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.     

The major histocompatibility complex-restricted antigen receptor on T cells. IX. Role of accessory molecules in recognition of antigen plus isolated IA

Antibody inhibition studies were done to determine which molecules on the surface of the T cell hybridomas other than their receptors for antigen plus IAd were involved in interaction with antigen-presenting B cells, with artificial IAd membranes on glass beads, or with anti-receptor antibodies coupled to Sepharose beads. We found that T cell LFA-1 was only involved when B cells were used to present antigen plus IAd, whereas T cell L3T4 was involved in the response of T cells to antigen plus IAd either on cells or in artificial membranes, but not if anti-receptor antibodies were used to stimulate the T cells. From these results we concluded that LFA-1 may be involved in the recognition of a ligand on cells that was not present in artificial membranes, but that L3T4 might interact with a nonpolymorphic portion of class II molecules present in both intact antigen-presenting cells and the antigen-presenting artificial membranes.     

A flow cytometry technique for measuring chromosome-mediated gene transfer

BACKGROUND: Using artificial chromosome expression systems (ACes), we have developed a unique and rapid screening technique to quantify delivery of foreign DNA into cells in vitro. Delivery was measured within 24 h after transfection, using flow cytometry to detect the transfer of ACes labeled with thymidine analogue. This technique can be used to optimize delivery parameters of ACes and heterologous DNA into cells and eventually tissue. METHOD: Chinese hamster ovary (CHO) cells carrying artificial chromosomes were grown in media supplemented with iododeoxyuridine (IdUrd). The 60-mb artificial chromosome was purified by flow cytometry sorting and transfected into Chinese hamster lung fibroblast cells (V79-4) or mouse connective tissue cells [LM(tk-)] using LipofectAMINE 2000trade mark, a cationic lipid, and Superfecttrade mark, a cationic dendrimer. The cells were incubated with an FITC-conjugated anti-bromodeoxyuridine (BrdUrd) antibody and analyzed by flow cytometry. IdUrd-incorporated artificial chromosome expressing green fluorescent protein (GFP) was transfected into V79-4 cells. Delivery was measured at 24 h and GFP expression was detected at 48 h. RESULTS: The delivery of intact artificial chromosomes into V79-4 and LMtk- cells was detected within 2 h and up to 48 h post-transfection. Maximum delivery rates of 20% and 14% were observed using LipofectAMINE 2000 and Superfect, respectively. Flow cytometry data correlated with microscopic observations. IdUrd incorporation resulted in less quenching after staining with Hoechst 33258 and chromomycin A3 than BrdUrd incorporation. The fluorescence intensity of the FITC-conjugated anti-BrdUrd antibody was greater with IdUrd-incorporated chromosomes than with BrdUrd-incorporated chromosomes. CONCLUSION: The results indicate that IdUrd-labeled artificial chromosomes can be detected 24 h after transfection. This efficient, sensitive, high-throughput detection technique is being used to evaluate and optimize other transfer technologies (e.g., electroporation and sonoporation), different delivery reagents, and protocols in a variety of cells in vitro. This work represents the first step in utilizing artificial chromosomes as nonviral vectors for gene therapy.     

PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo

Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.     

Artificial cells: building bioinspired systems using small-scale biology

Artificial cells have generated much interest since the concept was introduced by Aleksandr Oparin in the 1920s, and they have had an impact on the pharmaceutical and biotechnology industry in various areas, including potential therapeutic applications. Here, we discuss the development of small-scale, bio-inspired artificial cell components that recreate the function of key cellular and physiological systems. We describe artificial cells, selected current applications and how small-scale biology could be used to provide what might be a next-generation approach in this area. We believe that this type of work is in its infancy and that exploiting small-scale biological inspiration in the field of artificial cells has great potential for successes in the future.     

Motif-programmed artificial extracellular matrix

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.     

Movement of spermatozoa of Megaselia Scalaris (Diptera: Brachycera: Cyclorrhapha: Phoridae) in artificial and natural fluids

In artificial fluid, the spermatozoa move as linear cells or round up and rotate, propelled by spontaneous bending of their tails. Both linear and rounded cells can move forward and backward, but usually they move forward. The tails of all cells display, simultaneously, small primary bends and fewer, much larger secondary bends. Rounded cells form single secondary bends that remain unchanged as the cells rotate. They also form “node-like” primary bends that travel posteriorly or anteriorly as the cells rotate forward or backward, respectively. Linear cells move their anterior regions into and out of focus in a cyclic fashion. They form rather prominent primary bends, as well as two to four secondary bends that travel posteriorly as the cells move forward. Secondary bends change in shape continuously and are not sinusoidal. The cells follow approximately linear trajectories, but the distances traveled per cycle, speeds, and secondary bending patterns are variable. When methyl cellulose is added to artificial fluid, linear movement is improved, and forward speeds are approximately tripled. The movement of spermatozoa in natural fluid of the female reproductive tract is remarkably less stereotyped than that of cells in artificial fluid. The cells, usually resembling straight lines or arcs, are very flexible and active. They lack obvious cyclic activity and double bending patterns. They are capable of moving both forward and backward and of adjusting their bending activity and speed within rather wide limits. Their average forward speed is about nine times faster than that of cells in artificial fluid.     

Motility and orientation of a dinoflagellate, Gymnodinium, impaired by solar and ultraviolet radiation

Abstract The effects of solar and artificial ultraviolet radiation on the motility and orientation of the dinoflagellate Y-100 were studied. The cells show a weak photokinesis but a pronounced phototaxis which is consistently positive between 1 and 100 klx (= 4 mW m⁻² to 400 mW m⁻²); the precision of orientation increases with the fluence rate. Unfiltered solar radiation as well as artificial ultraviolet radiation reduce the percentage of motile cells increasingly with exposure time but the velocity of the still motile cells is less affected. Unirradiated control cells show a negative gravitaxis. After short exposure to solar or artificial ultraviolet radiation the precision of gravitaxis decreases and after prolonged exposure the cells start to actively move downward in the water column (positive gravitaxis). Phototaxis is also strongly impaired by ultraviolet radiation.     

Postoperative changes in vaginal smears after vaginal reconstruction with a free skin graft

Surgical vaginal reconstruction was performed by a free skin graft in two patients without a vagina. The postoperative changes in vaginal smears collected from the artificial vaginas were observed for about two years. Marked operation-induced inflammatory changes were observed until the second postoperative month. After the third postoperative month, the background became relatively clear. Cyanophilic and eosinophilic superficial cells, intermediate cells and D?derlein bacilli were observed occasionally in addition to keratotic cells. Six to 12 months after surgery, the vaginal smears showed little abnormality, except for the presence of keratotic cells. The changes in the vaginal smears after the third month show that the artificial vaginal epithelium changed cytologically to an almost normal vaginal mucosa that, although not histologically complete, responded to hormones. The presence of D?derlein bacilli suggests that the regional environment of the artificial vagina was almost the same as that of the normal vagina.     

Endothelialization of small-diameter vascular prostheses

In the field of arterial vascular reconstructions there is an increasing need for functional small-diameter artificial grafts (inner diameter < 6mm). When autologous replacement vessels are not available, for example because of the bad condition of the vascular system in the patient, the surgeon has no other alternative than to implant a synthetic polymer-based vessel. After implantation the initial major problem concerning these vessels is the almost immediate occlusion, due to blood coagulation and platelet deposition, under the relatively low flow conditions. As the search for the perfect bio-inert polymer has not revealed a material with suitable properties for this application, improved performance of small-diameter artificial blood vessels is now being sought in the biological field. The poor blood-compatibility of an artificial vascular graft is not simply because of its coagulation-stimulating or platelet-activating properties, but more due to its inability to actively participate in the prevention of blood coagulation and platelet deposition. As these functions are naturally performed by endothelial cells, the utilization of these cells seems inevitable for the construction of a functional small-diameter artificial blood vessels. This review describes the current status of the use of endothelial cells to improve the performance of artificial vascular prostheses.     

A mechanism of the toxicity of artificial ribonucleases for human cancer cells

The ability of artificial ribonucleases, low molecular weight compounds exhibiting RNA cleavage in vitro, to cause human cancer cell death in a concentration-dependent manner has been studied. The cytotoxic effect of artificial ribonucleases on cells appeared at rather low concentrations of these compounds (10⁻⁵ M). The study of mechanisms of the cytotoxic effect has shown that in addition to ribonuclease activity these compounds exhibit membranotropic activity. This activity allows the compounds to penetrate effectively inside cells. The cytotoxic effect of artificial ribonucleases involves damage of cell membrane, detachment of plasmalemma and impairments of its macromolecular organization. However, in the case of shortterm exposure to these compounds, cells survive even with damaged membrane.     

The effect of an artificial magnetic field on Salmonella infection in mice]

Experimental Salmonella infection in mice, developing simultaneously with the prolonged action of an artificial constant magnetic field with induction equal to 3 x 10(-4) T, was found to induce a pronounced decrease in nonspecific resistance in the animals. The study of Salmonella population structure revealed that the cells selected the animals subjected to the action of the artificial magnetic field had mostly a lesser number of signs of antibiotic resistance. By the end of the experiment Salmonella cultures isolated from the mice subjected to the action of the artificial magnetic field were characterized by greater virulence and resistance to the bactericidal action of blood serum. The use of sodium nucleinate under the conditions of the action of the artificial magnetic field enhanced the level of anti-infectious protection in the animals, which changed the direction of cell selection in Salmonella population towards cells with a greater number of markers of antibiotic resistance.     

PHOTOSYNTHETIC AND GROWTH RESPONSES TO SALINITY IN A MARINE ISOLATE OF NANNOCHLORIS BACILLARIS (CHLOROPHYCEAE)1

A marine unicellular alga, Nannochloris bacillaris Naumann, was studied with respect to growth, viability and photosynthesis during the steady-state and also subsequent to changes in the concentration of artificial seawater medium. Cells grew exponentially over the range of 2% to 300% artificial seawater, but more rapidly at lower salinities. In contrast to growth, photosynthesis as measured by both oxygen evolution and bicarbonate photoassimilation was not obviously inhibited for cells adapted within the range of 7% to 200% artificial seawater. In 300% artificial seawater, photosynthesis, especially bicarbonate photoassimilation, was inhibited. Osmotic shocks caused by transferring cells from 200% to 7% artificial seawater had little if any effect on growth, viability or photosynthesis. However, equal shocks in the upward direction (from 7% to 200% artificial seawater) caused long lag phases in growth, totally inhibited photosynthesis and very often led to cell death. Intermediate upward shocks were less deleterious, but did result in lags in growth.     

A novel mechanism of interaction between alpha-synuclein and biological membranes

Conformational abnormalities and aggregation of alpha-synuclein (alpha-syn) have been linked to the pathogenesis of Parkinson's (PD) and related diseases. It has been shown that alpha-syn can stably bind artificial phospholipid vesicles through alpha-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of alpha-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within alpha-syn, we demonstrated that the membrane interaction of alpha-syn in cells is also mediated by alpha-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of alpha-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of alpha-syn similar to the interaction with biological membranes. These results suggest that in cells, alpha-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of alpha-syn itself and by the cytoplasmic context.     

Phospholipids and cholesterol of liver nuclei during artificial hypobiosis of rats

The contents of lipids in the tissue and the nuclei of liver cells during artificial hypobiosis, as well as in the nuclei of liver cells for 3 days after the cessation of cooling in rats, were studied. In the artificial hypobiosis and in the state of normothermia 24 h after the cessation of cooling, the total phospholipid content of the liver cell nuclei increased by 20% due to minor phospholipids. The levels of sphingomyelin, phosphatidylinositol, phosphatidylserine, cardiolipin, and lysophosphatidylcholine were doubled in hypobiosis and then nonmonotonically returned to the normal level within 72 h. In the state of artificial hypobiosis, the levels of fatty acids, cholesterol, and diglycerides increased by 30–40%. The state of artificial hypobiosis did not affect the level of lipids in the liver tissue of Wistar rats. The increase of the lipid content in the liver cell nuclei of Wistar rats indicates the important role of lipids in functions of the nucleus when the energy supply and protein synthesis are inhibited under conditions of artificial hypobiosis.     

Assessing the use of artificial substrates to monitor Gambierdiscus populations in the Florida Keys

Four distinct coastal locations were sampled on a monthly basis near Long Key (Florida Keys, USA) over a 13-month period to study Gambierdiscus population dynamics on different substrates, including four macrophyte species (Dictyota spp., Halimeda spp., Laurencia spp., and Thalassia testudinum) and three artificial substrates (polyvinyl chloride (PVC) tiles, burlap, and fiberglass window screen). Cell densities of Gambierdiscus were generally lower on Dictyota versus Halimeda and Laurencia. Cell densities of Gambierdiscus were significantly correlated among macrophyte hosts in 54% of the comparisons, and between macrophyte hosts and artificial substrates in 72% of the comparisons. Predictive slopes determined from regression analyses between cell densities on artificial substrates and macrophyte hosts indicated that, on an areal basis, fewer cells were present on macrophytes versus artificial substrates (cells cm⁻²) and that slope variation (error) among the different macrophytes and sites ranged from 5% to 200%, averaging 61% overall. As the data required log-transformation prior to analyses, this level of error translates into two-orders of magnitude in range of estimation of the overall average abundance of Gambierdiscus cells on macrophytes (135 cells g⁻¹ wet weight); 20–2690 cells g⁻¹ ww. The lack of consistent correlation among Gambierdiscus cell densities on macrophytes versus artificial substrates, coupled with the high level of error associated with the predictive slope estimations, indicates that extreme caution should be taken when interpreting the data garnered from artificial substrate deployments, and that such deployments should be thoroughly vetted prior to routine use for monitoring purposes.     

Artificial mammalian gene regulation networks-novel approaches for gene therapy and bioengineering

Recently developed strategies for targeted molecular interventions in mammalian cells have created novel opportunities in biotechnological and biomedical research with huge economic and therapeutic impact: the design of mammalian cells with desired phenotypes for biopharmaceutical manufacturing, tissue engineering and gene therapy. These advances have been enabled by constructing artificial gene regulation systems with control modalities similar to those evolved in key regulatory networks of mammalian cells. This review highlights recurring cellular regulation strategies and artificial gene regulation technology currently in use for rational reprogramming of cellular key events including metabolism, growth, differentiation and cell death to achieve sophisticated bioprocess and therapeutic goals.     

新型人工红细胞的制备

目的:接枝淀粉包裹血红蛋白制备新型人造红细胞的代替品。方法:利用油酸接枝淀粉,在超声条件下下自组装,包裹天然牛血红蛋白,并鉴定其物理化学及生物学性能。测定包封率、红外光谱分析(FTIR)、电镜观察形态学及粒径,测定P50和Hill系数。结果:人工红细胞呈圆球形,平均粒径250nm,包封率高,具有良好的携氧、释氧能力。结论:成功制备了人工纳米红细胞,为进一步临床应用提供了基础。