GaN-based MIS-HEMTs with Al2O3 dielectric deposited by low-cost and environmental-friendly mist-CVD technique

We report on the fabrication and characterization of AlGaN/GaN metal-insulator-semiconductor (MIS) capacitors and high-electron-mobility transistors (MIS-HEMTs) using a 5 nm thick Al2O3 dielectric deposited by cost-effective and environmental-friendly mist chemical vapor deposition (mist-CVD) technique. Practically hysteresis-free capacitance–voltage profiles were obtained from the fabricated two-terminal MIS-capacitors indicating high quality of the mist-Al2O3/AlGaN interface. Compared with reference Schottky-gate HEMTs, mist MIS-HEMTs exhibited much improved performance including higher drain current on-to-off ratio, much lower gate leakage current in both forward and reverse directions and lower subthreshold swing. These results demonstrate the potential and viability of non-vacuum mist-CVD Al2O3 in the development of high-performance GaN-based MIS-HEMTs

Prognostic impact of clinical factors for immune checkpoint inhibitor with or without chemotherapy in older patients with non-small cell lung cancer and PD-L1 TPS ≥ 50%

IntroductionThe proportion of older patients diagnosed with advanced-stage non-small cell lung cancer (NSCLC) has been increasing. Immune checkpoint inhibitor (ICI) monotherapy (MONO) and combination therapy of ICI and chemotherapy (COMBO) are standard treatments for patients with NSCLC and programmed cell death ligand-1 (PD-L1) tumor proportion scores (TPS) ≥ 50%. However, evidence from the clinical trials specifically for older patients is limited. Thus, it is unclear which older patients benefit more from COMBO than MONO.MethodsWe retrospectively analyzed 199 older NSCLC patients of Eastern Cooperative Oncology Group performance status (ECOG PS) 0-1 and PD-L1 TPS ≥ 50% who were treated with MONO or COMBO. We analyzed the association between treatment outcomes and baseline patient characteristics in each group, using propensity score matching.ResultsOf the 199 patients, 131 received MONO, and 68 received COMBO. The median overall survival (OS; MONO: 25.2 vs. COMBO: 42.2 months, P = 0.116) and median progression-free survival (PFS; 10.9 vs. 11.8 months, P = 0.231) did not significantly differ between MONO and COMBO group. In the MONO group, OS was significantly shorter in patients without smoking history compared to those with smoking history [HR for smoking history against non-smoking history: 0.36 (95% CI: 0.16-0.78), P = 0.010]. In the COMBO group, OS was significantly shorter in patients with PS 1 than those with PS 0 [HR for PS 0 against PS 1: 3.84 (95% CI: 1.44-10.20), P = 0.007] and for patients with squamous cell carcinoma (SQ) compared to non-squamous cell carcinoma (non-SQ) [HR for SQ against non-SQ: 0.17 (95% CI: 0.06-0.44), P < 0.001]. For patients with ECOG PS 0 (OS: 26.1 months vs. not reached, P = 0.0031, PFS: 6.5 vs. 21.7 months, P = 0.0436) or non-SQ (OS: 23.8 months vs. not reached, P = 0.0038, PFS: 10.9 vs. 17.3 months, P = 0.0383), PFS and OS were significantly longer in the COMBO group.ConclusionsECOG PS and histological type should be considered when choosing MONO or COMBO treatment in older patients with NSCLC and PD-L1 TPS ≥ 50%

Claudin-2 Knockout by TALEN-Mediated Gene Targeting in MDCK Cells: Claudin-2 Independently Determines the Leaky Property of Tight Junctions in MDCK Cells

<div><p>Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000–4000 Ω·cm²) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the ‘leaky’ property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.</p></div

Effects of Osmolality on Paracellular Transport in MDCK II Cells.

Epithelia separate apical and basal compartments, and movement of substances via the paracellular pathway is regulated by tight junctions. Claudins are major constituents of tight junctions and involved in the regulation of tight junction permeability. On the other hand, the osmolality in the extracellular environment fluctuates in association with life activity. However, effects of osmotic changes on the permeaibility of claudins are poorly understood. Therefore, we investigated the effects of osmotic changes on the paracellular transport in MDCK II cells. Interestingly, apical hyposmolality decreased cation selectivity in the paracellular pathway gradually with time, and the elimination of the osmotic gradient promptly restored the cation selectivity. Apical hyposmolality also induced bleb formation at cell-cell contacts and changed the shape of cell-cell contacts from a jagged pattern to a slightly linear pattern. In claudin-2 knockout MDCK II cells, the decrease of cation selectivity, the bleb formation, nor the changes in the shape of cell-cell contacts was observed under the apical hyposmolality. Our findings in this study indicate that osmotic gradient between apical and basal sides is involved in the acute regulation of the cation selective property of claudin-2 channels

Localization of claudins at TJs between control and their respective knockout cells.

<p>(A–E) Immunofluorescence analysis of claudins and ZO-1 or ZO-3 in MDCK II cells transfected with the TALEN constructs for claudin-1, -2, -3, -4, or-7 gene knockouts. After transfection, cells were subcultured on filter inserts for 4 days before the analysis of claudin-2 and -3 (B and C), and for 2 days before the analysis of claudin-1, -4, and -7 (A, D, and E). (F) Immunofluorescence analysis of claudin-2 and ZO-1. TALEN constructs for the claudin-2 gene knockout were transfected into MDCK II cells, which were subcultured on filter inserts for 2 days before analysis. (G) Immunofluorescence analysis of claudin-2 and ZO-1 in a co-culture of wild-type and claudin-2 expressing MDCK I cells. Cells were cultured on filter inserts for 4 days before analysis. Scale bar = 10 μm.</p

Effects of claudin-2 knockout on the barrier properties of TJs.

<p>(A) Time course of TER and TER values at 6 days after seeding on filter inserts in control cells and claudin-2 knockout clones. Claudin-2 knockout clones showed much higher TER than control cells 1 day after seeding on filter inserts and the TER further increased with time. TER values of claudin-2 knockout clones at 6 days after seeding on filter inserts were more than 50-fold higher than control cells. (B) Charge selectivity (ratio of <i>P</i>_Na to <i>P</i>_Cl: <i>P</i>_Na/<i>P</i>_Cl) in control cells and claudin-2 knockout clones. Claudin-2 knockout clones showed much lower values of <i>P</i>_Na/<i>P</i>_Cl than control cells. (C) <i>P</i>_Na and <i>P</i>_Cl in control cells and claudin-2 knockout clones. <i>P</i>_Na in claudin-2 knockout clones was approximately 1% of that in control cells and <i>P</i>_Cl in claudin-2 knockout clones was approximately 12% of that in control cells. (D and E) Flux of fluorescein (D) and 4 kDa FITC-dextran (E) in control cells and claudin-2 knockout clones. Claudin-2 knockout had no significant effects on the flux of fluorescein and 4 kDa FITC-dextran. N = 3–5 for each experiment.</p

Effects of claudin-2 knockout on the localization of other claudins.

<p>(A) Immunofluorescence analysis of claudins in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1). Claudin-1, -3, -4, and -7 showed clearer and stronger signals at TJs in claudin-2 knockout cells than in control cells. Scale bar = 10 μm. (B) Quantification analysis of signal intensity of claudins at TJs in control MDCK II cells and claudin-2 knockout clones. The signal intensity of claudins at TJs in control cells and claudin-2 knockout clones was measured as described in <i>Materials and Methods</i>, and the relative signal intensity of each claudin was calculated as the ratio of the signal intensity in control cells (CTL) and claudin-2 knockout clones (KO 1 and 2) to the signal intensity in control cells. N = 4 for each experiment. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 compared with control.</p

Construction of TALENs and claudin-2 gene knockout in MDCK II cells.

<p>(A) TALEN binding sites in the claudin-2 gene. The left and right arms of TALEN targeting site are indicated in blue and the spacer region is indicated in red. The initiating codon within the spacer region is shaded. (B) Immunofluorescence analysis of claudin-2 and ZO-1 in MDCK II cells transfected with TALEN constructs for claudin-2 gene knockout. After transfection, cells were subcultured on filter inserts for 4 days before analysis. Claudin-2 staining was completely lost at cell-cell contacts in claudin-2 knockout cells. Scale bar = 10 μm.</p

Quantification analysis of the signal intensity of claudins and ZO proteins at TJs between control and their respective knockout cells.

<p>(A–C) Immunofluorescence analysis of ZO proteins in MDCK II cells transfected with TALEN constructs for ZO-1, -2, or-3 gene knockouts. After transfection, cells were subcultured on filter inserts for 4 days before analysis. ZO-1 knockout cells showed a straight shape of cell-cell contacts consistent with a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119869#pone.0119869.ref019" target="_blank">19</a>]. Scale bar = 10 μm. (D) Quantification analysis of the signal intensity of claudins and ZO proteins at TJs between control and their respective knockout cells. The signal intensity of these proteins between control cells and that between control and their respective knockout cells was measured, and the relative signal intensity of each protein was calculated as described in <i>Materials and Methods</i>. The relative intensity of claudins was compared with the average of the relative intensity of ZO proteins (ZOs). N = 3–6 for each experiment. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 compared with ZOs.</p

Localization of claudins in z-axis plane in control and claudin-2 knockout cells.

<p>Immunofluorescence analysis of claudins and occludin in co-culture of control MDCK II cells and claudin-2 knockout clone 1 (KO 1) in z-axis plane. The signals of claudin-1, -3, -4 and -7 were stronger at TJs in claudin-2 knockout cells compared with control cells, and lateral localization of these claudins was similar between control and claudin-2 knockout cells. Scale bar = 5 μm.</p