Bioinspirations: cell-inspired small-scale systems for enabling studies in experimental biomechanics

Biomechanical forces govern the behaviors of organisms and their environment and examining these behaviors to understand the underlying phenomena is an important challenge. One experimental approach for probing these interactions between organisms and their biomechanical environment uses biologically-inspired, artificial surrogates that reproduce organic mechanical systems. For the case of complex, multicellular organisms, robot surrogates have been particularly effective, such as in the analysis of the fins of fish and insects' wings. This biologically-inspired approach is also exciting when examining cell-scale responses as multicellular organisms' behavior is directly influenced by the integrated interactions of smaller-scale components (i.e., cells). In this review, we introduce the burgeoning field of engineering of artificial cells, which focuses on developing cell-scale entities replicating cellular behaviors. We describe both a bottom-up approach to constructing artificial cells, using molecular components to directly assemble artificial cells, as well as a top-down approach, in which living cells are encapsulated in a single entity whose behavior is determined by its constituent members. In particular, we discuss the potential role of these artificial cells as implantable controllers, designed to alter the mechanical behavior of a host organism. Eventually, artificial cells designed to function as small-scale controllers may help alter organisms' phenotypes.     

Production of nitrogen fertilizer with nitrogen-fixing blue - green algae

Heterocystous nitrogen-fixing blue-green algae consist of filaments containing two types of cells: the heterocysts, responsible for ammonia synthesis, and vegetative cells, which exhibit normal photosynthesis and reproductive growth. This unique biological system could be used for the conversion of solar energy into organic fertilizer, through cultivation of these algae in open ponds. The most immediately practical approach is the use of this process in conjunction with waste-water treatment. Initial experiments have involved the isolation of sewage effluents-adapted algae and their cultivation in small-scale ponds. Significant rates of biomass production and nitrogen fixation were achieved, but a substantial improvement is still needed for possible practical applications. The potential economics of such systems and the need for new sources of fertilizers are discussed.     

The therapeutic potential of stem cells

In recent years, there has been an explosion of interest in stem cells, not just within the scientific and medical communities but also among politicians, religious groups and ethicists. Here, we summarize the different types of stem cells that have been described: their origins in embryonic and adult tissues and their differentiation potential in vivo and in culture. We review some current clinical applications of stem cells, highlighting the problems encountered when going from proof-of-principle in the laboratory to widespread clinical practice. While some of the key genetic and epigenetic factors that determine stem cell properties have been identified, there is still much to be learned about how these factors interact. There is a growing realization of the importance of environmental factors in regulating stem cell behaviour and this is being explored by imaging stem cells in vivo and recreating artificial niches in vitro. New therapies, based on stem cell transplantation or endogenous stem cells, are emerging areas, as is drug discovery based on patient-specific pluripotent cells and cancer stem cells. What makes stem cell research so exciting is its tremendous potential to benefit human health and the opportunities for interdisciplinary research that it presents.     

ASSEMBLY AND OPTIMIZATION OF EXPRESSION OF SYNTHETIC GENES DERIVED FROM THE HUMAN ELASTIN REPEATED MOTIF

Naturally occurring proteins often possess interesting properties that make them attractive for the realization of innovative biomaterials. Repetitive artificial polypeptides have been modeled on the repeated domains of mammalian elastin, retaining and even enhancing their thermally responsive behavior. These protein polymers have been produced by recombinant biotechnology and due to their smart properties show a huge potential for a wide range of biomedical and biotechnological applications. For this reason, production of large quantities of highly purified material is a crucial step. We focused our attention on elastin-derived polypeptides based on the hexapeptidic motif typical of human elastin. Synthetic genes were assembled starting from a monomeric unit, and the different conditions were assayed to optimize the yield of the artificial polypeptides. Optimization of Escherichia coli strain and of the extraction procedure led to significant improvement in expression and recovery of the recombinant products. Electron micrographs of expressing bacteria under optimized conditions showed the accumulation of the recombinant product in the induced cells.     

Microalgae mass culture: the constraints of scaling-up

There is overwhelming evidence that microalgae would be the logical source of oils for biodiesel production, the best option for CO₂ sequestration and numerous other applications. However, this apparent lucrative approach is still in its infancy. In order to impact on global energy needs, bioremediation and other potential applications, vast quantities of biomass must be produced at a reliable rate and as cost-effective as possible. When extrapolating volumetric rates from laboratory or small-scale outdoor cultures to large-scale outdoor areal production rates, it becomes apparent that many of the potential claims are either misleading or still only a dream. Open raceway ponds are at present the only feasible culture system for the production of millions of tons of biomass. To date, at best photosynthetic efficiencies of around 1.2% have been achieved, but with present understanding and know-how efficiencies of double that should be achievable, especially when vertical mixing is increased in raceway ponds.     

A phenomenological model for chemico-mechanically induced cell shape changes during migration and cell–cell contacts

A phenomenological model for the evolution of shape transition of cells is considered. These transitions are determined by the emission of growth-factors, as well as mechanical interaction if cells are subjected to hard impingement. The originality of this model necessitates a formal treatment of the mathematical model, as well as the presentation of elementary cases in order to illustrate the consistence of the model. We will also show some small-scale relevant applications.     

Dendritic normalisation improves learning in sparsely connected artificial neural networks

Artificial neural networks, taking inspiration from biological neurons, have become an invaluable tool for machine learning applications. Recent studies have developed techniques to effectively tune the connectivity of sparsely-connected artificial neural networks, which have the potential to be more computationally efficient than their fully-connected counterparts and more closely resemble the architectures of biological systems. We here present a normalisation, based on the biophysical behaviour of neuronal dendrites receiving distributed synaptic inputs, that divides the weight of an artificial neuron’s afferent contacts by their number. We apply this dendritic normalisation to various sparsely-connected feedforward network architectures, as well as simple recurrent and self-organised networks with spatially extended units. The learning performance is significantly increased, providing an improvement over other widely-used normalisations in sparse networks. The results are two-fold, being both a practical advance in machine learning and an insight into how the structure of neuronal dendritic arbours may contribute to computation.     

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.     

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8(+) T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8(+) T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy.     

Real-Time Imaging of Electrical Signals with an Infrared FDA-Approved Dye

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.     

Haloarchaea: worth exploring for their biotechnological potential

Halophilic archaea are unique microorganisms adapted to survive under high salt conditions and biomolecules produced by them may possess unusual properties. Haloarchaeal metabolites are stable at high salt and temperature conditions that are useful for industrial applications. Proteins and enzymes of this group of archaea are functional under salt concentrations at which bacterial counterparts fail to be active. Such properties makes haloarchaeal enzymes suitable for salt-based applications and their use under dehydrating conditions. For example, bacteriorhodopsin or the purple membrane protein present in halophilic archaea has the most recognizable applications in photoelectric devices, artificial retinas, holograms etc. Haloarchaea are also useful for bioremediation of polluted hypersaline areas. Polyhydroxyalkanoates and exopolysccharides produced by these microorganisms are biodegradable and have the potential to replace commercial non-degradable plastics and polymers. Moreover, halophilic archaea have excellent potential to be used as drug delivery systems and for nanobiotechnology by virtue of their gas vesicles and S-layer glycoproteins. Despite of possible applications of halophilic archaea, laboratory-to-industrial transition of these potential candidates is yet to be established.     

DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors.

None of the vector systems currently available for gene therapy applications have been shown to be capable of both efficient gene transfer into nondividing cells and long-term expression through stable integration into host cell DNA. While integrating vectors based on adeno-associated virus are capable of mediating gene transfer into nondividing cells, this process is 200-fold less efficient than transduction of dividing cells. We demonstrate that the transduction efficiency of adeno-associated virus vectors can be increased by treatment with DNA-damaging agents. Nondividing cells are especially responsive, with increases in transduction efficiency of up to 750-fold. This finding has the potential to facilitate gene therapy applications requiring gene transfer to nondividing cells.     

Recent trends in stem cell-based therapies and applications of artificial intelligence in regenerative medicine

Stem cells are undifferentiated cells that can self-renew and differentiate into diverse types of mature and functional cells while maintaining their original identity. This profound potential of stem cells has been thoroughly investigated for its significance in regenerative medicine and has laid the foundation for cell-based therapies. Regenerative medicine is rapidly progressing in healthcare with the prospect of repair and restoration of specific organs or tissue injuries or chronic disease conditions where the body’s regenerative process is not sufficient to heal. In this review, the recent advances in stem cell-based therapies in regenerative medicine are discussed, emphasizing mesenchymal stem cell-based therapies as these cells have been extensively studied for clinical use. Recent applications of artificial intelligence algorithms in stem cell-based therapies, their limitation, and future prospects are highlighted.     

Developing microbial biofilm as a robust biocatalyst and its challenges

Biocatalysts, such as bacteria, yeast, fungi and the enzymes they produce, have been used for many industrial applications since they function as effective and environmentally friendly tools. Whole cells have also been used in many sophisticated bioprocesses since a number of sequential reactions can be catalyzed within the cells. However, the use of whole cells in suspension in batch, fed-batch and continuous processes has some limitations. For instance, the cultures are non-reusable, they are sometimes sensitive to the toxicity of substrates or products, there can be issues with short-term stability, and each of these issues can impede biocatalyst regeneration, perturbing the downstream process and causing complexity in running large scale continuous culture. Recently, biofilms have emerged as a new generation of biocatalysts to solve these limitations in the production of many bio-based materials, including chemicals, antibiotics, enzymes, bioethanol, biohydrogen, and electricity production via microbial fuel cells. The establishment of industrial processes using biofilms has the potential for high benefit in terms of low-cost cell immobilization without the necessity of added polymers or chemicals. Many small-scale biofilm reactors have been developed for the production of value-added products, and it may be challenging to establish it on an industrial scale.     

Engineering design principles for organelle size control systems

Organelle size is an important determinant of organelle function, and for this reason cells have evolved mechanisms to control and adjust organelle size in the face of intrinsic biological fluctuations. Size control systems have been found that employ a variety of distinct mechanisms, which fall into a small number of classes. Each class represents a design principle by which artificial size controllers could be developed for synthetic biology applications.     

Lipid bilayer arrays: Cyclically formed and measured

Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, and can potentially be applied in DNA sequencing. Droplet bilayers have significant technological potential for enabling many of these applications due to their compatibility with automation and array platforms. To further develop this potential, we have simplified the formation and electrical measurement of droplet bilayers using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and measurement over a 32‐element array with ～80% yield and no operator input following fluid addition. Cycling these arrays resulted in the formation and measurement of 96 out of 120 possible bilayers in 80 minutes, a sustainable rate that could significantly increase with automation and greater parallelization. This turn‐key, high‐yield approach to making artificial lipid bilayers requires no training, making the capability of creating and measuring lipid bilayers and ion channels accessible to a much wider audience. In addition, this approach is low‐cost, parallelizable, and automatable, allowing high‐throughput studies of ion channels and pore proteins in lipid bilayers for sensing or screening applications.     

Using System Emulation to Model Next-Generation Shared Virtual Memory Clusters

Recently much effort has been spent on providing a shared address space abstraction on clusters of small-scale symmetric multiprocessors. However, advances in technology will soon make it possible to construct these clusters with larger-scale cc-NUMA nodes, connected with non-coherent networks that offer latencies and bandwidth comparable to interconnection networks used in hardware cache-coherent systems. The shared memory abstraction can be provided on these systems in software across nodes and hardware within nodes.Recent simulation results have demonstrated that certain features of modern system area networks can be used to greatly reduce shared virtual memory (SVM) overheads [5,19]. In this work we leverage these results and we use detailed system emulation to investigate building future software shared memory clusters. We use an existing, large-scale hardware cache-coherent system with 64 processors to emulate a complete future cluster. We port our existing infrastructure (communication layer and shared memory protocol) on this system and study the behavior of a set of real applications. We present results for both 32- and 64-processor system configurations.We find that: (i) System emulation is invaluable in quantifying potential benefits from changes in the technology of commodity components. More importantly, it reveals potential problems in future systems that are easily overlooked in simulation studies. Thus, system emulation should be used along with other modeling techniques (e.g., simulation, implementation) to investigate future trends. (ii) Our work shows that current SVM protocols can only partially take advantage of faster interconnects and wider nodes due to operating system and architectural implications. We quantify the related issues and identify the areas where more research is required for future SVM clusters.     

Alginate as immobilization matrix for cells.

In recent years, entrapment of cells within spheres of Ca2+ alginate has become the most widely used technique for immobilizing living cells. This versatile method includes applications ranging from immobilization of living or dead cells in bioreactors, immobilization of plant protoplasts for micropropagation and immobilization of hybridoma cells for production of monoclonal antibodies, to entrapment of animal cells for implantation of artificial organs. This review evaluates the potential of this method on the basis of the current knowledge of structural and functional relationships in alginate gels.     

Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining

The repair of DNA by nucleotide excision repair (NER) and non-homologous end joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Examination of NHEJ and NER in vitro using cell-free extracts has led to a deeper understanding of the biochemical mechanisms that underlie these processes. Current methods for production of whole-cell extracts (WCEs) to investigate NER and NHEJ start with one or more liters of culture containing 1–5 × 10⁹ cells. Here, we describe a small-scale method for production of WCE that can be used to study NER. We also describe a rapid, small-scale method for the preparation of WCE that can be used in the study of NHEJ. These methods require less time, 20- to 1000-fold fewer cells than large-scale extracts, facilitate examination of numerous samples and are ideal for such applications as the study of host–virus interactions and analysis of mutant cell lines.