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Nitric oxide (NO) is a critical signaling molecule that has been implicated in the pathogenesis of neurocognitive diseases. Both excessive and insufficient NO production have been linked to pathology. Previously, we have shown that argininosuccinate lyase deficiency (ASLD) is a novel model system to investigate cell-autonomous, nitric oxide synthase-dependent NO deficiency. Humans with ASLD are at increased risk for developing hyperammonemia due to a block in ureagenesis. However, natural history studies have shown that individuals with ASLD have multisystem disease including neurocognitive deficits that can be independent of ammonia. Here, using ASLD as a model of NO deficiency, we investigated the effects of NO on brain endothelial cells in vitro and the blood-brain barrier (BBB) in vivo. Knockdown of ASL in human brain microvascular endothelial cells (HBMECs) led to decreased transendothelial electrical resistance, indicative of increased cell permeability. Mechanistically, treatment with an NO donor or inhibition of Claudin-1 improved barrier integrity in ASL-deficient HBMECs. Furthermore, in vivo assessment of a hypomorphic mouse model of ASLD showed increased BBB leakage, which was partially rescued by NO supplementation. Our results suggest that ASL-mediated NO synthesis is required for proper maintenance of brain microvascular endothelial cell functions as well as BBB integrity.

Radiographic mapping of hypoxia is needed to study a wide range of diseases. Complexes of Eu(II) are a promising class of molecules to fit this need, but they are generally limited by their rapid oxidation rates in vivo. Here, a perfluorocarbon-nanoemulsion perfused with N 2 , forms an interface with aqueous layers to hinder oxidation of a new perfluorocarbon-soluble complex of Eu(II). Conversion of the perfluorocarbon solution of Eu(II) into nanoemulsions results in observable differences between reduced and oxidized forms by magnetic resonance imaging both in vitro and in vivo. Oxidation in vivo occurrs over a period of ≈30 min compared to <5 min for a comparable Eu(II)-containing complex without nanoparticle interfaces. These results represent a critical step toward delivery of Eu(II)-containing complexes in vivo for the study of hypoxia., (© 2023 Wiley-VCH GmbH.)

Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using Eu II -based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and Eu II . Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated Eu II/III -containing probes. We studied three different Eu II/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time ( T 1 ) and 19 F signal of solutions containing different ratios of Eu II - and Eu III -containing complexes was plotted against the percentage of Eu II -containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.

The availability of high-fidelity animal models for oncology research has grown enormously in recent years, enabling preclinical studies relevant to prevention, diagnosis, and treatment of cancer to be undertaken. This has led to increased opportunities to conduct co-clinical trials, which are studies on patients that are carried out parallel to or sequentially with animal models of cancer that mirror the biology of the patients' tumors. Patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM) are considered to be the models that best represent human disease and have high translational value. Notably, one element of co-clinical trials that still needs significant optimization is quantitative imaging. The National Cancer Institute has organized a Co-Clinical Imaging Resource Program (CIRP) network to establish best practices for co-clinical imaging and to optimize translational quantitative imaging methodologies. This overview describes the ten co-clinical trials of investigators from eleven institutions who are currently supported by the CIRP initiative and are members of the Animal Models and Co-clinical Trials (AMCT) Working Group. Each team describes their corresponding clinical trial, type of cancer targeted, rationale for choice of animal models, therapy, and imaging modalities. The strengths and weaknesses of the co-clinical trial design and the challenges encountered are considered. The rich research resources generated by the members of the AMCT Working Group will benefit the broad research community and improve the quality and translational impact of imaging in co-clinical trials.

Relevant to co-clinical trials, the goal of this work was to assess repeatability, reproducibility, and bias of the apparent diffusion coefficient (ADC) for preclinical MRIs using standardized procedures for comparison to performance of clinical MRIs. A temperature-controlled phantom provided an absolute reference standard to measure spatial uniformity of these performance metrics. Seven institutions participated in the study, wherein diffusion-weighted imaging (DWI) data were acquired over multiple days on 10 preclinical scanners, from 3 vendors, at 6 field strengths. Centralized versus site-based analysis was compared to illustrate incremental variance due to processing workflow. At magnet isocenter, short-term (intra-exam) and long-term (multiday) repeatability were excellent at within-system coefficient of variance, wCV [±CI] = 0.73% [0.54%, 1.12%] and 1.26% [0.94%, 1.89%], respectively. The cross-system reproducibility coefficient, RDC [±CI] = 0.188 [0.129, 0.343] µm 2 /ms, corresponded to 17% [12%, 31%] relative to the reference standard. Absolute bias at isocenter was low (within 4%) for 8 of 10 systems, whereas two high-bias (>10%) scanners were primary contributors to the relatively high RDC. Significant additional variance (>2%) due to site-specific analysis was observed for 2 of 10 systems. Base-level technical bias, repeatability, reproducibility, and spatial uniformity patterns were consistent with human MRIs (scaled for bore size). Well-calibrated preclinical MRI systems are capable of highly repeatable and reproducible ADC measurements.

Hypoxia is a hallmark of many diseases, including cancer, arthritis, heart and kidney diseases, and diabetes, and it is often associated with disease aggressiveness and poor prognosis. Consequently, there is a critical need for imaging hypoxia in a noninvasive and direct way to diagnose, stage, and monitor the treatment and development of new therapies for these diseases. Eu-containing contrast agents for magnetic resonance imaging have demonstrated potential for in vivo imaging of hypoxia via changes in metal oxidation state from +2 to +3, but rapid oxidation in blood limits Eu II -containing complexes to studies compatible with direct injection to sites. Here, we report a new Eu II -containing complex that persists in oxygenated environments and is capable of persisting in blood long enough for imaging by magnetic resonance imaging. We describe the screening of a library of ligands that led to the discovery of the complex as well as a pH-dependent mechanism that hinders oxidation to enable usefulness in vivo. These studies of the first divalent lanthanide complex that persists in oxygenated solutions open the door to the use of Eu II -based contrast agents for imaging hypoxia in a wide range of diseases.

Of individuals who develop West Nile neuroinvasive disease (WNND), ~10% will die and >40% will develop long-term complications. Current treatment recommendations solely focus on supportive care; therefore, we urgently need to identify novel and effective therapeutic options. We observed a correlation between substance P (SP), a key player in neuroinflammation, and its receptor Neurokinin-1 (NK1R). Our study in a wild-type BL6 mouse model found that SP is upregulated in the brain during infection, which correlated with neuroinvasion and damage to the blood−brain barrier. Blocking the SP/NK1R interaction beginning at disease onset modestly improved survival and prolonged time to death in a small pilot study. Although SP is significantly increased in the brain of untreated WNND mice when compared to mock-infected animals, levels of WNV are unchanged, indicating that SP likely does not play a role in viral replication but may mediate the immune response to infection. Additional studies are necessary to define if SP plays a mechanistic role or if it represents other mechanistic pathways.

Anesthesia is often used in preclinical imaging studies that incorporate mouse or rat models. However, multiple reports indicate that anesthesia has significant physiological impacts. Thus, there has been great interest in performing imaging studies in awake, unanesthetized animals to obtain accurate results without the confounding physiological effects of anesthesia. Here, we describe a newly designed mouse holder that is interfaceable with existing MRI systems and enables awake in vivo mouse imaging. This holder significantly reduces head movement of the awake animal compared to previously designed holders and allows for the acquisition of improved anatomical images. In addition to applications in anatomical T 2 -weighted magnetic resonance imaging (MRI), we also describe applications in acquiring 31 P spectra, manganese-enhanced magnetic resonance imaging (MEMRI) transport rates and resting-state functional magnetic resonance imaging (rs-fMRI) in awake animals and describe a successful conditioning paradigm for awake imaging. These data demonstrate significant differences in 31 P spectra, MEMRI transport rates, and rs-fMRI connectivity between anesthetized and awake animals, emphasizing the importance of performing functional studies in unanesthetized animals. Furthermore, these studies demonstrate that the mouse holder presented here is easy to construct and use, compatible with standard Bruker systems for mouse imaging, and provides rigorous results in awake mice.

Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments.

Millions of people a year receive magnetic resonance imaging (MRI) contrast agents for the diagnosis of conditions as diverse as fatty liver disease and cancer. Gadolinium chelates, which provide preferred T 1 contrast, are the current standard but face an uncertain future due to increasing concerns about their nephrogenic toxicity as well as poor performance in high-field MRI scanners. Gadolinium-containing nanocrystals are interesting alternatives as they bypass the kidneys and can offer the possibility of both intracellular accumulation and active targeting. Nanocrystal contrast performance is notably limited, however, as their organic coatings block water from close interactions with surface Gadoliniums. Here, these steric barriers to water exchange are minimized through shape engineering of plate-like nanocrystals that possess accessible Gadoliniums at their edges. Sulfonated surface polymers promote second-sphere relaxation processes that contribute remarkable contrast even at the highest fields (r 1 = 32.6 × 10 -3 m Gd -1 s -1 at 9.4 T). These noncytotoxic materials release no detectable free Gadolinium even under mild acidic conditions. They preferentially accumulate in the liver of mice with a circulation half-life 50% longer than commercial agents. These features allow these T 1 MRI contrast agents to be applied for the first time to the ex vivo detection of nonalcoholic fatty liver disease in mice., (© 2021 Wiley-VCH GmbH.)

The complexes described here serve as contrast agents for magnetic resonance imaging thermometry. The complexes differentially enhance contrast between 275 and 325 K. The basis of the temperature response of the fluorinated contrast complex is the modulation of water exchange caused by trifluoromethyl groups that can be chemically controlled.

Mutations in the skeletal muscle Ca 2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to Ca 2+ driven increases in muscle metabolism. Lactate increases brown adipogenesis in both mouse and human brown preadipocytes. This study suggests that simple lifestyle modifications such as avoiding extreme temperatures and maintaining thermoneutrality could decrease the risk of life-threatening responses to heat and exercise in individuals with RYR1 pathogenic variants.

Growth differentiation factor (GDF) 11 levels decline with aging. The age-related loss of GDF 11 has been implicated in the pathogenesis of a variety of age-related diseases. GDF11 supplementation reversed cardiac hypertrophy, bone loss, and pulmonary dysfunction in old mice, suggesting that GDF11 has a rejuvenating effect. Less is known about the potential of GDF11 to improve recovery after an acute injury, such as stroke, in aged mice. GDF11/8 levels were assessed in young and aged male mice and in postmortem human brain samples. Aged mice were subjected to a transient middle cerebral artery occlusion (MCAo). Five days after MCAo, mice received and bromodeoxyuridine / 5-Bromo-2'-deoxyuridine (BrdU) and either recombinant GDF11 or vehicle for five days and were assessed for recovery for one month following stroke. MRI was used to determine cerebrospinal fluid (CSF) volume, corpus callosum (CC) area, and brain atrophy at 30 days post-stroke. Immunohistochemistry was used to assess gliosis, neurogenesis, angiogenesis and synaptic density. Lower GDF11/8 levels were found with age in both mice and humans (p<0.05). GDF11 supplementation reduced mortality and improved sensorimotor deficits after stroke. Treatment also reduced brain atrophy and gliosis, increased angiogenesis, improved white matter integrity, and reduced inflammation after stroke. GDF11 may have a role in brain repair after ischemic injury.

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) has been identified as an autosomal-dominant disorder characterized by a complex neurological phenotype, with high prevalence of intellectual disability and optic nerve atrophy/hypoplasia. The syndrome is caused by loss-of-function mutations in NR2F1, which encodes a highly conserved nuclear receptor that serves as a transcriptional regulator. Previous investigations to understand the protein's role in neurodevelopment have mostly used mouse models with constitutive and tissue-specific homozygous knockout of Nr2f1. In order to represent the human disease more accurately, which is caused by heterozygous NR2F1 mutations, we investigated a heterozygous knockout mouse model and found that this model recapitulates some of the neurological phenotypes of BBSOAS, including altered learning/memory, hearing defects, neonatal hypotonia and decreased hippocampal volume. The mice showed altered fear memory, and further electrophysiological investigation in hippocampal slices revealed significantly reduced long-term potentiation and long-term depression. These results suggest that a deficit or alteration in hippocampal synaptic plasticity may contribute to the intellectual disability frequently seen in BBSOAS. RNA-sequencing (RNA-Seq) analysis revealed significant differential gene expression in the adult Nr2f1+/- hippocampus, including the up-regulation of multiple matrix metalloproteases, which are known to be critical for the development and the plasticity of the nervous system. Taken together, our studies highlight the important role of Nr2f1 in neurodevelopment. The discovery of impaired hippocampal synaptic plasticity in the heterozygous mouse model sheds light on the pathophysiology of altered memory and cognitive function in BBSOAS., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background : Oxidative stress has been implicated in metabolic syndrome (MetS); however, antioxidants such as vitamin E have had limited success in the clinic. This prompts the question of what effects amore potent antioxidant might produce. A prime candidate is the recently developed bioengineered antioxidant, poly(ethylene glycol)-functionalizedhydrophilic carbon clusters (PEG-HCCs), which are capable of neutralizing the reactive oxygen species (ROS) superoxide anion and hydroxyl radical at10 6 /molecule of PEG-HCC. In this project, we tested the potential of PEG-HCCs as a possible therapeutic for MetS. Results : PEG-HCC treatment lessened lipid peroxidation, aspartate aminotransferase levels, non-fastingblood glucose levels, and JNK phosphorylation inob/ob mice. PEG-HCC-treated WT mice had an increased response to insulin by insulin tolerance tests and adecrease in blood glucose by glucose tolerance tests. These effects were not observed in HFD-fed mice, regardless of treatment. PEG-HCCs were observed in the interstitial space of liver, spleen, skeletal muscle, and adipose tissue. No significant difference was shown in gluconeogenesis or inflammatory gene expression between treatment and dietary groups. Expert Opinion : PEG-HCCs improved some parameters of disease possibly due to a resulting increase in peripheral insulin sensitivity. However, additional studies are needed to elucidate how PEG-HCCsare producing these effects.

Gene-environment interactions contribute to the risk for Autism Spectrum Disorder (ASD). Among environmental factors, prenatal exposure to stress may increase the risk for ASD. To examine if there is an interaction between exposure to maternal stress and reduced dosage or loss of Shank3, wild-type (WT), heterozygous (HET) and homozygous (HOM) female mice carrying a deletion of exons four through nine of Shank3 (Shank3ex4-9) were exposed to chronic unpredictable mild stress (CUMS) from prior to conception throughout gestation. This study examined maternal care of these dams and the white matter microstructure in the brains of their adult male offspring. Overall, our findings suggest that maternal exposure to CUMS increased pup-directed care for dams of all three genotypes. Compared to WT and HET dams, HOM dams also exhibited increased maternal care behaviors with increased time spent in the nest and reduced cage exploration, regardless of exposure to CUMS. Diffusion tensor imaging showed higher mean fractional anisotropy in the hippocampal stratum radiatum of WT and HOM male offspring from dams exposed to CUMS and HOM offspring from unexposed dams, compared to WT male offspring from unexposed dams. These data support that CUMS in Shank3-mutant dams results in subtle maternal care alterations and long-lasting changes in the white matter of the hippocampus of their offspring., Competing Interests: The authors have declared that no competing interests exist.

Fluorine-19 ( 19 F) magnetic resonance imaging (MRI) has the potential for a wide range of in vivo applications but is limited by lack of flexibility in exogenous probe formulation. Most 19 F MRI probes are composed of perfluorocarbons (PFCs) or perfluoropolyethers (PFPEs) with intrinsic properties which limit formulation options. Hydrophilic organofluorine molecules can provide more flexibility in formulation options. We report herein a hyperfluorinated hydrophilic organoflourine, ET1084 , with ∼24 wt. % 19 F content. It dissolves in water and aqueous buffers to give solutions with ≥8 M 19 F. 19 F MRI phantom studies at 9.4T employing a 10-minute multislice multiecho (MSME) scan sequence show a linear increase in signal-to-noise ratio (SNR) with increasing concentrations of the molecule and a detection limit of 5 mM. Preliminary cytotoxicity and genotoxicity assessments suggest it is safe at concentrations of up to 20 mM.

Subarachnoid hemorrhage (SAH) in 95% of cases results in long-term disabilities due to brain damage, pathogenesis of which remains uncertain. Hindrance of cerebrospinal fluid (CSF) circulation along glymphatic pathways is a possible mechanism interrupting drainage of damaging substances from subarachnoid space and parenchyma. We explored changes in CSF circulation at different time following SAH and possible role of brain tissue factor (TF). Fluorescent solute and fluorescent microspheres injected into cisterna magna were used to track CSF flow in mice. SAH induced by perforation of circle of Willis interrupted CSF flow for up to 30 days. Block of CSF flow did not correlate with the size of hemorrhage. Following SAH, fibrin deposits were observed on the brain surface including areas without visible blood. Block of astroglia-associated TF by intracerebroventricular administration of specific antibodies increased size of hemorrhage, decreased fibrin deposition and facilitated spread of fluorophores in sham/naïve animals. We conclude that brain TF plays an important role in localization of hemorrhage and also regulates CSF flow under normal conditions. Targeting of the TF system will allow developing of new therapeutic approaches to the treatment of SAH and pathologies related to CSF flow such as hydrocephalus.

Objective: Sympathetic predominance and ventricular repolarization abnormalities represent epilepsy-associated cardiac alterations and may underlie seizure-induced ventricular arrhythmias. Myocardial ion channel and electrical remodeling have been described early in epilepsy development and may contribute to ventricular repolarization abnormalities and excitability. Using the pilocarpine-induced acquired epilepsy model we sought to examine whether altered myocardial ion channel levels and electrophysiological changes also occur in animals with long-standing epilepsy., Methods: We examined myocardial adrenergic receptor and ion channel protein levels of epileptic and age-matched sham rats (9-20 months old) using western blotting. Cardiac electrical properties were examined using optical mapping ex vivo and electrophysiology in vivo. We investigated the propensity for ventricular tachycardia (VT) and the effects of β-adrenergic blockade on ventricular electrical properties and excitability in vivo., Results: In animals with long-standing epilepsy, we observed decreased myocardial voltage-gated K + channels Kv 4.2 and Kv 4.3 , which are known to underlie early ventricular repolarization in rodents. Decreased β1 and increased α1 A adrenergic receptor protein levels occurred in the myocardium of chronically epileptic animals consistent with elevated sympathetic tone. These animals exhibited many cardiac electrophysiological abnormalities, represented by longer QRS and corrected QT (QTc) intervals in vivo, slower conduction velocity ex vivo, and stimulation-induced VT. Administration of a β-adrenergic antagonist late in epilepsy was beneficial, as the therapy shortened the QTc interval and decreased stimulation-induced VT., Significance: Our findings demonstrate that myocardial ion channel remodeling and sympathetic predominance, risk factors for increased ventricular excitability and arrhythmias, persist in chronic epilepsy. The beneficial effects of β-adrenergic antagonist treatment late in the course of epilepsy suggest that attenuating elevated sympathetic tone may represent a therapeutic target for ameliorating epilepsy-associated cardiac morbidity.

Fluorine-19 ( 19 F) Magnetic Resonance Imaging (MRI) is an emerging modality for molecular imaging and cell tracking. The hydrophobicity of current exogenous probes, perfluorocarbons (PFCs) and perfluoropolyethers (PFPEs), limits the formulation options available for in vivo applications. Hydrophilic probes permit more formulation flexibility. Further, the broad Nuclear Magnetic Resonance (NMR) chemical shift range of organofluorine species enables multiple probes with unique 19 F MR signatures for simultaneous interrogation of distinct molecular targets in vivo. We report herein a flexible approach to stable liposomal formulations of hydrophilic fluorinated molecules (each bearing numerous magnetically equivalent 19 F atoms), with 19 F encapsulation of up to 22.7 mg/mL and a per particle load of 3.6 × 10 6 19 F atoms. Using a combination of such probes, we demonstrate, with no chemical shift artifacts, the simultaneous imaging of multiple targets within a given target volume by spectral 19 F MRI.

Hypothermia shows promise for stroke neuroprotection, but current cooling strategies cause undesirable side effects that limit their clinical applications. Increasing efforts have focused on pharmacological hypothermia as a treatment option for stroke. Previously, we showed that activation of a thermoregulatory ion channel, transient receptor potential vanilloid 1 (TRPV1), by dihydrocapsaicin (DHC) produces reliable hypothermia. In this study, we investigate the effects of TRPV1-mediated hypothermia by DHC on long-term ischemic stroke injury and functional outcome. Hypothermia initiated at 3.5 hours after stroke significantly reduced primary cortical injury. Interestingly, hypothermia by DHC also significantly reduced secondary thalamic injury, as DHC-treated stroke mice exhibited 53% smaller thalamic lesion size. DHC-treated stroke mice further demonstrated decreased neuronal loss and astrogliosis in the thalamus and less thalamic fiber loss by diffusion tensor imaging (DTI). Importantly, a single 8 hour treatment of hypothermia by DHC after stroke provided long-term improvement in functional outcome, as DHC-treated mice exhibited improved behavioral recovery at one month post-stroke. These findings indicate that TRPV1-mediated hypothermia is effective in reducing both primary cortical injury and remote secondary thalamic injury, and a single treatment can produce persistent effects on functional recovery. These data highlight the therapeutic potential for TRPV1 agonism for stroke treatment.

Magnetic resonance imaging (MRI) using redox-active, Eu II -containing complexes is one of the most promising techniques for noninvasively imaging hypoxia in vivo . In this technique, positive ( T 1 -weighted) contrast enhancement persists in areas of relatively low oxidizing ability, such as hypoxic tissue. Herein, we describe a fluorinated, Eu II -containing complex in which the redox-active metal is caged by intramolecular interactions. The position of the fluorine atoms enables temperature-responsive contrast enhancement in the reduced form of the contrast agent and detection of the oxidized contrast agent via MRI in vivo . Positive contrast is observed in 1 H-MRI with Eu in the +2 oxidation state, and chemical exchange saturation transfer and 19 F-MRI signal are observed with Eu in the +3 oxidation state. Contrast enhancement is controlled by the redox state of Eu, and modulated by the fluorous interactions that cage a bound water molecule reduce relaxivity in a temperature-dependent fashion. Together, these advancements constitute the first report of in vivo , redox-responsive imaging using 19 F-MRI.

This corrects the article DOI: 10.1038/ncomms14338.

In a rat model of right free wall replacement, the transplantation of an engineered multilayered myocardial patch fabricated from a polycaprolactone membrane supporting a chitosan/heart matrix hydrogel induces significant muscular and vascular remodeling and results in a significantly higher right ventricular ejection fraction compared to use of a commercially available pericardium patch., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases., Competing Interests: The authors declare no competing financial interests.

Background: Hypertrophic cardiomyopathy (HCM), defined as asymmetric left ventricular hypertrophy, is a leading cause of cardiac death in the young. Perturbations in calcium (Ca 2+ ) handling proteins have been implicated in the pathogenesis of HCM. JPH2 -encoded junctophilin 2 is a major component of the junctional membrane complex, the subcellular microdomain involved in excitation-contraction coupling. We hypothesized that a novel JPH2 mutation identified in patients with HCM is causally linked to HCM, and alters intracellular Ca 2+ signaling in a pro-hypertrophic manner., Objectives: To determine using a transgenic mouse model whether a JPH2 mutation found in a HCM patient is responsible for disease development., Methods: Genetic interrogation of a large cohort of HCM cases was conducted for all coding exons of JPH2 . Pseudo-knock-in (PKI) mice containing a novel JPH2 variant were subjected to echocardiography, cardiac MRI, hemodynamic analysis, and histology., Results: A novel JPH2 mutation, A405S, was identified in a genotype-negative proband with significant basal septal hypertrophy. Although initially underappreciated by traditional echocardiographic imaging, PKI mice with this JPH2 mutation (residue A399S in mice) were found to exhibit similar basal hypertrophy using a newly developed echo imaging plane, and this was confirmed using cardiac MRI. Histological analysis demonstrated cardiomyocyte hypertrophy and disarray consistent with HCM., Conclusions: Variant A405S is a novel HCM-associated mutation in JPH2 found in a proband negative for mutations in the canonical HCM-associated genes. Studies in the analogous mouse model demonstrated for the first time a causal link between a JPH2 defect and HCM. Moreover, novel imaging approaches identified subvalvular septal hypertrophy, specific findings also reported in the human JPH2 mutation carrier.

Key Points: Inhibiting Nox2 reactive oxygen species (ROS) production reduced in vivo calcium influx in dystrophic muscle. The lack of Nox2 ROS production protected against decreased in vivo muscle function in dystrophic mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was able to detect alterations in basal calcium levels in skeletal muscle and differentiate disease status. Administration of Mn 2+ did not affect muscle function or the health of the animal, and Mn 2+ was cleared from skeletal muscle rapidly. We conclude that MEMRI may be a viable, non-invasive technique to monitor molecular alterations in disease progression and evaluate the effectiveness of potential therapies for Duchenne muscular dystrophy., Abstract: Duchenne muscular dystrophy (DMD) is an X-linked progressive degenerative disease resulting from a mutation in the gene that encodes dystrophin, leading to decreased muscle mechanical stability and force production. Increased Nox2 reactive oxygen species (ROS) production and sarcolemmal Ca 2+ influx are early indicators of disease pathology, and eliminating Nox2 ROS production reduces aberrant Ca 2+ influx in young mdx mice, a model of DMD. Various imaging modalities have been used to study dystrophic muscle in vivo; however, they are based upon alterations in muscle morphology or inflammation. Manganese has been used for indirect monitoring of calcium influx across the sarcolemma and may allow detection of molecular alterations in disease progression in vivo using manganese-enhanced magnetic resonance imaging (MEMRI). Therefore, we hypothesized that eliminating Nox2 ROS production would decrease calcium influx in adult mdx mice and that MEMRI would be able to monitor and differentiate disease status in dystrophic muscle. Both in vitro and in vivo data demonstrate that eliminating Nox2 ROS protected against aberrant Ca 2+ influx and improved muscle function in dystrophic muscle. MEMRI was able to differentiate between different pathological states in vivo, with no long-term effects on animal health or muscle function. We conclude that MEMRI is a viable, non-invasive technique to differentiate disease status and might provide a means to monitor and evaluate the effectiveness of potential therapies in dystrophic muscle., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

In many human diseases, the presence of inflammation is associated with an increase in the level of reactive oxygen species (ROS). The resulting state of oxidative stress is highly detrimental and can initiate a cascade of events that ultimately lead to cell death. Thus, many therapeutic attempts have been focused on either modulating the immune system to lower inflammation or reducing the damaging caused by ROS. Berlin et al. reported the development of a novel nanoantioxidant known as poly(ethylene glycol)-functionalized-hydrophilic carbon clusters (PEG-HCCs). They showed that PEG-HCCs could be targeted to cancer cells, utilized as a drug delivery vector, and can even be visualized ex vivo. Our work here furthers this work and characterizes Gd-DTPA conjugated PEG-HCCs and explores the potential for in vivo tracking of T cells in live mice. We utilized a mouse model of delayed-type hypersensitivity (DTH) to assess the immunomodulatory effects of PEG-HCCs. The T1 -agent Gd-DTPA was then conjugated to the PEG-HCCs and T1 measurements, and T1 -weighted MRI of the modified PEG-HCCs was done to assess their relaxivity. We then assessed if PEG-HCCs could be visualized both ex vivo and in vivo within the mouse lymph node and spleen. Mice treated with PEG-HCCs showed significant improvements in the DTH assay as compared to the vehicle (saline)-treated control. Flow cytometry demonstrated that splenic T cells are capable of internalizing PEG-HCCs whereas fluorescent immunohistochemistry showed that PEG-HCCs are detectable within the cortex of lymph nodes. Finally, our nanoantioxidants can be visualized in vivo within the lymph nodes and spleen of a mouse after addition of the Gd-DTPA. PEG-HCCs are internalized by T cells in the spleen and can reduce inflammation by suppression of a recall immune response. PEG-HCCs can be modified to allow for both in vitro and in vivo visualization using MRI. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd., (© 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Autoimmune diseases mediated by a type of white blood cell-T lymphocytes-are currently treated using mainly broad-spectrum immunosuppressants that can lead to adverse side effects. Antioxidants represent an alternative approach for therapy of autoimmune disorders; however, dietary antioxidants are insufficient to play this role. Antioxidant carbon nanoparticles scavenge reactive oxygen species (ROS) with higher efficacy than dietary and endogenous antioxidants. Furthermore, the affinity of carbon nanoparticles for specific cell types represents an emerging tactic for cell-targeted therapy. Here, we report that nontoxic poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), known scavengers of the ROS superoxide (O 2 •- ) and hydroxyl radical, are preferentially internalized by T lymphocytes over other splenic immune cells. We use this selectivity to inhibit T cell activation without affecting major functions of macrophages, antigen-presenting cells that are crucial for T cell activation. We also demonstrate the in vivo effectiveness of PEG-HCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity and in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Our results suggest the preferential targeting of PEG-HCCs to T lymphocytes as a novel approach for T lymphocyte immunomodulation in autoimmune diseases without affecting other immune cells., Competing Interests: R.H., E.L.G.S., T.I., R.G.P., J.M.T., and C.B. are inventors on a patent application claiming PEG-HCCs as T lymphocyte immunomodulators for the treatment of inflammatory diseases. The patent rights are owned by Rice University and Baylor College of Medicine. These patents have been licensed to Acelerox, LLC in which J.M.T. owns stock. All potential conflicts of interest are managed through the respective institutional offices of research compliance. The other authors declare no competing financial interests.

The scope of this review focuses on recent applications in preclinical and clinical magnetic resonance imaging (MRI) toward accomplishing the goals of early detection and responses to therapy in animal models of Alzheimer's disease (AD). Driven by the outstanding efforts of the Alzheimer's Disease Neuroimaging Initiative (ADNI), a truly invaluable resource, the initial use of MRI in AD imaging has been to assess changes in brain anatomy, specifically assessing brain shrinkage and regional changes in white matter tractography using diffusion tensor imaging. However, advances in MRI have led to multiple efforts toward imaging amyloid beta plaques first without and then with the use of MRI contrast agents. These technological advancements have met with limited success and are not yet appropriate for the clinic. Recent developments in molecular imaging inclusive of high-power liposomal-based MRI contrast agents as well as fluorine 19 ((19)F) MRI and manganese enhanced MRI have begun to propel promising advances toward not only plaque imaging but also using MRI to detect perturbations in subcellular processes occurring within the neuron. This review concludes with a discussion about the necessity for the development of novel preclinical models of AD that better recapitulate human AD for the imaging to truly be meaningful and for substantive progress to be made toward understanding and effectively treating AD. Furthermore, the continued support of outstanding programs such as ADNI as well as the development of novel molecular imaging agents and MRI fast scanning sequences will also be requisite to effectively translate preclinical findings to the clinic., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background & Aims: Pediatric liver cancer is a rare but serious disease whose incidence is rising, and for which the therapeutic options are limited. Development of more targeted, less toxic therapies is hindered by the lack of an experimental animal model that captures the heterogeneity and metastatic capability of these tumors., Methods: Here we established an orthotopic engraftment technique to model a series of patient-derived tumor xenograft (PDTX) from pediatric liver cancers of all major histologic subtypes: hepatoblastoma, hepatocellular cancer and hepatocellular malignant neoplasm. We utilized standard (immuno) staining methods for histological characterization, RNA sequencing for gene expression profiling and genome sequencing for identification of druggable targets. We also adapted stem cell culturing techniques to derive two new pediatric cancer cell lines from the xenografted mice., Results: The patient-derived tumor xenografts recapitulated the histologic, genetic, and biological characteristics-including the metastatic behavior-of the corresponding primary tumors. Furthermore, the gene expression profiles of the two new liver cancer cell lines closely resemble those of the primary tumors. Targeted therapy of PDTX from an aggressive hepatocellular malignant neoplasm with the MEK1 inhibitor trametinib and pan-class I PI3 kinase inhibitor NVP-BKM120 resulted in significant growth inhibition, thus confirming this PDTX model as a valuable tool to study tumor biology and patient-specific therapeutic responses., Conclusions: The novel metastatic xenograft model and the isogenic xenograft-derived cell lines described in this study provide reliable tools for developing mutation- and patient-specific therapies for pediatric liver cancer., Lay Summary: Pediatric liver cancer is a rare but serious disease and no experimental animal model currently captures the complexity and metastatic capability of these tumors. We have established a novel animal model using human tumor tissue that recapitulates the genetic and biological characteristics of this cancer. We demonstrate that our patient-derived animal model, as well as two new cell lines, are useful tools for experimental therapies., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Type 2 diabetes (T2D) in lean individuals is not well studied and up to 26% of diabetes occurs in these individuals. Although the cause is not well understood, it has been primarily attributed to nutritional issues during early development., Objective: Our objective was to develop a lean T2D model using gestational low-protein (LP) programming., Study Design: Pregnant rats were fed control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery. Standard diet was given to dams after delivery and to pups after weaning. Glucose tolerance test was done at 2, 4, and 6 months of age. Magnetic resonance imaging of body fat for females was done at 4 months. Rats were sacrificed at 4 and 8 months of age and their perigonadal, perirenal, inguinal, and brown fat were weighed and expressed relative to their body weight. Euglycemic-hyperinsulinemic clamp was done around 6 months of age., Results: Male and female offspring exposed to a LP diet during gestation developed glucose intolerance and insulin resistance (IR). Further, glucose intolerance progressed with increasing age and occurred earlier and was more severe in females when compared to males. Euglycemic-hyperinsulinemic clamp showed whole body IR in both sexes, with females demonstrating increased IR compared to males. LP females showed a 4.5-fold increase in IR while males showed a 2.5-fold increase when compared to their respective controls. Data from magnetic resonance imaging on female offspring showed no difference in the subcutaneous, inguinal, and visceral fat content. We were able to validate this observation by sacrificing the rats at 4 and 8 months and measuring total body fat content. This showed no differences in body fat content between control and LP offspring in either males or females. Additionally, diabetic rats had a similar body mass index to that of the controls., Conclusion: LP gestational programming produces a progressively worsening T2D model in rats with a lean phenotype without obesity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: Current therapy for Alzheimer's disease (AD) focuses on delaying progression, illustrating the need for more effective therapeutic targets. Histone deacetylase 6 (HDAC6) modulates tubulin acetylation and has been implicated as an attractive target. HDAC6 is also elevated in postmortem tissue samples from patients. However, HDAC6 inhibitors have had limited success preclinically due to low blood-brain barrier penetration., Method: We investigated a specific, potent HDAC6 inhibitor (ACY-738) in a mouse model of AD. We determined the effects of ACY-738 treatment on axonal transport, behavior, and pathology in amyloid precursor protein/presenilin 1 mice., Results: We demonstrated improvements in in vivo axonal transport in two treatment groups as a result of ACY-738 brain levels. We also demonstrated recovery of short-term learning and memory deficits, hyperactivity, and modifications of tau and tubulin., Discussion: Our findings implicate specific, targeted HDAC6 inhibitors as potential therapeutics and demonstrate that further investigations are warranted into effects of HDAC6 inhibitors in AD.

Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Background: Accumulation and deposition of β-amyloid peptides (Aβ) in the brain is a central event in the pathogenesis of Alzheimer's disease (AD). Besides the parenchymal pathology, Aβ is known to undergo active transport across the blood-brain barrier and cerebral amyloid angiopathy (CAA) is a prominent feature in the majority of AD. Although impaired cerebral blood flow (CBF) has been implicated in faulty Aβ transport and clearance, and cerebral hypoperfusion can exist in the pre-clinical phase of Alzheimer's disease (AD), it is still unclear whether it is one of the causal factors for AD pathogenesis, or an early consequence of a multi-factor condition that would lead to AD at late stage. To study the potential interaction between faulty CBF and amyloid accumulation in clinical-relevant situation, we generated a new amyloid precursor protein (APP) knock-in allele that expresses humanized Aβ and a Dutch mutation in addition to Swedish/London mutations and compared this line with an equivalent knock-in line but in the absence of the Dutch mutation, both crossed onto the PS1M146V knock-in background., Results: Introduction of the Dutch mutation results in robust CAA and parenchymal Aβ pathology, age-dependent reduction of spatial learning and memory deficits, and CBF reduction as detected by fMRI. Direct manipulation of CBF by transverse aortic constriction surgery on the left common carotid artery caused differential changes in CBF in the anterior and middle region of the cortex, where it is reduced on the left side and increased on the right side. However these perturbations in CBF resulted in the same effect: both significantly exacerbate CAA and amyloid pathology., Conclusions: Our study reveals a direct and positive link between vascular and parenchymal Aβ; both can be modulated by CBF. The new APP knock-in mouse model recapitulates many symptoms of AD including progressive vascular and parenchymal Aβ pathology and behavioral deficits in the absence of APP overexpression.

Background: Axonal transport is vital for neurons and deficits in this process have been previously reported in a few mouse models of Alzheimer's disease prior to the appearance of plaques and tangles. However, it remains to be determined whether axonal transport is defective prior to the onset of neurodegeneration. The rTg4510 mouse, a fronto-temporal dementia and parkinsonism-17 (FTDP-17) tauopathy model, over-express tau-P301L mutation found in familial forms of FTDP-17, in the forebrain driven by the calcium-calmodulin kinase II promoter. This mouse model exhibits tau pathology, neurodegeneration in the forebrain, and associated behavioral deficits beginning at 4-5 months of age., Animal Model: rTg4510 transgenic mice were used in these studies. Mice were given 2 μL of MnCl2 in each nostril 1 h prior to Magnetic Resonance Imaging (MRI). Following MnCl2 nasal lavage, mice were imaged using Manganese enhanced Magnetic Resonance Imaging (MEMRI) Protocol with TE = 8.5 ms, TR = 504 ms, FOV = 3.0 cm, matrix size = 128 × 128 × 128, number of cycles = 15 with each cycle taking approximately 2 min, 9 s, and 24 ms using Paravision software (BrukerBioSpin, Billerica, MA). During imaging, body temperature was maintained at 37.0 °C using an animal heating system (SA Instruments, Stony Brook, NY)., Data Analysis: Resulting images were analyzed using Paravision software. Regions of interest (ROI) within the olfactory neuronal layer (ONL) and the water phantom consisting of one pixel (ONL) and 9 pixels (water) were selected and copied across each of the 15 cycles. Signal intensities (SI) of ONL and water phantom ROIs were measured. SI values obtained for ONL were then normalized the water phantom SI values. The correlation between normalized signal intensity in the ONL and time were assessed using Prism (GraphPad Software, San Diego, CA)., Results: Using the MEMRI technique on 1.5, 3, 5, and 10-month old rTg4510 mice and littermate controls, we found significant axonal transport deficits present in the rTg4510 mice beginning at 3 months of age in an age-dependent manner. Using linear regression analysis, we measured rates of axonal transport at 1.5, 3, 5, and 10 months of age in rTg4510 and WT mice. Axonal transport rates were observed in rTg4510 mice at 48% of WT levels at 3 months, 40% of WT levels at 5 months, and 30% of WT levels at 10 months of age. In order to determine the point at which tau appears in the cortex, we probed for phosphorylated tau levels, and found that pSer262 is present at 3 months of age, not earlier at 1.5 months of age, but observed no pathological tau species until 6 months of age, months after the onset of the transport deficits. In addition, we saw localization of tau in the ONL at 6 months of age., Discussion: In our study, we identified the presence of age-dependent axonal transport deficits beginning at 3 months of age in rTg4510 mice. We correlated these deficits at 3 months to the presence of hyperphosphorylated tau in the brain and the presence within the olfactory epithelium. We observed tau pathology not only in the soma of these neurons but also within the axons and processes of these neurons. Our characterization of axonal transport in this tauopathy model provides a functional time point that can be used for future therapeutic interventions.

Intestinal intussusception (ISS) commonly causes intestinal obstruction in children. One mechanism that has been proposed to cause ISS is inflammation-induced alteration of intestinal motility. We investigated whether innate inflammatory factors or altered motility is required for induction of ISS by LPS. We compared rates of ISS among BALB/c and C57BL/6 mice, mice lacking lymphocytes or depleted of phagocytes, or mice with defects in the Toll-like receptor 4 (TLR4) signaling pathway following administration of LPS or the Ca(2+) analog MnCl2. At 6 or 2 h after administration of LPS or MnCl2, respectively, mice underwent image analysis to assess intestinal contraction rate or laparotomy to identify ISS. LPS-induced ISS (LPS-ISS) was observed in BALB/c mice, but not in C57BL/6 mice or any BALB/c mice with disruptions of TLR4 signaling. LPS-induced serum TNF-α, IL-6, and nitric oxide (NO) and intestinal NO levels were similar in BALB/c and C57BL/6 mice. The rate of LPS-ISS was significantly reduced in phagocyte-depleted, but not lymphocyte-deficient, mice. Intestinal contraction rates were reduced in LPS-ISS-susceptible BALB/c mice, but not in LPS-ISS-resistant C57BL/6 or TLR4 mutant mice, suggesting a role for reduced intestinal contraction rate in LPS-ISS susceptibility. This was tested with MnCl2, a Ca(2+) antagonist that reduced intestinal contraction rates and induced ISS, irrespective of mouse strain. Therefore, LPS-ISS is initiated by innate immune signaling that requires TLR4 and phagocytes but may be independent of TNF-α, IL-6, and NO levels. Furthermore, alteration of intestinal motility, specifically, reduced intestinal contraction rate, is a key factor in the development of ISS.

Aim: We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase-associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer., Materials & Methods: Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the near-infrared (NIR) dye indocyanine green, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice., Results: Anti-NGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2-weighted MRI with higher tumor contrast than can be obtained using long-circulating, but nontargeted, PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro., Conclusion: TGNS with embedded NIR and magnetic resonance contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy.

Purpose: The purpose of this study was to determine if magnetization transfer contrast (MTC) imaging could be used to detect early macromolecular accumulation in a mouse model of early Alzheimer's disease., Methods: We obtained MTC images at 9.4 T at three different age points in the Tg2576 mouse model of Alzheimer's disease. The Tg2576 mouse exhibits increased amyloid beta deposition that eventually progresses into amyloid beta plaque formation, increased hyper-phosphorylated tau but does not exhibit neurodegeneration., Results: Our results show an increase in the MTC signal that predates plaque formation and reported learning and memory deficits in the Tg2576 mouse. This increase in the MTC signal was reversed in a model of antioxidant therapy., Conclusion: MTC magnetic resonance imaging can be used to detect early macromolecular changes in the Tg2576 mouse model of Alzheimer's disease. The source of the MTC contrast is likely complex and warrants further investigation in additional preclinical models that represent early and late stage Alzheimer's disease pathologies., (Copyright © 2013 Wiley Periodicals, Inc.)

A major limitation of exogenous vitamin D3 administration for the treatment of prostate cancer is the marginal, if any, clinical efficacy. We dissected the basis for the resistance to the vitamin D3 antitumor properties and specifically examined the effect of its major catabolic enzyme, CYP24A1, in prostate cancer. Local CYP24A1 expression levels and the effect of selective modulation were analyzed using tissue microarrays from needle core biopsy specimens and xenograft-bearing mouse models. CYP24A1 mRNA was elevated in malignant human prostate tissues compared to benign lesions. High CYP24A1 protein levels were seen in poorly differentiated and highly advanced stages of prostate cancer and correlated with parallel increase in the tumor proliferation rate. The use of CYP24A1 RNAi enhanced the cytostatic effects of vitamin D3 in human prostate cancer cells. Remarkably, subcutaneous and orthotopic xenografts of prostate cancer cells harboring CYP24A1 shRNA resulted in a drastic reduction in tumor volume when mice were subjected to vitamin D3 supplementation. CYP24A1 may be a predictive marker of vitamin D3 clinical efficacy in patients with advanced prostate cancer. For those with up-regulated CYP24A1, combination therapy with RNAi targeting CYP24A1 could be considered to improve clinical responsiveness to vitamin D3.

Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of Zic3 demonstrates an early role for Zic3 in gastrulation, CNS, cardiac and left-right axial development. The observation of multiple malformations in Zic3(null) mice and the relatively broad expression pattern of Zic3 suggest its important roles in multiple developmental processes. Here, we report that Zic3 is primarily required in epiblast derivatives to affect left-right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in Zic3 deficiency occur not because Zic3 is intrinsically required in the heart but rather because it functions early in the establishment of left-right body axis. In addition, we provide evidence supporting a role for Zic3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of Zic3 during left-right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of Zic3 with signaling pathways involved in the early establishment of laterality.

Neuronal network hyperexcitability underlies the pathogenesis of seizures and is a component of some degenerative neurological disorders such as Alzheimer's disease (AD). Recently, the microtubule-binding protein tau has been implicated in the regulation of network synchronization. Genetic removal of Mapt, the gene encoding tau, in AD models overexpressing amyloid-β (Aβ) decreases hyperexcitability and normalizes the excitation/inhibition imbalance. Whether this effect of tau removal is specific to Aβ mouse models remains to be determined. Here, we examined tau as an excitability modifier in the non-AD nervous system using genetic deletion of tau in mouse and Drosophila models of hyperexcitability. Kcna1(-/-) mice lack Kv1.1-delayed rectifier currents and exhibit severe spontaneous seizures, early lethality, and megencephaly. Young Kcna1(-/-) mice retained wild-type levels of Aβ, tau, and tau phospho-Thr(231). Decreasing tau in Kcna1(-/-) mice reduced hyperexcitability and alleviated seizure-related comorbidities. Tau reduction decreased Kcna1(-/-) video-EEG recorded seizure frequency and duration as well as normalized Kcna1(-/-) hippocampal network hyperexcitability in vitro. Additionally, tau reduction increased Kcna1(-/-) survival and prevented megencephaly and hippocampal hypertrophy, as determined by MRI. Bang-sensitive Drosophila mutants display paralysis and seizures in response to mechanical stimulation, providing a complementary excitability assay for epistatic interactions. We found that tau reduction significantly decreased seizure sensitivity in two independent bang-sensitive mutant models, kcc and eas. Our results indicate that tau plays a general role in regulating intrinsic neuronal network hyperexcitability independently of Aβ overexpression and suggest that reducing tau function could be a viable target for therapeutic intervention in seizure disorders and antiepileptogenesis.

Injury to the neurovasculature is a feature of brain injury and must be addressed to maximize opportunity for improvement. Cerebrovascular dysfunction, manifested by reduction in cerebral blood flow (CBF), is a key factor that worsens outcome after traumatic brain injury (TBI), most notably under conditions of hypotension. We report here that a new class of antioxidants, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), which are nontoxic carbon particles, rapidly restore CBF in a mild TBI/hypotension/resuscitation rat model when administered during resuscitation--a clinically relevant time point. Along with restoration of CBF, there is a concomitant normalization of superoxide and nitric oxide levels. Given the role of poor CBF in determining outcome, this finding is of major importance for improving patient health under clinically relevant conditions during resuscitative care, and it has direct implications for the current TBI/hypotension war-fighter victims in the Afghanistan and Middle East theaters. The results also have relevancy in other related acute circumstances such as stroke and organ transplantation.

Background: The transcription factor NFκB is an important mediator of cell survival and inflammation in the immune system. In the central nervous system (CNS), NFκB signaling has been implicated in regulating neuronal survival following acute pathologic damage such as traumatic brain injury (TBI) and stroke. NFκB is normally bound by the principal inhibitory protein, IκBα, and sequestered in the cytoplasm. Activation of NFκB requires the degradation of IκBα, thereby freeing NFκB to translocate to the nucleus and activate the target genes. Mice deficient in IκBα display deregulated and sustained NFκB activation and early postnatal lethality, highlighting a critical role of IκBα in NFκB regulation., Results: We investigated the role of IκBα in regulating NFκB activity in the brain and the effects of the NFκB/IκBα pathway in mediating neuroinflammation under both physiological and brain injury conditions. We report that astrocytes, but not neurons, exhibit prominent NFκB activity, and that basal NFκB activity in astrocytes is elevated in the absence of IκBα. By generating mice with brain-specific deletion of IκBα, we show that IκBα deficiency does not compromise normal brain development. However, basal neuroinflammation detected by GFAP and Iba1 immunoreactivity is elevated. This leads to impaired inflammatory responses following TBI and worsened brain damage including higher blood brain barrier permeability, increased injury volumes and enlarged ventricle volumes., Conclusions: We conclude that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation. We further demonstrate that IκBα plays an important role in regulating NFκB activity in the brain and a robust NFκB/IκBα-mediated neuroinflammatory response immediately following TBI is beneficial.

Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer's disease. We and others have shown that over expression of the mitochondrial antioxidant superoxide dismutase 2 (SOD-2) can improve many of the pathologies in the Tg2576 mouse model of Alzheimer's disease that harbors the Swedish mutation in the amyloid precursor protein. However, it is not clear if these improvements are due to functional improvements or structural/anatomical changes. To answer this question, we used diffusion tensor imaging (DTI) to assess the structural integrity of white matter tracts in the control mice, Tg2576 mouse and Tg2576 mice over expressing SOD-2. We observed minimal differences in diffusion parameters with SOD-2 over expression in this model indicating that the improvements we previously reported are due to functional changes and not any alterations to the white matter tractography.

Axonal pathology is a prevalent feature of Alzheimer's disease (AD) and is thought to occur predominantly due to the accumulation of amyloid beta (Aβ). However, it remains unclear whether therapeutics geared toward reducing Aβ improves axonal deficits. We have previously used Manganese Enhanced MRI to demonstrate that axonal transport deficits occur before plaque formation in the Tg2576 mouse model of Alzheimer's disease. Here we tested whether axonal transport deficits in the Tg2576 mouse model improve in response to the Aβ42 selective lowering agent R-Flurbiprofen (R-F). We demonstrated that in young animals (before Aβ plaque formation), R-F treatment reduced Aβ42 levels and coincided with a significant improvement in axonal transport (P = 0.0186). However, in older animals (after plaque formation had occurred), we observed that R-F treatment did not reduce Aβ42 levels although we still observed a significant improvement in axonal transport as assessed with MEMRI (P = 0.0329). We then determined that R-F treatment reduced tau hyper-phosphorylation in the older animals. These data indicate that both Aβ42 and tau comprise a role in axonal transport rate deficits in the Tg2576 model of Alzheimer's Disease., (Copyright © 2010 Wiley-Liss, Inc.)

Generation of reactive oxygen species (ROS) causes cellular oxidative damage and has been implicated in the etiology of Alzheimer's disease (AD). In contrast, multiple lines of evidence indicate that ROS can normally modulate long-term potentiation (LTP), a cellular model for memory formation. We recently showed that decreasing the level of superoxide through the overexpression of mitochondrial superoxide dismutase (SOD-2) prevents memory deficits in the Tg2576 mouse model of AD. In the current study, we explored whether AD-related LTP impairments could be prevented when ROS generation from mitochondria was diminished either pharmacologically or via genetic manipulation. In wild-type hippocampal slices treated with exogenous amyloid β peptide (Aβ1-42) and in slices from APP/PS1 mutant mice that model AD, LTP was impaired. The LTP impairments were prevented by MitoQ, a mitochondria-targeted antioxidant, and EUK134, an SOD and catalase mimetic. In contrast, inhibition of NADPH oxidase either by diphenyliodonium (DPI) or by genetically deleting gp91(phox), the key enzymatic component of NADPH oxidase, had no effect on Aβ-induced LTP blockade. Moreover, live staining with MitoSOX Red, a mitochondrial superoxide indicator, combined with confocal microscopy, revealed that Aβ-induced superoxide production could be blunted by MitoQ, but not DPI, in agreement with our electrophysiological findings. Finally, in transgenic mice overexpressing SOD-2, Aβ-induced LTP impairments and superoxide generation were prevented. Our data suggest a causal relationship between mitochondrial ROS imbalance and Aβ-induced impairments in hippocampal synaptic plasticity.