Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Positron Emission Tomography Imaging Agent

By dual labeling a targeting moiety with both nuclear and optical probes, the ability for noninvasive imaging and intraoperative guidance may be possible. Herein, the ability to detect metastasis in an immunocompetent animal model of human epidermal growth factor receptor 2 (HER-2)-positive cancer metastases using positron emission tomography (PET) and near-infrared (NIR) fluorescence imaging is demonstrated. METHODS: (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m was synthesized, characterized, and administered to female Balb/c mice subcutaneously inoculated with highly metastatic 4T1.2neu/R breast cancer cells. (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m (150 µg, 150 µCi, m = 2, n = 2) was administered through the tail vein at weeks 2 and 6 after implantation, and PET/computed tomography and NIR fluorescence imaging were performed 24 hours later. Results were compared with the detection capabilities of F-18 fluorodeoxyglucose (¹⁸FDG-PET). RESULTS: Primary tumors were visualized with ¹⁸FDG and (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m, but resulting metastases were identified only with the dual-labeled imaging agent. ⁶⁴Cu-PET imaging detected lung metastases, whereas ex vivo NIR fluorescence showed uptake in regions of lung, skin, skeletal muscle, and lymph nodes, which corresponded with the presence of cancer cells as confirmed by histologic hematoxylin and eosin stains. In addition to detecting the agent in lymph nodes, the high signal-to-noise ratio from NIR fluorescence imaging enabled visualization of channels between the primary tumor and the axillary lymph nodes, suggesting a lymphatic route for trafficking cancer cells. Because antibody clearance occurs through the liver, we could not distinguish between nonspecific uptake and liver metastases. CONCLUSION: (⁶⁴Cu-DOTA)_n-trastuzumab-(IRDye800)_m may be an effective diagnostic imaging agent for staging HER-2-positive breast cancer patients and intraoperative resection.     

Dynamic Near-Infrared Optical Imaging of 2-Deoxyglucose Uptake by Intracranial Glioma of Athymic Mice

Background

It is recognized that cancer cells exhibit highly elevated glucose metabolism compared to non-tumor cells. We have applied in vivo optical imaging to study dynamic uptake of a near-infrared dye-labeled glucose analogue, 2-deoxyglucose (2-DG) by orthotopic glioma in a mouse model.

Methodology and Principal Findings

The orthotopic glioma model was established by surgically implanting U87-luc glioma cells into the right caudal nuclear area of nude mice. Intracranial tumor growth was monitored longitudinally by bioluminescence imaging and MRI. When tumor size reached >4 mm diameter, dynamic fluorescence imaging was performed after an injection of the NIR labeled 2-DG, IRDye800CW 2-DG. Real-time whole body images acquired immediately after i.v. infusion clearly visualized the near-infrared dye circulating into various internal organs sequentially. Dynamic fluorescence imaging revealed significantly higher signal intensity in the tumor side of the brain than the contralateral normal brain 24 h after injection (tumor/normal ratio, TNR  = 2.8±0.7). Even stronger contrast was achieved by removing the scalp (TNR  = 3.7±1.1) and skull (TNR  = 4.2±1.1) of the mice. In contrast, a control dye, IRDye800CW carboxylate, showed little difference (1.1±0.2). Ex vivo fluorescence imaging performed on ultrathin cryosections (20 µm) of tumor bearing whole brain revealed distinct tumor margins. Microscopic imaging identified cytoplasmic locations of the 2-DG dye in tumor cells.

Conclusion and Significance

Our results suggest that the near-infrared dye labeled 2-DG may serve as a useful fluorescence imaging probe to noninvasively assess intracranial tumor burden in preclinical animal models.     

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats

Background

Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy.

Methods and Results

Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed.

Conclusions

These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.     

MRI of arterial flow reserve in patients with intermittent claudication: feasibility and initial experience

Objectives

The aim of this work was to develop a MRI method to determine arterial flow reserve in patients with intermittent claudication and to investigate whether this method can discriminate between patients and healthy control subjects.

Methods

Ten consecutive patients with intermittent claudication and 10 healthy control subjects were included. All subjects underwent vector cardiography triggered quantitative 2D cine MR phase-contrast imaging to obtain flow waveforms of the popliteal artery at rest and during reactive hyperemia. Resting flow, maximum hyperemic flow and absolute flow reserve were determined and compared between the two groups by two independent MRI readers. Also, interreader reproducibility of flow measures was reported.

Results

Resting flow was lower in patients compared to controls (4.9±1.6 and 11.1±3.2 mL/s in patients and controls, respectively (p<0.01)). Maximum hyperemic flow was 7.3±2.9 and 16.4±3.2 mL/s (p<0.01) and the absolute flow reserve was 2.4±1.6 and 5.3±1.3 mL/s (p<0.01), respectively in patients and controls. The interreader coefficient of variation was below 10% for all measures in both patients and controls.

Conclusions

Quantitative 2D MR cine phase-contrast imaging is a promising method to determine flow reserve measures in patients with peripheral arterial disease and can be helpful to discriminate patients with intermittent claudication from healthy controls.     

Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging

Background and Aims

Measuring the Al³⁺ uptake rate across the plasma membrane of intact root cells is crucial for understanding the mechanisms and time-course of Al toxicity in plants. However, a reliable method with the sufficient spatial and temporal resolution to estimate Al³⁺ uptake in intact root cells does not exist.

Methods

In the current study, fluorescent lifetime imaging (FLIM) analysis was used to quantify Al³⁺ uptake in the root-cell cytoplasm in vivo. This was performed via the estimation of the fluorescence lifetime of Al–lumogallion {5-chloro-3[(2,4-dihydroxyphenyl)azo]-2-hydroxybenzenesulfonic acid} complexes and measurements of intracellular pH while exposing arabidopsis seedlings to acidic and Al³⁺ stresses.

Key Results

The lifetime of Al–lumogallion complexes fluorescence is pH-dependent. The primary sites for Al³⁺ entry are the meristem and distal elongation zones, while Al³⁺ uptake via the cortex and epidermis of the mature root zone is limited. The maximum rates of Al uptake into the cytoplasm (2–3 µmol m⁻³ min⁻¹ for the meristematic root zone and 3–7 µmol m⁻³ min⁻¹ for the mature zone) were observed after a 30-min exposure to 100 µm AlCl₃ (pH 4·2). Intracellular Al concentration increased to 0·4 µm Al within the first 3 h of exposure to 100 µm AlCl₃.

Conclusions

FLIM analysis of the fluorescence of Al–lumogallion complexes can be used to reliably quantify Al uptake in the cytoplasm of intact root cells at the initial stages of Al³⁺ stress.Key words: Acid stress, Al³⁺, aluminium toxicity, Arabidopsis thaliana, low pH, fluorescent lifetime imaging (FLIM), lumogallion     

Ultrasound-Triggered Phase Transition Sensitive Magnetic Fluorescent Nanodroplets as a Multimodal Imaging Contrast Agent in Rat and Mouse Model

Ultrasound-triggered phase transition sensitive nanodroplets with multimodal imaging functionality were prepared via premix Shirasu porous glass (SPG) membrane emulsification method. The nanodroplets with fluorescence dye DiR and SPIO nanoparticles (DiR-SPIO-NDs) had a polymer shell and a liquid perfluoropentane (PFP) core. The as-formed DiR-SPIO-NDs have a uniform size of 385±5.0 nm with PDI of 0.169±0.011. The TEM and microscopy imaging showed that the DiR-SPIO-NDs existed as core-shell spheres, and DiR and SPIO nanoparticles dispersed in the shell or core. The MTT and hemolysis studies demonstrated that the nanodroplets were biocompatible and safe. Moreover, the proposed nanodroplets exhibited significant ultrasound-triggered phase transition property under clinical diagnostic ultrasound irradiation due to the vaporization of PFP inside. Meanwhile, the high stability and R₂ relaxivity of the DiR-SPIO-NDs suggested its applicability in MRI. The in vivo T₂-weighted images of MRI and fluorescence images both showed that the image contrast in liver and spleen of rats and mice model were enhanced after the intravenous injection of DiR-SPIO-NDs. Furthermore, the ultrasound imaging (US) in mice tumor as well as MRI and fluorescence imaging in liver of rats and mice showed that the DiR-SPIO-NDs had long-lasting contrast ability in vivo. These in vitro and in vivo findings suggested that DiR-SPIO-NDs could potentially be a great MRI/US/fluorescence multimodal imaging contrast agent in the diagnosis of liver tissue diseases.     

Exercise reduces cellular stress related to skeletal muscle insulin resistance

This study sought to evaluate the effects of a single session of exercise on the expression of Hsp70, of c-jun N-terminal kinase (JNK), and insulin receptor substrate 1 serine 612 (IRS^ser612) phosphorylation in the skeletal muscle of obese and obese insulin-resistant patients. Twenty-seven volunteers were divided into three experimental groups (eutrophic insulin-sensitive, obese insulin-sensitive, and obese insulin-resistant) according to their body mass index and the presence of insulin resistance. The volunteers performed 60 min of aerobic exercise on a cycle ergometer at 60 % of peak oxygen consumption. M. vastus lateralis samples were obtained before and after exercise. The protein expressions were evaluated by Western blot. Our findings show that compared with paired eutrophic controls, obese subjects have higher basal levels of p-JNK (100 ± 23 % vs. 227 ± 67 %, p = 0.03) and p-IRS-1^ser612 (100 ± 23 % vs. 340 ± 67 %, p < 0.001) and reduced HSP70 (100 ± 16 % vs. 63 ± 12 %, p < 0.001). The presence of insulin resistance results in a further increase in p-JNK (460 ± 107 %, p < 0.001) and a decrease in Hsp70 (46 ± 5 %, p = 0.006), but p-IRS-1^ser612 levels did not differ from obese subjects (312 ± 73 %, p > 0.05). Exercise reduced p-JNK in obese insulin-resistant subjects (328 ± 33 %, p = 0.001), but not in controls or obese subjects. Furthermore, exercise reduced p-IRS-1^ser612 for both obese (122 ± 44 %) and obese insulin-resistant (185 ± 36 %) subjects. A main effect of exercise was observed in HSP70 (p = 0.007). We demonstrated that a single session of exercise promotes changes that characterize a reduction in cellular stress that may contribute to exercise-induced increase in insulin sensitivity.     

Treatment with atorvastatin is associated with a better prognosis in chronic heart failure with systolic dysfunction: results from The Daunia Heart Failure Registry

Background

Few works have evaluated the effect of statins on left ventricular dysfunction in patients with chronic heart failure (CHF), by using tissue Doppler imaging (TDI). We therefore aimed to investigate whether atorvastatin treatment may influence prognosis and myocardial performance evaluated by TDI in subjects with CHF.

Methods

Five hundred thirty-two consecutive CHF outpatients enrolled in a local registry, the Daunia Heart Failure Registry, were prospectively analysed. 195 patients with CHF and left ventricular ejection fraction (LVEF) ≤40 %, either in treatment with atorvastatin (N: 114) or without statins (N: 81), underwent TDI examination. Adverse events were evaluated during follow-up.

Results

The atorvastatin group showed a lower incidence of adverse events (cardiac death: 0 % vs 7 %, p < 0.01), and better TDI performance (E/E’ 15 ± 5.7 vs 18 ± 8.3, p < 001) than controls. Ischaemic CHF patients in treatment with atorvastatin also showed a lower incidence of adverse events (death: 10 % vs 26 %, p < 0.05; sustained ventricular arrhythmias: 5 % vs 19 %, p < 0.05, cardiac death: 0 vs 8 %, p < 0.05) and better TDI performance (E/E’ ratio: 15.00 ± 5.68 vs 19.72 ± 9.14, p < 0.01; St: 353.70 ± 48.96 vs 303.33 ± 68.52 msec, p < 0.01) than controls. The association between atorvastatin and lower rates of cardiac death remained statistically significant even after correction in a multivariable analysis (RR 0.83, 95 % CI 0.71–0.96, p < 0.05 in CHF with LVEF ≤40 %; RR 0.77, 95 % CI 0.62–0.95, p < 0.05 in ischaemic CHF with LVEF ≤40 %).

Conclusions

Treatment with atorvastatin in outpatients with systolic CHF is associated with fewer cardiac deaths, and a better left ventricular performance, as assessed by TDI.     

Intraduodenal administration of intact pea protein effectively reduces food intake in both lean and obese male subjects

Background

Human duodenal mucosa secretes increased levels of satiety signals upon exposure to intact protein. However, after oral protein ingestion, gastric digestion leaves little intact proteins to enter the duodenum. This study investigated whether bypassing the stomach, through intraduodenal administration, affects hormone release and food-intake to a larger extent than orally administered protein in both lean and obese subjects.

Methods

Ten lean (BMI:23.0±0.7 kg/m²) and ten obese (BMI:33.4±1.4 kg/m²) healthy male subjects were included. All subjects randomly received either pea protein solutions (250 mg/kg bodyweight in 0.4 ml/kg bodyweight of water) or placebo (0.4 ml/kg bodyweight of water), either orally or intraduodenally via a naso-duodenal tube. Appetite-profile, plasma GLP-1, CCK, and PYY concentrations were determined over a 2 h period. After 2 h, subjects received an ad-libitum meal and food-intake was recorded.

Results

CCK levels were increased at 10(p<0.02) and 20(p<0.01) minutes after intraduodenal protein administration (IPA), in obese subjects, compared to lean subjects, but also compared to oral protein administration (OPA)(p<0.04). GLP-1 levels increased after IPA in obese subjects after 90(p<0.02) to 120(p<0.01) minutes, compared to OPA. Food-intake was reduced after IPA both in lean and obese subjects (-168.9±40 kcal (p<0.01) and −298.2±44 kcal (p<0.01), respectively), compared to placebo. Also, in obese subjects, food-intake was decreased after IPA (−132.6±42 kcal; p<0.01), compared to OPA.

Conclusions

Prevention of gastric proteolysis through bypassing the stomach effectively reduces food intake, and seems to affect obese subjects to a greater extent than lean subjects. Enteric coating of intact protein supplements may provide an effective dietary strategy in the prevention/treatment of obesity.     

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle

Background

The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.

Methodology/Principal Findings

To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and ¹H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H₂O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.

Conclusions

This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.     

Changes in aminoacidergic and monoaminergic neurotransmission in the hippocampus and amygdala of rats after ayahuasca ingestion

AIM: To evaluate changes in neurotransmission induced by a psychoactive beverage ayahuasca in the hippocampus and amygdala of naive rats. METHODS: The level of monoamines, their main metabolites and amino acid neurotransmitters concentrations were quantified using high performance liquid chromatography(HPLC). Four groups of rats were employed: saline-treated and rats receiving 250, 500 and 800 mg/kg of ayahuasca infusion(gavage). Animals were killed 40 min after drug ingestion and the structures stored at-80 ℃ until HPLC assay. The data from all groups were compared using Analysis of variance and Scheffé as post test and P 0.05 was accepted as significant. RESULTS: The results showed decreased concentrations of glycine(GLY)(0.13 ± 0.03 vs 0.29 ± 0.07, P 0.001) and γ-aminobutyric acid(GABA)(1.07 ± 0.14 vs 1.73 ± 0.25, P 0.001) in the amygdala of rats that received 500 of ayahuasca. Animals that ingested 800 mg/kg of ayahuasca also showed a reduction of GLY level(0.11 ± 0.01 vs 0.29 ± 0.07, P 0.001) and GABA(0.98 ± 0.06 vs 1.73 ± 0.25, P 0.001). In the hippocampus, increased GABA levels were found in rats that received all ayahuasca doses: 250 mg/kg(1.29 ± 0.19 vs 0.84 ± 0.21, P 0.05); 500 mg/kg(2.23 ± 038 vs 084 ± 0.21, P 0.05) and 800 mg/kg(1.98 ± 0.92 vs 0.84 ± 0.21, P 0.05). In addition, an increased utilization rate of all monoamines was found in the amygdala after ayahuasca administration in doses: 250 mg/kg(noradrenaline: 0.16 ± 0.02 vs 0.36 ± 0.06, P 0.01; dopamine: 0.39 ± 0.012 vs 2.39 ± 0.84, P 0.001; serotonin: 1.02 ± 0.22 vs 4.04 ± 0.91, P 0.001), 500 mg/kg(noradrenaline: 0.08 ± 0.02 vs 0.36 ± 0.06, P 0.001; dopamine: 0.33 ± 0.19 vs 2.39 ± 0.84, P 0.001; serotonin: 0.59 ± 0.08 vs 4.04 ± 0.91, P 0.001) and 800 mg/kg(noradrenaline: 0.16 ± 0.04 vs 0.36 ± 0.06, P 0.001; dopamine: 0.84 ± 0.65 vs2.39 ± 0.84, P 0.05; serotonin: 0.36 ± 0.02 vs 4.04 ± 0.91, P 0.001). CONCLUSION: Our data suggest increased release of inhibitory amino acids by the hippocampus and an increased utilization rate of monoamines by the amygdala after different doses of ayahuasca ingestion.     

Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity,immune regulation and therapy

Introduction

In autoimmune diseases, IL-17 producing T-cells (Th17), a pro-inflammatory subset of T-cells, are pathophysiologically involved. There is little knowledge on the role of Th17 cells in granulomatosis with polyangiitis (GPA). In the present study, we investigated Th17 cells, Tregs and subsets of circulating Th17 cells in GPA and related results to disease activity.

Methods

42 GPA patients in remission, 18 with active disease and 14 healthy controls (HC) were enrolled. Th17 cells, their subsets and regulatory T-cells were determined by intracellular fluorescence activated cell sorter (FACS). Data are given as mean percentage ±SD of total T-helper-cells.

Results

Th17 cells are expanded in active and quiescent GPA as compared to HC (1.7±1.4% vs. 0.7 ±0.3%, P = 0.006 and 1.9 ±1.5% vs. 0.7 ±0.3%, P<0.0001). Th17 expansion is stable over time and does not decline when remission is achieved. However, a negative association of Th17 cells and steroid dosage is observed (r=-0.46, P = 0.002). The Th17 expansion was not balanced by Tregs as indicated by skewed Th17/Treg ratios in active and quiescent GPA. Th17 subsets co-producing IFNγ or IL-10 are significantly increased in GPA. GPA patients in remission not receiving maintenance therapy have significantly more IL-10/IL-17A double positive T-cells than HC (0.0501 ±0.031% vs. 0.0282 ±0.016%, P = 0.007).

Conclusions

We provide evidence for a persistent, unbalanced expansion of Th17 cells and Th17 subsets which seems to be independent of disease activity. Maintenance therapy reduces -but does not normalize- Th17 expansion.     

Serial Non-Invasive Monitoring of Renal Disease Following Immune-Mediated Injury Using Near-Infrared Optical Imaging

Background

Non-invasive monitoring of disease progression in kidney disease is still a major challenge in clinical practice. In vivo near-infrared (NIR) imaging provides a new tool for studying disease mechanisms and non-invasive monitoring of disease development, even in deep organs. The LI-COR IRDye® 800CW RGD optical probe (RGD probe) is a NIR fluorophore, that can target integrin alpha v beta 3 (α_vβ₃) in tissues.

Objective

This study aims to monitor renal disease progression in an anti-glomerular basement membrane (GBM) nephritis mouse model.

Methods

Anti-GBM nephritis was induced in 129x1/svJ mice by anti-GBM serum challenge. The expression of integrin α_vβ₃ in the diseased kidney was examined by immunohistochemistry and quantitative polymerase chain reaction. The RGD probe and control fluorophores, the 800CW dye, and the BSA-conjugated 800CW dye, were administered into anti-GBM nephritic mice. LI-COR Pearl® Impulse imaging system was used for in vivo imaging; while ex vivo organ imaging was acquired using the Maestro^TM imaging system.

Results

Kidney tissue from anti-GBM nephritic mice showed higher levels of integrin α_vβ₃ expression at both the protein and the mRNA level compared to normal mice. The RGD probe allowed in vivo renal imaging and the fluorescent signal could be specifically captured in the diseased kidneys up to 14 days, reflecting longitudinal changes in renal function.

Conclusion

The infrared RGD molecular probe that tracks integrin expression can be successfully used to monitor renal disease progression following immune-mediated nephritis.     

Neurocardiological differences between musicians and control subjects

Background

Exercise training is beneficial in health and disease. Part of the training effect materialises in the brainstem due to the exercise-associated somatosensory nerve traffic. Because active music making also involves somatosensory nerve traffic, we hypothesised that this will have training effects resembling those of physical exercise.

Methods

We compared two groups of healthy, young subjects between 18 and 30 years: 25 music students (13/12 male/female, group M) and 28 controls (12/16 male/female, group C), peers, who were non-musicians. Measurement sessions to determine resting heart rate, resting blood pressure and baroreflex sensitivity (BRS) were held during morning hours.

Results

Groups M and C did not differ significantly in age (21.4 ± 3.0 vs 21.2 ± 3.1 years), height (1.79 ± 0.11 vs 1.77 ± 0.10 m), weight (68.0 ± 9.1 vs 66.8 ± 10.4 kg), body mass index (21.2 ± 2.5 vs 21.3 ± 2.4 kg∙m⁻²) and physical exercise volume (39.3 ± 38.8 vs 36.6 ± 23.6 metabolic equivalent hours/week). Group M practised music daily for 1.8 ± 0.7 h. In group M heart rate (65.1 ± 10.6 vs 68.8 ± 8.3 beats/min, trend P =0.08), systolic blood pressure (114.2 ± 8.7 vs 120.3 ± 10.0 mmHg, P = 0.01), diastolic blood pressure (65.0 ± 6.1 vs 71.0 ± 6.2 mmHg, P < 0.01) and mean blood pressure (83.7 ± 6.4 vs 89.4 ± 7.1, P < 0.01) were lower than in group C. BRS in groups M and C was 12.9 ± 6.7 and 11.3 ± 5.8 ms/mmHg, respectively (P = 0.17).

Conclusions

The results of our study suggest that active music making has training effects resembling those of physical exercise training. Our study opens a new perspective, in which active music making, additionally to being an artistic activity, renders concrete health benefits for the musician.     

Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans

In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL⁻¹; p < 0.05). In HST2, there was no change in plasma Hsp72 (REST: 0.94 ± 0.08, POST: 1.20 ± 0.15, 1 h POST: 1.17 ± 0.16 ng mL⁻¹; p > 0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p < 0.05). Exercise-induced increased intracellular Hsp72 expression was observed on HST1 (HST1: REST, 1 ± 0.02 vs. POST, 2.9 ± 0.9 density units, mean ± SE, p < 0.05) but was inhibited on HST2 (HST2: REST, 4.2 ± 1.2 vs. POST, 4.4 ± 1.1 density units, p > 0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = −0.85, p < 0.05). In conclusion, HA increased resting leukocyte Hsp72 levels and inhibited exercise-induced expression. This intracellular adaptation probably induces thermotolerance. In addition, the non-increase in plasma Hsp72 after HA may be related to lower stress at the cellular level in the acclimated individuals.     

Evaluation of the Metabolic Response to Cyclopamine Therapy in Pancreatic Cancer Xenografts Using a Clinical PET-CT System

OBJECTIVES: We analyzed the effects of anti-hedgehog signaling on the ¹⁸F-FDG uptake of pancreatic cancer xenografts (PCXs) using a clinically implemented positron emission tomography (PET)-computer tomography (CT) scanner with high-resolution reconstruction. METHODS: PCXs from two pancreatic cancer cell lines were developed subcutaneously in nude mice and injected intraperitoneally with a low dose of cyclopamine for 1 week. ¹⁸F-FDG PET-CT was performed using a new-generation clinical PET-CT scanner with minor modifications of the scanning protocol to adapt for small-animal imaging. The data set was reconstructed and quantified using a three-dimensional workstation. RESULTS: MiaPaCa-2 cells, which respond to cyclopamine, showed decreased ¹⁸F-FDG uptake without a change in tumor size. For hip tumors, the maximum standardized uptake value (SUV_max) was reduced by -24.5 ± 9.2%, the average SUV (SUV_avg) by -33.5 ± 7.0%, and the minimum SUV (SUV_min) by -54.4 ± 11.5% (P < .05). For shoulder tumors, SUV_max was reduced by -14.7 ± 7.5%, SUV_avg by -12.6 ± 6.3, and SUV_min by -30.3 ± 16.7% (P < .05). Capan-1 cells, which do not respond to cyclopamine, did not show significant SUV changes. CONCLUSIONS: The new generations of clinically implemented PET-CT scanners with high-resolution reconstruction detect a minimal response of PCX to low-dose short-term cyclopamine therapy without changes in tumor size and offer potential for preclinical translational imaging.     

Membrane Damage Induced by Supercritical Carbon Dioxide in Rhodotorula mucilaginosa

To clarify the mechanism of microbial inactivation by supercritical carbon dioxide (SCCO₂), membrane damage of Rhodotorula mucilaginosa was investigated within specific pressure (10 Mpa), temperature (37 °C), and treatment time (10–70 min) ranges, including cell morphological structure, membrane permeability and fluidity. SEM and TEM observations showed morphological changes in the cell envelope and intracellular organization after SCCO₂ treatment. Increase of membrane permeability was measured as increased uptake of the trypan blue dye with microscopy, and leakage of intracellular substances such as UV-absorbing materials and ions by determining the change of protein and electrical conductivity. The SCCO₂ mediated reduction in CFU ml⁻¹ was 0.5–1 log higher at 37 °C and 10 MPa for 60 min in Rose Bengal Medium containing 4 % sodium than a similar treatment in Rose Bengal Medium. Membrane fluidity analyzed by fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene showed that the florescence polarization and florescence anisotropy of the SCCO₂-treated cells were increased slightly and gently compared with the untreated cells. The correlation between membrane damage and death of cells under SCCO₂ was clear, and the membrane damage was a key factor induced the inactivation of cells.     

Pressure-accelerated dissociation of amyloid fibrils in wild-type hen lysozyme

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml⁻¹ fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (k_obs = 0.00193 min⁻¹ at 0.1 MPa, 0.0348 min⁻¹ at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV^0‡ = −32.9 ± 1.7 ml mol(monomer)⁻¹ and the activation compressibility Δκ^‡ = −0.0075 ± 0.0006 ml mol(monomer)⁻¹ bar⁻¹ for the dissociation reaction. The negative ΔV^0‡ and Δκ^‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.     

Impact of iron supply on the kinetics of recovery of photosynthesis in Cd-stressed poplar (Populus glauca)

Background and Aims

Cadmium (Cd) causes Fe-deficiency-like symptoms in plants, and strongly inhibits photosynthesis. To clarify the importance of Cd-induced Fe deficiency in Cd effects on photosynthesis, the recovery processes were studied by supplying excess Fe after the Cd symptoms had developed.

Methods

Fe-citrate at 10 µm or 50 µm was given with or without 10 µm Cd(NO₃)₂ to hydroponically cultured poplars (Populus glauca ‘Kopeczkii’) with characteristic Cd symptoms. Ion, chlorophyll and pigment contents, amount of photosynthetic pigment–protein complexes, chlorophyll fluorescence and carbon assimilation were measured together with the mapping of healing processes by fluorescence imaging.

Key Results

In regenerated leaves, the iron content increased significantly, while the Cd content did not decrease. As a result, the structural (increase in the amount of photosynthetic pigments and pigment–protein complexes, decrease in the F₆₉₀/F₇₄₀ ratio) and functional (elevation of CO₂ fixation activity and ΔF/F_m′) recovery of the photosynthetic machinery was detected. Cd-induced, light-stress-related changes in non-photochemical quenching, activity of the xanthophyll cycle, and the F₄₄₀?/F₅₂₀ ratio were also normalized. Imaging the changes in chlorophyll fluorescence, the recovery started from the parts adjacent to the veins and gradually extended to the interveinal parts. Kinetically, the rate of recovery depended greatly on the extent of the Fe supply, and chlorophyll a/b ratio and ΔF/F_m′ proved to be the most-rapidly reacting parameters.

Conclusions

Iron deficiency is a key factor in Cd-induced inhibition of photosynthesis.Key words: Cadmium, chlorophyll–protein, iron deficiency, poplar, Populus glauca Haines 1906 var. Kopeczkii, fluorescence imaging, chlorophyll fluorescence induction     

Circulating vascular progenitor cells and central arterial stiffness in polycystic ovary syndrome

Objective

Subjects with Polycystic ovarian syndrome (PCOS) are at increased risk of Type 2 diabetes mellitus (T2DM). The mechanism of this enhanced risk is unclear. Circulating vascular progenitor cells (VPC) are immature bone marrow derived cells capable of differentiating into mature endothelial cells. VPC number/function and central arterial stiffness predict cardio-metabolic disease in at-risk populations.

Design

We studied VPC and arterial stiffness measures in non-obese PCOS subjects as compared to age and body mass index (BMI) matched healthy controls in a cross–sectional study.

Methods

Fourteen subjects with PCOS and 12 controls of similar age, BMI (all <30 kg/m²) and metabolic profile were studied. VPC number and in vitro function were studied by flow cytometry and tube formation assays respectively. Augmentation index (AIx), a measure of central arterial stiffness, and central (aortic) blood pressures (BP) were measured by applanation tonometry.

Results

Subjects with PCOS had a reduced number, mean±SEM, of circulating CD34⁺133⁺ VPCs (317.5±51.0 vs. 558.3±101.2, p = 0.03) and impaired in vitro tube formation (completed tube area 1.0±0.06 vs. 1.2±0.05×10⁶ µm² p = 0.02). PCOS subjects had significantly higher AIx (18.4±1.9% vs. 4.9±2.0%) and this difference remained significant even after adjustments for age, BMI and smoking (p = 0.003) in multivariate analyses. Central systolic and pulse pressure were higher in PCOS subjects but these differences were not statistically significant after adjustment for age. Brachial systolic and pulse pressures were similar. VPC number/function and arterial stiffness or BP measures were not correlated.

Conclusions

Non-obese PCOS is characterized by a reduced VPC number, impaired VPC function and increased central arterial stiffness. These changes in novel vascular risk markers may explain the enhanced risk of T2DM and CVD in PCOS.