Biological Building Blocks Research Articles

A synthetic cell-free pathway for biocatalytic upgrading of one-carbon substrates.

Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA. The synthetic reductive formate pathway (ReForm) consists of five core enzymes catalyzing non-natural reactions that were established through a cell-free enzyme engineering platform. We demonstrate that ReForm works in a plug-and-play manner to accept diverse C1 substrates including CO2 equivalents. We anticipate that ReForm will facilitate efforts to build and improve synthetic C1 utilization pathways for a formate-based bioeconomy.

Biodielectrics: old wine in a new bottle?

Biodielectrics is a subset of biological and/or bioinspired materials that has brought a huge transformation in the advancement of medical science, such as localized drug delivery in cancer therapeutics, health monitoring, bone and nerve repair, tissue engineering and use in other nanoelectromechanical systems (NEMS). While biodielectrics has long been used in the field of electrical insulation for over a century, polar dielectric properties of biological building blocks have not been well understood at the fundamental building block level. In this review article, we provide a brief overview of dielectric properties of biological building blocks and its hierarchical organisations to include polar dielectric properties such as piezo, pyro, and ferroelectricity. This review article also discusses recent trends, scope, and potential applications of these dielectrics in science and technology. We highlight electromechanical properties embedded in rationally designed organic assemblies, and the challenges and opportunities inherent in mapping from molecular amino acid building blocks to macroscopic analogs of biological fibers and tissues, in pursuit of sustainable materials for next-generation technologies.

Preparation and temperature-controlled morphology of helical microrods composed of supramolecular α-cyclodextrin assemblies.

Significant efforts have been devoted so far to artificially fabricate supramolecular helical nano- and microstructures through the regulated assembly of biological and synthetic building blocks. However, the preparation of supramolecular helical structures with a regulated morphology remains challenging. Here, helical microrods composed of supramolecular α-cyclodextrin (α-CD) assemblies were fabricated by allowing an α-CD/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/2-pentanol mixture to stand at 30-60 °C under high humidity conditions. The morphology could be controlled by temperature to produce helical microrods with a regulated pitch and length. These helical rods can be applied as optical devices, chiral separation devices and asymmetric catalysts.

Misconception Analysis of Biology Education Students Using Diagnostic Three Tier Test on Genetics Material

Genetics is a branch of Biology that becomes the basic building blocks of Biology, because almost none of the development of biological science is based on the concept of genetics. However, until now Genetics is still considered difficult for students so that the opportunity for the emergence of an understanding of the concept of genetics that is different from the understanding put forward by experts. The difference in understanding can trigger misconceptions especially in Biology Education students as future teachers who need to master Genetics content. Therefore, efforts are needed to identify students' conceptions with diagnostic three tier test which is considered capable of providing information about misconceptions. This study aims to identify and analyze the misconceptions of Biology Education students on Genetics material. The sample used is a saturated sample, namely all Biology Education Semester 2 students of FKIP UHAMKA. The research method used is descriptive quantitative, which plays a role in data collection in the form of a percentage of student conceptions to be compared with predetermined criteria. The results of this study concluded that there were misconceptions from the 18th diagnostic three tier test of Genetics material by 50%. Of the four sub-materials, respectively, the highest misconceptions occurred in the substance of genetics by 54%, cell division by 51%, patterns of inheritance of traits by 49%, and mutations by 43%.

Temporal Enzymatic Treatment to Enhance the Remodeling of Multiple Cartilage Microtissues into a Structurally Organized Tissue.

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development.

Sound-based assembly of three-dimensional cellularized and acellularized constructs.

Herein we show an accessible technique based on Faraday waves that assist the rapid assembly of osteoinductive β-Tricalcium phosphate (β-TCP) particles as well as human osteoblast pre-assembled in spheroids. The hydrodynamic forces originating at 'seabed' of the assembly chamber can be used to tightly aggregate inorganic and biological entities at packing densities that resemble those of native tissues. Additionally, following a layer-by-layer assembly procedure, centimeter scaled osteoinductive three-dimensional and cellularized constructs have been fabricated. We showed that the intimate connection between biological building blocks is essential in engineering living system able of localized mineral deposition. Our results demonstrate, for the first time, the possibility to obtain three-dimensional cellularized and acellularized anisotropic constructs using Faraday waves.

Computational Study on the Water Corrosion Process at Schreibersite (Fe2NiP) Surfaces: from Phosphide to Phosphates.

Phosphorus (P) is a fundamental element for whatever form of life, in the same way as the other biogenic macroelements (SONCH). The prebiotic origin of P is still a matter of debate, as the phosphates present on earth are trapped in almost insoluble solid matrixes (apatites) and, therefore, hardly available for inclusion in living systems in the prebiotic era. The most accepted theories regard a possible exogenous origin during the Archean Era, through the meteoritic bombardment, when tons of reactive P in the form of phosphide ((Fe,Ni)3P, schreibersite mineral) reached the primordial earth, reacting with water and providing oxygenated phosphorus compounds (including phosphates). In the last 20 years, laboratory experiments demonstrated that the corrosion process of schreibersite by water indeed leads to reactive phosphates that, in turn, react with other biological building blocks (nucleosides and simple sugars) to form more complex molecules (nucleotides and complex sugars). In the present paper, we study the water corrosion of different crystalline surfaces of schreibersite by means of periodic DFT (density functional theory) simulations. Our results show that water adsorbs molecularly on the most stable (110) surface but dissociates on the less stable (001) one, giving rise to further reactivity. Indeed, subsequent water adsorptions, up to the water monolayer coverage, show that, on the (001) surface, iron and nickel atoms are the first species undergoing the corrosion process and, in a second stage, the phosphorus atoms also get involved. When adsorbing up to three and four water molecules per unit cell, the most stable structures found are the phosphite and phosphate forms of phosphorus, respectively. Simulation of the vibrational spectra of the considered reaction products revealed that the experimental band at 2423 cm-1 attributed to the P-H stretching frequency is indeed predicted for a phosphite moiety attached to the schreibersite (001) surface upon chemisorption of up to three water molecules.

Formation of Protein Nanoparticles in Microdroplet Flow Reactors.

Nanoparticles are increasingly being used for biological applications, such as drug delivery and gene transfection. Different biological and bioinspired building blocks have been used for generating such particles, including lipids and synthetic polymers. Proteins are an attractive class of material for such applications due to their excellent biocompatibility, low immunogenicity, and self-assembly characteristics. Stable, controllable, and homogeneous formation of protein nanoparticles, which is key to successfully delivering cargo intracellularly, has been challenging to achieve using conventional methods. In order to address this issue, we employed droplet microfluidics and utilized the characteristic of rapid and continuous mixing within microdroplets in order to produce highly monodisperse protein nanoparticles. We exploit the naturally occurring vortex flows within microdroplets to prevent nanoparticle aggregation following nucleation, resulting in systematic control over the particle size and monodispersity. Through combination of simulation and experiment, we find that the internal vortex velocity within microdroplets determines the uniformity of the protein nanoparticles, and by varying parameters such as protein concentration and flow rates, we are able to finely tune nanoparticle dimensional properties. Finally, we show that our nanoparticles are highly biocompatible with HEK-293 cells, and through confocal microscopy, we determine that the nanoparticles fully enter into the cell with almost all cells containing them. Due to the high throughput of the method of production and the level of control afforded, we believe that the approach described in this study for generating monodisperse protein-based nanoparticles has the potential for intracellular drug delivery or for gene transfection in the future.

Ferroelectricity in biological building blocks: Slipping on a banana peel?

Ferroelectricity in biological system has been anticipated both theoretically and experimentally over the past few decades. Claims of ferroelectricity in biological systems have given rise to confusion and methodological controversy. Over the years, a “loop” of induced polarization in response to a varying applied electrical field and a consequent polarization reversal has prompted many researchers to claim ferroelectricity in biological structures and their building blocks. Other observers were skeptical about the methodology adopted in generating the data and questioned the validity of the claimed ferroelectricity as such, “loop” can also be obtained from linear capacitors. In a paper with somewhat tongue-in-cheek title, Jim Scott showed that ordinary banana peels could exhibit closed loops of electrical charge which closely resemble and thus could be misinterpreted as ferroelectric hysteresis loops in barium sodium niobate, BNN paraphrasing it as “banana”. In this paper, we critically review ferroelectricity in biological system and argue that knowing the molecular and crystalline structure of biological building blocks and experimenting on such building blocks may be the way forward in revealing the “true” nature of ferroelectricity in biological systems.

Engineering High-Quality Cartilage Microtissues Using Hydrocortisone Functionalized Microwells.

Engineering clinically relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large-scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies which support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this article, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications. Impact statement This study addresses a key challenge facing emerging modular biofabrication strategies that use microtissues as biological building blocks. Namely, achieving the necessary robust and consistent differentiation of clinically relevant cell sources, for example, mesenchymal stem/stromal cells (MSCs), and the accumulation of sufficient tissue-specific extracellular matrix (ECM) to engineer tissue of scale. We achieved this by establishing hydrocortisone as a simple and potent method for improving MSC chondrogenesis, resulting in the biofabrication of high-quality (ECM rich) cartilage microtissues. These findings could enable the generation of more scalable engineered cartilage by ensuring the formation of high-quality microtissue building blocks generated using heterogeneous MSC populations.

Single-cell clinical chemistry: Exploring glucose uptake kinetics and HbA1c levels in individual red blood cells.

Single-molecule techniques have been a primary focus of many advances in biophysics over the last decades. However, the fact that many related technologies such as microfluidics, confocal microscopy, etc. are becoming standard laboratory tools makes it possible to pursue other goals such as single cell measurements. If single molecules are the building blocks of matter, single cells are the building blocks of biology, and the individual detection thereof offers similar benefits. Currently, most blood work is cuvette-based, averaging tens of millions of blood cells to obtain a single ensemble value. Measuring single blood cells yields a distribution of values, which provides more information about a given patient as well as how that patient may differ from the general populace. All of which is important in an era of big data which increasingly inclines medicine towards a more personalized approach. Our work focuses on the relationship between glucose, red blood cells (RBCs) and glycated hemoglobin, as quantifying the interplay of these parameters is important for a better understanding of diabetes. Using a microfluidic perfusion system allows the placement of RBCs and control over the solutions to which they are exposed. Using confocal microscopy and image analysis, we are able to observe the kinetics of fluorescently-labeled glucose passing into and out of individual RBCs and extract the relevant information, such as glucose influx and efflux rates, intracellular vs blood glucose levels, and more. Overall, we seek to quantify the relationship between bloodstream and intracellular glucose levels to inform a more personalized, and perhaps improved, diagnostic strategy for diabetes. We are also currently developing methods for quantifying %HbA1c at the single cell level utilizing Raman microscopy and mass spectrometry.

Interface-mediated protein aggregation.

The aggregation of proteins at interfaces has significant roles and can also lead to dysfunction of different physiological processes. The interfacial effects on the assembly and aggregation of biopolymers are not only crucial for a comprehensive understanding of protein biological functions, but also hold great potential for advancing the state-of-the-art applications of biopolymer materials. Recently, there has been remarkable progress in a collaborative context, as we strive to gain control over complex interfacial assembly structures of biopolymers. These biopolymer structures range from the nanoscale to mesoscale and even macroscale, and are attained through the rational design of interactions between biological building blocks and surfaces/interfaces. This review spotlights the recent advancements in interface-mediated assembly and properties of biopolymer materials. Initially, we introduce the solid-liquid interface (SIL)-mediated biopolymer assembly that includes the inorganic crystalline template effect and protein self-adoptive deposition through phase transition. Next, we display the advancement of biopolymer assembly instigated by the air-water interface (AWI) that acts as an energy conversion station. Lastly, we discuss succinctly the assembly of biopolymers at the liquid-liquid interface (LLI) along with their applications. It is our hope that this overview will stimulate the integration and progression of the science of interfacial assembled biopolymer materials and surfaces/interfaces.

Heteroatom-directed supramolecular helical-rich architectures in N-terminal protected pyridyl aromatic amino acids

Supramolecular helical structures formed by the assembly of biological and bio-inspired building blocks (typically amino acids and peptides) are an exciting class of materials with prospective applications in biomedical technologies and electronics.

Spatial patterning of phenotypically distinct microtissues to engineer osteochondral grafts for biological joint resurfacing

Modular biofabrication strategies using microtissues or organoids as biological building blocks have great potential for engineering replacement tissues and organs at scale. Here we describe the development of a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissues within an instructive 3D printed polymer framework. We first demonstrate that immature cartilage microtissues can spontaneously fuse to form homogeneous macrotissues, and that combining less cellular microtissues results in superior fusion and the generation of a more hyaline-like cartilage containing higher levels of sulphated glycosaminoglycans and type II collagen. Furthermore, temporally exposing developing microtissues to transforming growth factor-β accelerates their volumetric growth and subsequent capacity to fuse into larger hyaline cartilage grafts. Next, 3D printed polymeric frameworks are used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with zonal collagen organisation analogous to the structure seen in native articular cartilage. To engineer osteochondral grafts, hypertrophic cartilage microtissues are engineered as bone precursor tissues and spatially localised below phenotypically stable cartilage microtissues. Implantation of these engineered grafts into critically-sized caprine osteochondral defects results in effective defect stabilisation and histologically supports the restoration of a more normal articular surface after 6 months in vivo. These findings support the use of such modular biofabrication strategies for biological joint resurfacing.

Medial packing and elastic asymmetry stabilize the double-gyroid in block copolymers

Triply-periodic networks are among the most complex and functionally valuable self-assembled morphologies, yet they form in nearly every class of biological and synthetic soft matter building blocks. In contrast to simpler assembly motifs – spheres, cylinders, layers – networks require molecules to occupy variable local environments, confounding attempts to understand their formation. Here, we examine the double-gyroid network phase by using a geometric formulation of the strong stretching theory of block copolymer melts, a prototypical soft self-assembly system. The theory establishes the direct link between molecular packing, assembly thermodynamics and the medial map, a generic measure of the geometric center of complex shapes. We show that “medial packing” is essential for stability of double-gyroid in strongly-segregated melts, reconciling a long-standing contradiction between infinite- and finite-segregation theories. Additionally, we find a previously unrecognized non-monotonic dependence of network stability on the relative entropic elastic stiffness of matrix-forming to tubular-network forming blocks. The composition window of stable double-gyroid widens for both large and small elastic asymmetry, contradicting intuitive notions that packing frustration is localized to the tubular domains. This study demonstrates the utility of optimized medial tessellations for understanding soft-molecular assembly and packing frustration via an approach that is readily generalizable far beyond gyroids in neat block copolymers.

Electro-assembly of a dynamically adaptive molten fibril state for collagen.

Collagen is a biological building block that is hierarchically assembled into diverse morphological structures that, in some cases, is dynamically adaptive in response to external cues and in other cases forms static terminal structures. Technically, there is limited capabilities to guide the emergence of collagen’s hierarchical organization to recapitulate the richness of biological structure and function. Here, we report an electro-assembly pathway to create a dynamically adaptive intermediate molten fibril state for collagen. Structurally, this intermediate state is composed of partially aligned and reversibly associating fibrils with limited hierarchical structure. These molten fibrils can be reversibly reconfigured to offer dynamic properties such as stimuli-stiffening, stimuli-contracting, self-healing, and self-shaping. Also, molten fibrils can be guided to further assemble to recapitulate the characteristic hierarchical structural features of native collagen (e.g., aligned fibers with D-banding). We envision that the electro-assembly of collagen fibrils will provide previously unidentified opportunities for tailored collagen-based biomedical materials.

1,1,2‐Ethenetriol: The Enol of Glycolic Acid, a High‐Energy Prebiotic Molecule

AbstractAs low‐temperature conditions (e.g. in space) prohibit reactions requiring large activation energies, an alternative mechanism for follow‐up transformations of highly stable molecules involves the reactions of higher energy isomers that were generated in a different environment. Hence, one working model for the formation of larger organic molecules is their generation from high‐lying isomers of otherwise rather stable molecules. As an example, we present here the synthesis as well as IR and UV/Vis spectroscopic identification of the previously elusive 1,1,2‐ethenetriol, the higher energy enol tautomer of glycolic acid, a rather stable and hence unreactive biological building block. The title compound was generated in the gas phase by flash vacuum pyrolysis of tartronic acid at 400 °C and was subsequently trapped in argon matrices at 10 K. The spectral assignments are supported by B3LYP/6–311++G(2d,2p) computations. Upon photolysis at λ=180–254 nm, 1,1,2‐ethenetriol rearranges to glycolic acid and ketene.

1,1,2-Ethenetriol: The Enol of Glycolic Acid, a High-Energy Prebiotic Molecule.

As low‐temperature conditions (e.g. in space) prohibit reactions requiring large activation energies, an alternative mechanism for follow‐up transformations of highly stable molecules involves the reactions of higher energy isomers that were generated in a different environment. Hence, one working model for the formation of larger organic molecules is their generation from high‐lying isomers of otherwise rather stable molecules. As an example, we present here the synthesis as well as IR and UV/Vis spectroscopic identification of the previously elusive 1,1,2‐ethenetriol, the higher energy enol tautomer of glycolic acid, a rather stable and hence unreactive biological building block. The title compound was generated in the gas phase by flash vacuum pyrolysis of tartronic acid at 400 °C and was subsequently trapped in argon matrices at 10 K. The spectral assignments are supported by B3LYP/6–311++G(2d,2p) computations. Upon photolysis at λ=180–254 nm, 1,1,2‐ethenetriol rearranges to glycolic acid and ketene.

Biomimetic Nanozymes Based on Coassembly of Amino Acid and Hemin for Catalytic Oxidation and Sensing of Biomolecules.

Nanoassemblies based on self-assembly of biological building blocks are promising in mimicking the nanostructures, properties, and functionalities of natural enzymes. However, it remains a challenge to design of biomimetic nanozymes with tunable nanostructures and enhanced catalytic activities starting from simple biomolecules. Herein, the construction of nanoassemblies through coassembly of an amphiphilic amino acid and hemin is reported. The nanostructures and morphologies of the resulting nanoassemblies are readily controlled by tuning the molar ratio between the amino acid and hemin, thus leading to tailored peroxidase-mimicking activities of the nanoassemblies. Importantly, the optimized nanoassemblies exhibit a remarkable catalytic efficiency that is comparable to the natural counterpart when considering molecular mass along with good robustness in multiple catalytic cycles. The nanoassemblies are effectively integrated as biomimetic nanozymes in a sensing system for catalytic detection of glucose. Therefore, this work demonstrates that nanozymes with advanced catalytic capabilities can be constructed by self-assembly of minimalist biological building blocks and may thus promote the rational design and catalytic applications of biomimetic nanozymes.

Virus as a Nanocarrier for Drug Delivery Redefining Medical Therapeutics - A Status Report.

Over the last two decades, drug delivery systems have evolved at a tremendous pace. Synthetic nanoparticles have played an important role in vaccine design and delivery as these have shown improved safety and efficacy over conventional formulations. Nanocarriers formulated by natural, biological building blocks have become an important tool in biomedicine. A successful nanocarrier must possess specific properties like evading the host immune system, target specificity, cellular entry, escape from endosomes, and the ability to release the active material into the cytoplasm. The virus can perform some or all of these functions, making it a suitable candidate as a naturally occurring nanocarrier. Viruses could be made non-infectious and non-replicative without compromising their ability to penetrate cells, making them useful for a vast spectrum of applications. Currently, many such carrier molecules as bio-nanocapsules are at various development stages. This review covers the advances in the field of viruses as potential nanocarriers and discusses the related technologies and strategies to target specific cells by using virus-inspired nanocarriers. These virus-based nanocarriers could provide solutions to address pressing and emerging concerns in infectious diseases in the future.