Visualization of the distribution of covalently cross-linked hydrogels in CLARITY brain-polymer hybrids for different monomer concentrations.

CLARITY is a tissue preservation and optical clearing technique whereby a hydrogel is formed directly within the architectural confines of ex vivo brain tissue. In this work, the extent of polymer gel formation and crosslinking within tissue was assessed using Raman spectroscopy and rheology on CLARITY samples prepared with a range of acrylamide monomer (AAm) concentrations (1%, 4%, 8%, 12% w/v). Raman spectroscopy of individual neurons within hybrids revealed the chemical presence and distribution of polyacrylamide within the mouse hippocampus. Consistent with rheological measurements, lower %AAm concentration decreased shear elastic modulus G', providing a practical correlation with sample permeability and protein retention. Permeability of F(ab)'2 secondary fluorescent antibody changes from 9.3 to 1.4 µm2 s-1 going from 1 to 12%. Notably, protein retention increased linearly relative to standard PFA-fixed tissue from 96.6% when AAm concentration exceeded 1%, with 12% AAm samples retaining up to ~ 99.3% native protein. This suggests that though 1% AAm offers high permeability, additional %AAm may be required to enhance protein. Our quantitative results on polymer distribution, stability, protein retention, and macromolecule permeability can be used to guide the design of future CLARITY-based tissue-clearing solutions, and establish protocols for characterization of novel tissue-polymer hybrid biomaterials using chemical spectroscopy and rheology.

Noninvasive and Highly Multiplexed Five-Color Tumor Imaging of Multicore Near-Infrared Resonant Surface-Enhanced Raman Nanoparticles In Vivo.

In vivo multiplexed imaging aims for noninvasive monitoring of tumors with multiple channels without excision of the tissue. While most of the preclinical imaging has provided a number of multiplexing channels up to three, Raman imaging with surface-enhanced Raman scattering (SERS) nanoparticles was suggested to offer higher multiplexing capability originating from their narrow spectral width. However, in vivo multiplexed SERS imaging is still in its infancy for multichannel visualization of tumors, which require both sufficient multiplicity and high sensitivity concurrently. Here we create multispectral palettes of gold multicore-near-infrared (NIR) resonant Raman dyes-silica shell SERS (NIR-SERRS) nanoparticle oligomers and demonstrate noninvasive and five-plex SERS imaging of the nanoparticle accumulation in tumors of living mice. We perform the five-plex ratiometric imaging of tumors by varying the administered ratio of the nanoparticles, which simulates the detection of multiple biomarkers with different expression levels in the tumor environment. Furthermore, since this method does not require the excision of tumor tissues at the imaging condition, we perform noninvasive and longitudinal imaging of the five-color nanoparticles in the tumors, which is not feasible with current ex vivo multiplexed tissue analysis platforms. Our work surpasses the multiplicity limit of previous preclinical tumor imaging methods while keeping enough sensitivity for tumor-targeted in vivo imaging and could enable the noninvasive assessment of multiple biological targets within the tumor microenvironment in living subjects.

Vision, challenges and opportunities for a Plant Cell Atlas.

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

The Effect of Ethanol Consumption on Composition and Morphology of Femur Cortical Bone in Wild-Type and ALDH2*2-Homozygous Mice.

ALDH2 inactivating mutation (ALDH2*2) is the most abundant mutation leading to bone morphological aberration. Osteoporosis has long been associated with changes in bone biomaterial in elderly populations. Such changes can be exacerbated with elevated ethanol consumption and in subjects with impaired ethanol metabolism, such as carriers of aldehyde dehydrogenase 2 (ALDH2)-deficient gene, ALDH2*2. So far, little is known about bone compositional changes besides a decrease in mineralization. Raman spectroscopic imaging has been utilized to study the changes in overall composition of C57BL/6 female femur bone sections, as well as in compound spatial distribution. Raman maps of bone sections were analyzed using multilinear regression with these four isolated components, resulting in maps of their relative distribution. A 15-week treatment of both wild-type (WT) and ALDH2*2/*2 mice with 20% ethanol in the drinking water resulted in a significantly lower mineral content (p < 0.05) in the bones. There was no significant change in mineral and collagen content due to the mutation alone (p > 0.4). Highly localized islets of elongated adipose tissue were observed on most maps. Elevated fat content was found in ALDH2*2 knock-in mice consuming ethanol (p < 0.0001) and this effect appeared cumulative. This work conclusively demonstrates that that osteocytes in femurs of older female mice accumulate fat, as has been previously theorized, and that fat accumulation is likely modulated by levels of acetaldehyde, the ethanol metabolite.

Integrated Ca2+ flux and AFM force analysis in human iPSC-derived cardiomyocytes.

We developed a new approach for combined analysis of calcium (Ca2+) handling and beating forces in contractile cardiomyocytes. We employed human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from dilated cardiomyopathy (DCM) patients carrying an inherited mutation in the sarcomeric protein troponin T (TnT), and isogenic TnT-KO iPSC-CMs generated via CRISPR/Cas9 gene editing. In these cells, Ca2+ handling as well as beating forces and -rates using single-cell atomic force microscopy (AFM) were assessed. We report impaired Ca2+ handling and reduced contractile force in DCM iPSC-CMs compared to healthy WT controls. TnT-KO iPSC-CMs display no contractile force or Ca2+ transients but generate Ca2+ sparks. We apply our analysis strategy to Ca2+ traces and AFM deflection recordings to reveal maximum rising rate, decay time, and duration of contraction with a multi-step background correction. Our method provides adaptive computing of signal peaks for different Ca2+ flux or force levels in iPSC-CMs, as well as analysis of Ca2+ sparks. Moreover, we report long-term measurements of contractile force dynamics on human iPSC-CMs. This approach enables deeper and more accurate profiling of disease-specific differences in cardiomyocyte contraction profiles using patient-derived iPSC-CMs.

Salivary Thiocyanate as a Biomarker of Cystic Fibrosis Transmembrane Regulator Function.

Improved methods are needed to reliably assess Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in vivo in light of recent therapeutic developments targeting the CFTR protein. Oral fluid from patients with cystic fibrosis (CF) and healthy controls (HCs) were studied using colorimetry and nonresonant Raman spectroscopy. Colorimetry experiments showed only a 36% decrease in thiocyanate (SCN-) concentration, but a sharp Raman peak at 2068 cm-1, attributable to (SCN-) vibrations, normalized to C-H peak, was on average 18 times higher for HC samples. Samples from patients undergoing treatment with CFTR modulators including ivacaftor, lumacaftor, and tezacaftor showed a high normalized peak in response to therapy. The peak intensity was consistent in longitudinal samples from single donors and in stored samples. The Raman peak ratio is a more sensitive, convenient, noninvasive biomarker for assessments of the therapeutic efficacy of drugs targeting CFTR and provides a value that is in much better agreement with theoretical expectations of saliva SCN- concentrations compared to colorimetry. This insight may greatly facilitate assessments of CFTR modulator efficacy in individual patients.

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition.

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

Prolonged survival of transplanted stem cells after ischaemic injury via the slow release of pro-survival peptides from a collagen matrix.

Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen-dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.

PEG/Dextran Double Layer Influences Fe Ion Release and Colloidal Stability of Iron Oxide Nanoparticles.

Despite preliminary confidence on biosafety of polymer coated iron oxide nanoparticles (SPIONs), toxicity concerns have hampered their clinical translation. SPIONs toxicity is known to be due to catalytic activity of their surface and release of toxic Fe ions originating from the core biodegradation, leading to the generation of reactive oxygen species (ROS). Here, we hypothesized that a double-layer polymeric corona comprising of dextran as an interior, and polyethylene glycol (PEG) as an exterior layer better shields the core SPIONs. We found that ROS generation was cell specific and depended on SPIONs concentration, although it was reduced by sufficient PEG immobilization or 100 µM deferoxamine. 24 h following injection, PEGylated samples showed reduction of biodistribution in liver, heterogenous biodistribution profile in spleen, and no influence on NPs blood retention. Sufficient surface masking or administration of deferoxamine could be beneficial strategies in designing and clinical translation of future biomedical SPIONs.

Dynamic CT imaging of volumetric changes in pulmonary nodules correlates with physical measurements of stiffness.

BACKGROUND AND PURPOSE: A major challenge in CT screening for lung cancer is limited specificity when distinguishing between malignant and non-malignant pulmonary nodules (PN). Malignant nodules have different mechanical properties and tissue characteristics ('stiffness') from non-malignant nodules. This study seeks to improve CT specificity by demonstrating in rats that measurements of volumetric ratios in PNs with varying composition can be determined by respiratory-gated dynamic CT imaging and that these ratios correlate with direct physical measurements of PN stiffness. METHODS AND MATERIALS: Respiratory-gated MicroCT images acquired at extreme tidal volumes of 9 rats with PNs from talc, matrigel and A549 human lung carcinoma were analyzed and their volumetric ratios (δ) derived. PN stiffness was determined by measuring the Young's modulus using atomic force microscopy (AFM) for each nodule excised immediately after MicroCT imaging. RESULTS: There was significant correlation (p=0.0002) between PN volumetric ratios determined by respiratory-gated CT imaging and the physical stiffness of the PNs determined from AFM measurements. CONCLUSION: We demonstrated proof of concept that PN volume changes measured non-invasively correlate with direct physical measurements of stiffness. These results may translate clinically into a means of improving the specificity of CT screening for lung cancer and/or improving individual prognostic assessments based on lung tumor stiffness.

Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors.

Parasitic diseases cause ∼ 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

Filamentous Bacteriophage Promote Biofilm Assembly and Function.

Biofilms-communities of bacteria encased in a polymer-rich matrix-confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.

Transdermal Delivery of Functional Collagen Via Polyvinylpyrrolidone Microneedles.

Collagen makes up a large proportion of the human body, particularly the skin. As the body ages, collagen content decreases, resulting in wrinkled skin and decreased wound healing capabilities. This paper presents a method of delivering type I collagen into porcine and human skin utilizing a polyvinylpyrrolidone microneedle delivery system. The microneedle patches were made with concentrations of 1, 2, 4, and 8% type I collagen (w/w). Microneedle structures and the distribution of collagen were characterized using scanning electron microscopy and confocal microscopy. Patches were then applied on the porcine and human skin, and their effectiveness was examined using fluorescence microscopy. The results illustrate that this microneedle delivery system is effective in delivering collagen I into the epidermis and dermis of porcine and human skin. Since the technique presented in this paper is quick, safe, effective and easy, it can be considered as a new collagen delivery method for cosmetic and therapeutic applications.

Protein Corona Influences Cell-Biomaterial Interactions in Nanostructured Tissue Engineering Scaffolds.

Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo, demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.

[Pyr1]-Apelin-13 delivery via nano-liposomal encapsulation attenuates pressure overload-induced cardiac dysfunction.

Nanoparticle-mediated sustained delivery of therapeutics is one of the highly effective and increasingly utilized applications of nanomedicine. Here, we report the development and application of a drug delivery system consisting of polyethylene glycol (PEG)-conjugated liposomal nanoparticles as an efficient in vivo delivery approach for [Pyr1]-apelin-13 polypeptide. Apelin is an adipokine that regulates a variety of biological functions including cardiac hypertrophy and hypertrophy-induced heart failure. The clinical use of apelin has been greatly impaired by its remarkably short half-life in circulation. Here, we investigate whether [Pyr1]-apelin-13 encapsulation in liposome nanocarriers, conjugated with PEG polymer on their surface, can prolong apelin stability in the blood stream and potentiate apelin beneficial effects in cardiac function. Atomic force microscopy and dynamic light scattering were used to assess the structure and size distribution of drug-laden nanoparticles. [Pyr1]-apelin-13 encapsulation in PEGylated liposomal nanocarriers resulted in sustained and extended drug release both in vitro and in vivo. Moreover, intraperitoneal injection of [Pyr1]-apelin-13 nanocarriers in a mouse model of pressure-overload induced heart failure demonstrated a sustainable long-term effect of [Pyr1]-apelin-13 in preventing cardiac dysfunction. We concluded that this engineered nanocarrier system can serve as a delivery platform for treating heart injuries through sustained bioavailability of cardioprotective therapeutics.

Transdermal deferoxamine prevents pressure-induced diabetic ulcers.

There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

Use of bio-mimetic three-dimensional technology in therapeutics for heart disease.

Due to the limited self-renewal capacity of cardiomyocytes, the mammalian heart exhibits impaired regeneration and insufficient ability to restore heart function after injury. Cardiovascular tissue engineering is currently considered as a promising alternative therapy to restore the structure and function of the failing heart. Recent evidence suggests that the epicardium may play critical roles in regulation of myocardial development and regeneration. One of the mechanisms that has been proposed for the restorative effect of the epicardium is the specific physiomechanical cues that this layer provides to the cardiac cells. In this article we explore whether a new generation of epicardium-mimicking, acellular matrices can be utilized to enhance cardiac healing after injury. The matrix consists of a dense collagen scaffold with optimized biomechanical properties approaching those of embryonic epicardium. Grafting the epicardial patch onto the ischemic myocardium--promptly after the incidence of infarct--resulted in preserved contractility, attenuated ventricular remodeling, diminished fibrosis, and vascularization within the injured tissue in the adult murine heart.

Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles.

Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation.