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A critical stage of T cell development is β-selection; at this stage, the T cell receptor β (TCRβ) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through β-selection is assisted by low-affinity interactions between the nascent TCRβ chain and peptide presented on stromal major histocompatibility complex and cues provided by the niche. In this study, we identify a cue within the developing T cell niche that is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells, E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data suggest that E-cadherin facilitates interactions with the thymic niche to coordinate the β-selection stage of T cell development.

Ultrashort self-assembling peptides of typically less than 5 amino acids in length can be modified with highly aromatic protecting groups such as Fluorenylmethyloxycarbonyl (Fmoc) resulting in the rapid formation of nanofibrillar hydrogels. These hydrogels show great promise as scaffolds for tissue engineering and 3D cell culture. If further modified with bioactive peptide sequences, the hydrogels can be used to promote and control cell adhesion, and even support the ability for cultured mesenchymal stem cells (MSCs) to differentiate into therapeutically useful lineages, such as chondrocytes. Whilst there have been numerous examples of how the addition of such sequences can promote the bioactivity of cultured cells, the effect adding these sequences has on the nanoarchitecture, mechanical properties and stability of the resultant hydrogels is less well explored. Here, we have performed a series of experiments investigating the effect incorporating stoichiometric amounts of Fmoc-Arg-Gly-Asp (Fmoc-RGD) into Fmoc-Phe-Phe (Fmoc-FF) hydrogels has on nanofibril morphology, and the rheological properties and stability of the formed gels. Furthermore, we correlate these variations in nano-architectural and mechanical properties with their ability to support the culture of clinically valuable bone marrow derived human mesenchymal stem cells (hMSCs). Our results show that at relatively low concentrations Fmoc-RGD is incorporated into the self-assembled Fmoc-FF nanofibrils creating biocompatible hydrogels capable of supporting hMSC growth for up to a week. At increased concentrations of Fmoc-RGD, incorporation into the nanofibrils is lost and the hydrogels that form are no longer stable enough to support the culture of hMSCs for multiple days. These findings shed light on the importance of balancing the positive effects of adding bioactive ligands to 3D scaffolds with the negative effects these ligands can have on the stability and mechanical properties of these scaffolds. Further, the findings will have implications for the choice of ultrashort peptide scaffolds for tissue engineering applications to support MSCs towards chondrogenic differentiation, and their application as gel-cell constructs in cartilage tissue engineering for osteoarthritis.

A critical stage of T cell development is β-selection; at this stage the TCRβ chain is generated and the developing T cell starts to acquire antigenic specificity. Progression through β-selection is assisted by a low affinity interaction between the nascent TCRβ chain and peptide presented on stromal MHC and external cues provided by the niche, including Notch and CXCR4. In this study, we reveal the importance of a new cue within the murine developing T cell niche which is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data highlight a new aspect of the developing T cell niche and provide insights into the role of E-cadherin in the β-selection stage of T cell development.

COVID-19 is primarily known as a respiratory disease caused by the virus SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, cognitive and psychiatric issues, severe headaches, and even stroke are reported in as many as 30% of cases and can persist even after the infection is over (so-called ‘long COVID’). These neurological symptoms are thought to be caused by brain inflammation, triggered by the virus infecting the central nervous system of COVID-19 patients, however we still don’t fully understand the mechanisms for these symptoms. The neurological effects of COVID-19 share many similarities to neurodegenerative diseases such as Alzheimer’s and Parkinson’s in which the presence of cytotoxic protein-based amyloid aggregates is a common etiological feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we performed a bioinformatic scan of the SARS-CoV-2 proteome, detecting peptide fragments that were predicted to be highly amyloidogenic. We selected two of these peptides and discovered that they do rapidly self-assemble into amyloid. Furthermore, these amyloid assemblies were shown to be highly toxic to a neuronal cell line. We introduce and support the idea that cytotoxic amyloid aggregates of SARS-CoV-2 proteins are causing some of the neurological symptoms commonly found in COVID-19 and contributing to long COVID, especially those symptoms which are novel to long COVID in contrast to other post-viral syndromes.

During T cell development, the first step in creating a unique T Cell Receptor (TCR) is the genetic recombination of the TCRβ chain. The quality of the newly recombined TCRβ is assessed at the β-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of the complex events that control fate at the β-selection checkpoint is not yet understood. We shed new light on fate determination during β-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the β-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in β-selection, bookended by upregulation of the TCR co-receptors, CD28 and CD2 respectively. Within this ‘DN3bPre’stage, CD5 and Lef1 are upregulated to reflect pre-TCR signalling and their expression correlates with proliferation. During this phase, ACY1215-mediated disruption of the organisation of the β-selection immunological synapse is associated with a breakdown in connectivity of expression of pre-TCR, CD5 and Lef1. Subsequent deregulation of pre-TCR-induced proliferation leads to bypass of the β-selection checkpoint and subsequent failure to progress. We propose that the progressive expression of CD28, CD5 and Lef1, then CD2 reports and modulates the pre-TCR signal to orchestrate passage through the β-selection checkpoint. These findings suggest a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength, and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.

During T cell development, the first step in creating a unique T cell receptor (TCR) is genetic recombination of the TCRβ chain. The quality of the new TCRβ is assessed at the β-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of fate at the β-selection checkpoint is not yet understood. We shed new light on fate determination during β-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the β-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in β-selection, bookended by up-regulation of TCR co-receptors, CD28 and CD2, respectively. Within this “DN3bPre” stage, CD5 and Lef1 are up-regulated to reflect pre-TCR signalling, and their expression correlates with proliferation. These findings suggest a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.

Mesenchymal stem cell therapies show great promise in regenerative medicine. However, to generate clinically relevant numbers of these stem cells, significant in vitro expansion of the cells is required before transplantation into the affected wound or defect. The current gold standard protocol for recovering in vitro cultured cells involves treatment with enzymes such as trypsin which can affect the cell phenotype and ability to interact with the environment. Alternative enzyme free methods of adherent cell recovery have been investigated, but none match the convenience and performance of enzymatic detachment. In this work we have developed a synthetically simple, low cost cell culture substrate functionalized with gold nanorods that can support cell proliferation and detachment. When these nanorods are irradiated with biocompatible low intensity near infrared radiation (785 nm, 560 mWcm-2) they generate localized surface plasmon resonance induced nanoscale heating effects which trigger detachment of adherent mesenchymal stem cells. Through simulations and thermometry experiments we show that this localized heating is concentrated at the cell-nanorod interface, and that the stem cells detached using this technique show either similar or improved multipotency, viability and ability to differentiate into clinically desirable osteo and adipocytes, compared to enzymatically harvested cells. This proof-of-principle work shows that photothermally mediated cell detachment is a promising method for recovering mesenchymal stem cells from in vitro culture substrates, and paves the way for further studies to scale up this process and facilitate its clinical translation. STATEMENT OF SIGNIFICANCE: New non-enzymatic methods of harvesting adherent cells without damaging or killing them are highly desirable in fields such as regenerative medicine. Here, we present a synthetically simple, non-toxic, infra-red induced method of harvesting mesenchymal stem cells from gold nanorod functionalized substrates. The detached cells retain their ability to differentiate into therapeutically valuable osteo and adipocytes. This work represents a significant improvement on similar cell harvesting studies due to: its simplicity; the use of clinically valuable stem cells as oppose to immortalized cell lines; and the extensive cellular characterization performed. Understanding, not just if cells live or die but how they proliferate and differentiate after photothermal detachment will be essential for the translation of this and similar techniques into commercial devices.

During T cell development, genomic rearrangement must create a T cell receptor capable of transmitting signals. Allam et al. show that passage through the β-selection checkpoint requires the assembly of a platform to support TCR signaling, similar to the mature T cell immunological synapse., The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor β (TCRβ). Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro and in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell to allow transition beyond the β-selection checkpoint.

Nanostructured and bulk silicon carbide ($\mathrm{Si}\mathrm{C}$) materials are relevant for electronics, nano- and micromechanical systems, and biosensing applications. $\mathrm{Si}\mathrm{C}$ has recently emerged as an alternative platform for nanophotonics and quantum applications due to its intra-band-gap point defects, emitting from the visible to the near-infrared, which are ideal for photoluminescent probes. Here, we use a single-molecule localization microscope to study the photoluminescence (PL) properties of $\mathrm{Si}\mathrm{C}$ point defects in bulk, quantum dots, and nanoparticles of different sizes in the 3C and $4H\text{\ensuremath{-}}\mathrm{Si}\mathrm{C}$ polytypes using intra-band-gap excitation. We study the PL dynamics by using different excitation wavelengths, and we use the point-defect PL intermittency to achieve superresolved images, with a resolution of 20 nm and a minimum distance between emitters of 40 nm. We observe that, while 561 nm is an ideal excitation wavelength to obtain a sufficient blinking behavior, 638 nm is mostly quenching the PL. We further incubate $4H\text{\ensuremath{-}}\mathrm{Si}\mathrm{C}$ nanoparticles with MCF10A cells in vitro and observe superresolved images of the nanoparticles in cells by combining 561 and 638 nm excitation. This approach is very promising for the application of $\mathrm{Si}\mathrm{C}$ fluorescent nanocrystals and their quantum dots, hosting a combination of intra-band-gap color centers and surface defects, for quantum nanophotonics, magnetic sensing, and biomedical imaging, paving the way for single-particle tracking combined with spin sensing within a cellular environment using near-infrared emission.

Nanodiamonds containing the nitrogen vacancy centre (NV) have a significant role in biosensing, bioimaging, drug delivery, and as biomarkers in fluorescence imaging, due to their photo-stability and biocompatibility. The optical read out of the NV unpaired electron spin has been used in diamond magnetometry to image living cells and magnetically labelled cells. Diamond magnetometry is mostly based on the use of bulk diamond with a large concentration of NV centres in a wide field fluorescence microscope equipped with microwave excitation. It is possible to correlate the fluorescence maps with the magnetic field maps of magnetically labelled cells with diffraction limit resolution. Nanodiamonds have not as yet been implemented to image magnetic fields within complex biological systems at the nanometre scale. Here we demonstrate the suitability of nanodiamonds to correlate the fluorescence map with the magnetic imaging map of magnetically labelled cells. Nanoscale optical images with 17 nm resolution of nanodiamonds labelling fixed cells bound to iron oxide magnetic nanoparticles are demonstrated by using a single molecule localisation microscope. Nanoscale magnetic field images of the magnetised magnetic nanoparticles spatially assigned to individual cells are superresolved by the NV centres within nanodiamonds conjugated with the magnetic nanoparticles with 20 nm resolutions. Our method offers a new platform for the super-resolution of optical magnetic imaging in biological samples conjugated with nanodiamonds and iron-oxide magnetic nanoparticles.

The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T Cell Receptor β (TCRβ) receptor. Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro & in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the β-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the β-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell, to allow transition beyond the β-selection checkpoint.SummaryT cell development requires testing whether genomic rearrangement has produced a T cell receptor capable of transmitting signals. Most T cells fail this test. Here, we show that passage through the β-selection checkpoint requires assembly of a platform to support TCR signaling.

A fundamental question in biology is how single cells can reliably produce progeny of different cell types. Notch signalling frequently facilitates fate determination. Asymmetric cell division (ACD) often controls segregation of Notch signalling by imposing unequal inheritance of regulators of Notch. Here, we assessed the functional relationship between Notch and ACD in mouse T cell development. To attain immunological specificity, developing T cells must pass through a pivotal stage termed β-selection, which involves Notch signalling and ACD. We assessed functional interactions between Notch1 and ACD during β-selection through direct presentation of Notch ligands, DL1 and DL4, and pharmacological inhibition of Notch signalling. Contrary to prevailing models, we demonstrate that Notch signalling controls the distribution of Notch1 itself and cell fate determinants, α-adaptin and Numb. Furthermore, Notch and CXCR4 signalling cooperated to drive polarity during division. Thus, Notch signalling directly orchestrates ACD, and Notch1 is differentially inherited by sibling cells. This article has an associated First Person interview with the first author of the paper.

Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarity enables cells to sense and respond to the cues provided by the neighboring cells and the surrounding microenvironment. These responses play a critical role in regulating key physiological processes, including cell migration, proliferation, differentiation, vesicle trafficking and immune responses. The polarity protein complexes regulating these interactions are highly evolutionarily conserved between vertebrates and invertebrates. Interestingly, these polarity complexes interact with each other and key signaling pathways in a cell-polarity context-dependent manner. However, the exact mechanisms by which these interactions take place are poorly understood. In this review, we will focus on the roles of the key polarity complexes SCRIB, PAR and Crumbs in regulating different forms of cell polarity, including epithelial cell polarity, cell migration, asymmetric cell division and the T-cell immunological synapse assembly and signaling.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients., Scientific Reports, 8, ISSN:2045-2322

T cell precursors undergo asymmetric cell division after T cell receptor genomic recombination, with stromal cell cues controlling the differential inheritance of fate determinants Numb and α-Adaptin by the daughters of a dividing DN3a T cell precursor., During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal.

Networks of nanoscale fibrous coatings made from self-assembled peptides are promising candidates for biomaterials that can promote the growth of mammalian cells. One particularly attractive feature is the possibility of adding biofunctional sequences to peptides to promote cell attachment. We deconvolute the topographic and chemical effects of nanoscale fibrils on cells by depositing a plasma polymer film on TTR1-based fibrils decorated with a range of cell adhesive chemistries (RGD and cycloRGDfK), producing a surface that retains the nanoscale fibrous topography of surface-bound fibrils but lacks the fibril surface chemistry. The surface topography was found to influence cell toxicity and spreading, and the fibril surface chemistry influenced cell attachment and spreading. This study highlights the importance of considering both the chemical and physical features of novel biomaterials and illustrates the use of plasma polymer deposition as a tool for examining the relationship between amyloid fibril structure and function.

Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid supports can improve cell adhesion, spreading and differentiation compared to traditional flat, stiff 2D cell culture substrates like Tissue Culture Polystyrene (TCPS). This is due to the fibrous, nanotopographic nature of the amyloid fibril networks and the fact that they closely mimic the mechanical properties and architecture of the extracellular matrix (ECM). However, precise cell responses are strongly dependent on the nanostructure of the network at the cell culture interface, thus accurate characterization of the immobilized network is important. Due to its exquisite lateral resolution and simple sample preparation techniques, Atomic Force Microscopy (AFM) is an ideal technique to characterize the fibril network morphology. Thus, here we describe a detailed protocol, for the characterization of amyloid fibril networks by tapping mode AFM.

Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and β-lactoglobulin have material properties that mimic the extracellular microenvironment of many cell types. Cells cultured on such amyloid fibril networks show improved attachment, spreading and in the case of mesenchymal stem cells improved differentiation. Here we describe a detailed protocol for fabricating amyloid fibril networks suitable for eukaryotic cell culture applications.

Force spectroscopy is a means of obtaining mechanical information of individual nanometer-scale structures in composite materials, such as protein assemblies for use in consumer films or gels. As a recently developed force spectroscopy technique, bimodal force spectroscopy relates frequency shifts in cantilevers simultaneously excited at multiple frequencies to the elastic properties of the contacted material, yet its utility for quantitative characterization of biopolymer assemblies has been limited. In this study, a linear correlation between experimental frequency shift and Young's modulus of polymer films was used to calibrate bimodal force spectroscopy and quantify Young's modulus of two protein nanostructures: β-lactoglobulin fibrils and zein nanoparticles. Cross-sectional Young's modulus of protein fibrils was determined to be 1.6 GPa while the modulus of zein nanoparticles was determined as 854 MPa. Parallel measurement of β-lactoglobulin fibril by a competing pulsed-force technique found a higher cross-sectional Young's modulus, highlighting the importance of comparative calibration against known standards in both pulsed and bimodal force spectroscopies. These findings demonstrate a successful procedure for measuring mechanical properties of individual protein assemblies with potential use in biological or packaging applications using bimodal force spectroscopy.

Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila and C. elegans and has recently emerged as a mediator of T cell fate and hematopoiesis. The most appropriate methods for assessing ACD in T cells are still evolving. Here we describe the methods currently applied to monitor and measure ACD of developing and activated T cells. We provide an overview of approaches for capturing cells in the process of cytokinesis in vivo, ex vivo, or during in vitro culture. We provide methods for in vitro fixed immunofluorescent staining and for time-lapse analysis. We provide an overview of the different approaches for quantification of ACD of lymphocytes, discuss the pitfalls and concerns in interpretation of these analyses, and provide detailed methods for the quantification of ACD in our group.

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or β-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis.

Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila and C. elegans and has recently emerged as a mediator of T cell fate and hematopoiesis. The most appropriate methods for assessing ACD in T cells are still evolving. Here we describe the methods currently applied to monitor and measure ACD of developing and activated T cells. We provide an overview of approaches for capturing cells in the process of cytokinesis in vivo, ex vivo, or during in vitro culture. We provide methods for in vitro fixed immunofluorescent staining and for time-lapse analysis. We provide an overview of the different approaches for quantification of ACD of lymphocytes, discuss the pitfalls and concerns in interpretation of these analyses, and provide detailed methods for the quantification of ACD in our group.

Fibrous networks assembled from synthetic peptides are promising candidates for biomimetic cell culture platforms and implantable biomaterials. The ability of the materials to reproduce physiological cell-matrix interactions is essential. However, the synthetic complexity of such systems limits their applications, thus alternative materials are desirable. Here, we design lysozyme derived amyloid fibril networks with controllable topographies, and perform a comprehensive study of the response of cultured fibroblast and epithelial cells. At high surface coverage a favorable increase in spreading and the generation of focal adhesions was observed, due to a combination of biomimetic chemistry and morphology. Their ease of synthesis, makes the nanoscale fibrils presented here ideal materials for future clinical applications whereby large volumes of biomimetic biomaterials are required. Furthermore, the surface chemistry of the fibrils is sufficient for the promotion of focal adhesions with cultured cells, eliminating the need for complex protocols for fibril decoration with bioactive moieties.

Cancer is one of the most common causes of death worldwide. Consequently, important resources are directed towards bettering treatments and outcomes. Cancer is difficult to treat due to its heterogeneity, plasticity and frequent drug resistance. New treatment strategies should strive for personalized approaches. These should target neoplastic and/or activated microenvironmental heterogeneity and plasticity without triggering resistance and spare host cells. In this review, the putative use of increasingly physiologically relevant microfabricated cell-culturing systems intended for drug development is discussed. There are two main reasons for the use of miniaturized systems. First, scaling down model size allows for high control of microenvironmental cues enabling more predictive outcomes. Second, miniaturization reduces reagent consumption, thus facilitating combinatorial approaches with little effort and enables the application of scarce materials, such as patient-derived samples. This review aims to give an overview of the state-of-the-art of such systems while predicting their application in cancer drug development.

Designing surfaces with antifouling and antimicrobial properties has an important route to solve problems, such as infections and fouling, in healthcare and industrial applications. Recently, there has been considerable interest in developing surfaces with polymers because of their distinct properties, such as their length scale, their processability, low cost, tunable properties, and diverse functionalities. This article reviews the polymer systems developed as antifouling–antimicrobial surface coatings, termed as biopassive and bioactive polymers respectively. Many different types of bioactive and biopassive polymeric systems and their limitations are presented.

Biomaterials-associated-infections (BAI) are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while supporting tissue integration. Here, bacterial and cellular responses to poly(ethylene glycol) (PEG) brush-coatings on titanium oxide presenting the integrin-active peptide RGD (arginine-glycine-aspartic acid) (bioactive "PEG-RGD") were compared to monofunctional PEG brush-coatings (biopassive "PEG") and bare titanium oxide (TiO(2)) surfaces under flow. Staphylococcus epidermidis ATCC 35983 was deposited on the surfaces under a shear rate of 11 s(-1) for 2 h followed by seeding of U2OS osteoblasts. Subsequently, both S. epidermidis and U2OS cells were grown simultaneously on the surfaces for 48 h under low shear (0.14 s(-1)). After 2 h, staphylococcal adhesion was reduced to 3.6 +/- 1.8 x 10(3) and 6.0 +/- 3.9 x 10(3) cm(-2) on PEG and PEG-RGD coatings respectively, compared to 1.3 +/- 0.4 x 10(5) cm(-2) for the TiO(2) surface. When allowed to grow for 48 h, biofilms formed on all surfaces. However, biofilms detached from the PEG and PEG-RGD coatings when exposed to an elevated shear (5.6 s(-1)). U2OS cells neither adhered nor spread on PEG brush-coatings, regardless of the presence of biofilm. In contrast, in the presence of biofilm, U2OS cells adhered and spread on PEG-RGD coatings with a significantly higher surface coverage than on bare TiO(2). The detachment of biofilm and the high cell surface coverage revealed the potential significance of PEG-RGD coatings in the context of the "race for the surface" between bacteria and mammalian cells.

The aim of this work was to immobilize short melanocyte-stimulating hormone (MSH) anti-inflammatory peptide sequences onto a model surface using resorcinarenes, which are known to attach to a wide variety of hydrophilic materials. The first stage comprised the biological evaluation of a synthetic MSH peptide attached to different linking tethers (poly(ethylene glycol), PEG 350, octanoic acid or cholesterol) for the ability to inhibit inflammatory signaling. Findings showed that a glycinelysine-proline-D-valine sequence inhibited inflammatory signaling most effectively when attached to a PEG 350 tether. This molecule was selected for a second stage which comprised of synthesizing the MSH peptide attached to resorcinarene groups via a PEG tether and immobilizing it onto glass coverslips. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF–SIMS) indicated the presence of a surface attached peptide. The ability of the immobilized peptide to inhibit inflammatory signaling was then determined by culturing RN22 Schwann neuronal cells and human dermal fibroblast cells on functional surfaces and measuring NF-kB/p65 inflammatory transcription factor activation. Significant inhibition of inflammatory signaling was observed in cells cultured on functional surfaces. In conclusion, we therefore describe the development of a new approach enabling the rapid immobilization of short biologically active peptides. This work has potential for generating a ‘‘dip-and-dry’’ approach for altering the surface properties of biomaterial and medical devices. Many implantable medical devices can initiate an acute inflammatory response which in turn is detrimental to the function of the device. This can be triggered either by the initial implantation injury or by the biomaterial itself. Examples of this include coronary restenosis after stent implantation, the ‘stress cracking’ of pacemakers and the failure of peripheral nerve guides. Inflammation can be treated by steroids or non-steroidal anti-inflammatories (NSAIDs). However, these compounds frequently lack sensitivity or a specific targeted site of action, leading to side effects. Therefore the development of more specific and localized forms of treatment is important for controlling inflammation associated with biomaterials. Previous work has investigated modifying the material surface chemistry or immobilizing non-bio-fouling molecules, such as PEG. However, these techniques are not always appropriate because they do not specifically target inflammatory signaling. An alternative approach is through the exploitation of naturally occurring peptides to reduce inflammation. One potential peptide is a-MSH which has multiple roles in the body including the control of inflammation. An analogue of a-MSH, NleDPhe-a-MSH, has already been shown to increase allograft survival when injected into rats. Previous research has shown that the minimal sequence required for anti-inflammatory biology is a tripeptide at the carboxyl terminal consisting of lysine-proline-valine (KPV). This tripeptide mimics the anti-inflammatory effect of the full-length peptide, but is more specific as it does not exhibit melanotropic activity or stimulate dopa oxidase activity. This peptide has the ability to inhibit nuclear factor-kB (NF-kB) activation when stimulated by a wide range of proinflammatory agents, reducing the production of proinflammatory cytokines under its control. KPV can stimulate the direct production of the anti-inflammatory cytokine interleukin-10 (IL-10) and inhibit inflammation in vivo. Additionally, the peptide has been demonstrated to possess antimicrobial properties in vitro. The short tripeptide sequence makes it amenable for straightforward laboratory synthesis and this, in combination with its pharmacological potency, makes it an attractive candidate for immobilization on to medical device surfaces for creating a localized anti-inflammatory effect. a-MSH and its analogues have previously been immobilized using polyelectrolyte films and also as an analogue of the tripeptide (GKP-D-V), where it was attached to polystyrene beads. Both techniques demonstrated that the peptide retained anti-inflammatory properties when immobilized in vitro. Prostheses of titanium beads coated with polyelectrolyte multilayer films functionalized with a-MSH also retained the ability to inhibit inflammation when implanted into rats. The D-Val substitution in KP-D-V leads to greater anti-inflammatory properties than the KPV peptide and the parent molecule, a-MSH, probably due to reduced degradation. The present work describes a new methodology for immobilization of the tripeptide analogue GKP-D-V using resorcinarene group molecules. Resorcinarenes can be immobilized through hydrogen bonding of the hydroxylated bowl to hydrophilic materials through simple immersion of the material into a solution of the resorcinarene compound. This forms a new surface on that material, the properties of which then depend on the nature of the group covalently attached to the resorcinarene. Previous research has investigated the use of attaching alkyl chains and

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range ( 0.5–4.7 lm), and mean distance between peaks (RSm) gradually varying from 214 lm to 33 lm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra 1.53 lm/RSm 79 lm in Dex-deprived OI medium, and Ra 0.93 lm/RSm 135 lm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone., This work was supported by the Foundation for Science and Technology for the PhD Grant of A.B. Faia-Torres (SFRH/BD/36476/2007). We are also grateful to the Swiss National Science Foundation (SNSF) Grant P300P3_154664 for providing funds to MC.

The success of synthetic bone implants requires good interface between the material and the host tissue. To study the biological relevance of fi bronectin (FN) density on the osteogenic commitment of human bone marrow mesenchymal stem cells (hBMMSCs), human FN was adsorbed in a linear density gradient on the surface of PCL. The evolution of the osteogenic markers alkaline phosphatase and collagen 1 alpha 1 was monitored by immunohistochemistry, and the cytoskeletal organization and the cell-derived FN were assessed. The functional analysis of the gradient revealed that the lower FN-density elicited stronger osteogenic expression and higher cytoskeleton spreading, hallmarks of the stem cell commitment to the osteoblastic lineage. The identifi cation of the optimal FN density regime for the osteogenic commitment of hBM-MSCs presents a simple and versatile strategy to signifi cantly enhance the surface properties of polycaprolactone as a paradigm for other synthetic polymers intended for bone-related applications., This work was supported by the Foundation for Science and Technology for the PhD grant of A.B. Faia-Torres (SFRH/BD/36476/2007). We are also grateful to the Swiss National Science foundation (SNSF) grant P300P3_154664 for providing funds to MC.

Model lipid vesicle and supported lipid bilayer (SLB) systems are used in a variety of applications including biosensing, cell membrane mimics, and drug delivery. Exposure of a surface to a vesicle solution provides a straightforward method for creating such systems via vesicle adsorption and collapse. However, this process is complex and the relationship between the surface physicochemical properties and vesicle collapse is poorly understood. Plasma polymers are thin conformal films that can be applied to a variety of materials to modify surface properties. This paper uses quartz crystal microbalance with dissipation and fluorescence recovery after photobleaching (FRAP) to explore lipid vesicle interactions with plasma polymerized acrylic acid (ppAAc), allylamine (ppAAm), and ppAAc/ppAAm micropatterns. Vesicle interactions were dependent on plasma polymer chemistry and pH of the buffer solution. Vesicles readily and stably adsorbed to ppAAm over a wide pH range. ppAAc demonstrated limited interactions at pH 7 and vesicle adsorption at pH 4. Vesicle collapse and SLB formation could be induced using a pH change. FRAP was used to explore the fluidity of the lipid structures on both the patterned and unpatterned plasma polymer films. On ppAAm/ppAAc micropatterns, pH transitions combined with the presence of chemically distinct regions on the same substrate enabled immobile lipid islands on ppAAc to be surrounded by fluid lipid regions on ppAAm. This work demonstrates that plasma polymer films could enable spatially controlled vesicle adsorption and SLB formation on a wide variety of different substrates.

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data., PLoS ONE, 6 (12), ISSN:1932-6203

Background Increasing evidence shows that the cancer microenvironment affects both tumorigenesis and the response of cancer to drug treatment. Therefore in vitro models that selectively reflect characteristics of the in vivo environment are greatly needed. Current methods allow us to screen the effect of extrinsic parameters such as matrix composition and to model the complex and three-dimensional (3D) cancer environment. However, 3D models that reflect characteristics of the in vivo environment are typically too complex and do not allow the separation of discrete extrinsic parameters. Methodology/Principal Findings In this study we used a poly(ethylene glycol) (PEG) hydrogel-based microwell array to model breast cancer cell behavior in multilayer cell clusters that allows a rigorous control of the environment. The innovative array fabrication enables different matrix proteins to be integrated into the bottom surface of microwells. Thereby, extrinsic parameters including dimensionality, type of matrix coating and the extent of cell-cell adhesion could be independently studied. Our results suggest that cell to matrix interactions and increased cell-cell adhesion, at high cell density, induce independent effects on the response to Taxol in multilayer breast cancer cell clusters. In addition, comparing the levels of apoptosis and proliferation revealed that drug resistance mediated by cell-cell adhesion can be related to altered cell cycle regulation. Conversely, the matrix-dependent response to Taxol did not correlate with proliferation changes suggesting that cell death inhibition may be responsible for this effect. Conclusions/Significance The application of the PEG hydrogel platform provided novel insight into the independent role of extrinsic parameters controlling drug response. The presented platform may not only become a useful tool for basic research related to the role of the cancer microenvironment but could also serve as a complementary platform for in vitro drug development. ISSN:1932-6203

Traditional in vitro models used for the development of anti-cancer drugs are based on the monolayer culture of cells, which has a limited predictivity of in vivo efficacy. A number of cell culture platforms have been developed in recent years to improve predictivity and further to elucidate the mechanisms governing the differing responses observed in vitro versus in vivo. One detrimental aspect of current in vitro models is their inability to decouple the effect of different extrinsic factors on the responsiveness of the cells to drug treatment. Here, we have used an engineered poly(dimethylsiloxane) (PDMS) microwell array as a reductionist approach to study the effect of environmental parameters, independently of each other. It is observed for MCF-7 breast cancer cells, that culture within the three-dimensional (3D) environment of the microwells alone had an effect on the response to Taxol and results in a reduction of cell death in comparison to cells cultured on flat substrates. Additionally the microwells allowed the response of single versus multicell clusters to be differentiated. It was found that the formation of cell–cell contacts alters the drug response, depending on the type of adhesive protein present. Thus, with this microwell platform it is revealed that the presence of cell–cell contacts in addition to the dimensionality and the matrix composition of the environment are important mediators of altered drug responses. In conclusion the microwell array can not only serve as a platform to reveal which parameters of the extracellular environment affect drug response but further the interdependence of these parameters.

The melanocortin peptides α-MSH, Lys-Pro-Val and Lys-Pro- d -Val are known to be potent anti-inflammatory agents; however their role as antibacterial peptides is less clear. The aim of this study was to determine whether these peptides displayed antibacterial properties, and specifically whether the Lys-Pro- d -Val tripeptide was more potent than Lys-Pro-Val, consistent with their anti-inflammatory actions. α-MSH, Ac-Lys-Pro- d -Val-NH 2 and Ac-Lys-Pro-Val-NH 2 were found to be antibacterial against both Gram-positive and Gram-negative bacteria ( Staphylococcus aureus and Escherichia coli ) over a broad range of concentrations compared to a control peptide, Ac-Ala-Ala-Ala-NH 2 . However, the relative potency of α-MSH, Ac-Lys-Pro- d -Val-NH 2 , Ac-Lys-Pro-Val-NH 2 did not differ. Furthermore, it was found that the cationic charge on the lysine residue was not required for activity as a variant peptide Ac-Ala-Pro- d -Val-NH 2 was also antibacterial. We therefore describe a novel X-Pro- d / l -Val peptide sequence with similarity to the short melanocortin peptides, which possess antibacterial activity. The combined anti-inflammatory and antibacterial action of such peptides may also have potential value therapeutically.

The apicobasal polarisation of epithelial cells within an epithelium is critical for its function as a selective barrier. Microenvironmental parameters, including cell-matrix and cell-cell interactions, contribute to the initiation and orientation of this polarity. However, it is often non-trivial to decipher the differential effects of these parameters in a controlled manner using traditional in vitro platforms. A reductionist platform, consisting of E-cadherin coupled onto laterally mobile supported lipid bilayers, was utilised to mimic E-cadherin presentation in the cell membrane. These functionalised bilayers were generated either on flat 2D surfaces or the interior surfaces of round microwells. This platform enabled the study of E-cadherin adhesion and the initiation of polarisation in a controlled environment, where the dimensionality of the microenvironment, type of protein coating and cell shape could be independently studied. A high proportion of single epithelial cells interacted with and clustered cellular E-cadherin in the presence of E-cadherin functionalised bilayers, which was reduced in the presence of integrin-mediated adhesion. The differential response in E-cadherin clustering correlated with the polarisation of E-cadherin and Na,K-ATPase, a reporter for the induction of basolateral polarity. Neither the three-dimensional presentation of E-cadherin nor the cell shape affected E-cadherin clustering or polarisation in single cells. Thus, the mobile presentation of E-cadherin was sufficient to mimic a cell-cell contact and induce basolateral polarisation in single cells.

Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell culture substrates. Using standard microfabrication processes, biomaterials surfaces can be topographically patterned to comprise high-density arrays of micron-sized cavities with desirable geometry. Hundreds to thousands of individual cells or cell colonies with controlled size and shape can be trapped in these cavities by simple gravitational sedimentation. Efficient long-term cell confinement allows for parallel analyses and manipulation of cell fate during in vitro culture. These live-cell arrays have already found applications in cell biology, for example to probe the effect of cell colony size on embryonic stem cell differentiation, to dissect the heterogeneity in single cell proliferation kinetics of neural or hematopoietic stem/progenitor cell populations, or to elucidate the role of cell shape on cell function. Here, we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies.

Our aim was to develop a biodegradable fibrous dressing to act as a tissue guide for in situ wound repair while releasing Ibuprofen to reduce inflammation in wounds and reduce pain for patients on dressing changes. Dissolving the acid form of Ibuprofen (from 1% to 10% by weight) in the same solvent as 75% polylactide, 25% polyglycolide (PLGA) polymers gave uniformly loaded electrospun fibers which gave rapid release of drug within the first 8 h and then slower release over several days. Scaffolds with 10% Ibuprofen degraded within 6 days. The Ibuprofen released from these scaffolds significantly reduced the response of fibroblasts to major pro-inflammatory stimulators. Fibroblast attachment and proliferation on scaffolds was unaffected by the addition of 1-5% Ibuprofen. Scaffolds loaded with 10% Ibuprofen initially showed reduced cell attachment but this was restored by soaking scaffolds in media for 24 h. In summary, addition of Ibuprofen to electrospun biodegradable scaffolds can give acute protection of adjacent cells to inflammation while the scaffolds provide an open 3D fibrous network to which cells can attach and migrate. By 6 days, such scaffolds will have completely dissolved into the wound bed obviating any need for dressing removal.

We report on the optically detected magnetic resonance of nitrogen-vacancy centres in blinking nanodiamonds to measure magnetic fields from magnetically-labeled MCF10A cells. Magnetic fields imaging is reconstructed with a localization microscope down to 25 nm.