SiglecF(HI) Marks Late-Stage Neutrophils of the Infarcted Heart: A Single-Cell Transcriptomic Analysis of Neutrophil Diversification.

Background Neutrophils are thought to be short-lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single-cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady-state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late-emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecFHI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post-MI. Validation studies using quantitative polymerase chain reaction of fluorescent-activated cell sorter sorted Ly6G+SiglecFHI and Ly6G+SiglecFLO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA-deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G+SiglecFHI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G+SiglecFHI neutrophils expressed high levels of Myc-regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G+SigF+(Myc+NFÏ°B+) neutrophil that has been overlooked because of negative selection.

Macrophage calcium reporter mice reveal immune cell communication in vitro and in vivo.

Cell communication underlies emergent functions in diverse cell types and tissues. Recent evidence suggests that macrophages are organized in communicating networks, but new tools are needed to quantitatively characterize the resulting cellular conversations. Here, we infer cell communication from spatiotemporal correlations of intracellular calcium dynamics that are non-destructively imaged across cell populations expressing genetically encoded calcium indicators. We describe a hematopoietic calcium reporter mouse (Csf1rCreGCaMP5fl) and a computational analysis pipeline for inferring communication between reporter cells based on "excess synchrony." We observed signals suggestive of cell communication in macrophages treated with immune-stimulatory DNA in vitro and tumor-associated immune cells imaged in a dorsal window chamber model in vivo. Together, the methods described here expand the toolkit for discovery of cell communication events in macrophages and other immune cells.

The myeloid type I interferon response to myocardial infarction begins in bone marrow and is regulated by Nrf2-activated macrophages.

Sterile tissue injury is thought to locally activate innate immune responses via damage-associated molecular patterns (DAMPs). Whether innate immune pathways are remotely activated remains relatively unexplored. Here, by analyzing ~145,000 single-cell transcriptomes at steady state and after myocardial infarction (MI) in mice and humans, we show that the type I interferon (IFN) response, characterized by expression of IFN-stimulated genes (ISGs), begins far from the site of injury, in neutrophil and monocyte progenitors within the bone marrow. In the peripheral blood of patients, we observed defined subsets of ISG-expressing neutrophils and monocytes. In the bone marrow and blood of mice, ISG expression was detected in neutrophils and monocytes and their progenitors, intensified with maturation at steady-state and after MI, and was controlled by Tet2 and Irf3 transcriptional regulators. Within the infarcted heart, ISG-expressing cells were negatively regulated by Nrf2 activation in Ccr2- steady-state cardiac macrophages. Our results show that IFN signaling begins in the bone marrow, implicate multiple transcriptional regulators (Tet2, Irf3, and Nrf2) in governing ISG expression, and provide a clinical biomarker (ISG score) for studying IFN signaling in patients.

Neuronal HIF-1α in the nucleus tractus solitarius contributes to ventilatory acclimatization to hypoxia.

KEY POINTS: We hypothesized that hypoxia inducible factor 1α (HIF-1α) in CNS respiratory centres is necessary for ventilatory acclimatization to hypoxia (VAH); VAH is a time-dependent increase in baseline ventilation and the hypoxic ventilatory response (HVR) occurring over days to weeks of chronic sustained hypoxia (CH). Constitutive deletion of HIF-1α in CNS neurons in transgenic mice tended to blunt the increase in HVR that occurs in wild-type mice with CH. Conditional deletion of HIF-1α in glutamatergic neurons of the nucleus tractus solitarius during CH significantly decreased ventilation in acute hypoxia but not normoxia in CH mice. These effects are not explained by changes in metabolic rate, nor CO2 , and there were no changes in the HVR in normoxic mice. HIF-1α mediated changes in gene expression in CNS respiratory centres are necessary in addition to plasticity of arterial chemoreceptors for normal VAH. ABSTRACT: Chronic hypoxia (CH) produces a time-dependent increase of resting ventilation and the hypoxic ventilatory response (HVR) that is called ventilatory acclimatization to hypoxia (VAH). VAH involves plasticity in arterial chemoreceptors and the CNS [e.g. nucleus tractus solitarius (NTS)], although the signals for this plasticity are not known. We hypothesized that hypoxia inducible factor 1α (HIF-1α), an O2 -sensitive transcription factor, is necessary in the NTS for normal VAH. We tested this in two mouse models using loxP-Cre gene deletion. First, HIF-1α was constitutively deleted in CNS neurons (CNS-HIF-1α-/- ) by breeding HIF-1α floxed mice with mice expressing Cre-recombinase driven by the calcium/calmodulin-dependent protein kinase IIα promoter. Second, HIF-1α was deleted in NTS neurons in adult mice (NTS-HIF-1α-/- ) by microinjecting adeno-associated virus that expressed Cre-recombinase in HIF-1α floxed mice. In normoxic control mice, HIF-1α deletion in the CNS or NTS did not affect ventilation, nor the acute HVR (10-15 min hypoxic exposure). In mice acclimatized to CH for 1 week, ventilation in hypoxia was blunted in CNS-HIF-1α-/- and significantly decreased in NTS-HIF-1α-/- compared to control mice (P < 0.0001). These changes were not explained by differences in metabolic rate or CO2 . Immunofluorescence showed that HIF-1α deletion in NTS-HIF-1α-/- was restricted to glutamatergic neurons. The results indicate that HIF-1α is a necessary signal for VAH and the previously described plasticity in glutamatergic neurotransmission in the NTS with CH. HIF-1α deletion had no effect on the increase in normoxic ventilation with acclimatization to CH, indicating this is a distinct mechanism from the increased HVR with VAH.

Urocortin 2 Gene Transfer Reduces the Adverse Effects of a Western Diet on Cardiac Function in Mice.

Diabetes mellitus is associated with increased risk of heart failure. It has been previously demonstrated in mice that a single injection of adeno-associated virus 8 encoding urocortin 2 (AAV8.UCn2) increases glucose disposal in models of insulin resistance and improves the function of the failing heart. The present study tested the hypothesis that UCn2 gene transfer would reduce diabetes-related left ventricular (LV) dysfunction. Eight-week-old C57BL6 male mice were fed a Western diet (WD; 45% fat, 35% carbohydrate) for 40 weeks. At week 30, they received saline or AAV8.UCn2 (2 × 1013 genome copies/kg) via intravenous injection. Ten weeks after gene transfer, fasting blood glucose, glucose tolerance, and cardiac function were measured via echocardiography and in vivo measurement of LV contractile function, and the results were compared to those of mice fed normal chow (NC; 10% fat; 70% carbohydrate). The contents of key LV signaling proteins were also measured to probe mechanisms. WD increased 12 h fasting glucose (WD: 190 ± 11 mg/dL, n = 8; NC: 105 ± 12 mg/dL, n = 7; p = 0.0004). WD tended to reduce LV peak +dP/dt (p = 0.08) and LV peak -dP/dt (p = 0.05). LV ejection fraction was unchanged. Among WD-fed mice, UCn2 gene transfer reduced 12 h fasting glucose (WD-UCn2: 149 ± 6 mg/dL, n = 8; WD-Saline: 190 ± 11 mg/dL, n = 8; p = 0.012), increased LV peak +dP/dt (p < 0.001) and LV peak -dP/dt (p = 0.013), and reduced Tau (p < 0.02), indicating beneficial effects on systolic and diastolic LV function. In addition, among WD-fed mice, UCn2 gene transfer increased LV ejection fraction (p < 0.005) and the velocity of circumferential fiber shortening (p = 0.0005). Finally, a reduction was seen in fatty infiltration of the liver in WD-fed mice that had received UCn2 gene transfer. LV samples from WD-UCn2 mice showed increased phosphorylation of the protein kinase A catalytic domain (p = 0.03). In conclusion, UCn2 gene transfer increased LV systolic and diastolic function and reduced blood glucose in mice with diabetes-related LV dysfunction, indicating that UCn2 gene transfer may be of potential therapeutic benefit.

Transcriptomic analysis identifies a role of PI3K-Akt signalling in the responses of skeletal muscle to acute hypoxia in vivo.

KEY POINTS: Changes in gene expression that occur within hours of exposure to hypoxia in in vivo skeletal muscles remain unexplored. Two hours of hypoxia caused significant down-regulation of extracellular matrix genes followed by a shift at 6 h to altered expression of genes associated with the nuclear lumen while respiratory and blood gases were stabilized. Enrichment analysis of mRNAs classified by stability rates suggests an attenuation of post-transcriptional regulation within hours of hypoxic exposure, where PI3K-Akt signalling was suggested to have a nodal role by pathway analysis. Experimental measurements and bioinformatic analyses suggested that the dephosphorylation of Akt after 2 h of hypoxic exposure might deactivate RNA-binding protein BRF1, hence resulting in the selective degradation of mRNAs. ABSTRACT: The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia-tolerant tissues like skeletal muscles. We used RNA-seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down-regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down-regulated genes had mRNA half-lives longer than 2 h, suggesting a role for post-transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K-Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K-Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K-Akt targets, 75% could be explained by a deactivation of adenylate-uridylate-rich element (ARE)-binding protein BRF1, a target of PI3K-Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K-Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the dephosphorylation of ARE-binding protein BRF1.

Minocycline blocks glial cell activation and ventilatory acclimatization to hypoxia.

Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation, which persists upon return to normoxia and involves plasticity in both central nervous system respiratory centers and peripheral chemoreceptors. We investigated the role of glial cells in VAH in male Sprague-Dawley rats using minocycline, an antibiotic that inhibits microglia activation and has anti-inflammatory properties, and barometric pressure plethysmography to measure ventilation. Rats received either minocycline (45mg/kg ip daily) or saline beginning 1 day before and during 7 days of chronic hypoxia (CH, PiO2 = 70 Torr). Minocycline had no effect on normoxic control rats or the hypercapnic ventilatory response in CH rats, but minocycline significantly (P < 0.001) decreased ventilation during acute hypoxia in CH rats. However, minocycline administration during only the last 3 days of CH did not reverse VAH. Microglia and astrocyte activation in the nucleus tractus solitarius was quantified from 30 min to 7 days of CH. Microglia showed an active morphology (shorter and fewer branches) after 1 h of hypoxia and returned to the control state (longer filaments and extensive branching) after 4 h of CH. Astrocytes increased glial fibrillary acidic protein antibody immunofluorescent intensity, indicating activation, at both 4 and 24 h of CH. Minocycline had no effect on glia in normoxia but significantly decreased microglia activation at 1 h of CH and astrocyte activation at 24 h of CH. These results support a role for glial cells, providing an early signal for the induction but not maintenance of neural plasticity underlying ventilatory acclimatization to hypoxia.NEW & NOTEWORTHY The signals for neural plasticity in medullary respiratory centers underlying ventilatory acclimatization to chronic hypoxia are unknown. We show that chronic hypoxia activates microglia and subsequently astrocytes. Minocycline, an antibiotic that blocks microglial activation and has anti-inflammatory properties, also blocks astrocyte activation in respiratory centers during chronic hypoxia and ventilatory acclimatization. However, minocycline cannot reverse ventilatory acclimatization after it is established. Hence, glial cells may provide signals that initiate but do not sustain ventilatory acclimatization.

Spectroscopic Properties of Energy Transfer Effect in Sm3+/Eu3+ Doped LaF3 Nanocrystals.

The samples of LaF3∶Sm3+, LaF3∶Eu3+ and LaF3∶Sm3+/Eu3+nanocrystals with high quality mono-disperse and uniform sizes were synthesized with hydrothermal method. The crystallographic phase, surface morphology, crystalline sizes and fluorescence properties of Sm3+/Eu3+ sole- and co-doped nanocrystals were characterized with X-ray powder diffraction (XRD), transmission electron microscopy (TEM) and photoluminescence (PL) spectroscopic technique, respectively. The results of XRD and TEM show that the microstructure of the nanocrystals is hexagonal, with the average size about 40 nm. Using 442 nm He-Cd continuous wave (CW) laser to pump the Sm3+ ions doped in the LaF3∶Sm3+/Eu3+nanocrystals, the typical fluorescence emissions originating from the Eu3+ions were observed in the emission spectra, and that the energy transfer from Sm3+ions to Eu3+ions was completed. The mechanism and efficiency of the energy transfer from Sm3+ions to Eu3+ ions were investigated and discussed systematically based on the spectroscopic method. It is shown that the energy transfer of Sm3+→Eu3+ is attributed to the cross-relaxation between the excited state 4G5/2 of Sm3+ ion and the 5D1 and 5D0 states of Eu3+ ion. Meanwhile, the intensities of the characteristic fluorescence emissions of Eu3+ ions become stronger and stronger with the increase of the Eu3+ doping concentration, which suggest that the efficiency of energy transfer from Sm3+ ions to Eu3+ ions can be effectively improved by increasing the doping concentration of Eu3+ acceptor.

A Protocol to Collect Specific Mouse Skeletal Muscles for Metabolomics Studies.

Due to the highly sensitive nature of metabolic states, the quality of metabolomics data depends on the suitability of the experimental procedure. Metabolism could be affected by factors such as the method of euthanasia of the animals and the sample collection procedures. The effects of these factors on metabolites are tissue-specific. Thus, it is important to select proper methods to sacrifice the animal and appropriate procedures for collecting samples specific to the tissue of interest. Here, we present our protocol to collect specific mouse skeletal muscles with different fiber types for metabolomics studies. We also provide a protocol to measure lactate levels in tissue samples as a way to estimate the metabolic state in collected samples.

No evidence of a role for neuronal nitric oxide synthase in the nucleus tractus solitarius in ventilatory responses to acute or chronic hypoxia in awake rats.

When exposed to a hypoxic environment, the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR and is termed ventilatory acclimatization to hypoxia (VAH). This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. The mechanisms of HVR plasticity are currently poorly understood. We hypothesized that changes in neuronal nitric oxide synthase (nNOS) activity or expression in the nucleus tractus solitarius contribute to this plasticity and underlie VAH in rats. To test this, we treated rats held in normoxia or 10% O2 (CSH, PIO2 = 70 Torr) for 7-9 days and measured ventilation in conscious, unrestrained animals before and after microinjecting the general NOS antagonist L-NG-Nitroarginine methyl ester into the nucleus tractus solitarius (NTS) or systemically injecting the nNOS-specific antagonist S-methyl-l-thiocitrulline. Localization of injection sites in the NTS was confirmed by histology following the experiment. We found that 1) neither NTS-specific nor systemic nNOS antagonism had any effect on hypoxia-mediated changes in breathing or metabolism (P > 0.05), but 2) nNOS protein expression was increased in the middle and caudal NTS by CSH. A persistent HVR after nNOS blockade in the NTS contrasts with results in awake mice, and our findings do not support the hypotheses that nNOS in the NTS contribute to the HVR or VAH in awake rats.

Glutamate receptors in the nucleus tractus solitarius contribute to ventilatory acclimatization to hypoxia in rat.

When exposed to a hypoxic environment the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR. This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. Currently these mechanisms of HVR plasticity are unknown and we hypothesized that they involve glutamatergic synapses in the nucleus tractus solitarius (NTS), where afferent endings from arterial chemoreceptors terminate. To test this, we treated rats held in normoxia (CON) or 10% O2 (CSH) for 7 days and measured ventilation in conscious, unrestrained animals before and after microinjecting glutamate receptor agonists and antagonists into the NTS. In normoxia, AMPA increased ventilation 25% and 50% in CON and CSH, respectively, while NMDA doubled ventilation in both groups (P < 0.05). Specific AMPA and NMDA receptor antagonists (NBQX and MK801, respectively) abolished these effects. MK801 significantly decreased the HVR in CON rats, and completely blocked the acute HVR in CSH rats but had no effect on ventilation in normoxia. NBQX decreased ventilation whenever it was increased relative to normoxic controls; i.e. acute hypoxia in CON and CSH, and normoxia in CSH. These results support our hypothesis that glutamate receptors in the NTS contribute to plasticity in the HVR with CSH. The mechanism underlying this synaptic plasticity is probably glutamate receptor modification, as in CSH rats the expression of phosphorylated NR1 and GluR1 proteins in the NTS increased 35% and 70%, respectively, relative to that in CON rats.

Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung.

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection.

[Spectroscopic study on the energy transfer effect in Sm3+/Eu3+ codoped tetragonal LaOF nanocrystals system].

The powder samples of Sm3+/Eu3+ co-doped in LaOF nanocrystals were synthesized by using a hydrothermal-sintering technique. The characterization results of XRD and TE M suggests that t he structure of the nanocrystals is tetragonal,and its size is between 60-80 nm. Using 442 nm laser to pump the LaOF: Sm3+/Eu3+ nanocrystals samples, the energy transfer effect from Sm3+ ions to Eu3+ ions was completed, and the fluorescence emissions originating from the 5D0 level of Eu3+ were observed. The luminescence properties of the LaOF: Sm3+/Eu3+ nanocrystals system were analyzed with a spectroscopic method, and the mechanism and dynamic process of the energy transfer were explored. It was found that the efficiency of the energy transfer increase with the increase in the concentration of Eu3+ ions and the size of nanocrystals.

Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats.

During ventilatory acclimatization to hypoxia (VAH), time-dependent increases in ventilation lower Pco(2) levels, and this persists on return to normoxia. We hypothesized that plasticity in the caudal nucleus tractus solitarii (NTS) contributes to VAH, as the NTS receives the first synapse from the carotid body chemoreceptor afferents and also contains CO(2)-sensitive neurons. We lesioned cells in the caudal NTS containing the neurokinin-1 receptor by microinjecting the neurotoxin saporin conjugated to substance P and measured ventilatory responses in awake, unrestrained rats 18 days later. Lesions did not affect hypoxic or hypercapnic ventilatory responses in normoxic control rats, in contrast to published reports for similar lesions in other central chemosensitive areas. Also, lesions did not affect the hypercapnic ventilatory response in chronically hypoxic rats (inspired Po(2) = 90 Torr for 7 days). These results suggest functional differences between central chemoreceptor sites. However, lesions significantly increased ventilation in normoxia or acute hypoxia in chronically hypoxic rats. Hence, chronic hypoxia increases an inhibitory effect of neurokinin-1 receptor neurons in the NTS on ventilatory drive, indicating that these neurons contribute to plasticity during chronic hypoxia, although such plasticity does not explain VAH.

Ibuprofen blocks time-dependent increases in hypoxic ventilation in rats.

Recently, inflammatory processes have been shown to increase O(2)-sensitivity of the carotid body during chronic sustained hypoxia [Liu, X., He, L., Stensaas, L., Dinger, B., Fidone, S., 2009. Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. Am. J. Physiol. Lung Cell Mol. Physiol. 296, L158-L166]. We hypothesized that blocking inflammation with ibuprofen would reduce ventilatory acclimatization to hypoxia by blocking such increases in carotid body O(2) sensitivity. We tested this in conscious rats treated with ibuprofen (4mg/kg IP daily) or saline during acclimatization to hypoxia ( [Formula: see text] for 7 days). Ibuprofen blocked the increase in hypoxic ventilation observed in chronically hypoxic rats treated with saline; ibuprofen had no effects on ventilation in normoxic control rats. Ibuprofen blocked increases in inflammatory cytokines (IL-1ß, IL-6) in the brainstem with chronic hypoxia. The data supports our hypothesis and further analysis indicates that ibuprofen also blocks inflammatory processes in the central nervous system contributing to ventilatory acclimatization to hypoxia. Possible mechanisms linking inflammatory and hypoxic signaling are reviewed.

Delta-sarcoglycan gene therapy halts progression of cardiac dysfunction, improves respiratory failure, and prolongs life in myopathic hamsters.

BACKGROUND: The BIO14.6 hamster provides a useful model of hereditary cardiomyopathies and muscular dystrophy. Previous δ-sarcoglycan (δSG) gene therapy (GT) studies were limited to neonatal and young adult animals and prevented the development of cardiac and skeletal muscle dysfunction. GT of a pseudophosphorylated mutant of phospholamban (S16EPLN) moderately alleviated the progression of cardiomyopathy. METHODS AND RESULTS: We treated 4-month-old BIO14.6 hamsters with established cardiac and skeletal muscle diseases intravenously with a serotype-9 adeno-associated viral vector carrying δSG alone or in combination with S16EPLN. Before treatment at age 14 weeks, the left ventricular fractional shortening by echocardiography was 31.3% versus 45.8% in normal hamsters. In a randomized trial, GT halted progression of left ventricular dilation and left ventricular dysfunction. Also, respiratory function improved. Addition of S16EPLN had no significant additional effects. δSG-GT prevented severe degeneration of the transverse tubular system in cardiomyocytes (electron tomography) and restored distribution of dystrophin and caveolin-3. All placebo-treated hamsters, except animals removed for the hemodynamic study, died with heart failure between 34 and 67 weeks of age. In the GT group, signs of cardiac and respiratory failure did not develop, and animals lived for 92 weeks or longer, an age comparable to that reported in normal hamsters. CONCLUSION: GT was highly effective in BIO14.6 hamsters even when given in late-stage disease, a finding that may carry implications for the future treatment of hereditary cardiac and muscle diseases in humans.

The asparaginyl hydroxylase factor inhibiting HIF-1alpha is an essential regulator of metabolism.

Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, and improved glucose and lipid homeostasis and are resistant to high-fat-diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, and enhanced insulin sensitivity and are also protected against high-fat-diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.

Pulmonary artery pressure responses to increased cardiac output in chickens with raised metabolic rate.

Previous work has shown remarkable differences in the pressure-flow relations of the pulmonary circulation between birds and mammals. For example several studies suggest that the avian pulmonary blood vessels behave like rigid tubes, very different from the situation in mammalian lung. We therefore speculated that birds would develop high pulmonary artery pressures when the cardiac output was substantially increased during heavy exercise, for example during flight. However because of the technical difficulties of measuring pulmonary artery pressures in flight, the metabolic rate and cardiac output in anesthetized chickens were increased by infusing 2,4 Dinitrophenol (DNP) and the mean pressure was measured by means of a catheter in the pulmonary artery. Although the pulmonary artery pressure rose steadily as cardiac output increased, it remained below the high levels predicted from the previous studies for similar changes in pulmonary blood flow. Furthermore the increase in pressure was less than in mammals where recruitment and distension of pulmonary capillaries are known to occur. The reasons for this unexpected result are not clear.

Influence of crystal structure on the fluorescence emission of Eu3+:LaOF nanocrystals.

Optical transitions of Eu(3+)-doped LaOF nanocrystals with tetragonal and rhombohedral structures were studied experimentally using laser spectroscopic method. Fluorescence emissions from 5D1 and 5D0 of Eu3+ in LaOF nanocrystals were detected by exciting the sample with a 532 nm pulsed laser, and the influence of local symmetry on the fluorescence emission of doped ions was observed. The specific crystalline phase as well as the local symmetry on the fluorescence emission was investigated in both the frequency domain and time domain. It was found that Eu3+ ions doped in the tetragonal crystal structure present more efficient fluorescence emission, which was explained by lower symmetric distribution of local field at the doped ions.

Investigation of radiative and nonradiative relaxation by two-photon resonant excitation in Tm3+ doped LaF3 particles.

Nano-scaled and micro-scaled Tm(3+)-doped LaF3 were prepared by a hydrothermal technique. Two-photon resonant excitation from the ground state 3H6 to the excited state 1D2 was fulfilled by two pulsed dye lasers tuned at 656 nm and 643 nm, respectively, which were resonant with the ground state absorption of 3H6 --> 3F2 and the excited state absorption of 3H4 --> 1D2. The time evolution of the corresponding state was investigated with blue upconverted emission by tuning the delay of two excitation lights. The results showed that the nonradiative relaxation of the 3F2 state was mainly affected by the confinement effect, while the radiative relaxation of 3H4 state was significantly influenced by the Tm3+ concentration.