Remote sensing of emperor penguin abundance and breeding success.

Emperor penguins (Aptenodytes forsteri) are under increasing environmental pressure. Monitoring colony size and population trends of this Antarctic seabird relies primarily on satellite imagery recorded near the end of the breeding season, when light conditions levels are sufficient to capture images, but colony occupancy is highly variable. To correct population estimates for this variability, we develop a phenological model that can predict the number of breeding pairs and fledging chicks, as well as key phenological events such as arrival, hatching and foraging times, from as few as six data points from a single season. The ability to extrapolate occupancy from sparse data makes the model particularly useful for monitoring remotely sensed animal colonies where ground-based population estimates are rare or unavailable.

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry.

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 µm wide microfluidic channel. The fluid shear stress induces large, ear ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe [1] that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell-cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

Cells in the human body are viscoelastic: they have some of the properties of an elastic solid, like rubber, as well as properties of a viscous fluid, like oil. To carry out mechanical tasks ­ such as, migrating through tissues to heal a wound or to fight inflammation ­ cells need the right balance of viscosity and elasticity. Measuring these two properties can therefore help researchers to understand important cell tasks and how they are impacted by disease. However, quantifying these viscous and elastic properties is tricky, as both depend on the time-scale they are measured: when pressed slowly, cells appear soft and liquid, but they turn hard and thick when rapidly pressed. Here, Gerum et al. have developed a new system for measuring the viscosity and elasticity of individual cells that is fast, simple, and inexpensive. In this new method, cells are suspended in a specialized solution with a consistency similar to machine oil which is then pushed with high pressure through channels less than half a millimeter wide. The resulting flow of fluid shears the cells, causing them to elongate and rotate, which is captured using a fast camera that takes 500 images per second. Gerum et al. then used artificial intelligence to extract each cell's shape and rotation speed from these images, and calculated their viscosity and elasticity based on existing theories of how viscoelastic objects behave in fluids. Gerum et al. also investigated how the elasticity and viscosity of cells changed with higher rotation frequencies, which corresponds to shorter time-scales. This revealed that while higher frequencies made the cells appear more viscous and elastic, the ratio between these two properties remained the same. This means that researchers can compare results obtained from different experimental techniques, even if the measurements were carried out at completely different frequencies or time-scales. The method developed by Gerum et al. provides a fast an inexpensive way for analyzing the viscosity and elasticity of cells. It could also be a useful tool for screening the effects of drugs, or as a diagnostic tool to detect diseases that affect the mechanical properties of cells.

Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells.

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

Cu-releasing bioactive glass/polycaprolactone coating on Mg with antibacterial and anticorrosive properties for bone tissue engineering.

Bioactive glass nanoparticles containing copper (Cu-BGNs) were introduced into polycaprolactone (PCL) coating systems to improve the bioactivity, antibacterial properties, and corrosion resistance of vulnerable magnesium matrices under physiological conditions. The influence of different amounts of Cu-BGNs in PCL coatings was thoroughly investigated in determining the wettability, electrochemical properties, and antibacterial effects against Staphylococcus carnosus and Escherichia coli, as well as their cyto-compatibility. Cu-BGNs were observed randomly scattered in PCL coatings. Increasing the concentration of Cu-BGNs resulted in a slight decrease of the water contact angle, and a reduction in anticorrosion properties of the Cu-BGN composite coatings. Yet higher Cu-BGN content in coatings led to more calcium phosphate formation on the surface after 7 days of immersion in Dulbecco's modified Eagle's medium, which was confirmed by Fourier-transform infrared spectroscopy and x-ray photoelectron spectroscopy. The growth of S. carnosus and E. coli was inhibited by Cu2+ ions released from the Cu-BGN coatings. In addition, both direct and indirect cyto-compatibility experiments showed that the viability and proliferation of MG-63 cells on Cu-BGN coatings were highly increased compared to pure magnesium; however, an additional increase of Cu-BGN concentration showed a slight decrease of cell proliferation and cell activity. In summary, Cu-BGN/PCL composite coatings impart magnesium-based biomaterials with antibacterial and anticorrosive properties for clinical applications.

The role of focal adhesion anchoring domains of CAS in mechanotransduction.

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

Cell Adhesion on Surface-Functionalized Magnesium.

The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.

Biphasic response of cell invasion to matrix stiffness in three-dimensional biopolymer networks.

When cells come in contact with an adhesive matrix, they begin to spread and migrate with a speed that depends on the stiffness of the extracellular matrix. On a flat surface, migration speed decreases with matrix stiffness mainly due to an increased stability of focal adhesions. In a three-dimensional (3-D) environment, cell migration is thought to be additionally impaired by the steric hindrance imposed by the surrounding matrix. For porous 3-D biopolymer networks such as collagen gels, however, the effect of matrix stiffness on cell migration is difficult to separate from effects of matrix pore size and adhesive ligand density, and is therefore unknown. Here we used glutaraldehyde as a crosslinker to increase the stiffness of self-assembled collagen biopolymer networks independently of collagen concentration or pore size. Breast carcinoma cells were seeded onto the surface of 3-D collagen gels, and the invasion depth was measured after 3 days of culture. Cell invasion in gels with pore sizes >5 µm increased with higher gel stiffness, whereas invasion in gels with smaller pores decreased with higher gel stiffness. These data show that 3-D cell invasion is enhanced by higher matrix stiffness, opposite to cell behavior in two dimensions, as long as the pore size does not fall below a critical value where it causes excessive steric hindrance. These findings may be important for optimizing the recellularization of soft tissue implants or for the design of 3-D invasion models in cancer research.

Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes.

Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type.

Labeling of cancer cells with magnetic nanoparticles for magnetic resonance imaging.

PURPOSE: The process of invasion and metastasis formation of tumor cells can be studied by following the migration of labeled cells over prolonged time periods. This report investigates the applicability of iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent for cell labeling. METHODS: γFe2 O3 nanoparticles prepared with direct flame spray pyrolysis are biofunctionalized with poly-l-lysine (PLL). The nanoparticles within the cells were observed with transmission electron microscopy, bright-field microscopy, and magnetorelaxometry. MRI of labeled cells suspended in agarose was used to estimate the detection limit. RESULTS: PLL-coated particles are readily taken up, stored in intracellular clusters, and gradually degraded by the cells. During cell division, the nanoparticle clusters are divided and split between daughter cells. The MRI detection limit was found to be 25 cells/mm(3) for R2*, and 70 cells/mm(3) for R2. The iron specificity, however, was higher for R2 images. Due to the degradation of intracellular γFe2 O3 to paramagnetic iron ions within 13 days, the R1, R2, and R2* contrast gradually decreased over this time period to approximately 50% of its initial value. CONCLUSIONS: These results suggest that PLL-coated γFe2 O3 nanoparticles can be used as an MRI contrast agent for long-term studies of cell migration. Magn Reson Med 71:1896-1905, 2014. © 2013 Wiley Periodicals, Inc.

Biomechanical characterization of a desminopathy in primary human myoblasts.

Heterozygous mutations of the human desmin gene on chromosome 2q35 cause hereditary and sporadic myopathies and cardiomyopathies. The expression of mutant desmin brings about partial disruption of the extra sarcomeric desmin cytoskeleton and abnormal protein aggregation in the sarcoplasm of striated muscle cells. The precise molecular pathways and sequential steps that lead from a desmin gene defect to progressive muscle damage are still unclear. We tested whether mutant desmin changes the biomechanical properties and the intrinsic mechanical stress response of primary cultured myoblasts derived from a patient carrying a heterozygous R350P desmin mutation. Compared to wildtype controls, undifferentiated mutant desmin myoblasts revealed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, magnetic tweezer microrheometry of myoblasts using fibronectin-coated beads showed increased stiffness of diseased cells. Our findings provide the first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in desminopathies. We postulate that the expression of mutant desmin leads to increased mechanical stiffness, which results in excessive mechanical stress in response to strain and consecutively to increased mechanical vulnerability and damage of muscle cells.

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

BACKGROUND: Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication-deficient or more recently by conditionally replication-competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat-regulated transcription control as a promising tool for this purpose. METHODS AND RESULTS: We investigated the feasibility of a short fragment of the human hsp70B' promoter, with and without upstream insulator elements, for the regulation of transgene expression by replication-deficient or oncolytic adenoviruses. Two novel adenoviral vectors with an insulated hsp70B' promoter were developed and showed stringent heat-inducible gene expression with induction ratios up to 8000-fold. In contrast, regulation of gene expression from the hsp70B' promoter without insulation was suboptimal. In replication-competent/oncolytic adenoviruses regulation of the hsp70B' promoter was lost specifically during late replication in permissive cells and could not be restored by the insulators. CONCLUSIONS: We developed novel adenovirus gene transfer vectors that feature improved and stringent regulation of transgene expression from the hsp70B' promoter using promoter insulation. These vectors have potential for gene therapy applications that benefit from external modulation of therapeutic gene expression or for combination therapy with hyperthermia. Furthermore, our study reveals that vector replication can deregulate inserted cellular promoters, an observation which is of relevance for the development of replication-competent/oncolytic gene transfer vectors.

Combining adenoviral oncolysis with temozolomide improves cell killing of melanoma cells.

Oncolytic adenoviruses are emerging agents for treatment of cancer by tumor-restricted virus replication, cell lysis and virus spread. Clinical studies with first generation oncolytic adenoviruses have revealed that an increased potency is warranted in order to achieve therapeutic efficacy. One approach towards this end is to combine adenoviral oncolysis with chemotherapy. Here, a fundamental requirement is that chemotherapy does not interfere with adenovirus replication in cancer cells. We have previously developed a melanoma-targeted oncolytic adenovirus, Ad5/3.2xTyr, which features tyrosinase promoter regulated replication and enhanced cell entry into melanoma cells. In this study, we investigated a combination treatment of melanoma cells with Ad5/3.2xTyr and temozolomide (TMZ), which produces the same active metabolite as Dacarbazine/DTIC, the standard chemotherapy for advanced melanoma. We report that TMZ does not inhibit adenovirus replication in melanoma cells. Additive or synergistic cell killing of melanoma cells, dependent on the cell line used, was observed. Enhanced cell binding was not responsible for synergism of adenoviral oncolysis and TMZ treatment. We rather observed that higher numbers of virus genomes are produced in TMZ-treated cells, which also showed a cell cycle arrest in the G2 phase. Our results have important implications for the clinical implementation of adenoviral oncolysis for treatment of malignant melanoma. It suggests that such studies are feasible in the presence of TMZ or DTIC chemotherapy and recommends the investigation of a viro-chemo combination therapy.