Environmentally Assisted Cracking in Silicon Nitride Barrier Films on Poly(ethylene terephthalate) Substrates

A singular critical onset strain value has been used to characterize the strain limits of barrier films used in flexible electronics. However, such metrics do not account for time-dependent or environmentally assisted cracking, which can be critical in determining the overall reliability of these thin-film coatings. In this work, the time-dependent channel crack growth behavior of silicon nitride barrier films on poly­(ethylene terephthalate) (PET) substrates is investigated in dry and humid environments by tensile tests with in situ optical microscopy and numerical models. The results reveal the occurrence of environmentally assisted crack growth at strains well below the critical onset crack strain and in the absence of polymer-relaxation-assisted, time-dependent crack growth. The crack growth rates in laboratory air are about 1 order of magnitude larger than those tested in dry environments (dry air or dry nitrogen). In laboratory air, crack growth rates increase from ∼200 nm/s to 60 μm/s for applied stress intensity factors, <i>K</i>, ranging from 1.0 to 1.4 MPa·m^1/2, below the measured fracture toughness <i>K</i>_c of 1.8 MPa·m^1/2. The crack growth rates in dry environments were also strongly dependent on the prior storage of the specimens, with larger rates for specimens exposed to laboratory air (and therefore moisture) prior to testing compared to specimens stored in a dry environment. This behavior is attributed to moisture-assisted cracking, with a measured power law exponent of ∼22 in laboratory air. This study also reveals that much larger densities of channel cracks develop in the humid environment, suggesting an easier initiation of channel cracks in the presence of water vapor. The results obtained in this work are critical to address the time-dependent and environmental reliability issues of thin brittle barriers on PET substrates for flexible electronics applications

The representative protein expression profiles of the liver.

<p>Mouse liver tissues were harvested at the indicated circadian times and analyzed by western blotting. The circadian expression profiles of clock proteins in WT, <i>Bmal1</i>^{<i>+/GTΔC</i>}, <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>}, <i>Bmal1</i>^<i>+/—</i>and <i>Bmal1</i>^<i>-/-</i> mice (n = 3).</p

A Novel <i>Bmal1</i> Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms

<div><p>The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. <i>Bmal1</i> encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated <i>Bmal1</i> (<i>Bmal1</i>^<i>GTΔC</i>) allele. The homozygous mutant (<i>Bmal1</i>^{<i>GTΔC/GTΔC</i>}) mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (<i>Bmal1</i>^{<i>+/GTΔC</i>}) mice displayed a gradual loss of rhythms, in contrast to <i>Bmal1</i>^<i>+/-</i> mice where rhythms were sustained. <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1^GTΔC was unable to activate transcription of <i>Per1</i> promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of <i>Bmal1</i> has pivotal roles in the regulation of circadian rhythms and the <i>Bmal1</i>^<i>GTΔC</i> mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1.</p></div

Structures and genotyping of <i>Bmal1</i>^<i>GTΔC</i> mice.

<p>(A) The genomic structure of ES cells harboring <i>Bmal1</i>^<i>GTΔC</i> allele. Black and grey colors indicate the exon (E) and intron (I) region, respectively. Above panel represents intact organization of Bmal1 DNA and below panel show the insertion site of the gene-trap vector. Black triangle indicates the location of the primer sequence. (B) Domains of WT and BMAL1^GTΔC proteins. (C) The genotypes were determined by using three primers PCR method (Bmal1^wt-F, Bmal1^WT-R and Bmal1^GTΔC-R). (D) The genotypes were also determined by western blotting of β-GAL.</p

Underlying molecular mechanisms of <i>Bmal1</i>^<i>GTΔC</i>.

<p>(A) Effects of <i>Bmal1</i>^<i>GTΔC</i> on Per1 promoter activity. (B) BMAL1^wt, BMAL1^GTΔC and CLOCK were tagged with fluorescence proteins and the localizations were examined. The heterodimerization and cellular localization of CLOCK:BMAL1^wt and CLOCK:BMAL1^GTΔC were examined by BiFC assays (C) and IP experiments (D). (E) The dose dependent degradation of CLOCK by BMAL1^wt and BMAL1^GTΔC (n = 3).</p

The effects of C-terminal region of <i>Bmal1</i> on the molecular circadian rhythm.

<p>(A) The structures of BMAL1^wt and Bmal1 mutant constructs, Bmal1^GTΔC, Bmal1^ΔC and Bmal1 ^ΔN. (B) The representative circadian oscillation profiles of the contructs in (A). The constructs were trasfected to WT MEFs. The luciferase activities of <i>Bmal1</i> and <i>Per2</i> promoters were distinguished by the wavelength separation method (n = 3).</p

The progeny genotypes of <i>Bmal1</i> mutant and null mice.

<p>The progeny genotypes of <i>Bmal1</i> mutant and null mice.</p

Altered circadian gene expression of WT, <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice.

<p>(A) <i>In situ</i> hybridization results of <i>Per2</i> and <i>Bmal1</i> mRNA expression in the SCN of WT, <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>} mice. (B) The mRNA expression in the liver. Data are represented as the mean ± S.E.M. (n = 3~6 per group).</p

The circadian profiles of MEFs derived from WT, <i>Bmal1</i>^{<i>+/GTΔC</i>}, <i>Bmal1</i>^{<i>GTΔC/GTΔC</i>}, <i>Bmal1</i>^<i>+/-</i> and <i>Bmal1</i>^<i>-/-</i> mice.

<p><b>(A)</b> The representative real-time luminescence profiles of each genotype. Cells were synchronized by 2hr treatment of DEX and monitored by the real-time bioluminescence device. (B) FRP of WT, <i>Bmal1</i>^{<i>+/GTΔC</i>} and <i>Bmal1</i>^<i>+/-</i> cells. Asterisks indicate significant differences (*<i>p</i><0.05) compared with WT mice. Data are represented as the mean ± S.E.M. (n = 3).</p

Effects of one week of time-restrictive feeding on the phases of Per1, LDLR, and LDLR regulatory factors gene expression in the mouse liver.

<p>Young adult male mice, entrained to a 12∶12 photoperiodic cycle, were fed time-restrictively for seven consecutive days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044053#pone-0044053-g001" target="_blank">Figure 1</a>. On the 8^th day, mice were sacrificed at the indicated zeitgeber time (ZT) and liver samples were obtained. RNA isolation, reverse transcription, and real-time polymerase chain reaction were performed to measure specific messages for mouse <i>Per1</i>, <i>ldlr</i>, and LDLR regulatory factors. All mRNA levels were normalized to <i>tbp</i> mRNA levels. Data are expressed as mean ± S.E.M. (n = 4).</p