Deuteration of heptamethine cyanine dyes enhances their emission efficacy.

The design of bright short-wave infrared fluorophores remains a grand challenge. Here we investigate the impact of deuteration on the properties in a series of heptamethine dyes, the absorption of which spans near-infrared and SWIR regions. We demonstrate that it is a generally applicable strategy that leads to enhanced quantum yields of fluorescence, longer-lived singlet excited states and suppressed rates of non-radiative deactivation processes.

De novo design of energy transfer proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a "special pair" of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independent of complexities of native photosynthetic proteins, and as a first step towards synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that precisely position chlorophyll dimers. X-ray crystallography shows that one designed protein binds two chlorophylls in a binding orientation matching native special pairs, while a second positions them in a previously unseen geometry. Spectroscopy reveals excitonic coupling, and fluorescence lifetime imaging demonstrates energy transfer. We designed special pair proteins to assemble into 24-chlorophyll octahedral nanocages; the design model and cryo-EM structure are nearly identical. The design accuracy and energy transfer function of these special pair proteins suggest that de novo design of artificial photosynthetic systems is within reach of current computational methods.

Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

Decay-Associated Fourier Spectroscopy: Visible to Shortwave Infrared Time-Resolved Photoluminescence Spectra.

We describe and implement an interferometric approach to decay-associated photoluminescence spectroscopy, which we term decay-associated Fourier spectroscopy (DAFS). In DAFS, the emitted photon stream from a substrate passes through a variable path length Mach-Zehnder interferometer prior to detection and timing. The interferometer encodes spectral information in the intensity measured at each detector enabling simultaneous spectral and temporal resolution. We detail several advantages of DAFS, including wavelength-range insensitivity, drift-noise cancellation, and optical mode retention. DAFS allows us to direct the photon stream into an optical fiber, enabling the implementation of superconducting nanowire single photon detectors for energy-resolved spectroscopy in the shortwave infrared spectral window (λ = 1-2 µm). We demonstrate the broad applicability of DAFS, in both the visible and shortwave infrared, using two Förster resonance energy transfer pairs: a pair operating with conventional visible wavelengths and a pair showing concurrent acquisition in the visible and the shortwave infrared regime.

Obstructive sleep apnea: an unexpected cause of insulin resistance and diabetes.

Obstructive sleep apnea (OSA) is prevalent with type 2 diabetes. Conversely, nondiabetic patients with OSA are at increased risk of developing insulin resistance and diabetes. These disorders independently contribute to increased cardiovascular and cerebrovascular morbidity and mortality. The pathophysiology of OSA may help explain these associations. Evidence demonstrates that treatment of OSA with continuous positive airway pressure may lead to improvement in insulin sensitivity, hemoglobin A1c, systemic hypertension, and other components of the metabolic syndrome. Recognizing and treating OSA in patients with insulin resistance or diabetes ameliorates OSA-related symptoms and improves cardiometabolic risk.

Respiratory effort-related arousals contribute to sympathetic modulation of heart rate variability.

PURPOSE: Sympathetic activation induced by sleep-disordered breathing may contribute to cardiovascular morbidity. However, the apnea-hypopnea index (AHI) excludes respiratory effort-related arousals (RERAs) associated with inspiratory flow limitation without oxygen desaturation. We sought to determine whether RERAs are associated with sympathetic activation. METHODS: Twenty-five adults (12 males, 13 females) with AHI < 10/h and RERA index >5/h were included in this study. Power spectral density analysis was performed on two non-contiguous 10-min segments containing inspiratory flow limitation and arrhythmia-free electrocardiogram during N2 sleep. One segment contained RERA; the other did not, NO-RERA. Spectral power was described in a low-frequency domain (LF; 0.04-0.15 Hz), primarily sympathetic modulation, and a high frequency domain (HF; 0.15-0.4 Hz), parasympathetic modulation. RESULTS: Analyses of LF and HF powers were made using normalized and absolute values. LF power was greater during RERA compared to NO-RERA (50.3 vs. 30.1 %, p < 0.001) whereas HF power was greater during NO-RERA compared to RERA (69.9 vs. 49.7 %, p < 0.001). The LF/HF ratio was greater during RERA than NO-RERA (1.01 vs. 0.43, p < 0.001). Gender differences emerged using absolute values of power: The percentage increase in LF power during RERA relative to NO-RERA was significantly greater for females than males, 247.6 vs. 31.9 %, respectively (p < 0.02). CONCLUSIONS: RERAs are associated with a marked increase in cardiac sympathetic modulation, especially in females. Patients with a high RERA index, even in the setting of a low or normal AHI, may be exposed to elevated sympathetic tone during sleep.

Mg deficiency results in modulation of serum lipids, glutathione, and NO synthase isozyme activation in cardiovascular tissues: relevance to de novo synthesis of ceramide, serum Mg and atherogenesis.

The present work tested the hypothesis that short-term (S-T) dietary deficiency of magnesium (Mg) (21 days) in rats would: 1) result in reduction in serum(s) sphingomyelin (SM) and changes in several blood lipids, HDL-cholesterol (HDL-C) and phosphatidylcholine (PC) concomitant with elevations in s cholesterol (chol), s LDL+VLDL and trigycerides (TG), as well as reduction in the PC/cholesterol ratio; 2) lead to oxidative stress, characterized by reductions in glutathione (glut) content in the various chambers of the heart and activation of e-NOS and n-NOS in the atria, ventricles and aortic smooth muscle (ASM); 3) produce early cardiac damage characterized by leakage of creatine kinase (CK) and lactic dehydrogenase (LDH); and 4) demonstrate that these pathophysiological changes are a result of profound reductions in s ionized Mg (Mg(2+)) and activation of the SM-ceramide pathway. In addition, we hypothesized that: 1) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg(2+) would lead to de novo synthesis of ceramide and activation of NO synthase with reduction in glut, both of which would be attenuated by inhibition of sphingomyelinase (SMase) and serine palmitoyl CoA transferase (SPT); and 2) low levels of Mg(2+)added to the drinking water would either prevent or ameliorate these manifestations. Our data indicate that S-T Mg deficiency resulted in reductions in s Mg(2+), SM, PC, HDL-C and the PC/chol ratio concomitant with decreases in tissue levels of glut, leakage of cardiac CK and LDH, as well as activation of e-NOS and n-NOS in all chambers of the heart and ASM. The greater the reduction in s Mg(2+), the greater the effects on all parameters analyzed; very significant correlations to levels of s SM and Mg(2+) were found with all of the serum and tissue biochemical -molecular analytes measured. Our experiments also showed that VSMCs exposed to low Mg(2+)resulted in activation of NO synthase, loss of glut and de novo synthesis of ceramide which were attenuated by inhibitors of SMase and SPT. Low levels of drinking water Mg(2+)(e.g., 15 ppm) were cardio- and vascular protective. We believe these new findings support our concept of an important role for the SM-ceramide pathway in the manifestations of Mg deficiency and atherogenesis.

Correlation of pulmonary hypertension severity with metrics of comorbid sleep-disordered breathing.

PURPOSE: We performed nocturnal polysomnography in patients with pulmonary hypertension (PH) of varying etiologies to determine the association of metrics describing sleep-disordered breathing (SDB) with measures of PH severity. METHODS: Consecutive patients referred for evaluation of dyspnea on exertion and elevated pulmonary arterial pressure >30 mmHg on echocardiography, who underwent right and left heart catheterization and polysomnography, were included. Patients were not pre-selected for symptoms of sleep-disordered breathing. RESULTS: Twenty-eight patients including 22 females and six males with a mean age of 55.2 ± 11.9 years were evaluated. Etiologies of PH were idiopathic (32%) and PH associated with other diseases (68%). Most were World Health Organization (WHO) Functional class II (39%) and III (39%). The group mean pulmonary arterial pressure (mPAP) was 40.9 ± 15.1 mmHg. Diurnal resting and exercise arterial oxygen saturations (SaO(2)) were 94.9 ± 3.7% and 88.3 ± 8.9%. The mean apnea-hypopnea index (AHI) was 11.4 ± 19.8/h; 50% of all patients had an AHI ≥ 5/h; 30.6 ± 36.0% of total sleep time was spent with SaO(2) < 90% (T90%); 66% of subjects with an AHI ≥ 5/h of sleep reported snoring, and 60% noted daytime somnolence; however, only 29% had an Epworth Sleepiness Scale ≥10. Right atrial pressure and mPAP were significantly correlated with AHI and T90%. The best predictive model relating PH severity to metrics of SDB was a highly significant association (p = 0.005) between mPAP and a linear combination of AHI and T90%. CONCLUSIONS: SDB comprised of obstructive apneas, hypopneas, and nocturnal hypoxemia is prevalent in PH and cannot be accurately predicted by sleep apnea signs and symptoms or diurnal rest and exercise SaO(2). The association of AHI and T90% with mPAP suggests a potential relationship between the pathophysiology of sleep-disordered breathing and PH.

Short-term magnesium deficiency results in decreased levels of serum sphingomyelin, lipid peroxidation, and apoptosis in cardiovascular tissues.

The present study tested the hypothesis that short-term dietary deficiency of magnesium (Mg) (21 days) in rats would 1) result in decreased serum(s) [the present study tested the levels of Mg, sphingomyelin (SM), and phosphatidylcholine (PC)]; 2) promote DNA fragmentation, lipid peroxidation (LP), and activation of caspase-3 in cardiac (ventricular and atrial) and vascular(aortic) muscle; and 3) low levels of Mg(2+) added to drinking water would either prevent or greatly ameliorate these manifestations. The data indicate that short-term Mg deficiency (10% normal dietary intake) resulted in profound reductions in serum-ionized Mg and total Mg with an elevation in serum-ionized calcium (Ca(2+)), significant lowering of serum SM and serum PC, with concomitant LP, DNA fragmentation, and activation of caspase-3 in ventricular (right and left chambers), atrial (right and left chambers) and abdominal aortic smooth muscle. The greater the reduction in serum-ionized Mg, the greater the effects on DNA fragmentation, LP, and caspase-3 activity. The intake of water-borne Mg(2+) at all levels greatly attenuated or inhibited the reductions in serum SM and serum PC, activation of LP, DNA fragmentation, and the activation of caspase-3; even very low levels of Mg(2+) in drinking water (i.e., 15 parts.million(-1).day(-1)) were cardio- and vascular protective. In addition, we demonstrate that short-term dietary deficiency of Mg probably results in a downregulation of SM synthase and a decreased synthesis of PC.

Heart rate variability in conscious neonatal swine: spectral features and responses to short-term intermittent hypoxia.

BACKGROUND: Spectral analysis of the cardiac time series has been used as a tool for assessing levels of parasympathetic and sympathetic modulation of the sinoatrial node. In the present investigation we evaluated daily changes in heart rate variability spectra in conscious neonatal piglets that were either neurally intact (n = 5) or had undergone right stellate ganglionectomy (n = 5). The partial stellectomized animals and their intact litter mates were exposed to four days of intermittent hypoxia, each day comprising nine episodes of hypoxia alternating with nine episodes of normoxia. A time control group (n = 7) comprised animals from different litters that were not exposed to intermittent hypoxia. We hypothesized that exposure to intermittent hypoxia would increase sympathetic efferent neuronal modulation of heart rate variability spectra in neurally intact animals and in those with right stellate ganglionectomy, and that his effect would be observed in heart rate variability spectra computed from baseline recordings. RESULTS: Overall, heart rate variability spectra during baseline conditions were dominated by high frequency activity, a reflection of parasympathetic efferent neuronal innervation and linkage to the ventilatory cycle manifested as respiratory sinus arrhythmia. Exposure to intermittent hypoxia did not alter daily baseline spectral features that would indicate an increase of sympathetic cardiac activity: low frequency (0.05 - 0.15 Hz) activity was unaffected and the ratio of low- to -high frequency activity remained less than unity indicating a predominance of high frequency activity. The resultant spectra were remarkably similar despite differences in cardiac sympathetic efferent neuronal innervation and experimental treatment. When spectra were computed from cardiac time series during representative hypoxic episodes, significant increases in activity across the low frequency region (0.05 - 0.15 Hz) of heart rate variability spectra were noted and were comparable in neurally intact animals and in those with right stellate ganglionectomy. CONCLUSION: The findings of this investigation provided important information regarding sympathetic efferent neuronal innervation of the heart during the neonatal period. Both neurally intact animals and those with right stellate ganglionectomy had equivalent increases of activity in the low frequency region of heart rate variability spectra during hypoxic stimulation. Such a finding demonstrated the capability of residual cardiac sympathetic neuronal innervation to affect functionally appropriate changes in cardiac chronotropy.

The cardiac-related rhythm in preganglionic sympathetic activities of developing piglets.

This investigation was performed to determine whether partial spectral analysis of preganglionic sympathetic nerve discharges would reveal age-related differences in the distribution of baroreceptor afferent information to brainstem sympathetic-related neurons. Any influence of baroreceptor afferent activity on ordinary spectra of cervical sympathetic and splanchnic nerves was removed by partialization using the arterial blood pressure signal which represented baroreceptor activity. An absence of statistically significant coherence in partialized nerve spectra would indicate that sympathetic-related neurons receive peripheral baroreceptor afferent input, but are not interconnected, whereas the presence of significant coherence would mean that these neurons are interconnected. Ordinary spectral analysis did not demonstrate age-related differences in the relationship between nerve activity and baroreceptor afferent input. In many animals, large peaks, located at cardiac frequencies (range 2.75-5.6 Hz), were noted in ordinary nerve autopower spectra, and were significantly correlated in ordinary coherence spectra. Partialization of nerve spectra eliminated or reduced cardiac-related peaks in autopower spectra regardless of age, and, in 8 of 10 animals, reduced coherence estimates to non-significant values. In two animals, 19 and 36 days old, significant coherence values remained after partialization. These results demonstrated that cardiac-related peaks in coherence in spectra of preganglionic splanchnic and cervical sympathetic nerves were dependent upon peripheral afferent baroreceptor input in most animals. Further, the finding that significant residual coherence was absent in most cases suggested a paucity of intrabulbar pathways connecting brainstem sympathetic-related neurons.

Hypercapnia induces long-term changes in postganglionic renal nerve activity in the piglet.

In developing swine, time and frequency domain analyses were used to compare changes in discharge features of efferent phrenic and postganglionic renal nerve activities evoked by prolonged (1 h) exposure to severe hypercapnia (10% CO2, balance O2), before and after combined carotid sinus and aortic depressor nerve (CSN-AOD) sectioning. With intact CSN-AOD innervation, respiration-related activity in renal nerve discharge was rare (3 of 11 animals) during baseline periods with intact innervation, but was observed in most cases (10 of 11 animals) during baseline following denervation. Renal nerve respiration-related activity was recruited by hypercapnic stimulation in animals with intact CSN-AOD innervation, and was augmented in denervated animals with ongoing respiratory activity. Phrenic nerve discharge was markedly augmented during hypercapnia, whether CSN-AOD innervation was intact or not, and it did not exhibit a post-hypercapnic depression. Autopower spectra of renal nerve activity revealed the presence of two coexisting rhythms, 2-6 and 7-13 Hz, which were present whether CSN-AOD innervation was intact or not. The hypercapnic-induced increases of activity in the 2-6 and 7-13 Hz bands were not comparable, with the latter region exhibiting a much more robust response to hypercapnia, especially following CSN-AOD denervation. Thus, prolonged exposure to hypercapnia evoked changes in renal nerve discharge that involved increased coupling to neuronal ensembles shaping central inspiratory activity and those generating central sympathetic outflows, especially to networks generating 7-13 Hz rhythm. Such changes may permit more efficient modulation of innervated structures during exposure to stressors.

Developmental changes in heart rate variability during exposure to prolonged hypercapnia in piglets.

The hypothesis that hypercapnia-induced differences in heart rate variability (HRV) would emerge during early maturation was tested using a developing porcine model. Piglets were randomly assigned to either exposed (10% CO2 for 1 h) or control (100% O2) conditions, and then to one of three study groups: (a) 5-8 days old, (b) 13-15 days old, (c) 26-34 days old. Experiments were performed on pairs of age-and litter-matched animals that were anesthetized, paralyzed, and artificially ventilated. HRV was evaluated using power spectral analysis, SD of differences between successive RR intervals, and cardiac interval analysis. Statistical comparisons of simultaneously studied animals were made at baseline, 15 and 55 min after onset of hypercapnia, and 2 h after offset of hypercapnia. Our analyses revealed that only HRV of 26-34-day-old animals differed significantly from values of control animals. Cardiac intervals of those animals were distributed in such a manner that hypercapnia likely elicited coactivation of sympathetic and parasympathetic systems. Comparison of the distribution of cardiac intervals for other animals showed that 5-8-day-old animals had high frequency of balanced intervals at baseline that remained so during hypercapnia. Given that such coactivation may be neuroprotective, the paucity of balanced intervals in 13-15-day-old animals could mean that the end of the second postnatal week is associated with increased vulnerability.

Baroreceptor-related dysrhythmias in piglets with selective autonomic denervation of the heart.

This study was carried out to determine whether selective cardiac autonomic denervation performed on neonatal swine would evoke dysrhythmias later in development. Piglets (n = 27; 5-10 days old) underwent unilateral stellate ganglion ablation, or right cardiac vagotomy, or sham surgery. Fifty to sixty days after denervation, acute experiments were performed to evaluate responses to baroreceptor activation. Of all animals who exhibited prolonged R-R intervals, only those with right stellate ganglion ablation had prolonged corrected QT intervals. Despite findings suggesting an arrhythmogenic state (predominance of left-sided cardiac innervation), dysrhythmias occurred in all animals with stellate ganglion ablation, regardless of laterality, but in few vagotomized or control animals. Our results suggest that partial sympathetic innervation may alter cardiac function so that dysrhythmias are more likely to occur during baroreceptor activation.