A Dual-Enzyme Amplification Loop for the Sensitive Biosensing of Endopeptidases.

A rapid and sensitive approach for the detection of endopeptidases via a new analyte-triggered mutual emancipation of linker-immobilized enzymes (AMELIE) mechanism has been developed and demonstrated using a matrix metallopeptidase, a collagenase, as the model endopeptidase analyte. AMELIE involves an autocatalytic loop created by a pair of selected enzymes immobilized on solid substrates via linkers with specific sites that can be proteolyzed by one another. These bound enzymes are spatially separated so that they cannot act upon their corresponding substrates until the introduction of the target endopeptidase analyte that can also cleave one of the linkers. This triggers the self-sustained loop of enzymatic activities to emancipate all the immobilized enzymes. In this proof of concept, signal transduction was achieved by a colorimetric horseradish peroxidase-tetramethylbenzidine (HRP-TMB-H2O2) reaction with HRP that are also being immobilized by one of the linkers. The pair of immobilized enzymes were collagenase and alginate lyase, and they were immobilized by an alginate linker and a short peptide chain containing the amino acid sequence of Leu-Gly-Pro-Ala for collagenase. A detection limit of 2.5 pg collagenase mL-1 with a wide linear range up to 4 orders of magnitude was achieved. The AMELIE biosensor can detect extracellular collagenase in the supernatant of various bacteria cultures, with a sensitivity as low as 103 cfu mL-1 of E. coli. AMELIE can readily be adapted to provide the sensitive detection of other endopeptidases.

BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression.

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

Deconstructing, Replicating, and Engineering Tissue Microenvironment for Stem Cell Differentiation.

One of the most crucial components of regenerative medicine is the controlled differentiation of embryonic or adult stem cells into the desired cell lineage. Although most of the reported protocols of stem cell differentiation involve the use of soluble growth factors, it is increasingly evident that stem cells also undergo differentiation when cultured in the appropriate microenvironment. When cultured in decellularized tissues, for instance, stem cells can recapitulate the morphogenesis and functional specialization of differentiated cell types with speed and efficiency that often surpass the traditional growth factor-driven protocols. This suggests that the tissue microenvironment (TME) provides stem cells with a holistic "instructive niche" that harbors signals for cellular reprogramming. The translation of this into medical applications requires the decoding of these signals, but this has been hampered by the complexity of TME. This problem is often addressed by a reductionist approach, in which cells are exposed to substrates decorated with simple, empirically designed geometries, textures, and chemical compositions ("bottom-up" approach). Although these studies are invaluable in revealing the basic principles of mechanotransduction mechanisms, their physiological relevance is often uncertain. This review examines the recent progress of an alternative, "top-down" approach, in which the TME is treated as a holistic biological entity. This approach is made possible by recent advances in systems biology and fabrication technologies that enable the isolation, characterization, and reconstitution of TME. It is hoped that these new techniques will elucidate the nature of niche signals so that they can be extracted, replicated, and controlled. This review summarizes these emerging techniques and how the data they generated are changing our view on TME. Impact statement This review summarizes the current state of art of the understanding of instructive niche in the field of tissue microenvironment. Not only did we survey the use of different biochemical preparations as stimuli of stem cell differentiation and summarize the recent effort in dissecting the biochemical composition of these preparations, through the application of extracellular matrix (ECM) arrays and proteomics, but we also introduce the use of open-source, high-content immunohistochemistry projects in contributing to the understanding of tissue-specific composition of ECM. We believe this review would be highly useful for our peer researching in the same field. "Mr. Tulkinghorn is always the same… so oddly out of place and yet so perfectly at home." -Charles Dickens, Bleak House.

Rapid and Detergent-Free Decellularization of Cartilage.

The use of decellularized tissues or organs as cell culture scaffolds has proven to be a promising approach for tissue engineering and regenerative medicine, as these decellularized tissues can provide the instructive niche for cell differentiation and functions. Cartilage is a largely avascular tissue with limited regenerative capacity. Lesions caused by arthritis can lead to severe cartilage degeneration. Previous studies have indicated that decellularized cartilage can be used as scaffolds that support the chondrogenic differentiation of adult stem cells. However, these decellularization protocols all require the use of denaturing agents, such as high salt and detergents, that lead to the artifactual disruption of the chemical and physical integrity of the tissue microenvironment. Here, we established a new decellularization method for cartilage, through a combined effect of freezing-thawing, sectioning, and sonication in water. This protocol achieved the complete removal of cells within minutes, instead of hours or days required by existing procedures, and does not use any detergent. The resulting decellularized cartilage preserved the native ultrastructure and biochemical contents, including glycosaminoglycans, which is typically depleted by traditional decellularization methods. Human mesenchymal stem cells could readily adhere onto the decellularized cartilage. Together, this work unveils a simple new method for decellularizing cartilage, which will be useful in studying how tissue microenvironment supports chondrocyte growth and functions. Impact statement In this study, we develop a simple, fast cartilage decellularization method that does not require any detergent, so that the decellularized cartilage chemistry is preserved. Traditional detergent-based decellularization removes the tissue biochemical contents (i.e., glycosaminoglycans). In this new water decellularization protocol, the biochemical contents of cartilage can be preserved. This allows the study of biochemistry and physical content in extracellular matrix as a whole, and this protocol would definitely be useful for studying the effect of tissue microenvironment in supporting chondrocyte growth and functions.

Capturing instructive cues of tissue microenvironment by silica bioreplication.

Cells in tissues are enveloped by an instructive niche made of the extracellular matrix. These instructive niches contain three general types of information: topographical, biochemical and mechanical. While the combined effects of these three factors are widely studied, the functions of each individual one has not been systematically characterised, because it is impossible to alter a single factor in a tissue microenvironment without simultaneously affecting the other two. Silica BioReplication (SBR) is a process that converts biological samples into silica, faithfully preserving the original topography at the nano-scale. We explored the use of this technique to generate inorganic replicas of intact mammalian tissues, including tendon, cartilage, skeletal muscle and spinal cord. Scanning electron and atomic force microscopy showed that the resulting replicas accurately preserved the three-dimensional ultrastructure of each tissue, while all biochemical components were eradicated by calcination. Such properties allowed the uncoupling the topographical information of a tissue microenvironment from its biochemical and mechanical components. Here, we showed that human mesenchymal stem cells (MSC) cultured on the replicas of different tissues displayed vastly different morphology and focal adhesions, suggesting that the topography of the tissue microenvironment captured by SBR could profoundly affect MSC biology. MSC cultured on tendon replica elongated and expressed tenocytes marker, while MSC on the spinal cord replica developed into spheroids that resembled neurospheres, in morphology and in the expression of neurosphere markers, and could be further differentiated into neuron-like cells. This study reveals the significance of topographical cues in a cell niche, as tissue-specific topography was sufficient in initiating and directing differentiation of MSC, despite the absence of any biochemical signals. SBR is a convenient and versatile method for capturing this topographical information, facilitating the functional characterisation of cell niches. STATEMENT OF SIGNIFICANCE: Various studies have shown that three major factors, topographical, biochemical and mechanical, in a tissue microenvironment (TME) are essential for cellular homeostasis and functions. Current experimental models are too simplistic to represent the complexity of the TME, hindering the detailed understanding of its functions. In particular, the importance each factor in a tissue microenvironment have not been individually characterised, because it is challenging to alter one of these factors without simultaneously affecting the other two. Silica bioreplication (SBR) is a process that converts biological samples into silica replicas with high structural fidelity. SBR is a convenient and versatile method for capturing this topographical information on to a biologically inert material, allowing the functional characterisation of the architecture of a TME.

Replication of a Tissue Microenvironment by Thermal Scanning Probe Lithography.

Thermal scanning probe lithography (t-SPL) is a nanofabrication technique in which an immobilized thermolabile resist, such as polyphthalaldehyde (PPA), is locally vaporized by a heated atomic force microscope tip. Compared with other nanofabrication techniques, such as soft lithography and nanoimprinting lithography, t-SPL is more efficient and convenient as it does not involve time-consuming mask productions or complicated etching procedures, making it a promising candidate technique for the fast prototyping of nanoscale topographies for biological studies. Here, we established the direct use of PPA-coated surfaces as a cell culture substrate. We showed that PPA is biocompatible and that the deposition of allylamine by plasma polymerization on a silicon wafer before PPA coating can stabilize the immobilization of PPA in aqueous solutions. When seeded on PPA-coated surfaces, human mesenchymal stem cells (MSC) adhered, spread, and proliferated in a manner indistinguishable from cells cultured on glass surfaces. This allowed us to subsequently use t-SPL to generate nanotopographies for cell culture experiments. As a proof of concept, we analyzed the surface topography of bovine tendon sections, previously shown to induce morphogenesis and differentiation of MSC, by means of atomic force microscopy, and then "wrote" topographical data on PPA by means of t-SPL. The resulting substrate, matching the native tissue topography on the nanoscale, was directly used for MSC culture. The t-SPL substrate induced similar changes in cell morphology and focal adhesion formation in the MSC compared to native tendon sections, suggesting that t-SPL can rapidly generate cell culture substrates with complex and spatially accurate topographical signals. This technique may greatly accelerate the prototyping of models for the study of cell-matrix interactions.

Proteomic characterization of the interactions between fish serum proteins and waterborne bacteria reveals the suppression of anti-oxidative defense as a serum-mediated antimicrobial mechanism.

Fish blood is one of the crucial tissues of innate immune system, but the full repertoire of fish serum components involved in antibacterial defense is not fully identified. In this study, we demonstrated that turbot serum, but not the heat-inactivated control, significantly reduced the number of Edwardsiella tarda (E. tarda). By conjugating serum proteins with fluorescent dyes, we showed that E. tarda were coated with multiple fish proteins. In order to identify these proteins, we used E. tarda to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis. A total of 76 fish proteins were identified in high confidence, including known antimicrobial proteins such as immunoglobins and complement components. 34 proteins with no previously known immunological functions were also identified. The expression of one of these proteins, IQ motif containing H (IQCH), was exclusively in fish brain and gonads and was induced during bacterial infection. This approach also allowed the study of the corresponding proteomic changes in E. tarda exposed to turbot serum, which is a general decrease of bacterial protein expression except for an upregulation of membrane components after serum treatment. Interestingly, while most other known stresses stimulate bacterial antioxidant enzymes, fish serum induced a rapid suppression of antioxidant proteins and led to an accumulation of reactive oxygen species. Heat treatment of fish serum eliminated this effect, suggesting that heat labile factors in the fish serum overrode bacterial antioxidant defenses. Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity.

Stringent requirement for spatial arrangement of extracellular matrix in supporting cell morphogenesis and differentiation.

BACKGROUND: In vitro experiments on the functional roles of extracellular matrix (ECM) components usually involve the culture of cells on surfaces coated with purified ECM components. These experiments can seldom recuperate the spatial arrangement of ECM found in vivo. In this study, we have overcome this obstacle by using histological sections of bovine Achilles tendon as cell culture substrates. RESULTS: We found that tendon sections can be viewed as a pre-formed block of ECM in which the collagen fibrils exhibited a spatial regularity unraveled in any artificially constructed scaffold. By carving the tendon at different angles relative to its main axis, we created different surfaces with distinct spatial arrangements of collagen fibrils. To assess the cellular responses to these surfaces, human mesenchymal stem cells (MSCs) were directly cultured on these sections, hence exposed to the collagen with different spatial orientations. Cells seeded on longitudinal tendon sections adopted a highly elongated and aligned morphology, and expressed an increased level of tenomodulin, suggesting that the collagen fibrils present in this section provide a microenvironment that facilitates cell morphogenesis and differentiation. However, MSC elongation, alignment and induction of tenomodulin diminished dramatically even as the sectioned angle changed slightly. CONCLUSION: Our results suggest that cell functions are influenced not only by the type or concentration of ECM components, but also by the precise spatial arrangements of these molecules. The method developed in this study offers a simple and robust way for the studying of cell-ECM interactions, and opens many research avenues in the field of matrix biology.

Hierarchical coding of characters in the ventral and dorsal visual streams of Chinese language processing.

Functional and spatial hierarchical organization of increasingly language-like word forms has been proposed for alphabetic languages at the occipitotemporal cortex for visual word recognition. In the logographic Chinese language system, similar functional and spatial hierarchical presentations of brain responses to sublexical orthographic structure are beginning to be explored. In this study, we used whole-brain fMRI to show that a hierarchical coding of increasingly language-like character type is present in multiple Chinese language processing areas. Fluent Chinese readers were presented with Chinese synonyms/non-synonym pairs, identical/non-identical non-pronounceable pseudo-character pairs constructed with Chinese radicals, and identical/non-identical Korean character pairs. We observed the presence of a spatial gradient for increasing language-like character types in the ventral and dorsal visual streams of the cortex. At the left occipitotemporal cortex of the ventral visual stream, we observed a posterior-to-anterior gradient of character type selectivity with the anterior fusiform region being more selective for real Chinese characters and the posterior fusiform region being more selective for Korean characters. At the left and right intraparietal sulci of the dorsal visual stream, a medial-to-lateral gradient of character type selectivity was observed, with the lateral edge being more selective for real Chinese characters, the medial edge being more selective for pseudo-characters, and with less activation attributable to Korean characters. Spatial gradients of selecting character type were also identified in prefrontal cortex, dorsal striatum and lateral temporal cortex. The results suggest that the left occipitotemporal cortex and both left and right intraparietal sulci are tuned with a functional and spatial hierarchical sensitivity to the presence of semantic elements as well as different orthographic structures.

Can Cantonese rhymes be used in the assessment of hemispheric dominance for language?

English vowels had been proposed in previous studies to be used as a simple tool for the brain mapping of language. A proper fMRI study of Cantonese rhymes, each of which being a required and fundamental unit of a Cantonese syllable, remains to be carried out. Using an auditory task with Cantonese rhymes which carry no semantic meaning, we observed a minimal amount of positive BOLD signal at the caudate nucleus when Cantonese rhymes were contrasted with their corresponding filtered sounds. Typical language activating regions of the prefrontal cortex, the medial prefrontal cortex and the lateral temporal cortex on both left and right sides were not activated by Cantonese rhymes. Based on the absence of brain activation at the typical language areas in the contrast of Cantonese rhymes relative to filtered sounds, the auditory task with Cantonese rhymes may not be a robust tool for the individual clinical assessment of hemispheric dominance for language.