Phase Morphology, Mechanical, and Thermal Properties of Calcium Carbonate-Reinforced Poly(L-lactide)-<i>b</i>-poly(ethylene glycol)-<i>b</i>-poly(L-lactide) Bioplastics

Poly(L-lactide) (PLLA) is a promising candidate as a bioplastic because of its non-toxicity and biodegradability. However, the low flexibility of PLLA limits its use in many applications. Poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-b-PEG-b-PLLA) block copolymer is of interest for bioplastic applications due to its superior flexibility compared to PLLA. The aim of this work is to modify PLLA-b-PEG-b-PLLA using a low-cost calcium carbonate (CaCO3) filler to improve material properties compared to PLLA/CaCO3 composites. The addition of CaCO3 enhanced the crystallinity and thermal stability for the PLLA-b-PEG-b-PLLA matrix but not for the PLLA matrix, as determined by differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and thermogravimetric analysis (TGA). Phase morphology investigation using scanning electron microscopy (SEM) revealed that the interfacial adhesion between PLLA-b-PEG-b-PLLA and CaCO3 was stronger than between PLLA and CaCO3. Additionally, tensile testing was carried out to determine the mechanical properties of the composites. With the addition of CaCO3, the tensile stress and Young’s modulus of the PLLA-b-PEG-b-PLLA matrix were increased, whereas these properties of the PLLA matrix were significantly decreased. Thus, CaCO3 shows great promise as an inexpensive filler that can induce nucleation and reinforcing effects for PLLA-b-PEG-b-PLLA bioplastics

Thermally Induced Nanoimprinting of Biodegradable Polycarbonates Using Dynamic Covalent Cross-Links

The introduction of reversible covalent bonds into polymeric systems afford robust, yet dynamic, materials that can respond to external stimuli. A series of aliphatic polycarbonate polymers were synthesized via ring-opening polymerization of furanyl and maleimido-bearing cyclic carbonate monomers. These side chains undergo thermally induced Diels–Alder reactions to afford cross-linked films. Because both the diene and dienophile were incorporated into the same polymer backbone, a protected maleimido group, in the form of the furan adduct, was used. Both the forward and reverse Diels–Alder reaction are triggered thermally, which allows the deprotection of the maleimido group and the subsequent reaction with the furanyl side chains to form cross-links. Random copolymers and poly­(ethylene glycol) containing block copolymers were formed using diazabicyclo[5.4.0]­undec-7-ene as the catalyst and a thiourea cocatalyst. The polymers form uniform films that can be cross-linked in the bulk state. To further illustrate the dynamic nature of the covalent bonds within the cross-linked films, a patterned silicon mold was used to transfer a series of nanoscale patterns using a thermal nanoimprint process

MshA pilus production correlates with c-di-GMP.

<p>Expression of the DGC VCA0956 was induced with varying amounts of IPTG. Intracellular c-di-GMP level was determined with LC-MS/MS and normalized to total protein in the extract (grey bars, right Y axis). Four independent samples were used for each induction condition. Oneway ANOVA followed by Dunnett’s Multiple Comparison Test determined significance. Surface MshA pilin was determined by ELISA (black bars, left Y axis). Three biological replicates were tested in triplicate. Results were normalized to MshA production in the WT strain. Surface MshA pili were significantly increased in all conditions compared to WT (Oneway ANOVA, Dunnett’s Multiple Comparison Test.) Also, there was a significant correlation between increased c-di-GMP and increased surface MshA pili (Pearson correlation, p = 0.0025, R² = 0.9199).</p

C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in <i>Vibrio cholerae</i>

<div><p>In many bacteria, including <i>Vibrio cholerae</i>, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in <i>V</i>. <i>cholerae</i>. Previously, we reported that <i>cdgJ</i> encodes a c-di-GMP phosphodiesterase and a Δ<i>cdgJ</i> mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose<i>-</i>sensitive hemagglutinin <i>(</i>MshA<i>)</i> pilus biogenesis restores motility in the Δ<i>cdgJ</i> mutant. Mutations of the predicted ATPase proteins <i>mshE</i> or <i>pilT</i>, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A Δ<i>cdgJ</i> mutant has enhanced surface attachment, while the Δ<i>cdgJmshA</i> mutant phenocopies the high motility and low attachment phenotypes observed in a Δ<i>mshA</i> strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.</p></div

Mutations in <i>mshA</i>, <i>mshE</i>, <i>pilT</i>, and <i>cdgJ</i> affect near surface motility.

<p>Measured cell tracks at the indicated times post-inoculation. Bacterial populations were imaged for 82s with a 5 ms resolution, and a representative subset of individual cell tracks are presented for clarity. The field of view is 160μm x 160μm. Each line represents the motion of one cell and different colors are used to denote different cells. Non-motile cells have been filtered out of the data set at each time point for clarity.</p

MshE binds c-di-GMP.

<p>A. Isothermal Calorimetry was performed on purified proteins to determine the binding affinity for c-di-GMP. Three independent runs were included in the analysis, one representative run is depicted. VpsT is included as a positive control for c-di-GMP binding. B. Fluorescence thermal shift was utilized to investigate domains of MshE responsible for binding c-di-GMP. Fluorescence melt curves are displayed in the presence of 2mM ATP or c-di-GMP. Three independent experiments were performed in triplicate, with one representative experiment shown. C. Midpoints of melt curves are displayed in the presence of 2mM nucleotides or a buffer control. Three independent experiments were performed in triplicate, Significance was determined with a Oneway ANOVA followed by Dunnett’s Multiple Comparison test comparing to Buffer control. (*** p≤0.001, all others not significant).</p

Early surface interactions are reduced in <i>mshA</i>, <i>mshE</i>, and <i>pilT</i> mutants and increased in <i>cdgJ</i> mutant.

<p>A, C. The number of surface-adhered, stationary cells were counted at the indicated times after inoculation in a flow cell. B, D. Residence times were measured for each cell that interacted with the surface during three high-speed movies of 82s in length within the first 15 minutes post inoculation.</p