A novel culture system for modulating single cell geometry in 3D

Dedifferentiation of chondrocytes during in vitro expansion remains an unsolved challenge for repairing serious articular cartilage defects. In this study, a novel culture system was developed to modulate single cell geometry in 3D and investigate its effects on the chondrocyte phenotype. The approach uses 2D micropatterns followed by in situ hydrogel formation to constrain single cell shape and spreading. This enables independent control of cell geometry and extracellular matrix. Using collagen I matrix, we demonstrated the formation of a biomimetic collagenous “basket” enveloping individual chondrocytes cells. By quantitatively monitoring the production by single cells of chondrogenic matrix (e.g. collagen II and aggrecan) during 21-day cultures, we found that if the cell’s volume decreases, then so does its cell resistance to dedifferentiation (even if the cells remain spherical). Conversely, if the volume of spherical cells remains constant (after an initial decrease), then not only do the cells retain their differentiated status, but previously de-differentiated redifferentiate and regain a chondrocyte phenotype. The approach described here can be readily applied to pluripotent cells, offering a versatile platform in the search for niches toward either self-renewal or targeted differentiation

A one-step procedure to probe the viscoelastic properties of cells by Atomic Force Microscopy

The increasingly recognised importance of viscoelastic properties of cells in pathological conditions requires rapid development of advanced cell microrheology technologies. Here, we present a novel Atomic Force Microscopy (AFM)-microrheology (AFM2) method for measuring the viscoelastic properties in living cells, over a wide range of continuous frequencies (0.005 Hz ~ 200 Hz), from a simple stress-relaxation nanoindentation. Experimental data were directly analysed without the need for pre-conceived viscoelastic models. We show the method had an excellent agreement with conventional oscillatory bulk-rheology measurements in gels, opening a new avenue for viscoelastic characterisation of soft matter using minute quantity of materials (or cells). Using this capability, we investigate the viscoelastic responses of cells in association with cancer cell invasive activity modulated by two important molecular regulators (i.e. mutation of the p53 gene and Rho kinase activity). The analysis of elastic (G′(ω)) and viscous (G″(ω)) moduli of living cells has led to the discovery of a characteristic transitions of the loss tangent (G″(ω)/G′(ω)) in the low frequency range (0.005 Hz ~ 0.1 Hz) that is indicative of the capability for cell restructuring of F-actin network. Our method is ready to be implemented in conventional AFMs, providing a simple yet powerful tool for measuring the viscoelastic properties of living cells

External modulation method for generating accurate linear optical FMCW

Frequency modulation continuous wave (FMCW) lasers are key components in modern optical imaging. However, current intracavity modulation lasers do not exhibit low-frequency jitter rate and high linearity due to the inherent relaxation oscillations. Although this may be compensated in a direct modulation laser diode using an optoelectronic feedback loop, the available sweep speed is moderately small. In this letter, a special external modulation method is developed to improve the performance of FMCW. Since only the first sideband optical field is used during the entire generation process, phase noise is kept to a minimum and is also independent of the sweep speed. We demonstrate that the linearity and jitter rates do not deteriorate appreciably when the sweep speed is changed over three orders of magnitude, even up to the highest sweep speed of 2.5 GHz/ μs

Dissecting horizontal and vertical gene transfer of antibiotic resistance plasmid in bacterial community using microfluidics

The spread of antibiotic resistance genes (ARGs) has become an emerging threat to the global health. Although horizontal gene transfer (HGT) is regarded as one of the major pathways, more evidence has shown the significant involvement of vertical gene transfer (VGT). However, traditional cultivation-based methods cannot distinguish HGT and VGT, resulting in often contradictory conclusions. Here, single-cell microfluidics with time-lapse imaging has been successfully employed to dissect the contribution of plasmid-mediated HGT and VGT to ARG transmission in an environmental community. Using Escherichia coli with an ARG-coded plasmid pKJK5 with trimethoprim resistance as the donor, we quantified the effects of three representative antibiotics (trimethoprim, tetracycline and amoxicillin) on the ARG transfer process in an activated sludge bacterial community. It was found that HGT was influenced by the inhibitory mechanism of an antibiotic and its targets (donor, recipient alone or together), whereas VGT contributes significantly to the formation of transconjugants and consequently ARG spreading. Trimethoprim is highly resisted by the donor and transconjugants, and its presence significantly increased both the HGT and VGT rates. Although tetracycline and amoxicillin both inhibit the donor, they showed different effects on HGT rate as a result of different inhibitory mechanisms. Furthermore, we show the kinetics of HGT in a community can be described using an epidemic infection model, which in combination with quantitative measure of HGT and VGT on chip provides a promising tool to study and predict the dynamics of ARG spread in real-world communities

Bis(benzimidazol-1-yl)methane dihydrate

The bis­(benzimidazol-1-yl)methane mol­ecule of the title compound, C15H12N4·2H2O, displays a trans conformation with a twofold axis running through the methylene C atom. Two adjacent water mol­ecules are bonded to this mol­ecule through O—H⋯N hydrogen bonds, forming a trimer. Adjacent trimers are connected together via C—H⋯O inter­actions, forming a chain running along the b-axis direction. Two such chains are joined together via π–π inter­actions [centroid–centroid distance = 3.556 (2) Å], forming double chains, which are connected via the water mol­ecules through C—H⋯O associations, forming a sheet structure. The sheets are stacked on top of each other along the a-axis direction and connected through O—H⋯O and C—H⋯O inter­actions, forming a three-dimensional ABAB layer network structure

Editorial: Engineering probiotics for multiple interventions on intestinal diseases

No abstract available

Real-time study of rapid spread of antibiotic resistance plasmid in biofilm using microfluidics

Gene transfer in biofilms is known to play an important role in antibiotic resistance dissemination. However, the process remains poorly understood. In this study, microfluidics with time-lapse imaging was used for real-time monitoring of plasmid-mediated horizontal gene transfer (HGT) in biofilms. Pseudomonas putida KT2440 harboring an antibiotic resistance plasmid RP4 was chosen as the donor while Escherichia coli and activated sludge bacteria were used as the recipient cells. Dynamic features of the transfer process, including the transfer rate, cell growth rate and kinetic changes of the transfer frequency, were determined. It was found that the routes for gene transfer strongly depend on the structure and composition of a biofilm. While intraspecies HGT is essential to initiate a transfer event, the secondary retransfer from transconjugants to the same species is more efficient and can cause cascading gene spread in single-strain biofilms. For the activated sludge biofilm, only small and scattered colonies formed and vertical gene transfer appears to be the dominant route after initial intraspecies transfer. Furthermore, more than 46% of genera in the activated sludge were permissive to plasmid RP4, many of which are associated with human pathogens. These phenomena imply early prevention and interruptions to biofilm structure could provide an effect way to inhibit rapid antibiotic resistance gene spread and reduce the likelihood of catastrophic events associated with antibiotic resistance

Continuous cell sorting in a flow based on single cell resonance Raman spectra

Single cell Raman spectroscopy measures a spectral fingerprint of the biochemistry of cells, and provides a powerful method for label-free detection of living cells without the involvement of a chemical labelling strategy. However, as the intrinsic Raman signals of cells are inherently weak, there is a significant challenge in discriminating and isolating cells in a flowing stream. Here we report an integrated Raman-microfluidic system for continuous sorting of a stream of cyanobacteria, Synechocystis sp. PCC6803. These carotenoidcontaining microorganisms provide an elegant model system enabling us to determine the sorting accuracy using the subtly different resonance Raman spectra of microorganism cultured in a 12C or 13C carbon source. Central to the implementation of continuous flow sorting is the use of “pressure dividers” that eliminate fluctuations in flow in the detection region. This has enabled us to stabilise the flow profile sufficiently to allow automated operation with synchronisation of Raman acquisition, real-time classification and sorting at flow rates of ca. <100 μm/s, without the need to “trap” the cells. We demonstrate the flexibility of this approach in sorting mixed cell populations with the ability to achieve 96.3% purity of the selected cells at a speed of 0.5 Hz

Single-cell microfluidics to study the effects of genome deletion on bacterial growth behavior

By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population

Polarized cell motility induces hydrogen peroxide to inhibit cofilin via cysteine oxidation

Mesenchymal cell motility is driven by polarized actin polymerization [1]. Signals at the leading edge recruit actin polymerization machinery to promote membrane protrusion, while matrix adhesion generates tractive force to propel forward movement. To work effectively, cell motility is regulated by a complex network of signaling events that affect protein activity and localization. H2O2 has an important role as a diffusible second messenger [2], and mediates its effects through oxidation of cysteine thiols. One cell activity influenced by H2O2 is motility [3]. However, a lack of sensitive and H2O2-specific probes for measurements in live cells has not allowed for direct observation of H2O2 accumulation in migrating cells or protrusions. In addition, the identities of proteins oxidized by H2O2 that contribute to actin dynamics and cell motility have not been characterized. We now show, as determined by fluorescence lifetime imaging microscopy, that motile cells generate H2O2 at membranes and cell protrusions and that H2O2 inhibits cofilin activity through oxidation of cysteines 139 (C139) and 147 (C147). Molecular modeling suggests that C139 oxidation would sterically hinder actin association, while the increased negative charge of oxidized C147 would lead to electrostatic repulsion of the opposite negatively charged surface. Expression of oxidation-resistant cofilin impairs cell spreading, adhesion, and directional migration. These findings indicate that H2O2 production contributes to polarized cell motility through localized cofilin inhibition and that there are additional proteins oxidized during cell migration that might have similar roles