Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Real-time tissue perfusion assessment using fluorescence imaging topography scanning system: A preclinical investigation.

BACKGROUND AND OBJECTIVES: We previously developed a real-time fluorescence imaging topography scanning (RFITS) system for intraoperative multimodal imaging, image-guided surgery, and dynamic surgical navigation. The RFITS can capture intraoperative fluorescence, color reflectance, and surface topography concurrently and offers accurate registration of multimodal images. The RFITS prototype is a promising system for multimodal image guidance and intuitive 3D visualization. In the current study, we investigated the capability of the RFITS system in intraoperative fluorescence vascular angiography for real-time assessment of tissue perfusion. STUDY DESIGN/MATERIALS AND METHODS: We conducted ex vivo imaging of fluorescence perfusion in a soft casting life-sized human brain phantom. Indocyanine green (ICG) solutions diluted in dimethyl sulfoxide (DMSO) and human serum were injected into the brain phantom through the vessel simulating tube (2 ± 0.2 mm inner diameter) by an adjustable flow peristaltic pump. To demonstrate the translational potential of the system, an ICG/DMSO solution was perfused into blood vessels of freshly harvested porcine ears (n = 9, inner diameter from 0.56 to 1.27 mm). We subsequently performed in vivo imaging of fluorescence-perfused vascular structures in rodent models (n = 10). 5 mg/ml ICG solutions prepared in sterile water were injected via the lateral tail vein. All targets were imaged by the RFITS prototype at a working distance of 350-400 mm. RESULTS: 3D visualization of 10 µg/ml ICG-labeled continuous moving serum in the brain phantom was obtained at an average signal-to-background ratio (SBR) of 1.74 ± 0.03. The system was able to detect intravenously diffused fluorescence in porcine tissues with an average SBR of 2.23 ± 0.22. The RFITS prototype provided real-time monitoring of tissue perfusion in rats after intravenous (IV) administration of ICG. The maximum fluorescence intensity (average SBR = 1.94 ± 0.16, p < 0.001) was observed at Tpeak of ~30 seconds after the ICG signal was first detected (average SBR = 1.19 ± 0.13, p < 0.01). CONCLUSIONS: We have conducted preclinical studies to demonstrate the feasibility of applying the RFITS system in real-time fluorescence angiography and tissue perfusion assessment. Our system provides fluorescence/color composite images for intuitive visualization of tissue perfusion with 3D perception. The findings pave the way for future clinical translation.

Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma.

Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAFi) and histone deacetylase inhibitors (HDACi) to enhance the delivery of MC1R-targeted radiolabeled peptide ([212Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAFV600E cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [212Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAFi and/or HDACi was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAFi and HDACi significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAFV600E and BRAFWT cells. Combining [212Pb]DOTA-MC1L with BRAFi and/or HDACi improved the tumor response by increasing the delivery of 212Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDACi and BRAFi could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.

Musclin is an activity-stimulated myokine that enhances physical endurance.

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin-a peptide with high homology to natriuretic peptides (NP)-as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca(2+)-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

An unusual destination for magnetic foreign bodies.

Rare earth metal magnets (Buckyballs and similar products) remain an important public health risk for children. We report the presentation, course, and treatment of a boy who inserted a string of 30 magnets through his urethra into his bladder and review the diagnostic as well as the therapeutic options for foreign bodies inserted into the pediatric urogenital tract.

"Click"-cyclized (68)Ga-labeled peptides for molecular imaging and therapy: synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system.

Cyclization techniques are used often to impart higher in vivo stability and binding affinity to peptide targeting vectors for molecular imaging and therapy. The two most often used techniques to impart these qualities are lactam bridge construction and disulfide bond formation. While these techniques have been demonstrated to be effective, orthogonal protection/deprotection steps can limit achievable product yields. In the work described in this chapter, new α-melanocyte stimulating hormone (α-MSH) peptide analogs were synthesized and cyclized by copper-catalyzed terminal azide-alkyne cycloaddition "click" chemistry techniques. The α-MSH peptide and its cognate receptor (melanocortin receptor subtype 1, MC1R) represent a well-characterized model system to examine the effect of the triazole linkage for peptide cyclization on receptor binding in vitro and in vivo. Four new DOTA-conjugated α-MSH analogs were cyclized and evaluated by in vitro competitive binding assays, serum stability testing, and in vivo imaging by positron emission tomography (PET) of tumor-bearing mice. These new DOTA-conjugated click-cyclized analogs exhibited selective high binding affinity (<2 nM) for MC1R on melanoma cells in vitro, high stability in human serum, and produced high-contrast PET/CT images of tumor xenografts. (68)Ga-labeled DOTA bioconjugates displayed rapid pharmacokinetics with receptor-mediated tumor accumulation of up to 16 ± 5% ID/g. The results indicate that the triazole ring is an effective bioisosteric replacement for the standard lactam bridge assemblage for peptide cyclization. Radiolabeling results confirm that Cu catalyst is sufficiently removed prior to DOTA chelator addition to enable insertion of radio metals or stable metals for molecular imaging and therapy. Thus, these click-chemistry-cyclized variants show promise as agents for melanocortin receptor-targeted imaging and radionuclide therapy.

Using computed tomography to recover hidden medieval fragments beneath early modern leather bindings, first results.

Medieval bindings fragments have become increasingly interesting to Humanities researchers as sources for the textual and material history of medieval Europeans. Later book binders used these discarded and repurposed pieces of earlier medieval manuscripts to reinforce the structures of other manuscripts and printed books. That many of these fragments are contained within and obscured by decorative bindings that cannot be dismantled ethically has limited their discovery and description. Although previous attempts to recover these texts using IRT and MA-XRF scanning have been successful, the extensive time required to scan a single book, and the need to modify or create specialized IRT or MA-XRF equipment for this method are drawbacks. Our research proposes and tests the capabilities of medical CT scanning technologies (commonly available at research university medical schools) for making visible and legible these fragments hidden under leather bindings. Our research team identified three sixteenth-century printed codices in our university libraries that were evidently bound in tawed leather by one workshop. The damaged cover of one of these three had revealed medieval manuscript fragments on the book spine; this codex served as a control for testing the other two volumes to see if they, too, contain fragments. The use of a medical CT scanner proved successful in visualizing interior book-spine structures and some letterforms, but not all of the text was made visible. The partial success of CT-scanning points to the value of further experimentation, given the relatively wide availability of medical imaging technologies, with their potential for short, non-destructive, 3D imaging times.

Insulin sensitization by small molecules enhancing GLUT4 translocation.

Insulin resistance (IR) is the root cause of type II diabetes, yet no safe treatment is available to address it. Using a high throughput compatible assay that measures real-time translocation of the glucose transporter glucose transporter 4 (GLUT4), we identified small molecules that potentiate insulin action. In vivo, these insulin sensitizers improve insulin-stimulated GLUT4 translocation, glucose tolerance, and glucose uptake in a model of IR. Using proteomic and CRISPR-based approaches, we identified the targets of those compounds as Unc119 proteins and solved the structure of Unc119 bound to the insulin sensitizer. This study identifies compounds that have the potential to be developed into diabetes treatment and establishes Unc119 proteins as targets for improving insulin sensitivity.

Establishment of Novel Neuroendocrine Carcinoma Patient-Derived Xenograft Models for Receptor Peptide-Targeted Therapy.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

Three dimensional (3D) printed polylactic acid with nano-hydroxyapatite doped with europium(III) ions (nHAp/PLLA@Eu3+) composite for osteochondral defect regeneration and theranostics.

In the current research previously developed composites composed from poly (l-lactide) (PLLA) and nano-hydroxyapatite (10 wt% nHAp/PLLA) were functionalized with different concentrations of europium (III) (Eu3+). The aim of this study was to determine whether Eu3+ ions doped within the 10 wt% nHAp/PLLA scaffolds will improve the bioactivity of composites. Therefore, first set of experiments was designed to evaluate the effect of Eu3+ ions on morphology, viability, proliferation and metabolism of progenitor cells isolated from adipose tissue (hASC). Three different concentration were tested i.e. 1 mol%, 3 mol% and 5%mol. We identified the 10 wt% nHAp/PLLA@3 mol% Eu3+ scaffolds as the most cytocompatible. Further, we investigated the influence of the composites doped with 3 mol% Eu3+ ions on differentiation of hASC toward bone and cartilage forming cells. Our results showed that 10 wt% nHAp/PLLA@3 mol% Eu3+ scaffolds promotes osteogenesis and chondrogenesis of hASCs what was associated with improved synthesis and secretion of extracellular matrix proteins specific for bone and articular cartilage tissue. We also proved that obtained biomaterials have bio-imaging function and their integration with bone can be monitored using micro computed tomography (µCT).

Exposure to Static Magnetic and Electric Fields Treats Type 2 Diabetes.

Aberrant redox signaling underlies the pathophysiology of many chronic metabolic diseases, including type 2 diabetes (T2D). Methodologies aimed at rebalancing systemic redox homeostasis have had limited success. A noninvasive, sustained approach would enable the long-term control of redox signaling for the treatment of T2D. We report that static magnetic and electric fields (sBE) noninvasively modulate the systemic GSH-to-GSSG redox couple to promote a healthier systemic redox environment that is reducing. Strikingly, when applied to mouse models of T2D, sBE rapidly ameliorates insulin resistance and glucose intolerance in as few as 3 days with no observed adverse effects. Scavenging paramagnetic byproducts of oxygen metabolism with SOD2 in hepatic mitochondria fully abolishes these insulin sensitizing effects, demonstrating that mitochondrial superoxide mediates induction of these therapeutic changes. Our findings introduce a remarkable redox-modulating phenomenon that exploits endogenous electromagneto-receptive mechanisms for the noninvasive treatment of T2D, and potentially other redox-related diseases.

Management of pediatric penetrating oropharyngeal trauma.

Penetrating oropharyngeal trauma (OPT) is common in young children. Complications are rare but can be severe and with delayed onset. Controversy exists about the evaluation and management of OPT, although most injuries in the stable child can be managed in the outpatient setting. Two pediatric OPT cases and a brief review of the literature are presented.

Inhibition of glutamate cysteine ligase activity sensitizes human breast cancer cells to the toxicity of 2-deoxy-D-glucose.

It has been hypothesized that cancer cells increase glucose metabolism to protect against metabolic fluxes of hydroperoxides via glutathione-dependent peroxidases. 2-Deoxy-D-glucose, inhibits glucose metabolism and has been shown to cause cytotoxicity in cancer cells that is partially mediated by disruptions in thiol metabolism. In the current study, human breast cancer cells were continuously treated (24 hours) with 2-deoxy-D-glucose, and total glutathione content as well as the expression of the first enzyme in the glutathione synthetic pathway [glutamate cysteine ligase (GCL)] were found to be induced 2.0-fold. Inhibiting GCL activity during 2-deoxy-D-glucose exposure using l-buthionine-[S,R]-sulfoximine (BSO) significantly enhanced the cytotoxic effects of 2-deoxy-D-glucose and caused increases in endpoints indicative of oxidative stress, including % oxidized glutathione and steady-state levels of pro-oxidants as assayed using an oxidation-sensitive fluorescent probe. These results show that treatment of human breast cancer cells with 2-deoxy-d-glucose causes metabolic oxidative stress that is accompanied by increases in steady-state levels of GCL mRNA, GCL activity, and glutathione content. Furthermore, inhibition of 2-deoxy-D-glucose-mediated induction of GCL activity with BSO increases endpoints indicative of oxidative stress and sensitizes cancer cells to 2-deoxy-D-glucose-induced cytotoxicity. These results support the hypothesis that drug combinations capable of inhibiting both glucose and hydroperoxide metabolism may provide an effective biochemical strategy for sensitizing human cancer cells to metabolic oxidative stress.

Enhanced response of human head and neck cancer xenograft tumors to cisplatin combined with 2-deoxy-D-glucose correlates with increased 18F-FDG uptake as determined by PET imaging.

PURPOSE: To determine whether the response of human head and neck cancer xenografts to cisplatin (CIS) could be enhanced with 2-deoxy-D-glucose (2DG); whether 2-[(18)F]-fluoro-2-deoxy-D-glucose (FDG) uptake correlated with responses to this drug combination; and whether 2DG would enhance CIS-induced radiosensitization. METHODS AND MATERIALS: Clonogenic survival responses to CIS + 2DG were determined in FaDu and Cal-27 cells and reduced/oxidized glutathione levels were monitored as parameters indicative of oxidative stress. The efficacy of CIS + 2DG was determined in FaDu and Cal-27 xenografts, and FDG uptake was determined by using positron emission tomography. RESULTS: Use of CIS + 2DG enhanced cell killing of FaDu and Cal-27 cells compared with either drug alone while increasing the percentage of oxidized glutathione in vitro. Use of CIS + 2DG inhibited FaDu and Cal-27 tumor growth and increased disease-free survival compared with either drug alone. The Cal-27 tumors showed greater pretreatment FDG uptake and increased disease-free survival when treated with 2DG + CIS relative to FaDu tumors. Treatment with 2DG enhanced CIS-induced radiosensitization in FaDu tumor cells grown in vitro and in vivo and resulted in apparent cures in 50% of tumors. CONCLUSIONS: These results show the enhanced therapeutic efficacy of CIS + 2DG in human head and neck cancer cells in vitro and in vivo compared with either drug alone, as well as the potential for FDG uptake to predict tumor sensitivity to 2DG + CIS. These findings provide a strong rationale for evaluating 2DG + CIS in combined-modality head and neck cancer therapy with radiation in a clinical setting.

Demonstration of Nucleoside Transporter Activity in the Nose-to-Brain Distribution of [18F]Fluorothymidine Using PET Imaging.

To evaluate the role of nucleoside transporters in the nose-to-brain uptake of [18F]fluorothymidine (FLT), an equilibrative nucleoside transporter (ENT1,2) and concentrative nucleoside transporter (CNT1-3) substrate, using PET to measure local tissue concentrations. Anesthetized Sprague-Dawley rats were administered FLT by intranasal (IN) instillation or tail-vein injection (IV). NBMPR (nitrobenzylmercaptopurine riboside), an ENT1 inhibitor, was administered either IN or intraperitoneally (IP). Dynamic PET imaging was performed for up to 40 min. A CT was obtained for anatomical co-registration and attenuation correction. Time-activity curves (TACs) were generated for the olfactory bulb (OB) and remaining brain, and the area-under-the-curve (AUC) for each TAC was calculated to determine the total tissue exposure of FLT. FLT concentrations were higher in the OB than in the rest of the brain following IN administration. IP administration of NBMPR resulted in increased OB and brain FLT exposure following both IN and IV administration, suggesting that NBMPR decreases the clearance rate of FLT from the brain. When FLT and NBMPR were co-administered IN, there was a decrease in the OB AUC while an increase in the brain AUC was observed. The decrease in OB exposure was likely the result of inhibition of ENT1 uptake activity in the nose-to-brain transport pathway. FLT distribution patterns show that nucleoside transporters, including ENT1, play a key role in the distribution of transporter substrates between the nasal cavity and the brain via the OB.

FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms.

FGF21 is an endocrine hormone that regulates energy homeostasis and insulin sensitivity. The mechanism of FGF21 action and the tissues responsible for these effects have been controversial, with both adipose tissues and the central nervous system having been identified as the target site mediating FGF21-dependent increases in insulin sensitivity, energy expenditure, and weight loss. Here we show that, while FGF21 signaling to adipose tissue is required for the acute insulin-sensitizing effects of FGF21, FGF21 signaling to adipose tissue is not required for its chronic effects to increase energy expenditure and lower body weight. Also, in contrast to previous studies, we found that adiponectin is dispensable for the metabolic effects of FGF21 in increasing insulin sensitivity and energy expenditure. Instead, FGF21 acutely enhances insulin sensitivity through actions on brown adipose tissue. Our data reveal that the acute and chronic effects of FGF21 can be dissociated through adipose-dependent and -independent mechanisms.

Selective Deletion of Renin-b in the Brain Alters Drinking and Metabolism.

The brain-specific isoform of renin (Ren-b) has been proposed as a negative regulator of the brain renin-angiotensin system (RAS). We analyzed mice with a selective deletion of Ren-b which preserved expression of the classical renin (Ren-a) isoform. We reported that Ren-bNull mice exhibited central RAS activation and hypertension through increased expression of Ren-a, but the dipsogenic and metabolic effects in Ren-bNull mice are unknown. Fluid intake was similar in control and Ren-bNull mice at baseline and both exhibited an equivalent dipsogenic response to deoxycorticosterone acetate-salt. Dehydration promoted increased water intake in Ren-bNull mice, particularly after deoxycorticosterone acetate-salt. Ren-bNull and control mice exhibited similar body weight when fed a chow diet. However, when fed a high-fat diet, male Ren-bNull mice gained significantly less weight than control mice, an effect blunted in females. This difference was not because of changes in food intake, energy absorption, or physical activity. Ren-bNull mice exhibited increased resting metabolic rate concomitant with increased uncoupled protein 1 expression and sympathetic nerve activity to the interscapular brown adipose tissue, suggesting increased thermogenesis. Ren-bNull mice were modestly intolerant to glucose and had normal insulin sensitivity. Another mouse model with markedly enhanced brain RAS activity (sRA mice) exhibited pronounced insulin sensitivity concomitant with increased brown adipose tissue glucose uptake. Altogether, these data support the hypothesis that the brain RAS regulates energy homeostasis by controlling resting metabolic rate, and that Ren-b deficiency increases brain RAS activity. Thus, the relative level of expression of Ren-b and Ren-a may control activity of the brain RAS.