Living Materials Constructed with Dynamic Covalent Interface between Synthetic Polymers and B. subtilis

With advances in the field of synthetic biology increasingly allowing us to engineer living cells to perform intricate tasks, incorporating these engineered cells into the design of synthetic polymeric materials will enable programming materials with a wide range of biological functionalities. However, employable strategies for the design of synthetic polymers that form a well-defined interface with living cells and seamlessly integrate their functionalities in materials are still largely limited. Herein, we report the first example of living materials constructed with a dynamic covalent interface between synthetic polymers and living B. subtilis cells. We showedthat 3-acetamidophenylboronic acid (APBA) and polymers of APBA (pAPBA) form dynamic covalent bonds with available diols on the B. subtilis cell surface. Importantly, pAPBA binding to B. subtilis shows a multivalent effect with complete reversibility upon addition of competitive diol species, such as fructose and sorbitol. On the basis of these findings, we constructed telechelic block copolymers with pAPBA chain ends that crosslink B. subtilis cells and produced self- standing living materials. We further demonstrated that the encapsulated cells could be retrieved upon immersing these materials in solutions containing competitive diols and further subjected to biological analyses. This work establishes the groundwork for building a myriad of synthetic polymeric materials integrating engineered living cells and provides a platform for understanding the biology of cells confined within materials.

Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis

Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.

Evaluation of the In Vitro Cytotoxicity of Silver Nanoparticles on PBMC Cells Using MTT Assay

Background: Silver Nanoparticles are extensively studied by the scientific community for therapeutic applications. With respect to the fundamental pillars of bioethics “Primum non nocere” equal emphasis should be given to evaluate the toxicological perspectives of Silver nanoparticles. This study aims at evaluating the InVitro cytotoxic effects of Silver nanoparticles synthesized using hesperidin. Aim: To study the In Vitro cytotoxicity of silver nanoparticles on PBMC cells using (3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Methods: Synthesized silver nanoparticles at various concentrations are incubated with peripheral blood mononuclear cells (PBMC). After 24 hours MTT is added to the mixture to evaluate the cell viability post incubation. Yellow MTT (a tetrazole) which is reduced to purple formazan in the mitochondria of living cells. The absorbance of this colored solution can be quantified by measuring at 570 nm by a spectrophotometer. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells. Results: ?.Conclusion: Silver Nanoparticles do not exhibit any significant cytotoxicity on PBMCs and also there were no dose dependent trends in the results.

Decoding the Complex Crossroad of Tryptophan Metabolic Pathways

Among the 20 amino acids needed for protein synthesis, Tryptophan (Trp) is an aromatic amino acid fundamental not only for the synthesis of the major components of living cells (namely, the proteins), but also for the maintenance of cellular homeostasis [...]