Vapor Sublimation and Deposition to Fabricate a Porous Methyl Propiolate-Functionalized Poly-p-xylylene Material for Copper-Free Click Chemistry.

Conventional porous materials are mostly synthesized in solution-based methods involving solvents and initiators, and the functionalization of these porous materials usually requires additional and complex steps. In the current study, a methyl propiolate-functionalized porous poly-p-xylylene material was fabricated based on a unique vapor sublimation and deposition process. The process used a water solution and ice as the template with a customizable shape and dimensions, and the conventional chemical vapor deposition (CVD) polymerization of poly-p-xylylene on such an ice template formed a three-dimensional, porous poly-p-xylylene material with interconnected porous structures. More importantly, the functionality of methyl propiolate was well preserved by using methyl propiolate-substituted [2,2]-paracyclophane during the vapor deposition polymerization process and was installed in one step on the final porous poly-p-xylylene products. This functionality exhibited an intact structure and reactivity during the proposed vapor sublimation and deposition process and was proven to have no decomposition or side products after further characterization and conjugation experiments. The electron-withdrawing methyl propiolate group readily provided efficient alkynes as click azide-terminated molecules under copper-free and mild conditions at room temperature and in environmentally friendly solvents, such as water. The resulting methyl propiolate-functionalized porous poly-p-xylylene exhibited interface properties with clickable specific covalent attachment toward azide-terminated target molecules, which are widely available for drugs and biomolecules. The fabricated functional porous materials represent an advanced material featuring porous structures, a straightforward synthetic approach, and precise and controlled interface click chemistry, rendering long-term stability and efficacy to conjugate target functionalities that are expected to attract a variety of new applications.

Vapor-phased fabrication and modulation of cell-laden scaffolding materials.

Bottom-up approaches using building blocks of modules to fabricate scaffolds for tissue engineering applications have enabled the fabrication of structurally complex and multifunctional materials allowing for physical and chemical flexibility to better mimic the native extracellular matrix. Here we report a vapor-phased fabrication process for constructing three-dimensional modulated scaffold materials via simple steps based on controlling mass transport of vapor sublimation and deposition. We demonstrate the fabrication of scaffolds comprised of multiple biomolecules and living cells with built-in boundaries separating the distinct compartments containing defined biological configurations and functions. We show that the fabricated scaffolds have mass production potential. We demonstrate overall >80% cell viability of encapsulated cells and that modulated scaffolds exhibit enhanced cell proliferation, osteogenesis, and neurogenesis, which can be assembled into various geometric configurations. We perform cell co-culture experiments to show independent osteogenesis and angiogenesis activities from separate compartments in one scaffold construct.

Synergistic and Regulatable Bioremediation Capsules Fabrication Based on Vapor-Phased Encapsulation of Bacillus Bacteria and its Regulator by Poly-p-Xylylene.

A regulatable bioremediation capsule material was synthesized with isolated single-strain bacteria (Bacillus species, B. CMC1) and a regulator molecule (carboxymethyl cellulose, CMC) by a vapor-phased encapsulation method with simple steps of water sublimation and poly-p-xylylene deposition in chemical vapor deposition (CVD) process. Mechanically, the capsule construct exhibited a controllable shape and dimensions, and was composed of highly biocompatible poly-p-xylylene as the matrix with homogeneously distributed bacteria and CMC molecules. Versatility of the encapsulation of the molecules at the desired concentrations was achieved in the vapor-phased sublimation and deposition fabrication process. The discovery of the fabricated capsule revealed that viable living B. CMC1 inhabited the capsule, and the capsule enhanced bacterial growth due to the materials and process used. Biologically, the encapsulated B. CMC1 demonstrated viable and functional enzyme activity for cellulase activation, and such activity was regulatable and proportional to the concentration of the decorated CMC molecules in the same capsule construct. Impressively, 13% of cellulase activity increase was realized by encapsulation of B. CMC1 by poly-p-xylylene, and a further 34% of cellulase activity increase was achieved by encapsulation of additional 2.5% CMC. Accordingly, this synergistic effectiveness of the capsule constructs was established by combining enzymatic B. CMC1 bacteria and its regulatory CMC by poly-p-xylylene encapsulation process. This reported encapsulation process exhibited other advantages, including the use of simple steps and a dry and clean process free of harmful chemicals; most importantly, the process is scalable for mass production. The present study represents a novel method to fabricate bacteria-encapsulated capsule for cellulose degradation in bioremediation that can be used in various applications, such as wastewater treatment and transforming of cellulose into glucose for biofuel production. Moreover, the concept of this vapor-phased encapsulation technology can be correspondingly used to encapsulate multiple bacteria and regulators to enhance the specific enzyme functions for degradation of various organic matters.

Parylene-Based Porous Scaffold with Functionalized Encapsulation of Platelet-Rich Plasma and Living Stem Cells for Tissue Engineering Applications.

A scaffold was fabricated to synergistically encapsulate living human adipose-derived stem cells (hASCs) and platelet-rich plasma (PRP) based on a vapor-phase sublimation and deposition process. During the process, ice templates were prepared using sterile water as the solvent and were used to accommodate the sensitive living cells and PRP molecules. Under controlled processing conditions, the ice templates underwent vapor sublimation to evaporate water molecules, while at the same time, vapor-phase deposition of poly-p-xylylene (Parylene, USP Class VI highly biocompatible) occurred to replace the templates, and the final construction yielded a scaffold with Parylene as the matrix, with simultaneously encapsulated living hASCs and PRP molecules. Evaluation of the fabricated synergistic scaffold for the proliferation activities toward the encapsulated hASCs indicated significant augmentation of cell proliferation contributed by the PRP ingredients. In addition, osteogenic activity in the early stage by alkaline phosphatase expression and later stage with calcium mineralization indicated significant enhancement toward osteogenetic differentiation of the encapsulated hASCs, which were guided by the PRP molecules. By contrast, examinations of adipogenic activity by lipid droplet formation revealed an inhibition of adipogenesis with decreased intracellular lipid accumulation, and a statistically significant downregulation of adipogenic differentiation was postulated for the scaffold products when compared to the osteogenetic results and the control experiments. The reported fabrication method featured a clean and simple process to construct scaffolds that combined delicate living hASCs and PRP molecules inside the structure. The resultant synergistic scaffold and the selected commercially available hASCs and PRP are emerging as tissue engineering tools that provide multifunctionality for tissue repair and regeneration.

Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells.

Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that approximately 50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.

Characterization of a bi-directional promoter for divergent transcription of a PHD-zinc finger protein gene and a ran gene in the protozoan pathogen Giardia lamblia.

We showed previously that transcription of the ran gene in Giardia lamblia is regulated by an AT-rich initiator. In the present study, the ran initiator was found to regulate transcription of a neighbouring PHD zinc-finger protein gene. Deletion and scanning mutagenesis of the phd promoter in a firefly luciferase reporter system showed that the promoter activity is determined by multiple single-stranded T-tract DNA elements distributed into a distal domain spanning the ran initiator (-134/-103) and a proximal domain (-88/-48) spanning phd messenger RNA (mRNA) start sites (-74, -55 and -53 relative to the first ATG). The promoter activity is repressed by the single T-tract element on a non-template strand of the ran initiator, and is activated by closely spaced T-tract elements on the opposite strand. The T-tract elements in the phd and ran initiators compete for similar ssDNA binding proteins. Mutation of -47/-42 resulted in dramatic reduction of luciferase activity without changing luciferase mRNA levels, indicating the potential involvement of a regulatory mechanism in PHD protein translation. These findings suggest that G. lamblia uses multiple copies of a T-tract element as both core and distal elements in regulating transcription initiation, and that expression of the phd gene is regulated at multiple levels.