Mapping Full Conformational Transition Dynamics of Intrinsically Disordered Proteins Using a Single-Molecule Nanocircuit.

Intrinsically disordered proteins (IDPs) are emerging therapeutic targets for human diseases. However, probing their transient conformations remains challenging because of conformational heterogeneity. To address this problem, we developed a biosensor using a point-functionalized silicon nanowire (SiNW) that allows for real-time sampling of single-molecule dynamics. A single IDP, N-terminal transactivation domain of tumor suppressor protein p53 (p53TAD1), was covalently conjugated to the SiNW through chemical engineering, and its conformational transition dynamics was characterized as current fluctuations. Furthermore, when a globular protein ligand in solution bound to the targeted p53TAD1, protein-protein interactions could be unambiguously distinguished from large-amplitude current signals. These proof-of-concept experiments enable semiquantitative, realistic characterization of the structural properties of IDPs and constitute the basis for developing a valuable tool for protein profiling and drug discovery in the future.

Effects of warming on rice production and metabolism process associated with greenhouse gas emissions.

Evaluating the impact of global warming on rice production and greenhouse gas (GHG) emissions is critical for ensuring food security and mitigating the consequences of climate change. Nonetheless, the impacts of warming on crop production, GHG emissions, and microbial mechanisms in the single-cropping rice systems remain unclear. Here, a two-year field experiment was conducted to explore the effects of warming (increased by 2.7-3.0 °C on average) in the rice growing season on crop production and functional microorganisms associated with GHG emissions. Results showed that warming resulted in significant reduction (p < 0.01) in the aboveground biomass and grain yield as well as in grain weight, the number of spikelets per panicle, and the seed-setting rate. However, it caused a significant increase (p < 0.01) in the number of panicles by 15.6 % and 34.9 %, respectively. Furthermore, warming significantly increased (p < 0.01) seasonal methane (CH4) emissions but reduced nitrous oxide (N2O) emissions, particularly in 2022.The relative abundance of genes associated with CH4 metabolism and nitrogen metabolism was increased by 40.7 % and 32.7 %, respectively, in response to warming. Moreover, warming had a positive impact on the abundance of genes related to CH4 production and oxidation processes but did not affect the denitrification processes associated with N2O production. These results showed that warming decreased rice yield and biomass in the single cropping rice system but increased CH4 emissions and global warming potential. Taken together, to address the increasing food demand of a growing population and mitigate the impacts of global warming, it is imperative to duce GHG emissions and enhance crop yields.

Homolog of Human placenta-specific gene 8, PcPLAC8-10, enhances cadmium uptake by Populus roots.

Cadmium (Cd) pollution of soil occurs worldwide. Phytoremediation is an effective approach for cleaning up Cd polluted soil. Fast growing Populus species with high Cd uptake capacities are desirable for phytoremediation. Thus, it is important to elucidate the molecular functions of genes involved in Cd uptake by poplars. In this study, PcPLAC8-10, a homolog of Human placenta-specific gene 8 (PLAC8) implicated in Cd transport was functionally characterized in Populus × canescens. PcPLAC8-10 was transcriptionally induced in Cd-treated roots and it encoded a plasma membrane-localized transporter. PcPLAC8-10 exhibited Cd uptake activity when expressed in yeast cells. No difference in growth was observed between wild type (WT) and PcPLAC8-10-overexpressing poplars. PcPLAC8-10-overexpressing poplars exhibited increases in net Cd2+ influxes by 192% and Cd accumulation by 57% in the roots. However, similar reductions in biomass were found in WT and transgenic poplars when exposed to Cd. The complete motif of CCXXXXCPC in PcPLAC8-10 was essential for its Cd transport activity. These results suggest that PcPLAC8-10 is a plasma membrane-localized transporter responsible for Cd uptake in the roots and the complete CCXXXXCPC motif of PcPLAC8-10 plays a key role in its Cd transport activity in poplars.

Visualizing single-molecule conformational transition and binding dynamics of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) play crucial roles in cellular processes and hold promise as drug targets. However, the dynamic nature of IDPs remains poorly understood. Here, we construct a single-molecule electrical nanocircuit based on silicon nanowire field-effect transistors (SiNW-FETs) and functionalize it with an individual disordered c-Myc bHLH-LZ domain to enable label-free, in situ, and long-term measurements at the single-molecule level. We use the device to study c-Myc interaction with Max and/or small molecule inhibitors. We observe the self-folding/unfolding process of c-Myc and reveal its interaction mechanism with Max and inhibitors through ultrasensitive real-time monitoring. We capture a relatively stable encounter intermediate ensemble of c-Myc during its transition from the unbound state to the fully folded state. The c-Myc/Max and c-Myc/inhibitor dissociation constants derived are consistent with other ensemble experiments. These proof-of-concept results provide an understanding of the IDP-binding/folding mechanism and represent a promising nanotechnology for IDP conformation/interaction studies and drug discovery.

Long non-coding RNA XIST promotes the malignant features of oral squamous cell carcinoma (OSCC) cells through regulating miR-133a-5p/VEGFB.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) represents a frequently seen oral cavity malignancy, and the mechanisms of its occurrence and development remain unclear. The present work examined the expression and biological function of long non-coding RNA (lncNRA) XIST (X-inactive specific transcript) in OSCC cells and tissues. STUDY DESIGN: A total number of 50 OSCC and paired non-carcinoma tissue samples were collected in this study. Gene expression levels in cancer tissues and cells were quantified by RT-qPCR. In addition, gain- and loss-of-function experiments were conducted to investigate the biological roles of XIST as well as its downstream targets in OSCC cells. RESULTS: XIST was upregulated in OSCC cells and tissues, which predicted a poorer prognostic outcome in OSCC patients. Silencing XIST inhibited the growth and invasion of OSCC cells and triggered apoptosis. miR-133a-5p was identified as a downstream target of XIST, which was downregulated in OSCC tissues. miR-133a-5p mediated the effect of XIST by targeting VEGFB. VEGFB overexpression rescued the inhibitory effects of XIST silencing on cell growth, invasion and migration. CONCLUSION: Taken together, the above data indicates that XIST serves as an oncogenic factor to enhance the growth and invasion of OSCC cells by targeting the miR-133a/VEGFB axis.

Crystal idioblasts are involved in the anther dehiscence of Nicotiana tabacum.

In Nicotiana tabacum, the degeneration of connective tissue and stomium tissue (the stomium and circular cell cluster [CCC]) is essential for anther dehiscence. Both connective cells and CCC cells are crystal idioblasts, and these cells will undergo degeneration after accumulating calcium oxalate (CaOx) crystals. However, detailed data concerning this process are minimal. Therefore, this study used cellular biological and physiological methods to illustrate this relationship. Results demonstrated that tobacco anther dehiscence is a series of timed programmed cell death (PCD) processes that include the CCC, connective tissue, and stomium. The degenerating crystal idioblasts of the tobacco anther were found to possess two hallmark characteristics that distinguished them from normal PCD cells, namely dynamic changes in CaOx crystals and the appearance of numerous peroxisomes. The accumulation of CaOx and the production of H2 O2 occurred simultaneously or successively before PCD. The peak H2 O2 content was found to appear after the insoluble oxalate. Further, CeCl3 cytochemistry staining was used to detect subcellular H2 O2 , and the precipitate of H2 O2 was primarily present in peroxisomes and around CaOx crystals. These results show that anther dehiscence in N. tabacum is a PCD process in which crystal idioblasts play a vital role in CaOx degradation and H2 O2 production.

Effect of MicroRNA-138 on Tumor Necrosis Factor-Alpha-Induced Suppression of Osteogenic Differentiation of Dental Pulp Stem Cells and Underlying Mechanism.

High doses of tumor necrosis factor-α (TNF-α) suppress osteogenic differentiation of human dental pulp stem cells (hDPSCs). In the present study, we aimed to explore the role and potential regulatory mechanism of microRNA-138 (miR-138) in the osteogenic differentiation of hDPSCs after treatment with a high dose of TNF-α. The hDPSCs were cultured in osteogenic medium with or without 50 ng/ml TNF-α. The miR-138 levels were upregulated during osteogenic differentiation of the hDPSCs following TNF-α treatment. The miR-138 overexpression accelerated but miR-138 knockdown alleviated the TNF-α-induced suppression of the alkaline phosphatase activity, calcium deposition, and protein abundance of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and osteopontin during osteogenic differentiation induction of hDPSCs. Additionally, miR-138 overexpression accelerated but miR-138 knockdown alleviated the suppression of the focal adhesion kinase- (FAK-) extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway during osteogenic differentiation induction of hDPSCs under TNF-α treatment. In conclusion, miR-138 accelerates TNF-α-induced suppression of osteogenic differentiation of hDPSCs. Inactivation of the FAK-ERK1/2 signaling pathway may be one of the mechanisms underlying the effect of miR-138. Inhibition of miR-138 expression may be a strategy to weaken the inhibitory effect of high-dose TNF-α on the osteogenic differentiation of hDPSCs.

Downregulated microRNA-140-5p expression regulates apoptosis, migration and invasion of lung cancer cells by targeting zinc finger protein 800.

Despite advances in the diagnosis and treatment in recent years, lung cancer is still one of the primary causes of cancer-associated morbidity and mortality in globally. Abnormally expressed microRNAs (miRNAs/miRs) in tumor tissues serve vital roles in the pathological mechanism of tumors and have become prospective biomarkers for cancer diagnosis. The present study aimed to investigate the effects of the miR-140-5p/zinc finger protein 800 (ZNF800) axis in lung carcinoma, and determine its potential underlying molecular mechanisms. The degree of cell proliferation was assessed via the MTT assay, while the migratory and invasive abilities of lung cancer cells were determined via the Transwell and Matrigel assays. The expression levels of miR-140-5p and ZNF800 were detected via reverse transcription-quantitative PCR and western blot analyses. The results demonstrated that miR-140-5p expression was notably higher in normal human bronchial epithelial cells compared with the respective lung cancer cell lines, H292, PC-9, CL1-5 and H460. Furthermore, miR-140-5p expression increased in the lung cancer cells compared with the control cells following transfection with miR-140-5p mimic. Overexpressing miR-140-5p significantly suppressed the proliferative, invasive and migratory abilities of H460 and PC-9 cells, and stimulated cell apoptosis by upregulating the expression of cleaved-caspase-3. Notably, these effects were reversed following transfection with miR-140-5p inhibitor. miR-140-5p was predicted as a negative regulator of ZNF800, and ZNF800 knockdown significantly suppressed the proliferative and metastatic abilities of lung adenocarcinoma (LUAD) cells, which was comparable to the effects of miR-140-5p mimic. Taken together, these results suggest that miR-140-5p may block the malignant phenotype of LUAD by negatively regulating ZNF800 expression. Thus, the miR-140-5p/ZNF800 axis may be used as an alternative therapeutic target for lung carcinoma in general, and LUAD in particular.

Dissecting MicroRNA-mRNA Regulatory Networks Underlying Sulfur Assimilation and Cadmium Accumulation in Poplar Leaves.

The process of cadmium (Cd) accumulation and detoxification under different sulfur levels remains largely unknown in woody plants. To investigate the physiological and transcriptomic regulation mechanisms of poplars in response to different sulfate (S) supply levels and Cd exposure, we exposed Populus deltoides saplings to one of the low, moderate and high S levels together with either 0 or 50 µM Cd. Cd accumulation was decreased in low S-treated poplar leaves, and it tended to be increased in high S-supplied leaves under the Cd exposure condition. Sulfur nutrition was deficient in low S-supplied poplars, and it was improved in high S-treated leaves. Cd exposure resulted in lower sulfur level in the leaves supplied with moderate S, it exacerbated a Cd-induced sulfur decrease in low S-treated leaves and it caused a higher sulfur concentration in high S-supplied leaves. In line with the physiological changes, a number of mRNAs and microRNAs (miRNAs) involved in Cd accumulation and sulfur assimilation were identified and the miRNA-mRNA networks were dissected. In the networks, miR395 and miR399 members were identified as hub miRNAs and their targets were ATP sulfurylase 3 (ATPS3) and phosphate 2 (PHO2), respectively. These results suggest that Cd accumulation and sulfur assimilation are constrained by low and enhanced by high S supply, and Cd toxicity is aggravated by low and relieved by high S in poplar leaves, and that miRNA-mRNA regulatory networks play pivotal roles in sulfur-mediated Cd accumulation and detoxification in Cd-exposed poplars.

Sanbai Melon Seed Oil Exerts Its Protective Effects in a Diabetes Mellitus Model via the Akt/GSK-3ß/Nrf2 Pathway.

Traditional Chinese medicine (TCM) plays an important role in the treatment of type 2 diabetes mellitus (T2DM). However, the lack of adequate and scientifically rigorous evidence has limited its application in this disorder. Sanbai melon seed oil (SMSO) is used in folk medicine to treat DM; however, only few literature reports exist regarding its mechanism. Herein, we aimed to confirm the antidiabetic activity of SMSO in a T2DM model and further elucidate its possible mechanisms. The T2DM rat model was induced by high-fat and sugar diet and streptozocin (STZ, 40 mg/kg). SMSO was administered at doses of 0.7 g/kg, 1.4 g/kg, and 2.8 g/kg. Several biochemical parameters and antioxidant protein levels were measured to evaluate the hyperglycemic and antioxidant activities of SMSO. Western blotting was performed to determine its potential mechanism. Based on the results, SMSO treatment significantly reduced blood glucose levels, increased plasma insulin, and repaired islet tissue injury in diabetic rats (P < 0.05). To add, it markedly reduced MDA levels and increased that of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Western blot results showed that SMSO induced n-Nrf2 and HO-1 expression and Akt and GSK-3ß phosphorylation in a dose-dependent manner. Further studies showed that LY294002, aPI3K inhibitor, abolished the effects of SMSO on GSK-3ß phosphorylation and Nrf2 nuclear translocation as well as the protective effects on pancreatic ß cells. Together, these results suggest that SMSO regulates the Akt/GSK-3ß/Nrf2 pathway and induces the expression of antioxidant proteins to impede oxidative stress in rats with T2DM.

The ethylene receptor regulates Typha angustifolia leaf aerenchyma morphogenesis and cell fate.

MAIN CONCLUSION: Ethylene receptor is crucial for PCD and aerenchyma formation in Typha angustifolia leaves. Not only does it receive and deliver the ethylene signal, but it probably can determine the cell fate during aerenchyma morphogenesis, which is due to the receptor expression quantity. Aquatic plant oxygen delivery relies on aerenchyma, which is formed by a programmed cell death (PCD) procedure. However, cells in the outer edge of the aerenchyma (palisade cells and septum cells) remain intact, and the mechanism is unclear. Here, we offer a hypothesis: cells that have a higher content of ethylene receptors do not undergo PCD. In this study, we investigated the leaf aerenchyma of the aquatic plant Typha angustifolia. Ethephon and pyrazinamide (PZA, an inhibitor of ACC oxidase) were used to confirm that ethylene is an essential hormone for PCD of leaf aerenchyma cells in T. angustifolia. That the ethylene receptor was an indispensable factor in this PCD was confirmed by 1-MCP (an inhibitor of the ethylene receptor) treatment. Although PCD can be avoided by blocking the ethylene receptor, excessive ethylene receptors also protect cells from PCD. TaETR1, TaETR2 and TaEIN4 in the T. angustifolia leaf were detected by immunofluorescence (IF) using polyclonal antibodies. The result showed that the content of ethylene receptors in PCD-unsusceptible cells was 4-14 times higher than that one in PCD-susceptible cells, suggesting that PCD-susceptible cells undergo the PCD programme, while PCD-unsusceptible cells do not due to the content difference in the ethylene receptor in different cells. A higher level of ethylene receptor content makes the cells insensitive to ethylene, thereby avoiding cell death and degradation.

Abscisic acid enhances lead translocation from the roots to the leaves and alleviates its toxicity in Populus × canescens.

To shed light on physiological mechanisms underlying abscisic-acid (ABA)-mediated lead (Pb) uptake, translocation and detoxification, we exposed Populus × canescens saplings to either 0 or 3 mM Pb2+ in combination with either 0 or 10 µM exogenous ABA. Pb was taken up by the roots and accumulated mainly in the cortex. A fraction of the Pb in the roots was translocated to the leaves, thereby resulting in decreased photosynthesis and biomass. Pb accumulation caused a burst of reactive oxygen species (ROS), with higher concentrations of total thiols, glutathione, and ascorbate in the roots and/or leaves. Exogenous ABA stimulated Pb uptake, decreased Pb deposition in the cortex, and enhanced Pb vascular loading in the roots. Exogenous ABA alleviated the Pb-induced reductions in photosynthesis and root biomass, and decreased Pb-triggered ROS overproduction in the roots and/or leaves. Correspondingly, exogenous ABA stimulated the mRNA levels of a few genes involved in Pb uptake, transport, and detoxification, including NRAMP1.4, ABCG40, FRD3.1, PCS1.1, and ABCC1.1. These results suggest that exogenous ABA enhances Pb uptake and translocation, and alleviates Pb toxicity in poplars through the ABA-induced movement of Pb from the root cortex to the vascular stele, and transcriptionally regulated key genes involved in Pb tolerance.

[Trace Amounts of Phosphorus Removal Based on the in-suit Oxidation Products of Iron or Manganese in a Biofilter].

In order to remove trace amounts of phosphorus from water bodies, a lab-scale biofilter was constructed to investigate the capacity of in situ oxidation products of iron or manganese for phosphorus adsorption. SEM, EDS, BET, and zeta technologies were employed to reveal the adsorption mechanisms. The results indicated that phosphorus could be removed by the oxide products generated from the iron or manganese removal process, at 106.28 µg·mg-1 and 77.98 µg·mg-1, respectively, as shown by the linear relationships between phosphorus removal and the two oxides. SEM, EDS, and BET analysis demonstrated that the BET specific surface areas for the iron- and manganese-rich oxides were 96 m2·g-1 and 67 m2·g-1, respectively, with the former accumulated between the pore spaces of the filtering sand and easily washed out of the layer by backwashing, whereas the latter coated the surface of the filtering sand. Thus, backwashing was favorable for phosphorus adsorption in the iron oxidation process to avoid overaccumulation. Moreover, the zero point of charge of the two oxides indicated electrostatic attraction may have occurred between iron-rich oxide and phosphorus; however, inner-sphere complex reactions obviously occurred for the two oxides because the zero point of charge after phosphorus adsorption decreased to a lower level. In addition, other anions were negatively complexed with the phosphorus on the surface of the oxides, it demonstrated that phosphorus adsorption on the surface of the two oxides seemed to be a specific adsorption.

Programmed Cell Death and Aerenchyma Formation in Water-Logged Sunflower Stems and Its Promotion by Ethylene and ROS.

Previous studies have shown that waterlogging/ hypoxic conditions induce aerenchyma formation to facilitate gas exchange. Ethylene (ET) and reactive oxygen species (ROS), as regulatory signals, might also be involved in these adaptive responses. However, the interrelationships between these signals have seldom been reported. Herein, we showed that programmed cell death (PCD) was involved in aerenchyma formation in the stem of Helianthus annuus. Lysigenous aerenchyma formation in the stem was induced through waterlogging (WA), ethylene and ROS. Pre-treatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) partially suppressed aerenchyma formation in the seedlings after treatment with WA, ET and 3-amino-1, 2, 4-triazole (AT, catalase inhibitor). In addition, pre-treatment with the ethylene perception inhibitor 1-methylcyclopropene (1-MCP) partially suppressed aerenchyma formation induced through WA and ET in the seedlings, but barely inhibited aerenchyma formation induced through ROS. These results revealed that ethylene-mediated ROS signaling plays a role in aerenchyma formation, and there is a causal and interdependent relationship during WA, ET and ROS in PCD, which regulates signal networks in the stem of H. annuus.

Direct Measurement of Single-Molecule Adenosine Triphosphatase Hydrolysis Dynamics.

F1-ATPase (F1) is a bidirectional molecular motor that hydrolyzes nearly all ATPs to fuel the cellular processes. Optical observation of labeled F1 rotation against the α3ß3 hexamer ring revealed the sequential mechanical rotation steps corresponding to ATP binding/ADP release and ATP hydrolysis/Pi release. These substeps originate from the F1 rotation but with heavy load on the γ shaft due to fluorescent labeling and the photophysical limitation of an optical microscope, which hampers better understanding of the intrinsic kinetic behavior of ATP hydrolysis. In this work, we present a method capable of electrically monitoring ATP hydrolysis of a single label-free F1 in real time by using a high-gain silicon nanowire-based field-effect transistor circuit. We reproducibly observe the regular current signal fluctuations with two distinct levels, which are induced by the binding dwell and the catalytic dwell, respectively, in both concentration- and temperature-dependent experiments. In comparison with labeled F1, the hydrolysis rate of nonlabeled F1 used in this study is 1 order of magnitude faster (1.69 × 108 M-1 s-1 at 20 °C), and the differences between two sequential catalytic rates are clearer, demonstrating the ability of nanowire nanocircuits to directly probe the intrinsic dynamic processes of the biological activities with single-molecule/single-event sensitivity. This approach is complementary to traditional optical methods, offering endless opportunities to unravel molecular mechanisms of a variety of dynamic biosystems under realistic physiological conditions.

Exogenous glutathione enhances cadmium accumulation and alleviates its toxicity in Populus × canescens.

Glutathione (GSH) plays an important role in cadmium (Cd) tolerance in woody plants, but the underlying mechanisms remain largely unknown. To elucidate the physiological and transcriptional regulation mechanisms of GSH-mediated Cd tolerance in woody plants, we exposed Populus × canescens (Ait.) Smith saplings to either 0 or 75 µM Cd together with one of three external GSH levels. Glutathione treatments include buthionine sulfoximine (BSO, an inhibitor of GSH biosynthesis), no external GSH and exogenous GSH. External GSH resulted in higher Cd2+ uptake rate in the roots, greater Cd amount in poplars, lower Cd-induced H2O2 levels in the roots, and higher contents of endogenous GSH in Cd-treated roots and leaves. Furthermore, external GSH led to upregulated transcript levels of several genes including zinc/iron regulated transporter related protein 6.2 (ZIP6.2) and natural resistance-associated macrophage protein 1.3 (NRAMP1.3), which probably take part in Cd uptake, glutathione synthetase 2 (GS2) implicated in Cd detoxification, metal tolerance protein 1 (MTP1) and ATP-binding cassette transporter C3 (ABCC3) involved in Cd vacuolar accumulation in the roots, γ-glutamylcysteine synthetase (ECS) and phytochelatin synthetase family protein 1 (PCS1) involved in Cd detoxification, and oligopeptide transporter 7 (OPT7) probably implicated in Cd detoxification in the leaves of Cd-exposed P. × canescens. In contrast, BSO often displayed the opposite effects on Cd-triggered physiological and transcriptional regulation responses in poplars. These results suggest that exogenous GSH can enhance Cd accumulation and alleviate its toxicity in poplars. This is probably attributed to external-GSH-induced higher net Cd2+ influx in the roots, greater Cd accumulation in aerial parts, stronger scavenging of reactive oxygen species, and transcriptional overexpression of several genes involved in Cd uptake, detoxification and accumulation.

Root Canal Surface Strain and Canal Center Transportation Induced by 3 Different Nickel-Titanium Rotary Instrument Systems.

INTRODUCTION: Root canal anatomy and canal preparation instruments affect the outcome of endodontic treatment. The purpose of this study was to determine the root surface strain (SS) generated and the extent of canal center transportation during canal shaping using 3 different nickel-titanium instruments. METHODS: Simulated root canals in resin blocks (n = 10 per group) were prepared using adaptive rotary motion with twisted files (Twisted File Adaptive [TFA]; SybronEndo, Orange, CA), reciprocating rotary motion with WaveOne (WO; Dentsply Maillefer, Ballaigues, Switzerland) files, and continuous rotary motion with ProTaper Next files (PTN, Dentsply Maillefer). Electrical strain gauges at 3 sites recorded SS real time during canal shaping, and the blocks were scanned by micro-computed tomographic imaging to assess the canal center deviation at 3 sections after root canal instrumentation. The mean maximum SS and the canal center transportation for all groups and sites were derived and analyzed for a possible correlation between them. RESULTS: An overall increase in root SS was observed after root canal instrumentation. A significant difference in the induced mean maximum SS between TFA, WO, and PTN at specific sites of curved root canals was observed. A statistically significant difference in the mean distance of canal center transportation was observed among the 3 shaping techniques at the apical section. Finally, the mean maximum SS values induced during canal shaping strongly correlated with canal center transportation in the apical section and the coronal section. CONCLUSIONS: The curved canals prepared using TFA exhibited lower SS and less canal center transportation at the apical section than the WO and PTN systems. SS generated during canal shaping correlated with canal center transportation in a site-specific manner.

Endoglin-mediated suppression of prostate cancer invasion is regulated by activin and bone morphogenetic protein type II receptors.

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor ß (TGFß) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFß receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFß receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

Functions of lipopeptides bacillomycin D and fengycin in antagonism of Bacillus amyloliquefaciens C06 towards Monilinia fructicola.

In previous studies, Bacillus amyloliquefaciens C06 has been proven to be effective in controlling brown rot of stone fruit caused by Monilinia fructicola. When tested in vitro, cell-free filtrate of B. amyloliquefaciens C06 significantly inhibited mycelial growth and conidial germination of the fungal pathogen. This study aimed to determine the role of the antifungal compound(s) in the cell-free filtrate of B. amyloliquefaciens C06 by an approach combining a DNA-based suppression subtractive hybridization (SSH) method with MALDI-TOF-MS analysis. It was demonstrated that B. amyloliquefaciens C06 harbored two genes, bmyC and fenD, involved in biosynthesis of bacillomycin D and fengycin, two lipopeptides belonging to the iturin and fengycin family, respectively. To determine the roles of bacillomycin D and fengycin of B. amyloliquefaciens C06 in suppressing M. fructicola, the mutants of B. amyloliquefaciens C06 deficient in producing bacillomy- cin D, fengycin or both were constructed, and evaluated in vitro together with the wild-type B. amyloliquefaciens C06. The results indicated that bacillomycin D and fengycin jointly contributed to the inhibition of conidial germination of M. fructicola, and fengycin played a major role in suppressing mycelial growth of the fungal pathogen.

PDC1, a corn defensin peptide expressed in Escherichia coli and Pichia pastoris inhibits growth of Fusarium graminearum.

Plant defensin corn 1 (Pdc1) gene was amplified from corn genomic DNA with the primers designed from a corn EST sequence homologous to a barley defensin gene. The cloned gene contains two exons and an intron. The deduced 9KDa PDC1 peptide has a sequence that is identical to corn gamma2-zeathionin and has the typical features of a plant defensin, including a signal sequence of 35 amino acids, followed by a characteristic defensin domain of 47 amino acids containing 8 cysteines. The defensin protein was expressed from the cloned cDNA introduced into two different expression systems; prokaryotic, Escherichia coli and eukaryotic, yeast (Pichia pastoris). The PDC1 protein was purified with a nickel resin column and was tested for its antifungal activities using the pathogen Fusarium graminearum. The protein expressed in both E. coli and P. pastoris had antifungal activity, however the protein expressed in P. pastoris was more efficient in inhibiting growth of F. graminearum. FTIR analysis of PDC1 protein expressed in the two expression systems showed that expression in P. pastoris gave a product with more beta-sheets and less random unordered structure than when it was expressed in E. coli. In addition, removal of the His-tag used for purification increased the fungicidal activity of the PDC1 protein. The data presented here suggest that the defensin PDC1 peptide of corn could be effectively used to restrict the disease caused by F. graminearum.