant proteins for proteomic analysis.

[This corrects the article DOI: 10.1371/journal.pone.0202238.].

Modified TCA/acetone precipitation of plant proteins for proteomic analysis.

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

Accumulation Profiles of Embryonic Salt-Soluble Proteins in Maize Hybrids and Parental Lines Indicate Matroclinous Inheritance: A Proteomic Analysis.

Maize is one of the most widely cultivated crops. It accumulates a large quantity of seed storage proteins, which are important for seed development and germination, and contribute to the nutritional quality of seeds. Based on solubility, the storage proteins are divided into albumins (water-soluble), globulins (salt-soluble), prolamins (alcohol-soluble), and glutelins (acid- or alkali-soluble). Maize hybrids are cultivated due to the superior performance of F1 hybrids than that of their parents, a phenomenon known as heterosis. However, the accumulation patterns of seed storage proteins in maize embryos between the hybrids and their parental inbred lines have not been compared. In the present study, two elite inbred lines of China, Zheng 58 and Chang 7-2, and their reciprocal hybrids (Zheng 58 × Chang 7-2 and Chang 7-2 × Zheng 58) were used to explore parental influences on the accumulation patterns of seed storage proteins in maize embryos. For this purpose, we focused on seed salt-soluble proteins (SSPs) in our experiments. The SSPs were selectively extracted from maize mature embryos after extensive removal of water-soluble albumin and separated using two-dimensional gel electrophoresis (2-DE), followed by mass spectrometry analysis. Our results indicated that the 2-DE SSP profiles of hybrids closely resembled those of their maternal parent rather than the paternal parent. In other words, 2-DE SSP profiles of Zheng 58 × Chang 7-2 were more similar those of Zheng 58 whereas such profiles of Chang 7-2 × Zheng 58 were more similar to those of Chang 7-2 although the 2-DE profiles of all four maize types were quite similar. In total, 12 relatively abundant SSPs spots representing five kinds of proteins were identified, of which nine protein spots displayed non-additive accumulation in at least one hybrid. This study provided additional data on dominance and partial dominance effects on maize hybrids embryos. Besides, earlier studies on accumulation profiles of globulin-1 (also known as vicilin), which is one of the most abundant globulins in maize embryos, also support the above results. This study would be helpful in revealing the mechanisms underlying SSPs accumulation patterns in the hybrids.