Applicability of 2PI under conditions of altered R^ap or R^bl.

<p><b>A)</b> Nyquist plot of HT-29/B6 impedance spectrum after the application of forskolin. Arrows indicate estimates of R^sub (see inset) and R^T using the three methods, M1 (light grey), M2 (dark grey) and ANN (black). R^T = R^epi+R^sub. <b>B,C)</b> 2PI: Plotting epithelial conductance G^epi = 1/R^epi (♦, in the absence; ♦, in the presence of forskolin; ⋄, after EGTA application) against transepithelial fluorescein flux allows estimate of transcellular conductance, G^trans<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062913#pone.0062913-Krug1" target="_blank">[5]</a>. (B) Experiment without forskolin application. G^trans equals y-intercept (arrow) of the linear regression (grey line, G^trans = 0.64 mS/cm²). (C) G^trans in the presence of forskolin is obtained from the y-intercept (black arrow) of the linear regression (black line, G^trans = 1.69 mS/cm²). Shifting the linear regression to pass through the values obtained before the application of forskolin (grey line) allows estimate of G^trans in the absence of forskolin (grey arrow, G^trans = 0.65 mS/cm²). <b>D)</b> Comparison of R^trans values obtained from 30 experiments (15 without and 15 with forskolin or nystatin application, black and grey bars, respectively), using the three methods to estimate R^epi. Four experiments (three without and one with nystatin application) yielded negative or unreasonably high R^trans values when evaluated with methods M1 and M2. <b>E)</b> Same as (D) but after omission of these four experiments. Remaining estimates from experiments without forskolin or nystatin application were very similar for all three methods, estimates from experiments with forskolin or nystatin application showed lowest variance when evaluated by ANN.</p

Estimation of apical and basolateral parameters.

<p><b>A)</b> HT-29/B6 cell layers were divided in two groups, with R^epi values <450 Ω·cm² (354±16 Ω·cm², n = 12) and R^epi values >450 Ω·cm² (757±65 Ω·cm², n = 14), respectively. These two groups differed significantly in R^para (p<0.01, Student’s t-test) but not in R^trans. <b>B)</b> Set of four impedance spectra obtained in the absence (two larger curves) or presence of forskolin (two smaller curves). Experimental data (♦) were fitted using the six component circuit (○) as described in the methods section, to obtain R^ap, R^bl, C^ap and C^bl. <b>C,D)</b> R^epi, R^para, R^trans, R^ap, R^bl (C) and C^ap, C^bl (D) values from 11 experiments with apical or baslolateral nystatin application and 7 experiments with forskolin application. For forskolin purely apical effects were assumed. None of the parameters differed significantly between the two groups. <b>E,F)</b> Same data as in C and D, but regrouped for R^epi <450 Ω·cm² (9 values) and R^epi >450 Ω·cm² (9 values). As in (A), R^para (p<0.05, Student’s t-test) but not R^trans was significantly different between these two groups. However, both, R^ap and C^ap were significantly larger in cell layers with R^epi >450 Ω·cm² (p<0.01, Student’s t-test). <b>G, H)</b> R^epi, R^para, R^trans, R^ap and R^bl (G), and C^ap and C^bl (H) values for three IPEC-J2 cell layers treated with nystatin.</p

Parameter ranges for HT-29/B6 and IPEC-J2 cell-appropriate training data.

<p>Δ values denote fixed step sizes used for the generation of model impedance spectra; in all other cases, step sizes were chosen dynamically in order to yield 10 steps per parameter. Values <i>in italics</i> denote absolute interval constraints rather than variables with varied values. Ranges for HT-29/B6 circuit parameters were based on published estimates: R^trans between 380 and 1500 Ω·cm², R^para between 1500 and 4 000 Ω·cm² which was reduced to between 20 and 100 Ω·cm² at low [Ca²⁺]_o, C^epi between 2.1 and 3.5 µF/cm², R^sub (resistance of filter supports) between 2 and 10 Ω·cm²<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062913#pone.0062913-Krug1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062913#pone.0062913-Gitter1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062913#pone.0062913-Gitter2" target="_blank">[25]</a>. IPEC-J2 cells possess very high R^epi (several k Ω·cm², <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062913#pone.0062913-Nossol1" target="_blank">[47]</a>). Literature values for any further parameter values were not available.</p

Parameters estimated for HT-29/B6 and IPEC-J2 cells.

<p>Parameters estimated for HT-29/B6 and IPEC-J2 cells.</p

Nyquist diagrams and equivalent electrical circuits.

<p><b>A)</b> Nyquist diagram of an impedance spectrum calculated for circuits depicted in (C) and (D). Data points, •, were calculated using 42 different frequencies between 1.3 Hz to 16 kHz. x-intercepts at low frequencies (f→0) correspond to the total epithelial resistance (R^T, also called “TER”). x-intercepts at high frequencies (f→∞) correspond to the subepithelial resistance, R^sub, as under these conditions the reactance of the membrane capacitor (1/(ω·C^epi)) approaches zero and thus short-circuits R^epi. Note that circuits (C) and (D) always yield semicircular spectra. <b>B)</b> Example for a Nyquist diagram of a non-semicircular impedance spectrum calculated for the circuit depicted in (E). Data points, <b>▴</b>, were calculated using 42 different frequencies between 1.3 Hz to 16 kHz. Spectra calculated for this model are the sum of two semicircles. Again, x-intercepts at low frequencies (f→0) correspond to the total epithelial resistance (R^T), x-intercepts at high frequencies (f→∞) correspond to the subepithelial resistance, R^sub. <b>C-E)</b> Equivalent electric circuits of epithelia. Components contributing to Repi are drawn in red, R^sub is highlighted in blue. Components contributing to R^T (sum of R^epi and R^sub) are joint by grey lines. <b>C)</b> Simplest form of an equivalent electric circuit describing epithelial and subepithelial resistance (R^epi, R^sub) and epithelial capacitance (C^epi). <b>D)</b> Equivalent electric circuit as in (C), but R^epi consists of two resistors in parallel, the transcellular (R^trans) and the paracellular resistance (R^para). <b>E)</b> Equivalent electric circuit as in (D), but the transcellular pathway is devided into an apical and a basolateral RC unit (R^ap, C^ap and R^bl, C^bl, respectively). </p

Missing Amide I Mode in Gap-Mode Tip-Enhanced Raman Spectra of Proteins

Tip-enhanced Raman spectroscopy is a surface sensitive analytical method that combines the advantages of scanning probe microscopy and Raman spectroscopy. It holds great promises for imaging of biological samples with high spatial resolution (10–50 nm), well below the optical diffraction limit. It offers the opportunity to directly localize and identify proteins and their conformation in a complex (e.g., native) environment. Tip-enhanced Raman (TER) spectra in the so-called “gap-mode” configuration with a metal tip in scanning tunnelling feedback with a metal substrate coated with different proteins (bovine serum albumin, immunoglobulin G, trypsin, and β-lactoglobulin) as well as of model octapeptides (with and without an aromatic amino acid residue) are presented. The goal was to determine if it is possible to reliably assign marker bands for proteins and if different secondary structures of proteins can be distinguished in their gap-mode TER spectra as reliably as by IR and conventional Raman spectroscopy. It is shown that contrary to the presented conventional Raman spectra of proteins the amide I mode, which is widely used to identify secondary structure motifs of proteins, is not visible in gap-mode TERS. Aromatic modes are prominent and can be used as reliable marker bands for imaging of proteins in a complex environment

Full Spectroscopic Tip-Enhanced Raman Imaging of Single Nanotapes Formed from β‑Amyloid(1–40) Peptide Fragments

This study demonstrates that spectral fingerprint patterns for a weakly scattering biological sample can be obtained reproducibly and reliably with tip-enhanced Raman spectroscopy (TERS) that correspond well with the conventional confocal Raman spectra collected for the bulk substance. These provided the basis for obtaining TERS images of individual self-assembled peptide nanotapes using an automated, objective procedure that correlate with the simultaneously obtained scanning tunneling microscopy (STM) images. TERS and STM images (64 × 64 pixels, 3 × 3 μm²) of peptide nanotapes are presented that rely on marker bands in the Raman fingerprint region. Full spectroscopic information in every pixel was obtained, allowing post-processing of data and identification of species of interest. Experimentally, the “gap-mode” TERS configuration was used with a solid metal (Ag) tip in feedback with a metal substrate (Au). Confocal Raman data of bulk nanotapes, TERS point measurements with longer acquisition time, atomic force microscopy images, and an infrared absorption spectrum of bulk nanotapes were recorded for comparison. It is shown that the unique combination of topographic and spectroscopic data that TERS imaging provides reveals differences between the STM and TERS images, for example, nanotapes that are only weakly visible in the STM images, a coverage of the surface with an unknown substance, and the identification of a patch as a protein assembly that could not be unambiguously assigned based on the STM image alone

Multicenter Phase II Study Evaluating Two Cycles of Docetaxel, Cisplatin and Cetuximab as Induction Regimen Prior to Surgery in Chemotherapy-Naive Patients with NSCLC Stage IB-IIIA (INN06-Study)

<div><p>Background</p><p>Different strategies for neoadjuvant chemotherapy in patients with early stage NSCLC have already been evaluated. The aim of this study was to evaluate the tolerability and efficacy of a chemoimmunotherapy when limited to two cycles.</p><p>Methods</p><p>Between 01/2007 and 03/2010 41 patients with primarily resectable NSCLC stage IB to IIIA were included. Treatment consisted of two cycles cisplatin (40 mg/m² d1+2) and docetaxel (75 mg/m² d1) q3 weeks, accompanied by the administration of cetuximab (400 mg/m² d1, then 250 mg weekly). The primary endpoint was radiological response according to RECIST.</p><p>Results</p><p>40 patients were evaluable for toxicity, 39 for response. The main grade 3/4 toxicities were: neutropenia 25%, leucopenia 11%, febrile neutropenia 6%, nausea 8% and rash 8%. 20 patients achieved a partial response, 17 a stable disease, 2 were not evaluable. 37 patients (95%) underwent surgery and in three of them a complete pathological response was achieved. At a median follow-up of 44.2 months, 41% of the patients had died, median progression-free survival was 22.5 months.</p><p>Conclusions</p><p>Two cycles of cisplatin/ docetaxel/ cetuximab showed promising efficacy in the neoadjuvant treatment of early-stage NSCLC and rapid operation was possible in 95% of patients. Toxicities were manageable and as expected.</p><p>Trial Registration</p><p>EU Clinical Trials Register; Eudract-Nr: <a href="https://www.clinicaltrialsregister.eu/ctr-search/search?query=2006-004639-31" target="_blank">2006-004639-31</a></p></div

Participant diagram.

<p>Paricipant diagram describing patients analyzed in the INN-06 study.</p

Adverse Events (n = 40).

<p>Adverse Events (n = 40).</p