PDK1 is important lipid kinase for RANKL-induced osteoclast formation and function via the regulation of the Akt-GSK3ß-NFATc1 signaling cascade.

Perturbations in the balanced process of osteoblast-mediated bone formation and osteoclast-mediated bone resorption leading to excessive osteoclast formation and/or activity is the cause of many pathological bone conditions such as osteoporosis. The osteoclast is the only cell in the body capable of resorbing and degrading the mineralized bone matrix. Osteoclast formation from monocytic precursors is governed by the actions of two key cytokines macrophage-colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Binding of RANKL binding to receptor RANK initiates a series of downstream signaling responses leading to monocytic cell differentiation and fusion, and subsequent mature osteoclast bone resorption and survival. The phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) signaling cascade is one such pathway activated in response to RANKL. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), is considered the master upstream lipid kinase of the PI3K-Akt cascade. PDK1 functions to phosphorylate and partially activate Akt, triggering the activation of downstream effectors. However, the role of PDK1 in osteoclasts has yet to be clearly defined. In this study, we specifically deleted the PDK1 gene in osteoclasts using the cathepsin-K promoter driven Cre-LoxP system. We found that the specific genetic ablation of PDK1 in osteoclasts leads to an osteoclast-poor osteopetrotic phenotype in mice. In vitro cellular assays further confirmed the impairment of osteoclast formation in response to RANKL by PDK1-deficient bone marrow macrophage (BMM) precursor cells. PDK1-deficient BMMs exhibited reduced ability to reorganize actin cytoskeleton to form a podosomal actin belt as a result of diminished capacity to fuse into giant multinucleated osteoclasts. Notably, biochemical analyses showed that PDK1 deficiency attenuated the phosphorylation of Akt and downstream effector GSK3ß, and reduced induction of NFATc1. GSK3ß is a reported negative regulator of NFATc1. GSK3ß activity is inhibited by Akt-dependent phosphorylation. Thus, our data provide clear genetic and mechanistic insights into the important role for PDK1 in osteoclasts.

miR-136-5p Regulates the Inflammatory Response by Targeting the IKKß/NF-κB/A20 Pathway After Spinal Cord Injury.

BACKGROUND/AIMS: miR-136-5p participates in recovery after spinal cord injury (SCI) via an unknown mechanism. We investigated the mechanism underlying the involvement of miR-136-5p in the inflammatory response in a rat model of SCI. METHODS: Sprague-Dawley rat astrocytes were cultured in vitro to construct a reporter plasmid. Luciferase assays were used to detect the ability of miR-136-5p to target the IKKß and A20 genes. Next, recombinant lentiviral vectors were constructed, which either overexpressed miR-136-5p or inhibited its expression. The influence of miR-136-5p overexpression and miR-136-5p silencing on inflammation was observed in vivo in an SCI rat model. The expression of IL-1ß, IL-6, TNF-α, IFN-α, and related proteins (A20, IKKß, and NF-κB) was detected. RESULTS: In vitro studies showed that luciferase activity was significantly activated in the presence of the 3' untranslated region (UTR) region of the IKKß gene after stimulation of cells with miR-136-5p. However, luciferase activity was significantly inhibited in the presence of the 3'UTR region of the A20 gene. Thus, miR-136-5p may act directly on the 3'UTR regions of the IKKß and A20 genes to regulate their expression. miR-136-5p overexpression promoted the production of related cytokines and NF-κB in SCI rats and inhibited the expression of A20 protein. CONCLUSION: Overexpression of miR-136-5p promotes the generation of IL-1ß, IL-6, TNF-α, IFN-α, IKKß, and NF-κB in SCI rats but inhibits the expression of A20. Under these conditions, inflammatory cell infiltration into the rat spinal cord increases and injury is significantly aggravated. Silencing of miR-136-5p significantly reduces the protein expression results described after miR-136-5p overexpression and ameliorates the inflammatory cell infiltration and damage to the spinal cord. Therefore, miR-136-5p might be a new target for the treatment of SCI.

Effect of OH-/Al3+ and Si/Al molar ratios on the coagulation performance and residual Al speciation during surface water treatment with poly-aluminum-silicate-chloride (PASiC).

Coagulation performance, mechanism of poly-aluminum-silicate-chloride (PASiC) and residual Al speciation in the effluent with respect to a specific surface water treatment in China were comprehensively investigated in this study. The impact of OH(-)/Al(3+) and Si/Al molar ratios on the coagulation performance, mechanism and residual Al speciation of PASiC in surface water treatment was discussed as a function of coagulant dosage. It was intended to provide an insight into the relationship between coagulation performance and residual Al. Experimental results revealed that when OH(-)/Al(3+) molar ratio = 2.00 and Si/Al molar ratio = 0.0500 in PASiC coagulant, PASiC exhibited beneficial coagulation property and relatively lower content of residual Al. Surface bridging and entrapment was more effective compared with charge neutralization during the specific surface water treatment. The majority of residual Al in the effluent existed in the form of insoluble suspended or particulate Al. Dissolved organically bound Al was almost the major speciation in dissolved Al and dissolved inorganically bound monomeric Al was the only component in dissolved monomeric Al. Al in PASiC remained abundant at lower dosages and residual Al concentration could be effectively reduced at the dosages of 12.0-15.0mg/L as Al.

[Treatment of simulated produced wastewater from polymer flooding in oil production using dithiocarbamate-type flocculant].

A dithiocarbamate flocculant, DTC (T403), was prepared by the reaction of amine-terminated polyoxypropane-ether compound known as Jeffamine-T403 and carbon disulfide in alkaline solution. The oil removal efficiency of DTC (T403) for simulated produced wastewater from polymer flooding in oil production was studied by Jar-test. The effect of the dosage of DTC (T403), hydrolyzed polyacrylamide (HPAM), Fe2+ and Fe3+ ions, and pH on the oil removal efficiency of DTC (T403) was investigated. The results showed that the chelate polymer formed by DTC (T403) and Fe2+ ion has good oil removal performance by net capturing mechanism. HPAM had a negative effect on oil removal efficiency of DTC (T403). For the treatment of the simulated wastewater containing 0-900 mg/L of HPAM and 300 mg/L of oil, the residual oil concentrations in water samples decreased below 10 mg/L when the dosage of Fe2+ and DTC (T403) was 10 mg/L and 25 mg/L, respectively. The oil removal efficiency of DTC (T403) was affected by pH and good oil removal efficiency was obtained when the pH was below 7.5. DTC (T403) is appropriate for the treatment of oily wastewater containing Fe2+ ion.