Subtle Regulation of Scaffold Stiffness for the Optimized Control of Cell Behavior

The rigidity of extracellular matrices can impact cell fate, guide tissue development, and initiate tumor formation. Scaffolds such as hydrogels with tunable levels of stiffness have been developed to control cell adhesion, migration, and differentiation, providing suitable microenvironments for different tissue outcomes. However, studies of cell–material interactions are largely confined to biomaterials with stiffness values that are coarsely regulated, so refinements in sensitive cellular responses and optimal stiffness values that determine cell fate remain elusive. Here, a freezing temperature, as a tunable regulating factor, was introduced to freeze-drying processes to form silk fibroin (SF) scaffolds with refined control of stiffness values. Due to this control of intermediate structural conformations of SF, the scaffolds exhibited differences in stiffness values to permit refined assessments of impact on cell behavior on cell-friendly surfaces. Both in vitro and in vivo results with these scaffolds exhibited gradually changeable cell migration and differentiation outcomes, as well as differences in tissue ingrowth, demonstrating the sensitivity of cellular responses to such refined mechanical cues. The optimal vascularization capacity of these SF scaffolds was in the 3–7.4 kPa range, suggesting a key range to develop bioactive biomaterials. Systematic fine regulation of scaffold rigidity based on the present strategy provides a platform for an improved understanding of cell–material interactions and also for generating optimized microenvironments for tissue regeneration

Cross-resistance among flumorph, dimethomorph and iprovalicarb.

<p>Log-transformed EC₅₀ values (the effective concentration for causing 50% inhibition of mycelial growth inhibition of <i>Phytophthora melonis</i>) for isolates of <i>P. melonis</i> were compared among the three carboxylic acid amide fungicides using Spearman’s rank correlation coefficients. (A), (B), and (C) indicate positive cross-resistance among flumorph, dimethomorph, and iprovalicarb; (D-F) include only the higher EC₅₀ values from (A-C), i.e., EC₅₀ values from CAA-resistant isolates. (D) reveals a positive correlation between the EC₅₀ values for dimethomorph and flumorph among CAA-resistant isolates, while (E) and (F) reveals a negative correlation between iprovalicarb and flumorph and between iprovalicarb and dimethomorph among CAA-resistant isolates.</p

Structure and site of mutation in the <i>PmCesA3</i> gene associated with carboxylic acid amide (CAA) fungicide resistance.

<p>(A) Intron/exon structure of the <i>PmCesA3</i> gene. Numbers represent the size in base pairs. Point mutations in CAA-resistant mutants and the predicted amino acid substitution in the mutant gene products are indicated. (B) Alignment of partial amino acid sequences of CesA3 in <i>P. melonis</i> (PmCesA3), <i>P. infestans</i> (PiCesA3), and <i>P. viticola</i> (PvCesA3). TJ-90, TX-21, and TX-33 were wild-type isolates. D63-1 and D70-3 were dimethomorph-resistant mutants. F58-4 and F63-11 were flumorph-resistant mutants. I63-2 and I70-5 were iprovalicarb-resistant mutants. Mutations in CAA-resistant mutants of <i>P. infestans</i>, <i>P. viticola</i> and <i>P. melonis</i> are indicated by asterisks.</p

Isolates of <i>Phytophthora melonis</i> used for RAPD analysis and their sensitivities to flumorph, dimethomorph and iprovalicarb.

a<p>One isolate of <i>Phytophthora drechsleri</i> was used as an outgroup control.</p>b<p>EC₅₀ values, the effective concentration for causing 50% inhibition of mycelial growth inhibition of <i>P. melonis</i>.</p>c<p>Number represents a different field in the same district.</p

Frequency distributions of EC₅₀ values (the effective concentration causing 50% inhibition of mycelial growth of <i>Phytophthora melonis</i>) for flumorph, dimethomorph and iprovalicarb.

<p>In total, 80 isolates of <i>P. melonis</i> were collected from areas never exposed to carboxylic acid amide fungicides.</p

Results of the experiments conducted to induce resistance against flumorph, dimethomorph, and iprovalicarb in <i>Phytophthora melonis</i>.

a<p>SM, spontaneous mutation. UV, UV-mutagenesis.</p>b<p>Survival frequency, number of mutants/total number of zoospores used for mutant generation.</p>c<p>EC₅₀, the effective concentration for causing 50% inhibition of mycelial growth inhibition of <i>P. melonis</i>.</p>d<p>Resistance factor  =  EC₅₀ of resistant isolates at the 10^th transfer/EC₅₀ of its parent.</p

Purification, Characterization, and Mode of Action of Plantaricin GZ1-27, a Novel Bacteriocin against <i>Bacillus cereus</i>

Bacillus cereus is an opportunistic pathogen that causes foodborne diseases. We isolated a novel bacteriocin, designated plantaricin GZ1-27, and elucidated its mode of action against B. cereus. Plantaricin GZ1-27 was purified using ammonium sulfate precipitation, gel-filtration chromatography, and RP-HPLC. MALDI-TOF/MS revealed that its molecular mass was 975 Da, and Q-TOF-MS/MS analysis predicted the amino acid sequence as VSGPAGPPGTH. Plantaricin GZ1-27 showed thermostability and pH stability. The antibacterial mechanism was investigated using flow cytometry, confocal laser-scanning microscopy, scanning and transmission electron microscopy, and RT-PCR, which revealed that GZ1-27 increased cell membrane permeability, triggered K+ leakage and pore formation, damaged cell membrane integrity, altered cell morphology and intracellular organization, and reduced the expression of genes related to cytotoxin production, peptidoglycan synthesis, and cell division. These results suggest that plantaricin GZ1-27 effectively inhibits B. cereus at both the cellular and the molecular levels and is a potential natural food preservative targeting B. cereus

Fitness of CAA-resistant and -sensitive isolates of <i>Phytophthora melonis in vitro</i>.

a<p>Isolates in bold font are parents of the resistant isolates listed under them in regular font. Isolates starting with the letter F, D, and I, are flumorph-resistant mutants, dimethomorph-resistant mutants, and iprovalicarb-resistant mutants, respectively.</p>b<p>For each parent and its resistant progeny, means followed by same letters are not significantly different according to Fisher’s least significance difference (α = 0.05).</p>c<p>CFI (compound fitness index)  =  mycelial growth × zoospore production × lesion area on cucumber leaves.</p

Genetic relationships among 15 isolates of <i>Phytophthora melonis</i>.

<p>The denrogram (UPGMA) shows the relationships among the isolates of <i>P. melonis</i> based on randomly amplified polymorphic DNA (RAPD) analysis with 16 decamer primers. Scale at the bottom depicts the genetic distance.</p

Spearman rank correlation for cross-resistance in <i>Phytophthora capsici</i> between pyrimorph and other fungicides.

<p>(A) dimethomorph; (B) flumorph; (C) mandipropamid; (D) azoxystrobin; (E) cyazofamid; (F) cymoxanil; (G) chlorothalonil; (H) etridiazole; (I) fluazinam; (J) metalaxyl; and (K) zoxamide. Data points are the logarithmic values of effective concentrations for 50% mycelial growth inhibition (log EC₅₀) among <i>Phytophthora capsici</i> isolates for the indicated fungicide combinations.</p