Quantification of soybean seed germination response to seed deterioration under PEG-induced water stress using hydrotime concept

This experiment was arranged to investigate the ability of hydrotime model (θ_H) for estimating soybean seed germination (cv. ‘JK’) under different accelerated aging periods (AAP, 0, 24, 48, and 72 h) at each of the following water potentials (ψ, 0, ??0.12, ??0.24, and ??0.36 MPa). Results indicated that both germination percentage (GP) and germination rate (GR) significantly influenced by ψ, AAP, and their interactions (P?<?0.01). GP and GR decreased by 62.6 and 47.3% with longer AAP from 0 to 72 h and by 90.7 and 81.5% with lower ψ from zero to ??0.36 MPa as compared to the control, respectively. Therefore, the effect of ψ on GP and GR was more than AAP. The θ_H value was constant (~?6.71 MPa h^?1) till 50.6 h AAP and then linearly declined with the rate of 0.1545 MPa h^?1 per hour increase in AAP until 72 h (~?50% lower than its initial value). The ψ_b(50) value was ? 0.343 MPa in the control and then increased (became more positive) by ~?70% until 72 h AAP (? 0.104 MPa). In general, GP and GR of soybean declined with the increasing ψ_b(50) which can be due to cell membrane damage and reduce the activity of enzymes and organelles during AAP. Based on our findings, the θ_H model could describe well these relationships and their parameters can nicely be used for simulating soybean seed germination under this condition.     

High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria

Tungstate (W) is recognized as an agent of environmental pollution and a substitute to depleted uranium. According to some preliminary studies, tungstate toxicity is related to the formation of reactive oxygen species (ROS) under abnormal pathological conditions. The kidneys and liver are the main tungstate accumulation sites and important targets of tungstate toxicity. Since the mitochondrion is the main ROS production site, we evaluated the mechanistic toxicity of tungstate in isolated mitochondria for the first time, following a two‐step ultracentrifugation method. Our findings demonstrated that tungstate‐induced mitochondrial dysfunction is related to the increased formation of ROS, lipid peroxidation, and potential membrane collapse, correlated with the amelioration of adenosine triphosphate and glutathione contents. The present study indicated that mitochondrial dysfunction was associated with disruptive effects on the mitochondrial respiratory chain and opening of mitochondrial permeability transition (MPT) pores, which is correlated with cytochrome c release. Our findings suggest that high concentrations of tungstate (2 mM)‐favored MPT pore opening in the inner membranes of liver and kidney mitochondria of rats. Besides, the results indicated higher tungstate susceptibility in the kidneys, compared with the liver.     

TGF-alpha regulates TLR expression and function on epidermal keratinocytes

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-alpha, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-alpha regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human beta-defensins. These findings provide evidence that TGF-alpha up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.     

Simple benzene derivatives adsorption on defective single-walled carbon nanotubes: a first-principles van der Waals density functional study

We have investigated the interaction between open-ended zig-zag single-walled carbon nanotube (SWCNT) and a few benzene derivatives using the first-principles van der Waals density functional (vdW-DF) method, involving full geometry optimization. Such sp ²-like materials are typically investigated using conventional DFT methods, which significantly underestimate non-local dispersion forces (vdW interactions), therefore affecting interactions between respected molecules. Here, we considered the vdW forces for the interacting molecules that originate from the interacting π electrons of the two systems. The ?0.54 eV adsorption energy reveals that the interaction of benzene with the side wall of the SWCNT is typical of the strong physisorption and comparable with the experimental value for benzene adsorption onto the graphene sheet. It was found that aromatics are physisorbed on the sidewall of perfect SWCNTs, as well as at the edge site of the defective nanotube. Analysis of the electronic structures shows that no orbital hybridization between aromatics and nanotubes occurs in the adsorption process. The results are relevant in order to identify the potential applications of noncovalent functionalized systems.

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Taxadien‐5α‐hydroxylase and taxadien‐5α‐ol O‐acetyltransferase catalyze the oxidation of taxadiene to taxadien‐5α‐ol and subsequent acetylation to taxadien‐5α‐yl‐acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High‐throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien‐5α‐ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography‐mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien‐5α‐yl‐acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7‐fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.     

Dopamine and cAMP‐regulated phosphoprotein 32kDa (DARPP‐32), protein phosphatase‐1 and cyclin‐dependent kinase 5 expression in ovarian cancer

Dopamine and cyclic‐AMP activated phosphoprotein Mr32kDa (DARPP‐32) is a central signalling protein in neurotransmission. Following DARPP‐32 phosphorylation by protein kinase A (PKA), DARPP‐32 becomes a potent protein phosphatase 1 (PP1) inhibitor. DARPP‐32 can itself inhibit PKA following DARPP‐32 phosphorylation by cyclin‐dependent kinase 5 (Cdk5). Increasing evidence indicates a role for DARPP‐32 and its associated signalling pathways in cancer; however, its role in ovarian cancer remains unclear. Using immunohistochemistry, expression of DARPP‐32, PP1 and Cdk5 was determined in a large cohort of primary tumours from ovarian cancer patients (n = 428, 445 and 434 respectively) to evaluate associations between clinical outcome and clinicopathological criteria. Low cytoplasmic and nuclear DARPP‐32 expression was associated with shorter patient overall survival and progression‐free survival (P = .001, .001, .004 and .037 respectively). Low nuclear and cytoplasmic DARPP‐32 expression remained significantly associated with overall survival in multivariate Cox regression (P = .045, hazard ratio (HR) = 0.734, 95% confidence interval (CI) = 0.542‐0.993 and P = .001, HR = 0.494, 95% CI = 0.325‐0.749, respectively). High cytoplasmic and nuclear PP1 expression was associated with shorter patient overall survival and high cytoplasmic PP1 expression with shorter progression‐free survival (P = .005, .033, and .037, respectively). High Cdk5 expression was associated with shorter progression‐free survival (P = .006). These data suggest a role for DARPP‐32 and associated signalling kinases as prognostic markers with clinical utility in ovarian cancer.     

Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.     

A mechanistic insight into SMAC peptide interference with XIAP-Bir2 inhibition of executioner caspases

X-linked inhibitor of apoptosis protein (XIAP) inhibits apoptosis mainly through inhibition of caspase-9 and executioner caspases of -3 and -7. The inhibition of the former protease is implemented through the bacculoviral inhibitory repeat-3 (Bir3) domain, while the inhibition of the latter is accomplished by the interaction of the linker region located between the Bir1 and the Bir2 domains with their active sites. Both modes of inhibition are antagonized by SMAC, which is released from mitochondria during the initiation of the intrinsic apoptosis pathway. Although the mechanism of SMAC interference in Bir3 inhibition of caspase-9 is clearly established, the mechanism by which SMAC interferes with the inhibition of the executioner caspases by XIAP remains largely unknown. To address this issue, we performed a limited proteolysis of glutathione S-transferase (GST)-tagged XIAP-Bir2 by trypsin in the presence and in the absence of SMAC peptide. Under these conditions, the proteolysis of the linker region was diminished considerably. Furthermore, the rate of association of caspase-3 and -7 with XIAP in the presence of the SMAC peptide was reduced drastically, suggesting that SMAC peptide restricts the exposure of the linker region. A limited proteolysis of caspase-7 in the presence of GST-Bir2 and GST-NBir3 (the Bir3 domain of human NAIP) as negative controls was also performed. Matrix-assisted laser desorption/ionization time-of-flight analysis of the fragments revealed the identity of protected sites, suggesting that the Bir2 domain makes numerous contacts with the large subunit of caspase-7. These, combined with the results from Far-Western experiments, strongly suggest that the groove for the inhibitor(s)-of-apoptosis-protein-binding motif on the Bir2 favors binding to the N-terminus of the large subunit rather than to the small subunit of caspase-7. Our results further show that the active-site pocket of caspase-7 is first occupied by the linker region, followed by the interaction of the N-terminus of the enzyme with the SMAC-binding site of the Bir2 domain.