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Pulmonary embolism (PE), along with the closely related condition of deep vein thrombosis, affect an estimated 600,000 patients in the US per year. Untreated, PE carries a mortality rate of 30%. Because many patients experience mild or non-specific symptoms, imaging studies are necessary for definitive diagnosis of PE. Iodinated CT pulmonary angiography (CTPA) is recommended for most patients, while nuclear medicine-based ventilation/perfusion (V/Q) scans are reserved for patients in whom the use of iodine is contraindicated. Magnetic particle imaging (MPI) is an emerging tracer imaging modality with high image contrast (no tissue background signal) and sensitivity to superparamagnetic iron oxide (SPIO) tracer. Importantly, unlike CT or nuclear medicine, MPI uses no ionizing radiation. Further, MPI is not derived from magnetic resonance imaging (MRI); MPI directly images SPIO tracers via their strong electronic magnetization, enabling deep imaging of anatomy including within the lungs, which is very challenging with MRI. Here, the first high-contrast in vivo MPI lung perfusion images of rats are shown using a novel lung perfusion agent, MAA-SPIOs.

PurposeTo provide a comprehensive understanding of how the selection of radiation dose affects the temporal and spatial progression of radiation-induced necrosis in the rat model.Materials and methodsNecrosis was induced with a single fraction of radiation exposure, at doses ranging between 20 and 60 Gy, to the right hemisphere of 8-week-old Fischer rats from a linear accelerator. The development and progression of necrosis in the rats was monitored and quantified every other week with T1- and T2-weighted gadolinium contrast-enhanced MRI studies.ResultsThe time to onset of necrosis was found to be dose-dependent, but after the initial onset, the necrosis progression rate and total volume generated was constant across different doses ranging between 30 and 60 Gy. Radiation doses less than 30 Gy did not develop necrosis within 33 weeks after treatment, indicating a dose threshold existing between 20 and 30 Gy.ConclusionThe highest dose used in this study led to the shortest time to onset of radiation-induced necrosis, while producing comparable disease progression dynamics after the onset. Therefore, for the radiation-induced necrosis rat model using a linear accelerator, the most optimum results were generated from a dose of 60 Gy.

Efficient muscle-tendon performance during cyclical tasks is dependent on both active and passive mechanical tissue properties. Here we examine whether age-related changes in the properties of muscle-tendon units (MTUs) compromise their ability to do work and utilize elastic energy storage. We empirically quantified passive and active properties of the medial gastrocnemius muscle and material properties of the Achilles tendon in young (∼6 mo) and old (∼32 mo) rats. We then used these properties in computer simulations of a Hill-type muscle model operating in series with a Hookean spring. The modeled MTU was driven through sinusoidal length changes and activated at a phase that optimized muscle-tendon tuning to assess the relative contributions of active and passive elements to the force and work in each cycle. In physiologically realistic simulations where young and old MTUs started at similar passive forces and developed similar active forces, the capacity of old MTUs to store elastic energy and produce positive work was compromised. These results suggest that the observed increase in the metabolic cost of locomotion with aging may be in part due to the recruitment of additional muscles to compensate for the reduced work at the primary MTU. Furthermore, the age-related increases in passive stiffness coupled with a reduced active force capacity in the muscle can lead to shifts in the force-length and force-velocity operating range that may significantly impact mechanical and metabolic performance. Our study emphasizes the importance of the interplay between muscle and tendon mechanical properties in shaping MTU performance during cyclical contractions.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

Recurrent synaptic connections between neighboring neurons are a key feature of mammalian cortex, accounting for the vast majority of cortical inputs. Although computational models indicate that reorganization of recurrent connectivity is a primary driver of experience-dependent cortical tuning, the true biological features of recurrent network plasticity are not well identified. Indeed, whether rewiring of connections between cortical neurons occurs during behavioral training, as is widely predicted, remains unknown. Here, we probe M1 recurrent circuits following motor training in adult male rats and find robust synaptic reorganization among functionally related layer 5 neurons, resulting in a 2.5-fold increase in recurrent connection probability. This reorganization is specific to the neuronal subpopulation most relevant for executing the trained motor skill, and behavioral performance was impaired following targeted molecular inhibition of this subpopulation. In contrast, recurrent connectivity is unaffected among neighboring layer 5 neurons largely unrelated to the trained behavior. Training-related corticospinal cells also express increased excitability following training. These findings establish the presence of selective modifications in recurrent cortical networks in adulthood following training. SIGNIFICANCE STATEMENT Recurrent synaptic connections between neighboring neurons are characteristic of cortical architecture, and modifications to these circuits are thought to underlie in part learning in the adult brain. We now show that there are robust changes in recurrent connections in the rat motor cortex upon training on a novel motor task. Motor training results in a 2.5-fold increase in recurrent connectivity, but only within the neuronal subpopulation most relevant for executing the new motor behavior; recurrent connectivity is unaffected among adjoining neurons that do not execute the trained behavior. These findings demonstrate selective reorganization of recurrent synaptic connections in the adult neocortex following novel motor experience, and illuminate fundamental properties of cortical function and plasticity.

Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite’s replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite., Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis rat to Toxoplasma. Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro, Δgra35, Δgra42, and Δgra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane.

Ketamine is a glutamate N-methyl-D-aspartate receptor antagonist that is a rapid acting dissociative anesthetic. It has been proposed to be an adjuvant treatment along with other drugs (atropine, midazolam, pralidoxime) used in the current standard of care (SOC) for organophosphate and nerve agent exposures. Ketamine is a pharmaceutical agent that is readily available to most clinicians in emergency departments and possesses a broad therapeutic index with well characterized effects in humans. The objective of this study was to determine the pharmacokinetic profile of ketamine and its active metabolite, norketamine, in F344 rats following single or repeated intramuscular administrations of subanesthetic levels (7.5 mg/kg or 30 mg/kg) of ketamine with or without the standard of care. Following administration, plasma and brain tissues were collected and analyzed using a liquid chromatography-mass spectrometry method to quantitate ketamine and norketamine. Following sample analysis, the pharmacokinetics were determined using non-compartmental analysis. The addition of the current SOC had a minimal impact on the pharmacokinetics of ketamine following intramuscular administration and repeated dosing at 7.5 mg/kg every 90 min allows for sustained plasma concentrations above 100 ng/mL. The pharmacokinetics of ketamine with and without the SOC in rats supports further investigation of the efficacy of ketamine co-administration with the SOC following nerve agent exposure in animal models.

Key points Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. Abstract TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

Retinal diseases can be induced by a variety of factors, including gene mutations, environmental stresses and dysmetabolic processes. The result is a progressive deterioration of visual function, which sometimes leads to blindness. Many treatments are under investigation, though results are still mostly unsatisfactory and restricted to specific pathologies, particularly in the case of gene therapy. The majority of treatments have been tested in animal models, but very few have progressed to human clinical trials. A relevant approach is to study the relation between the type of treatments and the degenerative characteristics of the animal model to better understand the effectiveness of each therapy. Here we compare the results obtained from different animal models treated with natural compounds (saffron and naringenin) to anticipate the potentiality of a single treatment in different pathologies.

Linoleic acid (LA, 18:2n-6) is a precursor to arachidonic acid (AA, 20:4n-6), which can be converted by brain lipoxygenase and cyclooxygenase (COX) enzymes into various lipid mediators involved in the regulation of brain immunity. Brain AA metabolism is activated in rodents by the bacterial endotoxin, lipopolysaccharide (LPS). This study tested the hypothesis that dietary LA lowering, which limits plasma supply of AA to the brain, reduces LPS-induced upregulation in brain AA metabolism. Male Fischer CDF344 rats fed an adequate LA (5.2% energy (en)) or low LA (0.4% en) diet for 15weeks were infused with LPS (250ng/h) or vehicle into the fourth ventricle for 2days using a mini-osmotic pump. The incorporation rate of intravenously infused unesterified 14C-AA into brain lipids, eicosanoids, and activities of phospholipase A2 and COX-1 and 2 enzymes were measured. Dietary LA lowering reduced the LPS-induced increase in prostaglandin E2 concentration and COX-2 activity (P

Skeletal muscles power a broad diversity of animal movements, despite only being able to produce high forces over a limited range of velocities. Pennate muscles use a range of gear ratios, the ratio of muscle shortening velocity to fiber shortening velocity, to partially circumvent these force–velocity constraints. Muscles operate with a high gear ratio at low forces; fibers rotate to greater angles of pennation, enhancing velocity but compromising force. At higher forces, muscles operate with a lower gear ratio; fibers rotate little so limiting muscle shortening velocity, but helping to preserve force. This ability to shift gears is thought to be due to the interplay of contractile force and connective tissue constraints. In order to test this hypothesis, gear ratios were determined in the medial gastrocnemius muscles of both healthy young rats, and old rats where the interaction between contractile and connective tissue properties was assumed to be disrupted. Muscle fiber and aponeurosis stiffness increased with age (P

Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength.

// Marta Anna Kowalik 1 , Andrea Perra 1 , Giovanna Maria Ledda-Columbano 1 , Giuseppe Ippolito 3 , Mauro Piacentini 2,3 , Amedeo Columbano 1 and Laura Falasca 3 1 Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy 2 Department of Biology, University of Rome “Tor Vergata”, Rome, Italy 3 National Institute for Infectious Disease, IRCCS “Lazzaro Spallanzani”, Rome, Italy Correspondence to: Mauro Piacentini, email: // Amedeo Columbano, email: // Keywords : preneoplastic nodules, autophagy, amiodarone, chloroquine, miR-224 Received : November 18, 2015 Accepted : December 23, 2015 Published : December 31, 2015 Abstract Although inhibition of autophagy has been implicated in the onset and progression of cancer cells, it is still unclear whether its dysregulation at early stages of tumorigenesis plays an oncogenic or a tumor suppressor role. To address this question, we employed the Resistant-Hepatocyte rat model to study the very early stages of hepatocellular carcinoma (HCC) development. We detected a different autophagy-related gene expression and changes in the ultrastructural profile comparing the most aggressive preneoplastic lesions, namely those positive for the putative progenitor cell marker cytokeratin-19 (KRT-19) with the negative ones. The ultrastructural and immunohistochemical analyses of KRT-19-positive preneoplastic hepatocytes showed the presence of autophagic vacuoles which was associated with p62, Ambra1 and Beclin1 protein accumulation suggesting that a differential modulation of autophagy occurs at early stages of the oncogenesis in KRT-19-positive vs negative lesions. We observed an overall decrease of the autophagy-related genes transcripts and a strong up-regulation of miR-224 in the KRT-19-positive nodules. Interestingly, the treatment with the autophagy inducer, Amiodarone, caused a marked increase in the proliferation of KRT-19 positive preneoplastic lesions associated with a strong increase of their size; by contrast, Chloroquine, an inhibitor of the autophagic process, led to their reduction. These results show that autophagy modulation is a very early event in hepatocarcinogenesis and is restricted to a hepatocytes subset in the most aggressive preneoplastic lesions. Our findings highlight the induction of autophagy as a permissive condition favouring cancer progression indicating in its inhibition a therapeutic goal to interfere with the development of HCC.

The theta rhythm during waking has been associated with voluntary motor activity and learning processes involving the hippocampus. Theta also occurs continuously during rapid eye movement (REM) sleep where it likely serves memory consolidation. Theta amplitude builds across wakefulness and is the best indicator of the homeostatic need for non-REM (NREM) sleep. Although REM sleep is homeostatically regulated independently of NREM sleep, the drivers of REM sleep regulation are under debate. The dynamics of theta within REM sleep bouts have not been thoroughly explored. We equipped 20 male rats with sleep instrumentation and hippocampal electrodes to measure theta across normal sleep/waking periods over the first 4 h of the sleep phase on two consecutive days. We found that theta power decreased by a third, on average, within individual REM sleep bouts, but recovered between bouts. Thus, there was no general decline in theta power across the duration of the recording period or between days. The time constant of theta power decline within a REM sleep bout was the same whether the bout was short, midlength, or long, and did not predict the behavioral state immediately following the REM sleep bout. Interestingly, the more time spent in NREM sleep prior to REM sleep, the larger the decline in theta power during REM sleep, indicating that REM sleep theta may be homeostatically driven by NREM sleep just as NREM delta power is driven by the length of prior waking and by waking theta. Potential causes and implications for this phenomenon are discussed.

Background Most of the animal models commonly used for preclinical research into Alzheimer’s disease (AD) largely fail to address the pathophysiology, including the impact of known risk factors, of the widely diagnosed sporadic form of the disease. Here, we use a transgenic rat (APP21) that does not develop AD-like pathology spontaneously with age, but does develop pathology following vascular stress. To further the potential of this novel rat model as a much-needed pre-clinical animal model of sporadic AD, we characterize APP21 transgenic rats behaviorally and histologically up to 19 months of age. Methods The open field test was used as a measure of activity; and the Morris water maze was used to assess learning, memory, and strategy shift. Neuronal loss and microglia activation were also assessed throughout the brain. Results APP21 transgenic rats showed deficits in working memory from an early age, yet memory recall performance after 24 and 72 h was equal to that of wildtype rats and did not deteriorate with age. A deficit in strategy shift was observed at 19 months of age in APP21 transgenic rats compared to Fischer wildtype rats. Histologically, APP21 transgenic rats demonstrated accelerated white matter inflammation compared to wildtype rats, but interestingly no differences in neuron loss were observed. Conclusions The combined presence of white matter pathology and executive function deficits mirrored what is often found in patients with mild cognitive impairment or early dementia, and suggests that this rat model will be useful for translationally meaningful studies into the development and prevention of sporadic AD. The presence of widespread white matter inflammation as the only observed pathological correlate for cognitive deficits raises new questions as to the role of neuroinflammation in cognitive decline. Electronic supplementary material The online version of this article (10.1186/s12974-018-1273-7) contains supplementary material, which is available to authorized users.

Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites – a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II) – associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins and lipids from isolated nuclei, Site IDAY 4targets (KD[APPARENT]≈ 2-4 x 10−6M and BMAX[APPARENT]≈ 6 pmols/106cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4targets (KD[APPARENT]≈ 1.5 x 10−3M and BMAX[APPARENT]≈ 350 pmols/106cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

Axon regeneration after spinal cord injury (SCI) is attenuated by growth inhibitory molecules associated with myelin. We report that rat myelin stimulated the growth of axons emerging from rat neural progenitor cells (NPCs) transplanted into sites of SCI in adult rat recipients. When plated on a myelin substrate, neurite outgrowth from rat NPCs and from human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) was enhanced threefold. In vivo, rat NPCs and human iPSC-derived NSCs extended greater numbers of axons through adult central nervous system white matter than through gray matter and preferentially associated with rat host myelin. Mechanistic investigations excluded Nogo receptor signaling as a mediator of stem cell-derived axon growth in response to myelin. Transcriptomic screens of rodent NPCs identified the cell adhesion molecule neuronal growth regulator 1 (Negr1) as one mediator of permissive axon-myelin interactions. The stimulatory effect of myelin-associated proteins on rodent NPCs was developmentally regulated and involved direct activation of the extracellular signal-regulated kinase (ERK). The stimulatory effects of myelin on NPC/NSC axon outgrowth should be investigated further and could potentially be exploited for neural repair after SCI.

Previously, we and others have shown that rodent neural progenitor cells (NPCs) can support functional recovery after cervical and thoracic transection injuries. To extend these observations to a more clinically relevant model of spinal cord injury, we performed unilateral midcervical contusion injuries in Fischer 344 rats. Two-weeks later, E14-derived syngeneic spinal cord-derived multi-potent NPCs were implanted into the lesion cavity. Control animals received either no grafts or fibroblast grafts. The NPCs differentiated into all three neural lineages (neurons, astrocytes, oligodendrocytes) and robustly extended axons into the host spinal cord caudal and rostral to the lesion. Graft-derived axons grew into host gray matter and expressed synaptic proteins in juxtaposition with host neurons. Animals that received NPC grafts exhibited significant recovery of forelimb motor function compared with the two control groups (analysis of variance p

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 μM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.

Anatomical connectivity and lesion studies reveal distinct functional heterogeneity along the dorsal-ventral axis of the hippocampus. The immediate early gene Arc is known to be involved in neural plasticity and memory and can be used as a marker for cell activity that occurs, for example, when hippocampal place cells fire. We report here, that Arc is expressed in a greater proportion of cells in dorsal CA1, CA3, and dentate gyrus (DG), following spatial behavioral experiences compared to ventral hippocampal subregions (dorsal CA1=33%; ventral CA1=13%; dorsal CA3=23%; ventral CA3=8%; and dorsal DG=2.5%; ventral DG=1.2%). The technique used here to obtain estimates of numbers of behavior-driven cells across the dorsal-ventral axis, however, corresponds quite well with samples from available single unit recording studies. Several explanations for the two- to-threefold reduction in spatial behavior-driven cell activity in the ventral hippocampus can be offered. These include anatomical connectivity differences, differential gain of the self-motion signals that appear to alter the scale of place fields and the proportion of active cells, and possibly variations in the neuronal responses to non-spatial information within the hippocampus along its dorso-ventral axis.

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression and pharmacological properties inside living hepatocytes ‒ the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106cells/day). Five ring-hydroxylated and one deacylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT]= 1.64 × 10−7M and VMAX[APPARENT]= 0.1 nmols/106cells/day; and, System II (low-affinity and high-velocity), Km[APPARENT]= 3.25 × 10−5M and VMAX[APPARENT]= 1000 nmols/106cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT]and VMAX[APPARENT]constants of 9.6 × 10−5M and 4.7 nmols/106cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

A majority of patients with orofacial cleft deformity requires cleft repair through a bone graft. However, elevated amount of bone resorption and subsequent bone graft failure remains a significant clinical challenge. Bisphosphonates (BPs), a class of anti-resorptive drugs, may offer great promise in enhancing the clinical success of bone grafting. In this study, we compared the effects of systemic and local delivery of BPs in an intraoral bone graft model in rats. We randomly divided 34 female 20-week-old Fischer F344 Inbred rats into four groups to repair an intraoral critical-sized defect (CSD): (1) Control: CSD without graft (n = 4); (2) Graft/Saline: bone graft with systemic administration of saline 1 week post-operatively (n = 10); (3) Graft/Systemic: bone graft with systemic administration of zoledronic acid 1 week post-operatively (n = 10); and (4) Graft/Local: bone graft pre-treated with zoledronic acid (n = 10). At 6-weeks post-operatively, microCT volumetric analysis showed a significant increase in bone fraction volume (BV/TV) in the Graft/Systemic (62.99 ±14.31%) and Graft/Local (69.35 ±13.18%) groups compared to the Graft/Saline (39.18±10.18%). Similarly, histological analysis demonstrated a significant increase in bone volume in the Graft/Systemic (78.76 ±18.00%) and Graft/Local (89.95 ±4.93%) groups compared to the Graft/Saline (19.74±18.89%). The local delivery approach resulted in the clinical success of bone grafts, with reduced graft resorption and enhanced osteogenesis and bony integration with defect margins while avoiding the effects of BPs on peripheral osteoclastic function. In addition, local delivery of BPs may be superior to systemic delivery with its ease of procedure as it involves simple soaking of bone graft materials in BP solution prior to graft placement into the defect. This new approach may provide convenient and promising clinical applications towards effectively managing cleft patients.

Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC(50) for ∆F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous ∆F508-CFTR-expressing cells or primary cultures of ∆F508/∆F508 human bronchial epithelia. The ΔF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the ∆F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action.

In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.

International audience; The end products of polyunsaturated fatty acid (PUFA) peroxidation, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and isoprostanes (8-iso-PGF2alpha), are widely used as systemic lipid oxidation/oxidative stress biomarkers. However, some of these compounds have also a dietary origin. Thus, replacing dietary saturated fat by PUFAs would improve health but could also increase the formation of such compounds, especially in the case of a pro-oxidant/antioxidant imbalanced diet. Hence, the possible impact of dietary fatty acids and pro-oxidant compounds was studied in rats given diets allowing comparison of the effects of heme iron vs. ferric citrate and of omega-6- vs. omega-3-rich oil on the level of lipid peroxidation/oxidative stress biomarkers. Rats given a heme iron-rich diet without PUFA were used as controls. The results obtained have shown that MDA and the major urinary metabolite of HNE (the mercapturic acid of dihydroxynonane, DHN-MA) were highly dependent on the dietary factors tested, while 8-iso-PGF2alpha was modestly but significantly affected. Intestinal inflammation and tissue fatty acid composition were checked in parallel and could only explain the differences we observed to a limited extent. Thus, the differences in biomarkers were attributed to the formation of lipid oxidation compounds in food or during digestion, their intestinal absorption, and their excretion into urine. Moreover, fecal extracts from the rats fed the heme iron or fish oil diets were highly toxic for immortalized mouse colon cells. Such toxicity can eventually lead to promotion of colorectal carcinogenesis, supporting the epidemiological findings between red meat intake and colorectal cancer risk. Therefore, the analysis of these biomarkers of lipid peroxidation/oxidative stress in urine should be used with caution when dietary factors are not well controlled, while control of their possible dietary intake is needed also because of their pro-inflammatory, toxic, and even cocarcinogenic effects.

We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.

BackgroundBone structure and strength are rapidly lost during conditions of decreased mechanical loading, and aged bones have a diminished ability to adapt to increased mechanical loading. This is a concern for older patients that experience periods of limited mobility or bed rest, but the acute effects of disuse on the bones of aged patients have not been thoroughly described. Previous animal studies have primarily examined the effect of mechanical unloading on young animals. Those that have studied aged animals have exclusively focused on bone loss during unloading and not bone recovery during subsequent reloading. In this study, we investigated the effect of decreased mechanical loading and subsequent reloading on bone using a hindlimb unloading model in Adult (9month old) and Aged (28month old) male rats.MethodsAnimals from both age groups were subjected to 14days of hindlimb unloading followed by up to 7days of reloading. Additional Aged rats were subjected to 7days of forced treadmill exercise during reloading or a total of 28days of reloading. Trabecular and cortical bone structure of the femur were quantified using ex vivo micro-computed tomography (μCT), and mechanical properties were quantified with mechanical testing.ResultsWe found that Adult rats had substantially decreased trabecular bone volume fraction (BV/TV) following unloading (- 27%) while Aged animals did not exhibit significant bone loss following unloading. However, Aged animals had lower trabecular BV/TV after 3days of reloading (- 20% compared to baseline), while trabecular BV/TV of Adult rats was not different from baseline values after 3days of reloading. Trabecular BV/TV of Aged animals remained lower than control animals even with exercise during 7days of reloading and after 28days of reloading.ConclusionsThese data suggest that aged bone is less responsive to both increased and decreased mechanical loading, and that acute periods of disuse may leave older subjects with a long-term deficit in trabecular bone mass. These finding indicate the need for therapeutic strategies to improve the skeletal health of elderly patients during periods of disuse.

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC‐/MSC‐derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold‐perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT‐PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC‐/EV‐treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up‐regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as "globulomers", which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aβ oligomers yielded signs of the disease, including cell culture age-dependent binding of Aβ and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain, Aβ-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease. © 2013 Elsevier Inc.