Hybrid simultaneous laser- and ultrasonic-assisted machining of Ti-6Al-4V alloy

The machinability of Ti-6Al-4V alloy has been a constant challenge in the industry, although the material is widely used in the aerospace and medical industries due to its mechanical properties, particularly its strength-to-weight ratio. The current research presents a hybrid laser- and ultrasonic-assisted machining (LUAM) technique to improve the machinability of Ti-6Al-4V alloy in a turning process. This is compared with ultrasonic-assisted machining (UAM), laser-assisted machining (LAM), and convectional turning (CT). The results reveal that UAM and LAM can reduce the cutting forces and surface roughness (Ra) compared to the CT. However, these are achieved mainly at the lowest range of cutting speeds. The hybrid LUAM process demonstrates process improvement with wider range of cutting speeds and depths of cut, which is achieved due to the combined force reduction and thermal softening effect by the hybrid process.</p

Structure of PDI in the absence or presence of CTA1.

<p>Curve fitting (left panels) and second derivatives (right panels) for the FTIR spectrum of PDI recorded in the absence (<b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>) or presence (<b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>) of ¹³C-labeled CTA1 are shown. For curve fitting, the dotted line represents the sum of all deconvoluted components (solid lines) from the measured spectrum (dashed line). Unless otherwise noted, all experiments were performed with sodium borate buffer (pH 7.0) containing 1 mM GSH. (<b>A</b>, <b>B</b>) PDI structure at 10°C. (<b>C</b>, <b>D</b>) PDI structure at 10°C in the absence of reductant. (<b>E</b>, <b>F</b>) PDI structure at 37°C. (<b>G</b>, <b>H</b>) PDI structure at 37°C in pH 6.5 buffer.</p

Refolding of disordered PDI.

<p>PDI was placed at 10°C in sodium borate buffer (pH 7.0) containing ¹³C-labeled CTA1 and 1 mM GSH. The temperature was then raised to 37°C for 60 min. (<b>A</b>) The FTIR spectra of PDI+CTA1 were recorded at 10°C (solid line) and at 37°C (dotted line). (<b>B</b>) Curve fitting (left panel) and second derivatives (right panel) for the FTIR spectrum of PDI at 37°C are shown. For curve fitting, the dotted line represents the sum of all deconvoluted components (solid lines) from the measured spectrum (dashed line).</p

Loss of PDI structure in the presence of CTA1.

<p>Far-UV CD spectra of PDI (green), CTA1 (blue), and both proteins in the same sample at a 1∶1 molar ratio (red) are shown. The black dotted line was obtained by spectral subtraction of the individual CTA1 and PDI spectra from the spectrum of both proteins together. All measurements were taken at 10°C in pH 7.0 buffer containing 1 mM GSH.</p

PDI unfolding but not oxidoreductase activity is required for disassembly of the CT holotoxin.

<p>SPR was used to monitor the real-time PDI-mediated disassembly of CT. A baseline measurement corresponding to the mass of the sensor-bound CT holotoxin established the 0 MicroRIU signal. The time course was then initiated with perfusion of PDI (<b>A</b>), EDC-treated PDI (<b>B</b>), or bacitracin-treated PDI (<b>C</b>) over the CT-coated sensor. The perfusion buffer contained either 30 mM GSH (left panels) or 1 mM GSH (right panels); non-reducing SDS-PAGE with Coomassie staining found the CTA1/CTA2 disulfide bond was reduced at 30 mM GSH but not 1 mM GSH (<i>inset</i>, right panel of <b>A</b>). PDI was removed from the perfusion buffer at time intervals denoted by asterisks and was replaced with sequential additions of anti-PDI, anti-CTA1, and anti-CTB antibodies as indicated by the arrowheads. One of two representative experiments is shown for each condition.</p

The chaperone activity of PDI is required for CT intoxication.

<p>(<b>A</b>) Untreated and ribostamycin-treated CHO cells were challenged with the stated concentrations of CT for 2 hr before cAMP levels were quantified. The averages ± ranges of 2 independent experiments with triplicate samples are shown. (<b>B</b>) CHO cells were transfected with a plasmid encoding a CTA1 construct that is co-translationally inserted into the ER. Dislocation of this CTA1 construct back to the cytosol of untreated or ribostamycin-treated cells was detected by the rise in intracellular cAMP at 4 hr post-transfection. Cells transfected with an empty plasmid (Mock) were used to establish the resting levels of cAMP. Data are presented as the averages ± standard deviations of three replicate samples per condition. One of three representative experiments is shown. (<b>C</b>) CHO cells were pulse-labeled at 4°C with 1 µg/mL of CT. Untreated or drug-treated cells were then chased in toxin-free medium for 2 hr at 37°C. Membrane fractions from digitonin-permeabilized cells were resolved by non-reducing SDS-PAGE and probed by Western blot with an anti-CTA antibody. (<b>D</b>) Untreated or ribostamycin-treated CHO cells were pulse-labeled at 4°C with 1 µg/mL of CT and then chased in toxin-free medium for 2 hr at 37°C. Cytosolic fractions from digitonin-permeabilized cells were then perfused over an SPR sensor coated with an anti-CTA1 monoclonal antibody. Known quantities of CTA were perfused over the sensor as positive controls, and the cytosolic fraction from unintoxicated cells was perfused over the sensor as a negative control. At the end of each perfusion, bound ligand was stripped from the sensor slide.</p

The chaperone activity of PDI is required for disassembly of the CT holotoxin.

<p>(<b>A</b>, <b>B</b>) A baseline SPR measurement corresponding to the mass of the sensor-bound CT holotoxin established the 0 MicroRIU signal. The time course was then initiated with perfusion of S-nitrosylated PDI (<b>A</b>) or ribostamycin-treated PDI (<b>B</b>) over the CT-coated sensor in buffer containing 30 mM GSH. In (<b>B</b>), PDI was removed from the perfusion buffer after 600 sec and replaced with sequential additions of anti-PDI, anti-CTA1, and anti-CTB antibodies as indicated by the arrowheads. One of two representative experiments is shown for each condition. (<b>C</b>, <b>D</b>) Curve fitting (left panels) and second derivatives (right panels) for the FTIR spectrum of ribostamycin-treated PDI recorded in the absence (<b>C</b>) or presence (<b>D</b>) of ¹³C-labeled CTA1 are shown. For curve fitting, the dotted line represents the sum of all deconvoluted components (solid lines) from the measured spectrum (dashed line).</p

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds

<div><p>Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.</p></div

Phenolic compounds do not affect the thermal unfolding or ER-to-cytosol translocation of CTA1.

<p>(A) A purified CTA1/CTA2 heterodimer was placed in 20 mM sodium phosphate buffer (pH 7.4) containing 10 mM β-mercaptoethanol. Aliquots (1 μg) of the toxin were either left untreated (lanes 1–2), treated with 100 μg/mL of grape seed extract (lane 3), or treated with 10 μg/mL of a specific grape compound: caftaric acid (lane 4), quercitrin (lane 5), gallic acid (lane 6), or PB1 (lane 7). All samples were incubated at 37°C for 1 h. The samples were then shifted to 4°C and exposed to the protease thermolysin for 1 h, with the exception of the untreated toxin sample in lane 1. Samples were visualized by SDS-PAGE with Coomassie staining. Previous control experiments demonstrated that grape seed extract does not directly inhibit the proteolytic activity of thermolysin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166477#pone.0166477.ref015" target="_blank">15</a>]. (B) Using a plasmid-based transfection system, CTA1 was co-translationally inserted into the ER lumen before export back into the cytosol. The intracellular distribution of CTA1 was determined by immunoprecipitation of organelle (O) and cytosol (C) fractions from transfected cells radiolabeled for 1 h in the absence of compound (control), in the presence of a phenolic cocktail (100 μg/mL) containing all CT hit compounds other than petunidin and resveratrol (10C), or in the presence of 0.1 μM GA. The percentage of radiolabeled CTA1 found in the cytosol was calculated from two independent experiments (averages ± ranges).</p

Polyphenolic compounds disrupt CT adherence to the host plasma membrane.

<p>(A) Vero cells were incubated at 4°C for 30 min with 1 μg/mL of FITC-CTB. Unbound toxin was removed from the medium and replaced with 100 μg/mL of grape seed extract, 100 μg/mL of a cocktail containing all 12 CT hit compounds (12C), 17 μg/mL of a cocktail containing PB2 and EGCG (2C), 10 μg/mL of PB2, or 10 μg/mL of EGCG. After an additional 30 min at 4°C, FITC-CTB fluorescence was recorded with a plate reader. Values were standardized to the FITC-CTB signal from control cells incubated in the absence of grape compounds. (B) Vero cells were incubated at 4°C for 1 h in the combined presence of FITC-CTB and 100 μg/mL of grape seed extract, various concentrations of the 12C cocktail, or various concentrations of the 2C cocktail. FITC-CTB fluorescence was then recorded, with values standardized to the FITC-CTB signal from control cells incubated in the absence of grape compounds. Data from both panels represent the means ± SEMs of 4 independent experiments with 6 replicate samples per condition.</p