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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

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 0.4). 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

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., National Science Foundation 1916797 David W Ehrhardt, Kenneth D Birnbaum, Seung Yon Rhee; National Science Foundation 2052590 Seung Yon Rhee

Lyme disease caused by theBorrelia burgdorferi(Bb or B. burgdorferi) is a most common vector-borne, multi-systemic disease in USA. Although, most Lyme disease patients can be cured with a course of antibiotic treatment, a significant percent of patient population fail to be disease-free post-treatment, necessitating the development of more effective therapeutics. We previously found several drugs including disulfiram having with good activity againstB. burgdorferi. In current study, we evaluated the potential of repurposing the FDA approved disulfiram drug for itsborreliacidal activity. Ourin vitroresults indicate disulfiram shows excellentborreliacidal activity against both the log and stationary phaseB. burgdorferi. Subsequent mice studies have determined that the disulfiram eliminatedB. burgdorfericompletely from hearts and urinary bladder by day 28 post infection, demonstrating the practical application and efficacy of disulfiram againstB. burgdorferi in vivo. Moreover, disulfiram treated mice showed reduced expression of inflammatory markers and protected against histopathology and organ damage. Furthermore, disulfiram treated mice showed significantly lower amounts of total antibody titers (IgM and IgG) at day 21 and total IgG2b at day 28 post infection. Mechanistically, cellular analysis of lymph nodes revealed a decrease in percentage of CD19+ B cells and increase in total percentage of CD3+ T cells, CD3+ CD4+ T helpers, and naïve and effector memory cells in disulfiram-treated mice. Together, we demonstrate that disulfiram has the potential and could be repurposed as an effective antibiotic for treating Lyme disease in near future.

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 tympanic membrane (TM) is a dynamic structure that separates the middle ear from the external auditory canal. It is also integral for the transmission of sound waves. In this study, we demonstrate the feasibility of using Raman spectroscopy to identify early chemical changes resulting from inflammation in the TM that can serve as an indicator of acute otitis media. Bacterial lipopolysaccharide (LPS) was injected trans-tympanicaly in a murine model. Presence of inflammatory response was assessed with binocular microscopy, confirmed with histopathology and immunofluorescence staining. Successful discrimination suggesting spectral differences among the control and LPS treated groups was achieved using principal component analysis. Raman imaging revealed major differences in collagen distribution and nucleic acid content. Image segmentation analysis on the trichrome stained tissue sections was performed to corroborate the Raman spectra. The spectral co-localization study suggests changes in the expression of collagen IV specific signals in LPS treated samples. The overall findings of the study support prospective application of RS in the diagnosis and therapeutic monitoring of otitis media.

Extracellular matrix (ECM) properties affect multiple cellular processes such as cell survival, proliferation, and protein synthesis. Thus, a polymeric‐cell delivery system with the ability to manipulate the extracellular environment can act as a fundamental regulator of cell function. Given the promise of stem cell therapeutics, a method to uniformly enhance stem cell function, in particular trophic factor release, can prove transformative in improving efficacy and increasing feasibility by reducing the total number of cells required. Herein, a click‐chemistry powered 3D, single‐cell encapsulation method aimed at synthesizing a polymeric coating with the optimal thickness around neural progenitor cells is introduced. Polymer encapsulation of neural stem cells significantly increases the release of neurotrophic factors such as VEGF and CNTF. Cell encapsulation with a soft extracellular polymer upregulates the ADCY8‐cAMP pathway, suggesting a mechanism for the increase in paracrine factors. Hence, the described single‐cell encapsulation technique can emerge as a translatable, nonviral cell modulation method and has the potential to improve stem cells' therapeutic effect., Through the use of cell glycoengineering techniques and click chemistry, single‐cell polymer encapsulation is possible. This polymeric extracellular matrix can modify the trophic factor production of neural progenitor cells. The cyclic adenosine monophosphate pathway and actin interactions are important in trophic factor regulation, specifically for VEGFB. The ability to encapsulate cells has implications for optimizing stem cell therapeutics.

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.

Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.

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.

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.

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.

Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

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.

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.

To determine whether the therapeutic activity of αB crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73-92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.

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 (Trypanosomareceptor 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., Graphical abstract, Highlights • Identified unique short sequences conserved in parasite but not in host orthologue. • Peptides corresponding to these sequences are active anti-parasitic drug lead. • Cyclization of the peptides generates drug leads for in vivo proof of concept.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with many types of chronic infection. At sites of chronic infection, such as the airways of people with cystic fibrosis (CF), P. aeruginosa forms biofilm-like aggregates. These are clusters of bacterial cells encased in a polymer-rich matrix that shields bacteria from environmental stresses and antibiotic treatment. When P. aeruginosa forms a biofilm, large amounts of filamentous Pf bacteriophage (phage) are produced. Unlike most phage that typically lyse and kill their bacterial hosts, filamentous phage of the genus Inovirus, which includes Pf phage, often do not, and instead are continuously extruded from the bacteria. Here, we discuss the implications of the accumulation of filamentous Pf phage in the biofilm matrix, where they interact with matrix polymers to organize the biofilm into a highly ordered liquid crystal. This structural configuration promotes bacterial adhesion, desiccation survival, and antibiotic tolerance – all features typically associated with biofilms. We propose that Pf phage make structural contributions to P. aeruginosa biofilms and that this constitutes a novel form of symbiosis between bacteria and bacteriophage.

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.

Systemic therapies for inflammatory bowel disease are associated with increased risk of infections and malignancies. Topical therapies reduce systemic exposure, but can be difficult to retain or have limited proximal distribution. To mitigate these issues, we developed a thermo-sensitive platform, using a polymer-based system that is liquid at room temperature but turns into a viscous gel upon reaching body temperature. Following rectal administration to mice with dextran sulphate sodium-induced colitis, the platform carrying budesonide or mesalamine becomes more viscoelastic near body temperature. Mice given the drug-containing platform gained more weight and had reduced histologic and biologic features of colitis than mice given the platform alone or liquid drugs via enema. Image analysis showed that enemas delivered with and without the platform reached similar distances in the colons of mice, but greater colonic retention was achieved by using the platform.

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.

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.

by hyperemia and mucosal thickness and weight lose. On day 3 after colitis induction, 66% and 16% of Desnosumab-DSS-treated mice had weight loss and mucosal thickening respectively, compared to 100% and 66% of vehicle DSS-treated mice (p