Cell-Free Protein Expression in Polymer Materials.

While synthetic biology has advanced complex capabilities such as sensing and molecular synthesis in aqueous solutions, important applications may also be pursued for biological systems in solid materials. Harsh processing conditions used to produce many synthetic materials such as plastics make the incorporation of biological functionality challenging. One technology that shows promise in circumventing these issues is cell-free protein synthesis (CFPS), where core cellular functionality is reconstituted outside the cell. CFPS enables genetic functions to be implemented without the complications of membrane transport or concerns over the cellular viability or release of genetically modified organisms. Here, we demonstrate that dried CFPS reactions have remarkable tolerance to heat and organic solvent exposure during the casting processes for polymer materials. We demonstrate the utility of this observation by creating plastics that have spatially patterned genetic functionality, produce antimicrobials in situ, and perform sensing reactions. The resulting materials unlock the potential to deliver DNA-programmable biofunctionality in a ubiquitous class of synthetic materials.

Genetic and epigenetic alterations in MGMT gene and correlation with concomitant chemoradiotherapy (CRT) in cervical cancer.

PURPOSE: The MGMT (O6-methylguanine-DNA methyltransferase) gene plays a crucial role in repairing DNA damage caused by alkylating agents, including those used in chemotherapy. Genetic and epigenetic alterations can influence the regulation of MGMT gene, which in turn may impact the response to concomitant chemoradiotherapy (CRT) in cervical cancer. The present study was undertaken to evaluate the correlation of such variations in MGMT gene with the treatment outcome of concomitant chemoradiotherapy (CRT) in cervical cancer. METHODS: A total of 460 study subjects (240 controls and 220 patients) were subjected to genotypic analysis of MGMT gene variants rs12917(T/C) and rs2308327(A/G) by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). Out of them, 48 each of controls and patients were analyzed for promoter methylation and expression by methylation-specific PCR and real-time PCR, respectively. Patients (n = 48) were followed up and evaluated for treatment (CRT) outcome. Statistical analyses were done using GraphPad (9.0) and SPSS version 18.0. RESULTS: Individuals with GG genotype, G allele of rs2308327, and haplotype 'TA' of both variants showed a significant increase in the development of cervical cancer (P ≤ 0.05). In epigenetic regulation, there was a significant hypermethylation of MGMT gene and down-regulation of their expression in patients compared to control individuals. In treatment outcome of CRT, GG genotype of rs2308327(A/G) gene variant showed better response and GG + AG was significantly associated with vital status (alive). Unmethylated MGMT gene showed better median overall survival up to 25 months significant in comparison to methylated MGMT promoter. CONCLUSION: Gene variant rs2308327(A/G) and promoter hypermethylation regulated MGMT gene can be a good prognostic for treatment response in cervical cancer patients.

Iridescent biofilms of Cellulophaga lytica are tunable platforms for scalable, ordered materials.

Nature offers many examples of materials which exhibit exceptional properties due to hierarchical assembly of their constituents. In well-studied multi-cellular systems, such as the morpho butterfly, a visible indication of having ordered submicron features is given by the display of structural color. Detailed investigations of nature's designs have yielded mechanistic insights and led to the development of biomimetic materials at laboratory scales. However, the manufacturing of hierarchical assemblies at industrial scales remains difficult. Biomanufacturing aims to leverage the autonomy of biological systems to produce materials at lower cost and with fewer carbon emissions. Earlier reports documented that some bacteria, particularly those with gliding motility, self-assemble into biofilms with polycrystalline structures and exhibit glittery, iridescent colors. The current study demonstrates the potential of using one of these bacteria, Cellulophaga lytica, as a platform for the large scale biomanufacturing of ordered materials. Specific approaches for controlling C. lytica biofilm optical, spatial and temporal properties are reported. Complementary microscopy-based studies reveal that biofilm color variations are attributed to changes in morphology induced by cellular responses to the local environment. Incorporation of C. lytica biofilms into materials is also demonstrated, thereby facilitating their handling and downstream processing, as would be needed during manufacturing processes. Finally, the utility of C. lytica as a self-printing, photonic ink is established by this study. In summary, autonomous surface assembly of C. lytica under ambient conditions and across multiple length scales circumvent challenges that currently hinder production of ordered materials in industrial settings.

Microbially Induced Calcium Carbonate Precipitation by Sporosarcina pasteurii: a Case Study in Optimizing Biological CaCO3 Precipitation.

Current production of traditional concrete requires enormous energy investment that accounts for approximately 5 to 8% of the world's annual CO2 production. Biocement is a building material that is already in industrial use and has the potential to rival traditional concrete as a more convenient and more environmentally friendly alternative. Biocement relies on biological structures (enzymes, cells, and/or cellular superstructures) to mineralize and bind particles in aggregate materials (e.g., sand and soil particles). Sporosarcina pasteurii is a workhorse organism for biocementation, but most research to date has focused on S. pasteurii as a building material rather than a biological system. In this review, we synthesize available materials science, microbiology, biochemistry, and cell biology evidence regarding biological CaCO3 precipitation and the role of microbes in microbially induced calcium carbonate precipitation (MICP) with a focus on S. pasteurii. Based on the available information, we provide a model that describes the molecular and cellular processes involved in converting feedstock material (urea and Ca2+) into cement. The model provides a foundational framework that we use to highlight particular targets for researchers as they proceed into optimizing the biology of MICP for biocement production.

Higher order genes interaction in DNA repair and cytokine genes polymorphism and risk to lung cancer in North Indians.

Context: Lung cancer pathological process involves cumulative effects exerted by gene polymorphism(s), epigenetic modifications, and alterations in DNA repair machinery. Further, DNA damage due to oxidative stress, chronic inflammation, and the interplay between genetic and environmental factors is also an etiologic milieu of this malignant disease. Aims: The present study aims to assess the prognostic value of DNA repair, cytokines, and GST gene polymorphism in lung cancer patients who had not received any neoadjuvant therapy. Materials and Methods: In this case-control study, 127 cases and 120 controls were enrolled. DNA from the blood samples of both patients and controls was used to genotype XRCC1Arg399Gln, XPDLys751Gln, and interleukin-1 (IL-1ß) genes by polymerase chain reaction (PCR)-restriction fragment length polymorphism method, whereas multiplex PCR was performed to genotype GSTT1 and GSTM1. Results: Binary logistic regression analysis showed that XRCC1Arg399Gln-mutant genotype (Gln/Gln, odds ratio [OR] = 4.6, 95% confidence interval [CI]: 2.2-9.6) and GSTT1 null (OR = 2.7, 95% CI: 1.6-4.5) were linked to cancer susceptibility. Generalized multidimensional reduction analysis of higher order gene-gene interaction using cross-validation testing (CVT) accuracy showed that GSTT1 (CVT 0.62, P = 0.001), XPD751 and IL-1ß (CVT 0.6, P = 0.001), and XRCC1399, XPD751, and interleukin-1 receptor antagonists (IL-1RN) (CVT 0.98, P = 0.001) were single-, two-, and three-factor best model predicted, respectively, for lung cancer risk. Classification and regression tree analysis results showed that terminal nodes which contain XRCC1399-mutant genotype (AA) had increased the risk to lung cancer. Conclusion: The present study demonstrated that XRCC1399 (Gln/Gln), GSTT1, and IL-1RN allele I, I/II served as the risk genotypes. These genes could serve as the biomarkers to predict lung cancer risk.

Particle-Based Microrheology As a Tool for Characterizing Protein-Based Materials.

Microrheology based on video microscopy of embedded tracer particles has the potential to be used for high-throughput protein-based materials characterization. This potential is due to a number of characteristics of the techniques, including the suitability for measurement of low sample volumes, noninvasive and noncontact measurements, and the ability to set up a large number of samples for facile, sequential measurement. In addition to characterization of the bulk rheological properties of proteins in solution, for example, viscosity, microrheology can provide insight into the dynamics and self-assembly of protein-based materials as well as heterogeneities in the microenvironment being probed. Specifically, passive microrheology in the form of multiple particle tracking and differential dynamic microscopy holds promise for applications in high-throughput characterization because of the lack of user interaction required while making measurements. Herein, recent developments in the use of multiple particle tracking and differential dynamic microscopy are reviewed for protein characterization and their potential to be applied in a high-throughput, automatable setting.

Engineering Gelation Kinetics in Living Silk Hydrogels by Differential Dynamic Microscopy Microrheology and Machine Learning.

Microbes embedded in hydrogels comprise one form of living material. Discovering formulations that balance potentially competing for mechanical and biological properties in living hydrogels-for example, gel time of the hydrogel formulation and viability of the embedded organisms-can be challenging. In this study, a pipeline is developed to automate the characterization of the gel time of hydrogel formulations. Using this pipeline, living materials comprised of enzymatically crosslinked silk and embedded E. coli-formulated from within a 4D parameter space-are engineered to gel within a pre-selected timeframe. Gelation time is estimated using a novel adaptation of microrheology analysis using differential dynamic microscopy (DDM). In order to expedite the discovery of gelation regime boundaries, Bayesian machine learning models are deployed with optimal decision-making under uncertainty. The rate of learning is observed to vary between artificial intelligence (AI)-assisted planning and human planning, with the fastest rate occurring during AI-assisted planning following a round of human planning. For a subset of formulations gelling within a targeted timeframe of 5-15 min, fluorophore production within the embedded cells is substantially similar across treatments, evidencing that gel time can be tuned independent of other material properties-at least over a finite range-while maintaining biological activity.

Biomorphic Ceramics: Synthesis and Characterization of Preceramic Polymer-Modified Melanin.

Recent efforts have demonstrated that the morphology of ceramics can be manipulated to control both their deformation mechanism and mechanical performance. However, precise control of the ceramic nanostructure is still difficult to achieve. Biotemplating, leading to biomorphic materials, provides a facile route to manipulate the nanostructure of the resulting materials, and the use of melanin as a coating provides a new route to biotemplated materials. Melanin is underutilized for structural materials partly due to the cost of procuring it from natural sources and the inability to control the shape and sizes of melanin particles. Taking a combined synthetic biology and chemical synthesis approach, we report the melanization of Escherichia coli and its subsequent silanization and functionalization with preceramic polymers to make novel biomorphic silicon-based ceramic materials. Graft-to and graft-from reactions were used to append preceramic polymers to the melanin, followed by pyrolysis under argon. Samples were analyzed by FTIR, XRD, XPS, and TEM and found to retain the shape and size of the original cells with high fidelity. The homogeneity of coverage and yield of the resulting ceramic materials depended on the type of grafting reaction. This work provides a promising proof-of-concept that bacterial-templated ceramics can be readily made and opens a host of possibilities for further studies and applications.

Cytokine gene variants and socio-demographic characteristics as predictors of cervical cancer: A machine learning approach.

Cervical cancer is still one of the most prevalent cancers in women and a significant cause of mortality. Cytokine gene variants and socio-demographic characteristics have been reported as biomarkers for determining the cervical cancer risk in the Indian population. This study was designed to apply a machine learning-based model using these risk factors for better prognosis and prediction of cervical cancer. This study includes the dataset of cytokine gene variants, clinical and socio-demographic characteristics of normal healthy control subjects, and cervical cancer cases. Different risk factors, including demographic details and cytokine gene variants, were analysed using different machine learning approaches. Various statistical parameters were used for evaluating the proposed method. After multi-step data processing and random splitting of the dataset, machine learning methods were applied and evaluated with 5-fold cross-validation and also tested on the unseen data records of a collected dataset for proper evaluation and analysis. The proposed approaches were verified after analysing various performance metrics. The logistic regression technique achieved the highest average accuracy of 82.25% and the highest average F1-score of 82.58% among all the methods. Ridge classifiers and the Gaussian Naïve Bayes classifier achieved the highest sensitivity-85%. The ridge classifier surpasses most of the machine learning classifiers with 84.78% accuracy and 97.83% sensitivity. The risk factors analysed in this study can be taken as biomarkers in developing a cervical cancer diagnosis system. The outcomes demonstrate that the machine learning assisted analysis of cytokine gene variants and socio-demographic characteristics can be utilised effectively for predicting the risk of developing cervical cancer.

Marine Structural Protein Stability Induced by Hofmeister Salt Annealing and Enzymatic Cross-Linking.

The Humboldt squid is one of the fiercest marine predators thanks in part to its sucker ring teeth that are biopolymer blends of a protein isoform family called suckerin with compression strength that rivals silkworm silk. Here, we focus on the popular suckerin-12 isoform to understand what makes the secondary structure of this biopolymer different in water and the potential role of diverse physical and chemical cross-linkings. By choosing a salt post-treatment, in accordance with the Hofmeister series, we achieved film stability with salt annealing that is comparable to chemical cross-links. By correlating the film morphology with the protein secondary structure changes, suckerin-12 films were shown to contract upon treatment with kosmotropic salts and exhibited increased stability in water. These changes are related to the rearrangement of suckerin-12 secondary structure from random coils and helices to ß-sheets. Overall, understanding secondary structure changes caused by aqueous and ionic environments can be instructive for the tuning of the suckerin film sclerotization, its conversion to a tough biological material, and to ultimately produce the natural squid sucker ring teeth.

Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12.

Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined ß-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ∼100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and ß-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

Silk fibroin as an additive for cell-free protein synthesis.

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

Lyophilized Cell-Free Systems Display Tolerance to Organic Solvent Exposure.

Cell-free systems offer a powerful way to deliver biochemical activity to the field without cold chain storage. These systems are capable of sensing as well as biosynthesis of useful molecules at the point of need. So far, cell-free protein synthesis (CFPS) reactions have been studied as aqueous solutions in test tubes or absorbed into paper or cloth. Embedding biological functionality into broadly used materials, such as plastic polymers, represents an attractive goal. Unfortunately, this goal has for the most part remained out of reach, presumably due to the fragility of biological systems outside of aqueous environments. Here, we describe a surprising and useful feature of lyophilized cell-free lysate systems: tolerance to a variety of organic solvents. Screens of individual CFPS reagents and different CFPS methods reveal that solvent tolerance varies by CFPS reagent composition. Tolerance to suspension in organic solvents may facilitate the use of polymers to deliver dry cell-free reactions in the form of coatings or fibers, or allow dosing of analytes or substrates dissolved in nonaqueous solvents, among other processing possibilities.

Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG).

Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

Corrigendum to "3D printed bionic nanodevices" [Nano Today 11 (2016) 330-350].

[This corrects the article PMC5016035.].

Disulfide Crosslinked Hydrogels Made From the Hydra Stinging Cell Protein, Minicollagen-1.

Minicollagens from cnidarian nematocysts are attractive potential building blocks for the creation of strong, lightweight and tough polymeric materials with the potential for dynamic and reconfigurable crosslinking to modulate functionality. In this study, the Hydra magnipapillata minicollagen-1 isoform was recombinantly expressed in bacteria, and a high throughput purification protocol was developed to generate milligram levels of pure protein without column chromatography. The resulting minicollagen-1 preparation demonstrated spectral properties similar to those observed with collagen and polyproline sequences as well as the ability to self-assemble into oriented fibers and bundles. Photo-crosslinking with Ru(II) ( bpy ) 3 2 + was used to create robust hydrogels that were analyzed by mechanical testing. Interestingly, the minicollagen-1 hydrogels could be dissolved with reducing agents, indicating that ruthenium-mediated photo-crosslinking was able to induce disulfide metathesis to create the hydrogels. Together, this work is an important first step in creating minicollagen-based materials whose properties can be manipulated through static and reconfigurable post-translational modifications.

Anion-Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels.

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin-12, is used to create enzymatically crosslinked, macro-scale hydrogels. Upon exposure to specific salt conditions, suckerin-12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin-12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin-12 hydrogels. The potential for suckerins to add new properties to materials based on naturally-derived proteins is highlighted.

Programmable Mechanical Properties from a Worm Jaw-Derived Biopolymer through Hierarchical Ion Exposure.

Mechanisms of biomaterial sclerotization in natural systems promise new insights into how the mechanical properties of engineered materials may be dynamically modulated. One such example involves the proteinaceous jaw of the marine sandworm, Nereis virens. Previously, the mechanical properties of the N. virens jaw were shown to be modulated by Zn binding, a property that was proposed to be enabled by the presence of the histidine-rich jaw protein, Nvjp-1. Here we demonstrate the creation of Nvjp-1-based hydrogels and show that progressive sclerotization of these hydrogels can be accomplished with hierarchical exposure to metal cations and anions. Divalent Zn cations are capable of reversibly sclerotizing the hydrogels through the formation of coordinate cross-links, an effect that is shown to be remarkably specific for Zn. Additionally, the degree of Zn-induced sclerotization is strongly influenced by the identity of the anion present in the hydrogel. Thus, the viscoelastic properties of Nvjp-1 hydrogels can be modulated through programmed, hierarchical exposure to specific cations and anions present in the sclerotizing salts. These observations have resulted in new hydrogel capabilities, such as the creation of anion-controlled shape-memory polymers, and will add to the number of control parameters that can be used to tune the properties of functional hydrogels in a dynamic manner.

Bio-Optics and Bio-Inspired Optical Materials.

Through the use of the limited materials palette, optimally designed micro- and nanostructures, and tightly regulated processes, nature demonstrates exquisite control of light-matter interactions at various length scales. In fact, control of light-matter interactions is an important element in the evolutionary arms race and has led to highly engineered optical materials and systems. In this review, we present a detailed summary of various optical effects found in nature with a particular emphasis on the materials and optical design aspects responsible for their optical functionality. Using several representative examples, we discuss various optical phenomena, including absorption and transparency, diffraction, interference, reflection and antireflection, scattering, light harvesting, wave guiding and lensing, camouflage, and bioluminescence, that are responsible for the unique optical properties of materials and structures found in nature and biology. Great strides in understanding the design principles adapted by nature have led to a tremendous progress in realizing biomimetic and bioinspired optical materials and photonic devices. We discuss the various micro- and nanofabrication techniques that have been employed for realizing advanced biomimetic optical structures.