Ursoricin, a bacteriocin of Streptococcus ursoris, has potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.

The emergence of drug-resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has increased the need to discover novel antimicrobial agents that are effective against these species. Here, we describe the identification and purification of the mutacin BHT-B-like gene locus and bacteriocin peptide from Streptococcus ursoris, which is closely related to Streptococcus ratti; hence, we named this bacteriocin ursoricin. Ursoricin is a cationic, chromosome-encoded peptide that has potent antimicrobial effects against Gram-positive pathogens, including MRSA and VRE, with minimum inhibitory concentrations in the micromolar range. Ursoricin also inhibits the biofilm formation of high biofilm-forming S. aureus. Antibacterial activity was retained after treatment at 100°C for 60 min at a pH range of 3-9 and was partially reduced by treatment with proteinase K for 2 h (63% residual activity). The potent anti-MRSA, anti-VRE, and antibiofilm effects of ursoricin suggest that it is a possible candidate for the treatment of MRSA, VRE, and biofilm-associated infections. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has posed a significant public health threat and economic burdens that make the identification and development of novel antimicrobial agents urgent. Bacteriocins are promising new agents that exhibit antibacterial activity against a wide range of human pathogens. In this study, we report that the bacteriocin produced by Streptococcus ursoris showed good antibacterial activity against a wide range of Staphylococcus aureus and enterococcus strains, particularly methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and high biofilm-forming S. aureus. Interestingly, this bacteriocin had a stronger effect on S. aureus than on Staphylococcus epidermidis, which is a major commensal bacterium in human skin; this result is important when considering the disturbance of bacterial flora, especially on the skin, mediated by the application of antibacterial agents.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

Polarity Does Not Matter: Molecular Weight Reverses the Photoisomerization-Induced Phase Separation of an Azobenzene-Bearing Polymer.

The non-canonical photoisomerization-induced phase separation of an azobenzene-bearing polymer is found. The polymer composed of acrylate-based azobenzene (AzoAA) and N,N-dimethylacrylamide (DMA), namely poly(AzoAA-r-DMA), phase separates under visible light-induced cis-to-trans isomerization at high molecular weight, whereas the phase separation is realized under UV light-induced trans-to-cis isomerization at low molecular weight. Conventionally, the origin of photoisomerization-induced phase separation is believed to arise from the difference in polarity between the apolar trans and polar cis states; thereby the direction of phase changes, either to separate or dissolute, is uniquely determined by the polarity changes during the isomerization of azobenzene. Contrary to this common perception, the poly(AzoAA-r-DMA) in this study phase separates through both trans and cis isomerization, depending on the molecular weight. The non-canonical phase separation of poly(AzoAA-r-DMA) reported herein suggests that molecular weight plays a significant role in determining the phase behavior of azobenzene-bearing polymers. This study provides a platform for the development of spatial-temporally controlled delivery vehicles and microreactors.

Relationship between CD4+ T-cell counts at baseline and initial periodontal treatment efficacy in patients undergoing treatment for HIV infection: A retrospective observational study.

AIM: To retrospectively investigate the relationship between the CD4+ T-cell counts at baseline and the efficacy of the initial periodontal treatment of patients undergoing treatment for human immunodeficiency virus (HIV) infection using the periodontal inflamed surface area (PISA). MATERIALS AND METHODS: Thirty-three patients with chronic periodontitis who had undergone periodontal examination at baseline and after the initial periodontal treatment were enrolled. PISA was calculated from the periodontal probing depth and bleeding on probing, and the ratio of PISA after treatment to that at baseline (PISA response ratio) was calculated. Groups with a response ratio of <1 and ≥1 were defined as the improvement and the non-improvement groups, respectively. RESULTS: PISA after the initial periodontal treatment significantly decreased compared with that at baseline (p < .05). A weak negative correlation was found between the PISA response ratio and CD4+ T-cell counts at baseline (p < .05). The CD4+ T-cell counts at baseline were significantly higher in the improvement group than in the non-improvement group (p < .05). Multivariate analysis revealed that the CD4+ T-cell counts at baseline was an independent factor that affects the PISA (p < .05). CONCLUSIONS: The higher the CD4+ T-cell counts at baseline in patients undergoing treatment for HIV infection, the more effective the initial periodontal treatment.

Freeze-dried noncoagulating platelet-derived factor concentrate is a safe and effective treatment for early knee osteoarthritis.

PURPOSE: While a wide variety of platelet-rich plasma (PRP) solutions has been developed, innovation continues. In this case, the freeze-dried platelet factor concentrate (PFC-FD) represents another step in PRP refinement. The preparation of PFC-FD at a central laboratory with freeze drying for shelf stabilization should provide additional quality improvements if clinical effectiveness can be demonstrated. Therefore, this study was undertaken to assess the safety and effectiveness of PFC-FD in a prospective open-label trial of patients suffering from knee osteoarthritis (OA). METHODS: 312 consecutive knee OA patients (67% female, mean age 63 ± 10 years), were prospectively recruited in an outpatient knee clinic in Japan. Of these, 10 (3.2%) were lost to follow-up at < 12 months and 17 (5.5%) sought additional knee therapy during the follow-up period. The primary outcome of interest was achievement of the OMERACT-OARSI responder criteria with secondary outcomes of adverse events and PROMs scores 1, 3, 6, 12 months following a single PFC-FD injection. RESULTS: 285 patients (91%) completed 12 month PROMs. The 17 who sought additional therapy were considered failures leaving an effective sample size of 302 for our primary outcome in which 62% of patients achieved OMERACT-OARSI responder status by 12 months. This varied by OA class with Kellgren-Lawrence grade 4 patients 3.6 times less likely to be responders than grade 1-2 patients. 6% of patients experienced a non-serious adverse event, primarily pain or swelling at the injection site. CONCLUSIONS: PFC-FD provides an observable clinical improvement in 62% of knee OA patients at 12 months post-injection with very little risk of any clinically relevant adverse event. Of course, nearly 40% of patients did not experience an observable clinical improvement, primarily among those with worse KL grades. LEVEL OF EVIDENCE: Therapeutic, Level II.

Photoactivatable surfaces resolve the impact of gravity vector on collective cell migratory characteristics.

Despite considerable interest in the impact of space travel on human health, the influence of the gravity vector on collective cell migration remains unclear. This is primarily because of the difficulty in inducing collective migration, where cell clusters appear in an inverted position against gravity, without cellular damage. In this study, photoactivatable surfaces were used to overcome this challenge. Photoactivatable surfaces enable the formation of geometry-controlled cellular clusters and the remote induction of cellular migration via photoirradiation, thereby maintaining the cells in the inverted position. Substrate inversion preserved the circularity of cellular clusters compared to cells in the normal upright position, with less leader cell appearance. Furthermore, the inversion of cells against the gravity vector resulted in the remodeling of the cytoskeletal system via the strengthening of external actin bundles. Within the 3D cluster architecture, enhanced accumulation of active myosin was observed in the upper cell-cell junction, with a flattened apical surface. Depending on the gravity vector, attenuating actomyosin activity correlates with an increase in the number of leader cells, indicating the importance of cell contractility in collective migration phenotypes and cytoskeletal remodeling.

Mapping stress inside living cells by atomic force microscopy in response to environmental stimuli.

The response of cells to environmental stimuli, under either physiological or pathological conditions, plays a key role in determining cell fate toward either adaptive survival or controlled death. The efficiency of such a feedback mechanism is closely related to the most challenging human diseases, including cancer. Since cellular responses are implemented through physical forces exerted on intracellular components, more detailed knowledge of force distribution through modern imaging techniques is needed to ensure a mechanistic understanding of these forces. In this work, we mapped these intracellular forces at a whole-cell scale and with submicron resolution to correlate intracellular force distribution to the cytoskeletal structures. Furthermore, we visualized dynamic mechanical responses of the cells adapting to environmental modulations in situ. Such task was achieved by using an informatics-assisted atomic force microscope (AFM) indentation technique where a key step was Markov-chain Monte Carlo optimization to search for both the models used to fit indentation force-displacement curves and probe geometry descriptors. We demonstrated force dynamics within cytoskeleton, as well as nucleoskeleton in living cells which were subjected to mechanical state modulation: myosin motor inhibition, micro-compression stimulation and geometrical confinement manipulation. Our results highlight the alteration in the intracellular prestress to attenuate environmental stimuli; to involve in cellular survival against mechanical signal-initiated death during cancer growth and metastasis; and to initiate cell migration.

Precise Tuning and Characterization of Viscoelastic Interfaces for the Study of Early Epithelial-Mesenchymal Transition Behaviors.

There is growing evidence that cellular functions are regulated by the viscoelastic nature of surrounding matrices. This study aimed to investigate the impact of interfacial viscoelasticity on adhesion and epithelial-mesenchymal transition (EMT) behaviors of epithelial cells. The interfacial viscoelasticity was manipulated using spin-coated thin films composed of copolymers of ε-caprolactone and d,l-lactide photo-cross-linked with benzophenone, whose mechanical properties were characterized using atomic force microscopy and a rheometer. The critical range for the morphological transition of epithelial Madin-Darby canine kidney (MDCK) cells was of the order of 102 ms relaxation time, which was 1-2 orders of magnitude smaller than the relaxation times reported (10-102 s). An analysis of strain rate-dependent viscoelastic properties revealed that the difference was caused by the different strain rate/frequency used for the mechanical characterization of the interface and bulk. Furthermore, decoupling of the interfacial viscous and elastic terms demonstrated that E/N-cadherin expression levels were regulated differently by interfacial relaxation and elasticity. These results confirm the significance of precise manipulation and characterization of interfacial viscoelasticity in mechanobiology studies on EMT progression.

Oxidative stress impairs the calcification ability of human dental pulp cells.

BACKGROUND: The relationship between internal root resorption and oxidative stress has not yet been reported. This study aimed to add molecular insight into internal root resorption. The present study was conducted to investigate the effect of hydrogen peroxide (H2O2) as an inducer of oxidative stress on the calcification ability of human dental pulp cells (hDPCs) and the involvement of inositol 1, 4, 5-trisphosphate (IP3). MATERIAL AND METHODS: hDPCs (Lonza, Basel, Switzerland) were exposed to H2O2. Cell viability and reactive oxygen species (ROS) production were then evaluated. To investigate the effect of H2O2 on the calcification ability of hDPCs, real-time PCR for alkaline phosphatase (ALP) mRNA expression, ALP staining, and Alizarin red staining were performed. Data were compared with those of hDPCs pretreated with 2-aminoethyldiphenylborate (2-APB), which is an IP3 receptor inhibitor. RESULTS: H2O2 at concentrations above 250 µM significantly reduced cell viability (P < 0.01). More ROS production occurred in 100 µM H2O2-treated hDPCs than in control cells (P < 0.01). 2-APB significantly decreased the production (P < 0.05). H2O2-treated hDPCs showed significant reductions in ALP mRNA expression (P < 0.01), ALP activity (P < 0.01), and mineralized nodule deposition compared with negative control cells (P < 0.01). 2-APB significantly inhibited these reductions (P < 0.01, P < 0.05 and P < 0.01, respectively). Data are representative of three independent experiments with three replicates for each treatment and values are expressed as means ± SD. CONCLUSION: To the best of our knowledge, this is the first study documenting the involvement of IP3 signaling in the calcification ability of human dental pulp cells impaired by H2O2.

Improved anti-cancer effect of epidermal growth factor-gold nanoparticle conjugates by protein orientation through site-specific mutagenesis.

Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs.

Modulation of Mesenchymal Stem Cells Mechanosensing at Fluid Interfaces by Tailored Self-Assembled Protein Monolayers.

Mechanical cues of cellular microenvironments can modulate cell functions including cell spreading and differentiation. Most studies of cellular functions are performed using a solid substrate, and it is thought that cells cannot spread on fluid substrates because of rapid relaxation, which cannot resist against actomyosin-based cell contractility. Here, the spreading and growth of anchorage-dependent cells such as human mesenchymal stem cells at the liquid interface between a perfluorocarbon fluid and the culture medium are observed. It is demonstrated that a monomolecular protein nanosheet self-assembled at a fluid interface is sufficiently rigid to support cell spreading without additional treatment. Fine tuning of the packing of these proteins at the liquid interface permits tailoring of the mechanics of the protein layer, ultimately allowing for the regulation of cell spreading. The greater stiffness of the protein nanosheets triggers cell spreading, adhesion growth, and yes-associated protein nuclear translocation. Cell behavior at the fluid interface is explained within the framework of the molecular clutch model. In addition, the freestanding ultrathin protein nanosheets are extremely flexible, easily deformed, and perceived by cells as being much softer. The findings are expected to provide a new perspective for insights into cell-material interactions.

Photoactivatable Hydrogel Interfaces for Resolving the Interplay of Chemical, Mechanical, and Geometrical Regulation of Collective Cell Migration.

Collective migration is the mechanobiological interplay within migrating cell clusters and against extracellular matrixes (ECMs) underneath, mediating various physiological and pathological processes. Therefore, it is crucial to develop a robust platform on which collective migration can be studied under standardized conditions to understand how cells migrate differently between normal and disease states. We herein demonstrated phtotoactivatable hydrogel interfaces as suitable candidates for such applications. The substrate was composed of a poly(acrylamide) (PAAm) hydrogel whose surface was sequentially functionalized with poly-d-lysine (PDL) and photocleavable poly(ethylene glycol) (PEG). On the surface of the gel substrates, cell clusters with any given geometries can be prepared by controlling the irradiation patterns (geometrical cue), and their collective migration can be induced by the subsequent irradiation of the surrounding regions. Moreover, the substrate mechanical properties can be controlled by changing the composition of the PAAm hydrogel (mechanical cue), and the chemical properties were controlled by changing the amount of immobilized PDL, thereby altering the adsorbed amount of ECM proteins (chemical cue). The photoactivatable gel substrates were characterized by fluorescence microscopy, ζ-potential measurements, and the protein adsorption test. Through the study of the interplay of chemical, mechanical, and geometrical cues in the regulation of collective characteristics, we found additive effects of chemical and mechanical cues on the suppression of circular expansion by up-regulating the epithelial morphology. Also, the impact of geometrical cues became more significant by decreasing the chemical cue. We believe the present platform will be a useful research tool for the comprehensive mechanobiological analysis of collective cell migration.

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.

Photoactivatable Substrates: A Material-Based Approach for Dissecting Cell Migration.

Photoactivatable substrates, which show changes in surface cell adhesiveness in response to photoirradiation, are promising platforms for cell manipulation with high spatiotemporal resolution. In addition to having applications in cell and tissue engineering, these materials are unique tools for basic biological sciences research, and they complement conventional genetic engineering technologies. One of the most useful applications is in the study of cell migration, which occurs in various physiological and pathological processes. In this personal account, I provide a brief overview of the development of photoactivatable substrates and their applications, highlighting in particular the contributions of our research group to collective cell migration studies. This material-based approach is useful for dissecting the molecular biological and mechanobiological aspects of the regulatory mechanisms in the cellular social activities.

Facile preparation of a photoactivatable surface on a 96-well plate: a versatile and multiplex cell migration assay platform.

Cell migration is an essential cellular activity in various physiological and pathological processes, such as wound healing and cancer metastasis. Therefore, in vitro cell migration assays are important not only for fundamental biological studies but also for evaluating potential drugs that control cell migration activity in medical applications. In this regard, robust control over cell migrating microenvironments is critical for reliable and quantitative analysis as cell migration is highly dependent upon the microenvironments. Here, we developed a facile method for making a commercial glass-bottom 96-well plate photoactivatable for cell adhesion, aiming to develop a versatile and multiplex cell migration assay platform. Cationic poly-d-lysine was adsorbed to the anionic glass surface via electrostatic interactions and, subsequently, functionalized with poly(ethylene glycol) (PEG) bearing a photocleavable reactive group. The initial PEGylated surface is non-cell-adhesive. However, upon near-ultraviolet (UV) irradiation, the photorelease of PEG switches the surface from non-biofouling to cell-adhesive. With this platform, we assayed cell migration in the following procedure: (1) create cell-attaching regions of precise geometries by controlled photoirradiation, (2) seed cells to allow them to attach selectively to the irradiated regions, (3) expose UV light to the remaining PEGylated regions to extend the cell-adhesive area, (4) analyse cell migration using microscopy. Surface modification of the glass surface was characterized by ζ-potential and contact angle measurements. The PEGylated surface showed cell-resistivity and became cell-adhesive upon releasing PEG by near-UV irradiation. The method was applied for parallelly evaluating the effect of model drugs on the migration of epithelial MDCK cells in the multiplexed platform. The dose-response relationship for cytochalasin D treatment on cell migration behavior was successfully evaluated with high reproducibility. Interestingly, the impact of blebbistatin on cell migration was dependent upon the widths of the migrating regions, resulting in both cases of migration acceleration and deceleration. These results clearly demonstrate that the cellular response to certain drugs is highly affected by their migrating geometries. Therefore, the obtained novel photoactivatable 96-well plate serves as a useful high-throughput platform for the identification of drug candidates that have an effect on cell migration behavior.

α-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma.

Invasive lobular carcinoma (ILC) is more frequently lymph node positive than is invasive ductal carcinoma (IDC), and ILC cell infiltration shows distinctive histological characteristics, suggesting the action of ILC-specific invasion molecules. To identify such a molecule, we used a proteomic approach in the pseudopodia of MDA-MB-231 breast cancer cells. A pseudopodial constituent was identified using excimer laser ablation, two-dimensional difference gel electrophoresis, mass spectroscopy, and immunocytofluorescence. MDA-MB-231 cells were modified to express various levels of this constituent by transient transfection and were examined for pseudopodia formation and migratory abilities using wound healing and two-chamber assays. Immunohistochemical positivity of human breast cancer cells (56 ILCs and 21 IDCs) was compared with clinicopathological variables. An actin-binding adaptor protein, α-parvin, was found to localize to pseudopodia and to form focal adhesions in cells not induced to extend pseudopodia. Pseudopodial length and density and migratory abilities correlated with α-parvin expression. Twenty-one (37.5 %) ILCs stained positive for α-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). α-Parvin positivity in ILC was significantly associated with lymphatic invasion (P = 0.038) and lymph node metastasis (P = 0.003) in univariate analyses and to lymph node metastasis (P = 0.020) in multivariate analyses. α-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that α-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.

Ultimately Adaptive Fluid Interfacial Phospholipid Membranes Unveiled Unanticipated High Cellular Mechanical Work.

Living cells actively interact biochemically and mechanically with the surrounding extracellular matrices (ECMs) and undergo dramatic morphological and dimensional transitions, concomitantly remodeling ECMs. However, there is no suitable method to quantitatively discuss the contribution of mechanical interactions in such mutually adaptive processes. Herein, a highly deformable "living" cellular scaffold is developed to evaluate overall mechanical energy transfer between cell and ECMs. It is based on the water-perfluorocarbon interface decorated with phospholipids bearing a cell-adhesive ligand and fluorescent tag. The bioinert nature of the phospholipid membranes prevents the formation of solid-like protein nanofilms at the fluid interface, enabling to visualize and quantify cellular mechanical work against the ultimately adaptive model ECM. A new cellular wetting regime is identified, wherein interface deformation proceeds to cell flattening, followed by its eventual restoration. The cellular mechanical work during this adaptive wetting process is one order of magnitude higher than those reported with conventional elastic platforms. The behavior of viscous liquid drops at the air-water interface can simulate cellular adaptive wetting, suggesting that overall viscoelasticity of the cell body predominates the emergent wetting regime and regulates mechanical output. Cellular-force-driven high-energy states on the adaptive platform can be useful for cell fate manipulation.

Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteins.

In cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells that move through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECM protein types on leader cells and migrating clusters is unknown. To address this, we investigated the response of leader cells and migrating clusters upon moving from one ECM protein to another using a photoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since they are abundant in two distinct regions in living tissues, namely basement membrane and connective tissue. Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areas was achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled the deposition of the second ECM protein in the new irradiated regions, resulting in region-selective heterogeneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation from laminin into laminin compared to those migrating from laminin into collagen were observed. The formation of focal adhesion and actin structures for cells within the same cluster in the ECM proteins responded according to the underlying ECM protein type. Finally, integrin ß1 was crucial for the appearance of leader cells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin, integrin ß1 was not responsible. This highlights the correlation between leader cells in collective migration and the biochemical signals that arise from underlying extracellular matrix proteins.

Ionic Liquid Interface as a Cell Scaffold.

In sharp contrast to conventional solid/hydrogel platforms, water-immiscible liquids, such as perfluorocarbons and silicones, allow the adhesion of mammalian cells via protein nanolayers (PNLs) formed at the interface. However, fluorocarbons and silicones, which are typically used for liquid cell culture, possess only narrow ranges of physicochemical parameters and have not allowed for a wide variety of cell culturing environments. In this paper, it is proposed that water-immiscible ionic liquids (ILs) are a new family of liquid substrates with tunable physicochemical properties and high solvation capabilities. Tetraalkylphosphonium-based ILs are identified as non-cytotoxic ILs, whereon human mesenchymal stem cells are successfully cultured. By reducing the cation charge distribution, or ionicity, via alkyl chain elongation, the interface allows cell spreading with matured focal contacts. High-speed atomic force microscopy observations of the PNL formation process suggest that the cation charge distribution significantly altered the protein adsorption dynamics, which are associated with the degree of protein denaturation and the PNL mechanics. Moreover, by exploiting dissolution capability of ILs, an ion-gel cell scaffold is fabricated. This enables to further identify the significant contribution of bulk subphase mechanics to cellular mechanosensing in liquid-based culture scaffolds.