Microindentation of fresh soft biological tissue: A rapid tissue sectioning and mounting protocol.

Microindentation of fresh biological tissues is necessary for the creation of 3D biomimetic models that accurately represent the native extracellular matrix microenvironment. However, tissue must first be precisely sectioned into slices. Challenges exist in the preparation of fresh tissue slices, as they can tear easily and must be processed rapidly in order to mitigate tissue degradation. In this study, we propose an optimised mounting condition for microindentation and demonstrate that embedding tissue in a mixture of 2.5% agarose and 1.5% gelatin is the most favourable method of tissue slice mounting for microindentation. This protocol allows for rapid processing of fresh biological tissue and is applicable to a variety of tissue types.

Collagen-I influences the post-translational regulation, binding partners and role of Annexin A2 in breast cancer progression.

Introduction: The extracellular matrix (ECM) has been heavily implicated in the development and progression of cancer. We have previously shown that Annexin A2 is integral in the migration and invasion of breast cancer cells and in the clinical progression of ER-negative breast cancer, processes which are highly influenced by the surrounding tumor microenvironment and ECM. Methods: We investigated how modulations of the ECM may affect the role of Annexin A2 in MDA-MB-231 breast cancer cells using western blotting, immunofluorescent confocal microscopy and immuno-precipitation mass spectrometry techniques. Results: We have shown that the presence of collagen-I, the main constituent of the ECM, increases the post-translational phosphorylation of Annexin A2 and subsequently causes the translocation of Annexin A2 to the extracellular surface. In the presence of collagen-I, we identified fibronectin as a novel interactor of Annexin A2, using mass spectrometry analysis. We then demonstrated that reducing Annexin A2 expression decreases the degradation of fibronectin by cancer cells and this effect on fibronectin turnover is increased according to collagen-I abundance. Discussion: Our results suggest that Annexin A2's role in promoting cancer progression is mediated by collagen-I and Annexin A2 maybe a therapeutic target in the bi-directional cross-talk between cancer cells and ECM remodeling that supports metastatic cancer progression.

Impact of Propeptide Cleavage on the Stability and Activity of a Streptococcal Immunomodulatory C5a Peptidase for Biopharmaceutical Development.

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AStreptococcus pyogenesC5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group AS. pyogenesknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed inEscherichia colicells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.

Exclusive Human Milk Diet for Extremely Premature Infants: A Novel Fortification Strategy That Enhances the Bioactive Properties of Fresh, Frozen, and Pasteurized Milk Specimens.

Background: Human milk (HM) fortification has been recommended for the nutritional optimization of very low-birthweight infants. This study analyzed the bioactive components of HM and evaluated fortification choices that could accentuate or attenuate the concentration of such components, with special reference to human milk-derived fortifier (HMDF) offered to extremely premature infants as an exclusive human milk diet. Materials and Methods: An observational feasibility study analyzed the biochemical and immunochemical characteristics of mothers' own milk (MOM), both fresh and frozen, and pasteurized banked donor human milk (DHM), each supplemented with either HMDF or cow's milk-derived fortifier (CMDF). Gestation-specific specimens were analyzed for macronutrients, pH, total solids, antioxidant activity (AA), α-lactalbumin, lactoferrin, lysozyme, and α- and ß-caseins. Data were analyzed for variance applying general linear model and Tukey's test for pairwise comparison. Results: DHM exhibited significantly lower (p < 0.05) lactoferrin and α-lactalbumin concentrations than fresh and frozen MOM. HMDF reinstated lactoferrin and α-lactalbumin and exhibited higher protein, fat, and total solids (p < 0.05) in comparison to unfortified and CMDF-supplemented specimens. HMDF had the highest (p < 0.05) AA, suggesting the potential capability of HMDF to enhance oxidative scavenging. Conclusion: DHM, compared with MOM, has reduced bioactive properties, and CMDF conferred the least additional bioactive components. Reinstatement and further enhancement of bioactivity, which has been attenuated through pasteurization of DHM, is demonstrated through HMDF supplementation. Freshly expressed MOM fortified with HMDF and given early, enterally, and exclusively (3E) appears an optimal nutritional choice for extremely premature infants.

Comparison of macroscale and microscale mechanical properties of fresh and fixed-frozen porcine colonic tissue.

Mechanical changes to the microenvironment of the extracellular matrix (ECM) in tissue have been hypothesised to elicit a pathogenic response in the surrounding cells. Hence, 3D scaffolds are a popular method of studying cellular behaviour under conditions that mimic in vivo microenvironment. To create a 3D biomimetic scaffold that captures the in vivo ECM microenvironment a robust mechanical characterisation of the whole ECM at the microscale is necessary. This study examined the multiscale methods of characterising the ECM microenvironment using porcine colon tissue. To facilitate fresh tissue microscale mechanical characterisation, a protocol for sectioning fresh, unfixed, soft biological tissue was developed. Four experiments examined both the microscale and macroscale mechanics of both fresh (Fr) and fixed-frozen (FF) porcine colonic tissue using microindentation for microscale testing and uniaxial compression testing for macroscale testing. The results obtained in this study show a significant difference in elastic modulus between Fr and FF tissue at both the macroscale and microscale. There was an order of magnitude difference between the Fr and FF tissue at the microscale between each of the three layers of the colon tested i.e. the muscularis propria (MP), the submucosa (SM) and the mucosa (M). Macroscale testing cannot capture these regional differences. The findings in this study suggest that the most appropriate method for mechanically characterising the ECM is fresh microscale mechanical microindentation. These methods can be used on a range of biological tissues to create 3D biomimetic scaffolds that are more representative of the in vivo ECM, allowing for a more in-depth characterisation of the disease process.

Identification of the common neurobiological process disturbed in genetic and non-genetic models for autism spectrum disorders.

Autism spectrum disorders (ASD) are neurodevelopmental disorders. Genetic factors, along with non-genetic triggers, have been shown to play a causative role. Despite the various causes, a triad of common symptoms defines individuals with ASD; pervasive social impairments, impaired social communication, and repeated sensory-motor behaviors. Therefore, it can be hypothesized that different genetic and environmental factors converge on a single hypothetical neurobiological process that determines these behaviors. However, the cellular and subcellular signature of this process is, so far, not well understood. Here, we performed a comparative study using "omics" approaches to identify altered proteins and, thereby, biological processes affected in ASD. In this study, we mined publicly available repositories for genetic mouse model data sets, identifying six that were suitable, and compared them with in-house derived proteomics data from prenatal zinc (Zn)-deficient mice, a non-genetic mouse model with ASD-like behavior. Findings derived from these comparisons were further validated using in vitro neuronal cell culture models for ASD. We could show that a protein network, centered on VAMP2, STX1A, RAB3A, CPLX2, and AKAP5, is a key convergence point mediating synaptic vesicle release and recycling, a process affected across all analyzed models. Moreover, we demonstrated that Zn availability has predictable functional effects on synaptic vesicle release in line with the alteration of proteins in this network. In addition, drugs that target kinases, reported to regulate key proteins in this network, similarly impacted the proteins' levels and distribution. We conclude that altered synaptic stability and plasticity through abnormal synaptic vesicle dynamics and function may be the common neurobiological denominator of the shared behavioral abnormalities in ASD and, therefore, a prime drug target for developing therapeutic strategies.

Synthesis of Poly(acrylic acid)-Cysteine-Based Hydrogels with Highly Customizable Mechanical Properties for Advanced Cell Culture Applications.

The fabrication of highly customizable scaffolds is a key enabling technology in the development of predictive in vitro cell models for applications in drug discovery, cancer research, and regenerative medicine. Naturally derived and synthetic hydrogels are good candidates for in vitro cell growth studies, owing to their soft and biocompatible nature; however, they are often hindered by limited ranges of stiffness and the requirement to modify the gel with additional extracellular matrix (ECM) proteins for cell adherence. Here, we report on the synthesis of a printable synthetic hydrogel based on cysteine-modified poly(acrylic acid) (PAA-Cys) with tuneable mechanical and swelling properties by incorporating acrylic acid into the PAA-Cys network and subsequent photoinitiated thiol-acrylate cross-linking. Control of the acrylic acid concentration and UV curing time produces a series of hydrogels with swelling ratios in excess of 100% and Young's modulus values ranging from ∼2 to ∼35 kPa, of which most soft tissues fall within. Biocompatibility studies with RPE1 cells showed excellent cell adhesion and cell viability without the need for further modification with ECM proteins, but still can be modified as needed. The versatility of the hydrogel tuneable properties is demonstrated by culturing with RPE1 cells, which in vivo perform an important function in the visual process and the dysfunction of which may lead to various retinal abnormalities, such as glaucoma.

Electrosynthesis of Biocompatible Free-Standing PEDOT Thin Films at a Polarized Liquid|Liquid Interface.

Conducting polymers (CPs) find applications in energy conversion and storage, sensors, and biomedical technologies once processed into thin films. Hydrophobic CPs, like poly(3,4-ethylenedioxythiophene) (PEDOT), typically require surfactant additives, such as poly(styrenesulfonate) (PSS), to aid their aqueous processability as thin films. However, excess PSS diminishes CP electrochemical performance, biocompatibility, and device stability. Here, we report the electrosynthesis of PEDOT thin films at a polarized liquid|liquid interface, a method nonreliant on conductive solid substrates that produces free-standing, additive-free, biocompatible, easily transferrable, and scalable 2D PEDOT thin films of any shape or size in a single step at ambient conditions. Electrochemical control of thin film nucleation and growth at the polarized liquid|liquid interface allows control over the morphology, transitioning from 2D (flat on both sides with a thickness of <50 nm) to "Janus" 3D (with flat and rough sides, each showing distinct physical properties, and a thickness of >850 nm) films. The PEDOT thin films were p-doped (approaching the theoretical limit), showed high π-π conjugation, were processed directly as thin films without insulating PSS and were thus highly conductive without post-processing. This work demonstrates that interfacial electrosynthesis directly produces PEDOT thin films with distinctive molecular architectures inaccessible in bulk solution or at solid electrode-electrolyte interfaces and emergent properties that facilitate technological advances. In this regard, we demonstrate the PEDOT thin film's superior biocompatibility as scaffolds for cellular growth, opening immediate applications in organic electrochemical transistor (OECT) devices for monitoring cell behavior over extended time periods, bioscaffolds, and medical devices, without needing physiologically unstable and poorly biocompatible PSS.

Zinc is a key regulator of gastrointestinal development, microbiota composition and inflammation with relevance for autism spectrum disorders.

Gastrointestinal (GI) problems and microbiota alterations have been frequently reported in autism spectrum disorders (ASD). In addition, abnormal perinatal trace metal levels have been found in ASD. Accordingly, mice exposed to prenatal zinc deficiency display features of ASD-like behavior. Here, we model GI development using 3D intestinal organoids grown under zinc-restricted conditions. We found significant morphological alterations. Using proteomic approaches, we identified biological processes affected by zinc deficiency that regulate barrier permeability and pro-inflammatory pathways. We confirmed our results in vivo through proteomics studies and investigating GI development in zinc-deficient mice. These show altered GI physiology and pro-inflammatory signaling, resulting in chronic systemic and neuroinflammation, and gut microbiota composition similar to that reported in human ASD cases. Thus, low zinc status during development is sufficient to compromise intestinal barrier integrity and activate pro-inflammatory signaling, resulting in changes in microbiota composition that may aggravate inflammation, altogether mimicking the co-morbidities frequently observed in ASD.

Cell specific variation in viability in suspension in in vitro Poiseuille flow conditions.

The influence of Poiseuille flow on cell viability has applications in the areas of cancer metastasis, lab-on-a-chip devices and flow cytometry. Indeed, retaining cell viability is important in the emerging field of adoptive cell therapy, as cells need to be returned to patients' bodies, while the viability of other cells, which are perhaps less accustomed to suspension in a fluidic environment, is important to retain in flow cytometers and other such devices. Despite this, it is unclear how Poiseuille flow affects cell viability. Following on from previous studies which investigated the viability and inertial positions of circulating breast cancer cells in identical flow conditions, this study investigated the influence that varying flow rate, and the corresponding Reynolds number has on the viability of a range of different circulating cells in laminar pipe flow including primary T-cells, primary fibroblasts and neuroblastoma cells. It was found that Reynolds numbers as high as 9.13 had no effect on T-cells while the viabilities of neuroblastoma cells and intestinal fibroblasts were significantly reduced in comparison. This indicates that in vitro flow devices need to be tailored to cell-specific flow regimes.

The Metallome as a Link Between the "Omes" in Autism Spectrum Disorders.

Metal dyshomeostasis plays a significant role in various neurological diseases such as Alzheimer's disease, Parkinson's disease, Autism Spectrum Disorders (ASD), and many more. Like studies investigating the proteome, transcriptome, epigenome, microbiome, etc., for years, metallomics studies have focused on data from their domain, i.e., trace metal composition, only. Still, few have considered the links between other "omes," which may together result in an individual's specific pathologies. In particular, ASD have been reported to have multitudes of possible causal effects. Metallomics data focusing on metal deficiencies and dyshomeostasis can be linked to functions of metalloenzymes, metal transporters, and transcription factors, thus affecting the proteome and transcriptome. Furthermore, recent studies in ASD have emphasized the gut-brain axis, with alterations in the microbiome being linked to changes in the metabolome and inflammatory processes. However, the microbiome and other "omes" are heavily influenced by the metallome. Thus, here, we will summarize the known implications of a changed metallome for other "omes" in the body in the context of "omics" studies in ASD. We will highlight possible connections and propose a model that may explain the so far independently reported pathologies in ASD.

Cdk5 and GSK3ß inhibit fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

Gradients in the in vivo intestinal stem cell compartment and their in vitro recapitulation in mimetic platforms.

Intestinal tissue, and specifically its mucosal layer, is a complex and gradient-rich environment. Gradients of soluble factor (BMP, Noggin, Notch, Hedgehog, and Wnt), insoluble extracellular matrix proteins (laminins, collagens, fibronectin, and their cognate receptors), stromal stiffness, oxygenation, and sheer stress induced by luminal fluid flow at the crypt-villus axis controls and supports healthy intestinal tissue homeostasis. However, due to current technological challenges, very few of these features have so far been included in in vitro intestinal tissue mimetic platforms. In this review, the tightly defined and dynamic microenvironment of the intestinal tissue is presented in detail. Additionally, the authors introduce the current state-of-the-art intestinal tissue mimetic platforms, as well as the design drawbacks and challenges they face while attempting to capture the complexity of the intestinal tissue's physiology. Finally, the compositions of an "idealized" mimetic system is presented to guide future developmental efforts.

Citric acid functionalized nitinol stent surface promotes endothelial cell healing.

While drug-eluting stents containing anti-proliferative agents inhibit proliferation of smooth muscle cells (SMCs), they also delay the regrowth of the endothelial cells which can result in subsequent development of restenosis. Acidic extracellular environments promote cell anchorage and migration by inducing conformational change in integrins, the main cell adhesion proteins. This study addresses the feasibility of a citric acid (CA) functionalized nitinol stent for improving vascular biocompatibility, specifically enhancing endothelialization. CA functionalized nitinol vascular stents are compared to commercial bare metal (Zilver Flex) and paclitaxel eluting stents (Zilver PTX) in terms of re-endothelialization. To study the effect of stent coatings, a stent conditioned media methodology was developed in an attempt to represent in vivo conditions. Overall, distinct advantages of the CA functionalized nitinol stent over commercial Zilver PTX DES and Zilver Flex BMS stents in terms of endothelial cell adhesion, migration, and proliferation are reported. These novel findings indicate the potential of a CA functionalized stent to serve as a bioactive and therapeutic surface for re-endothelialization, perhaps in combination with a SMC proliferation inhibitor coating, to prevent restenosis.

The influence of cell elastic modulus on inertial positions in Poiseuille microflows.

Microchannels are used as a transportation highway for suspended cells both in vivo and ex vivo. Lymphatic and cardiovascular systems transfer suspended cells through microchannels within the body, and microfluidic techniques such as lab-on-a-chip devices, flow cytometry, and CAR T-cell therapy utilize microchannels of similar sizes to analyze or separate suspended cells ex vivo. Understanding the forces that cells are subject to while traveling through these channels are important because certain applications exploit these cell properties for cell separation. This study investigated the influence that cytoskeletal impairment has on the inertial positions of circulating cells in laminar pipe flow. Two representative cancer cell lines were treated using cytochalasin D, and their inertial positions were investigated using particle streak imaging and compared between benign and metastatic cell lines. This resulted in a shift in inertial positions between benign and metastatic as well as treated and untreated cells. To determine and quantify the physical changes in the cells that resulted in this migration, staining and nanoindentation techniques were then used to determine the cells' size, circularity, and elastic modulus. It was found that the cells' exposure to cytochalasin D resulted in decreased elastic moduli of cells, with benign and metastatic cells showing decreases of 135 ± 91 and 130 ± 60 Pa, respectively, with no change in either size or shape. This caused benign, stiffer cancer cells to be more evenly distributed across the channel width than metastatic, deformable cancer cells; additionally, a decrease in the elastic moduli of both cell lines resulted in increased migration toward the channel center. These results indicate that the elastic modulus may play more of a part in the inertial migration of such cells than previously thought.

Measuring Endocytosis During Proliferative Cell Quiescence.

Quiescence (also called "G0") is the state in which cells have exited the cell cycle but are capable to reenter as required. Though poorly understood, it represents one of the most prevalent cell states across all life. Many biologically important cell types reside in quiescence including mature hepatocytes, endothelial cells, and dormant adult stem cells. Furthermore, the quiescence program occurs in both short- and long-term varieties, depending on the physiological environments. A barrier slowing our understanding of quiescence has been a scarcity of available in vitro model systems to allow for the exploration of key regulatory pathways, such as endocytosis. Endocytosis, the internalization of extracellular material into the cell, is a fundamental and highly regulated process that impacts many cell biological functions. Accordingly, we have developed an in vitro model of deep quiescence in hTERT-immortalized RPE1 cells, combining both long-term contact inhibition and mitogen removal, to measure endocytosis. In addition, we present an analytical approach employing automated high-throughput microscopy and image analysis that yields high-content data allowing for meaningful and statistically robust interpretation. Importantly, the methods presented herein provide a suitable platform that can be easily adapted to investigate other regulatory processes across the cell cycle.

Expression of Annexin A2 Promotes Cancer Progression in Estrogen Receptor Negative Breast Cancers.

When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.

Author Correction: The in vitro inertial positions and viability of cells in suspension under different in vivo flow conditions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enzymatic Biofuel Cells for Self-Powered, Controlled Drug Release.

Self-powered drug-delivery systems based on conductive polymers (CPs) that eliminate the need for external power sources are of significant interest for use in clinical applications. Osmium redox polymer-mediated glucose/O2 enzymatic biofuel cells (EBFCs) were prepared with an additional CP-drug layer on the cathode. On discharging the EBFCs in the presence of glucose and dioxygen, model drug compounds incorporated in the CP layer were rapidly released with negligible amounts released when the EBFCs were held at open circuit. Controlled and ex situ release of three model compounds, ibuprofen (IBU), fluorescein (FLU), and 4',6-diamidino-2-phenylindole (DAPI), was achieved with this self-powered drug-release system. DAPI released in situ in cell culture media was incorporated into retinal pigment epithelium (RPE) cells. This work demonstrates a proof-of-concept responsive drug-release system that may be used in implantable devices.

The in vitro inertial positions and viability of cells in suspension under different in vivo flow conditions.

The influence of Poiseuille flow on cell viability has applications in the areas of cancer metastasis, lab-on-a-chip devices and flow cytometry. Indeed, retaining cell viability is important in the emerging field of cell therapy as cells need to be returned to patients' bodies. Despite this, it is unclear how this fundamental fluid regime affects cell viability. This study investigated the influence that varying flow rate, and the corresponding wall shear stress (τw) has on the viability and inertial positions of circulating cells in laminar pipe flow. The viability of two representative cell lines under different shear stresses in two different systems were investigated while particle streak imaging was used to determine their inertial positions. It was found that peristaltic pumps have a negative effect on cell viability in comparison to syringe pumps. Increasing shear stress in a cone and plate above 3 Pa caused an increase in cell death, however, τw as high as 10 Pa in circulation has little to no effect on cell viability. Inertial lift forces that move cells towards the centre of the channel protect them from experiencing detrimental levels of τw, indicating that τw in Poiseuille flow is not a good predictor of cell viability during advection.