Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate.

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first ß-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.

Postoperative Quality of Life Following Conventional Endodontic Intracanal Irrigation Compared with Laser-Activated Irrigation: A Randomized Clinical Study.

Objective: The aim of this randomized clinical study was to evaluate the effect of laser-activated irrigation using a photon-induced photoacoustic streaming (PIPS) technique on postoperative pain following completion of root canal obturation. Methods: Fifty-six patients were enrolled in this randomized clinical trial. Fifty-six healthy premolars or molars with asymptomatic irreversible pulpitis, symptomatic irreversible pulpitis, or symptomatic pulpal necrosis, with or without apical periodontitis, were mechanically prepared for endodontic treatment and divided into two groups. Patients were randomly allocated to treatment groups. In the positive control group G1, the final irrigation with 2 cc of 5.25% sodium hypochlorite (NaOCl) was achieved using a 27G needle, introduced into the canal to a distance of 5 mm from the predetermined working length. In the experimental group G2, the root canals were irrigated with 17% ethyldiamine tetric acid (EDTA) and 5.25% NaOCl following the PIPS protocol, using an Er:YAG 2940 nm laser (LightWalker ATS®; Fotona, Slovenia) with a 600 µm diameter tip and operating parameters of 20 mJ per pulse, 15 Hz frequency, 0.3 W average power, and a 50-µs pulse duration. Postoperatively, the patients were advised to take a minor analgesic (ibuprofen 400 mg) in the event of pain perception. Postoperative pain levels were assessed after 24, 48, and 72 h and 7 days through the use of a Visual Analogue Scale questionnaire, completed by each patient. Data were analyzed using Kolmogorov-Smirnov, Fisher Exact, Chi square, Mann-Whitney test, and Friedman's test. The level of significance was set at α = 0.05. Results: There was no significant difference between the laser-irradiated group and the control group (p < 0.5). Laser activation of irrigating solutions did not increase postoperative pain. Conclusions: The outcome of this investigation indicated that PIPS was as effective as conventional irrigation in relation to postoperative pain, making this activation technique interesting to use for supplementary root canal disinfection.

Evaluating the abnormal ossification in tibiotarsi of developing chick embryos exposed to 1.0ppm doses of platinum group metals by spectroscopic techniques.

Platinum group metals (PGMs), i.e., palladium (Pd), platinum (Pt) and rhodium (Rh), are found at pollutant levels in the environment and are known to accumulate in plant and animal tissues. However, little is known about PGM toxicity. Our previous studies showed that chick embryos exposed to PGM concentrations of 1mL of 5.0ppm (LD50) and higher exhibited severe skeletal deformities. This work hypothesized that 1.0ppm doses of PGMs will negatively impact the mineralization process in tibiotarsi. One milliliter of 1.0ppm of Pd(II), Pt(IV), Rh(III) aqueous salt solutions and a PGM-mixture were injected into the air sac on the 7th and 14th day of incubation. Control groups with no-injection and vehicle injections were included. On the 20th day, embryos were sacrificed to analyze the PGM effects on tibiotarsi using four spectroscopic techniques. 1) Micro-Raman imaging: Hyperspectral Raman data were collected on paraffin embedded cross-sections of tibiotarsi, and processed using in-house-written MATLAB codes. Micro-Raman univariate images that were created from the ν1(PO4(3-)) integrated areas revealed anomalous mineral inclusions within the bone marrow for the PGM-mixture treatment. The age of the mineral crystals (ν(CO3(2-))/ν1(PO4(3-))) was statistically lower for all treatments when compared to controls (p≤0.05). 2) FAAS: The percent calcium content of the chemically digested tibiotarsi in the Pd and Pt groups changed by ~45% with respect to the no-injection control (16.1±0.2%). 3) Micro-XRF imaging: Abnormal calcium and phosphorus inclusions were found within the inner longitudinal sections of tibiotarsi for the PGM-mixture treatment. A clear increase in the mineral content was observed for the outer sections of the Pd treatment. 4) ICP-OES: PGM concentrations in tibiotarsi were undetectable (<5ppb). The spectroscopic techniques gave corroborating results, confirmed the hypothesis, and explained the observed pathological (skeletal developmental abnormalities) and histological changes (tibiotarsus ischemia and nuclear fragmentation in chondrocytes).