Concentration and distribution of silica nanoparticles in colon cancer cells assessed by synchrotron based X-ray techniques.

The quantitative uptake of Silica nanoparticles (SiNPs), although representing an essential prerequisite for their theranostic use, is difficult to address and it is still not utterly investigated. In this study, we tested the uptake and toxicity of two different types of luminescent core-shell silica-PEG (polyethylene glycol) nanoparticles SiNP and their carboxylate analogues on human adenocarcinoma cell line LoVo. We assessed the intracellular spatial distribution and concentration of Si element in the cell by a state-of-the-art approach merging synchrotron-based X-ray techniques (XRFM) with scanning transmission X-Ray microscopy (STXM). The concentration maps of Si obtained reflect the distribution of the SiNPs. In addition, we calculated the number of SiNPs per volume unit in each single cell, quantitating the exact amount of conveyed particles. The absence of effects on proliferation and cell death was confirmed by viability assays, morphological analysis and cytofluorimetric evaluation of ROS content. The three-dimensional analysis of intracellular uptake of both types of nanoparticles (with different surface charge) was performed by confocal fluorescence microscopy, which showed a main localization in the cytosolic region with no sign of nuclear uptake.

p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity.

The role of pH on the thermodynamics and kinetics of muscle biochemistry: an in vivo study by (31)P-MRS in patients with myo-phosphorylase deficiency.

In this study we assessed ΔG'(ATP) hydrolysis, cytosolic [ADP], and the rate of phosphocreatine recovery using Phosphorus Magnetic Resonance Spectroscopy in the calf muscle of a group of patients affected by glycogen myo-phosphorylase deficiency (McArdle disease). The goal was to ascertain whether and to what extent the deficit of the glycogenolytic pathway would affect the muscle energy balance. A typical feature of this pathology is the lack of intracellular acidosis. Therefore we posed the question of whether, in the absence of pH decrease, the rate of phosphocreatine recovery depends on the amount of phosphocreatine consumed during exercise. Results showed that at the end of exercise both [ADP] and ΔG'(ATP) of patients were significantly higher than those of matched control groups reaching comparable levels of phosphocreatine concentration. Furthermore, in these patients we found that the rate of phosphocreatine recovery is not influenced by the amount of phosphocreatine consumed during exercise. These outcomes provide experimental evidence that: i) the intracellular acidification occurring in exercising skeletal muscle is a protective factor for the energy consumption; and ii) the influence of pH on the phosphocreatine recovery rate is at least in part related to the kinetic mechanisms of mitochondrial creatine kinase enzyme.

Pitfalls and advantages of different strategies for the absolute quantification of N-acetyl aspartate, creatine and choline in white and grey matter by 1H-MRS.

This study extensively investigates different strategies for the absolute quantitation of N-acetyl aspartate, creatine and choline in white and grey matter by (1)H-MRS at 1.5 T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water-unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post-processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T(2), of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole-metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T(2)s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T(2) estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time-consuming alternative to IP in the quantitation of brain metabolites by (1)H-MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water-suppressed and one water-unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using WhoM, particularly at longer echo times.

Versatile coil design and positioning of transverse-field RF surface coils for clinical 1.5-T MRI applications.

Clinical MRI/MRS applications require radio frequency (RF) surface coils positioned at an arbitrary angle alpha with respect to B(0). In these experimental conditions the standard circular loop (CL) coil, producing an axial RF field, shows a large signal loss in the central region of interest (ROI). We demonstrate that transverse-field figure-of-eight (FO8) RF surface coils design are not subject to the same amount of signal loss in the central ROI as loop coils when their orientations are changed. The 1.5-T CL and FO8 prototypes (diameter = 10 cm) were built on Plexiglas using copper strips (width = 4 mm, thickness = 100 mum). The two linear elements of the FO8 coil were 1 cm apart. Axial spoiled gradient echo (SPGR) images of a phantom containing doped water were acquired with the coil plane at alpha=0 degrees , 45 degrees , and 90 degrees . As alpha increases, the CL images show, in the central ROI, a signal that decreases from a maximum value to zero. Whereas the FO8 images show, in the same ROI, a signal that varies little from the maximum value (20%). Optimized FO8 coils can be oriented with the coil plane positioned along any direction with respect to B(0) without significant signal loss. Transverse RF coil design should be useful for clinical MRS studies and also for parallel imaging techniques where versatile RF coils disposed along arbitrary directions are required.

In vivo evidence of neuronal loss in the hypothalamus of narcoleptic patients.

A dysfunction of the orexin (hypocretin) system in the hypothalamus has recently been linked to the pathogenesis of narcolepsy. The authors used in vivo proton MR spectroscopy to assess the N-acetylaspartate (NAA) content in the hypothalamus of narcoleptic patients. Hypothalamic NAA/creatine-phosphocreatine was reduced in narcoleptic patients compared with control subjects (p < 0.01). Hypothalamic neuronal loss/damage is a central pathogenetic feature in narcolepsy.

Diffusion MRI shows increased water apparent diffusion coefficient in the brains of cirrhotics.

BACKGROUND: Brain edema and increased intracranial pressure worsen prognosis in patients with end-stage chronic cirrhosis. OBJECTIVE: To use diffusion-weighted imaging (DWI) to quantify water apparent diffusion coefficient (ADC) in different brain regions of patients with chronic liver failure with or without hepatic encephalopathy. METHODS: The authors studied 14 patients with viral liver cirrhosis and 12 sex- and age-matched healthy volunteers. Seven patients had no clinical evidence of hepatic encephalopathy; six had grade I hepatic encephalopathy; and one had grade II hepatic encephalopathy. Brain DWI was obtained using a single-shot echo-planar imaging sequence, and four gradient strengths (b values = 0, 300, 600, and 900 s/mm(2)) were applied to calculate the average diffusivity maps. RESULTS: Mean ADC values in the brains of patients with cirrhosis were significantly increased in all selected regions of interest (caudate, putamen, and pallidus nuclei; occipital, parietal, and frontal lobe white matter) except in the thalamus. Venous ammonia was linearly related to ADC values in deep gray and white matter regions of interest. CONCLUSIONS: Brain water apparent diffusion coefficient is increased in patients with chronic liver disease and may be useful in monitoring patients with hepatic encephalopathy.

The mono-exponential pattern of phosphocreatine recovery after muscle exercise is a particular case of a more complex behaviour.

A mathematical model is proposed showing that the mono-exponential recovery of phosphocreatine (PCr) after exercise is an approximation of a more complex pattern, which is identified by a second-order differential equation. The model predicts the possibility of three different patterns of PCr recovery: bi-exponential, oscillatory damped, and critically damped; the mono-exponential pattern being a particular case of the functions which are solutions of the differential equation. The model was tested on a sample of recovery data from 50 volunteers, checking whether the recovery patterns predicted by the model lead to a significant improvement of fit (IF) compared with the mono-exponential pattern. Results show that the IF is linked to pH. Bi-exponential solutions showed an IF in the pH range 6.65-6.85, and the oscillatory solutions at pH>6.9. Critically damped solutions displayed a poor IF. Oscillation frequencies found in the oscillatory recoveries increase at increasing pH. These results show that pH has a pivotal role on the pattern of PCr recovery and implications on the regulation of oxidative phosphorylation are discussed.

Abnormal brain energy metabolism shown by in vivo phosphorus magnetic resonance spectroscopy in patients with chronic liver disease.

We used phosphorus magnetic resonance spectroscopy (31P-MRS) to assess in vivo the brain bioenergetics of 28 patients with liver cirrhosis. Seven had clinical hepatic encephalopathy (HE), nine hepatocellular carcinoma. 31P-MRS was performed by the DRESS localisation technique on occipital lobes. Brain phosphocreatine was significantly reduced in patients with or without overt HE, and inorganic phosphate was increased in both groups of patients. The cytosolic phosphorylation potential (PP), the relative rate of oxidative metabolism and the regulatory [ADP] were all abnormal. Brain PP was inversely correlated with serum ammonia concentration only in patients without liver cancer. The degree of bioenergetic failure was significantly higher in the presence of overt encephalopathy. We conclude that patients with liver cirrhosis had a derangement of brain energy metabolism, and that 31P-MRS offers a non-invasive method for investigating the underlying mechanisms of HE, with relevant implications in the identification and management of this condition.

Phosphorus MR spectroscopy shows a tissue specific in vivo distribution of biochemical expression of the G3460A mutation in Leber's hereditary optic neuropathy.

Occipital lobe and calf muscle energy metabolism were studied in vivo by magnetic resonance spectroscopy (31P-MRS) in four members of a family harbouring the mitochondrial DNA G3460A mutation causing Leber's hereditary optic neuropathy (LHON). Three siblings carried 100% mutated mitochondrial DNA (homoplasmy), while their mother had coexistence of mutated and wild-type mitochondrial DNA (heteroplasmy). Indices of brain energy metabolism on 31P-MRS were abnormal in all subjects examined, but the muscle oxidative phosphorylation rate was normal. These findings indicate a tissue specific distribution of the biochemical expression of the G3460A LHON mutation and suggest that extramitochondrial factors, such as nuclear genes, may influence expression of this mutation in vivo.

Deficient energy metabolism is associated with low free magnesium in the brains of patients with migraine and cluster headache.

We used phosphorus magnetic resonance spectroscopy to assess in vivo the brain cytosolic free magnesium concentration and the free energy released by the reaction of adenosine triphosphate (ATP) hydrolysis (DeltaG(ATPhyd)), the latter being an index of the cell's bioenergetics condition. We studied 78 patients with migraine in attack-free periods (7 with migraine stroke, 13 with migraine with prolonged aura, 37 with migraine with typical aura or basilar migraine, and 21 with migraine without aura), and 13 patients with cluster headache. In the occipital lobes of all subgroups of migraine and in cluster headache patients cytosolic free [Mg(2+)] as well as the free energy released by the reaction of ATP hydrolysis were significantly reduced. Among migraine patients, the level of free energy released by the reaction of ATP hydrolysis and the cytosolic free [Mg(2+)] showed a trend in keeping with the severity of clinical phenotype, both showing the lowest values in patients with migraine stroke and the highest in patients with migraine without aura. These results support our current hypothesis that the reduction in free [Mg(2+)] in tissues with mitochondrial dysfunction is secondary to the bioenergetics deficit, and are against a primary role of low brain cytosolic free [Mg(2+)] in causing the bioenergetics deficit in headache.

Deficit of brain and skeletal muscle bioenergetics in progressive supranuclear palsy shown in vivo by phosphorus magnetic resonance spectroscopy.

Brain and muscle energy metabolism was assessed in vivo in five patients with progressive supranuclear palsy (PSP) using phosphorous magnetic resonance spectroscopy (31P MRS). 31P MRS disclosed a reduced phosphocreatine (PCr) and an increased calculated free adenosine diphosphate (ADP) in the occipital lobes of all patients. In our patients with PSP, inorganic phosphate (Pi) was significantly increased and Mg2+ was reduced. In the gastrocnemius muscle, Pi at rest was increased in four patients, and the three patients who were able to perform an incremental exercise showed a rate of PCr postexercise recovery slower than control subjects. Our findings show that multisystemic deficit of energy metabolism occurs in PSP and suggest that it may play a role in the pathogenesis of this disorder.

'Secondary' 4216/ND1 and 13708/ND5 Leber's hereditary optic neuropathy mitochondrial DNA mutations do not further impair in vivo mitochondrial oxidative metabolism when associated with the 11778/ND4 mitochondrial DNA mutation.

The pathogenic role of 'secondary' mitochondrial DNA (mtDNA) point mutations, when occurring in patients with Leber's hereditary optic neuropathy (LHON) in association with 'primary' mutations, is still controversial. We used phosphorus magnetic resonance spectroscopy to establish whether two of these 'secondary' LHON mtDNA mutations, 4216/ND1 and 13708/ND5 (haplogroup J), further affect in vivo mitochondrial oxidative metabolism in subjects with the 'primary' 11778/ND4 mtDNA mutation. Brain and skeletal muscle energy metabolism was assessed in 10 subjects homoplasmic for the 11778/ND4 mtDNA mutation and 10 subjects homoplasmic for the same mutation occurring on the haplogroup J mtDNA background. Brain phosphocreatine concentration and phosphorylation potential were significantly reduced and brain inorganic phosphate concentration was significantly increased compared with controls in both groups of 11778/ND4-positive subjects. The degree of reduction in the phosphocreatine concentration and phosphorylation potential and of increase in the inorganic phosphate concentration was, however, similar in the two groups with the 11778/ND4 mtDNA mutation with or without the haplogroup J. Similarly, the rate of muscle phosphocreatine resynthesis after exercise, a sensitive index of the rate of mitochondrial ATP production, was reduced by the same extent in both groups of LHON subjects. This in vivo study does not support synergism of the 4216/ND1 and 13708/ND5 'secondary' mutations with the 11778/ND4 'primary' mutation in determining the deficit of energy metabolism in LHON.

In vivo (31)P-MRS assessment of cytosolic [Mg(2+)] in the human skeletal muscle in different metabolic conditions.

Cytosolic free [Mg(2+)] can be assessed in vivo by (31)P-MRS from the chemical shift of beta-ATP. The reliability of in vivo measurements depends on the availability of appropriate in vitro calibration curves obtained by using solutions that mimic the in vivo cytosolic conditions as far as possible. We build a semi-empiric equation that correlates the chemical shift of beta-ATP to free [Mg(2+)] taking into account the amount of Mg(2+) bound to all other ligands in solution. Our experiments resulted in a reliable ten-parameters equation directly usable to assess the cytosolic free [Mg(2+)] of human skeletal muscle at rest, during work and recovery. Our experiments also resulted in a new equation that allows the assessment of cytosolic pH from the chemical shift of Pi taking into account the measured free [Mg(2+)]. To perform simultaneous calculation of free [Mg(2+)] and pH in the skeletal muscle in different metabolic conditions we developed a specific software package available on Internet (http://www.unibo.it/bioclin) together with another program based on the equation previously obtained to calculate cytosolic free [Mg(2+)] in the human brain. The reliability and effectiveness of our equations and software were tested on the calf muscles of healthy volunteers at rest, during work and recovery.

Improved brain and muscle mitochondrial respiration with CoQ. An in vivo study by 31P-MR spectroscopy in patients with mitochondrial cytopathies.

We used in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and delta G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six-months of treatment with CoQ (150 mg/day), all brain MRS-measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.

Low brain intracellular free magnesium in mitochondrial cytopathies.

The authors studied, by in vivo phosphorus magnetic resonance spectroscopy (31P-MRS), the occipital lobes of 19 patients with mitochondrial cytopathies to clarify the functional relation between energy metabolism and concentration of cytosolic free magnesium. All patients displayed defective mitochondrial respiration with low phosphocreatine concentration [PCr] and high inorganic phosphate concentration [Pi] and [ADP]. Cytosolic free [Mg2+] and the readily available free energy (defined as the actual free energy released by the exoergonic reaction of ATP hydrolysis, i.e., deltaG(ATPhyd)) were abnormally low in all patients. Nine patients were treated with coenzyme Q10 (CoQ), which improved the efficiency of the respiratory chain, as shown by an increased [PCr], decreased [Pi] and [ADP], and increased availability of free energy (more negative value of deltaG(ATPhyd)). Treatment with CoQ also increased cytosolic free [Mg2+] in all treated patients. The authors findings demonstrate low brain free [Mg2+] in our patients and indicate that it resulted from failure of the respiratory chain. Free Mg2+ contributes to the absolute value of deltaG(ATPhyd). The results also are consistent with the view that cytosolic [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.

Phosphorus magnetic resonance spectroscopy in multiple system atrophy and Parkinson's disease.

We performed in vivo phosphorus magnetic resonance spectroscopy on the occipital lobes of 15 patients with multiple system atrophy (MSA; eight with olivopontocerebellar atrophy [OPCA] and seven with the striatonigral degeneration variant [SND]), 13 patients with idiopathic Parkinson's disease (PD), and 16 age-matched healthy subjects. The MSA group showed significantly reduced phosphocreatine (PCr), increased inorganic phosphate (Pi), and unchanged cytosolic free [Mg2+], and pH. We did not find any significant difference between the OPCA and SND variants. However, patients with PD showed significantly increased content of Pi, decreased cytosolic free [Mg2+], and unchanged [PCr] and pH. Comparing the MSA and PD groups, [PCr] was significantly lower in MSA than in PD, whereas cytosolic free [Mg2+] was significantly lower in PD. Despite a certain degree of overlap of [PCr] and [Mg2+] values between the two groups, by considering both variables at the same time it was possible to classify correctly 93% of cases by discriminant analysis. We conclude that phosphorus magnetic resonance spectroscopy discloses abnormal phosphate metabolite and ion contents in both MSA and PD, respectively, and may provide noninvasive diagnostic help to differentiate MSA from PD.

Aspects of human bioenergetics as studied in vivo by magnetic resonance spectroscopy.

We outline the relevant capabilities of in vivo phosphorus MR spectroscopy by discussing some aspects of normal human biochemistry as studied by this technique. The transport of inorganic phosphate from cytosol into mitochondria in the human skeletal muscle was studied by exploiting a new experimental protocol. We found that Pi was transported into mitochondria in the absence of ATP biosynthesis and in the presence of a pH gradient. The control of CoQ on the efficiency of oxidative phosphorylation in the skeletal muscle and brain was studied by administering CoQ to patients with mitochondrial cytopathies due to known enzyme defects. Before CoQ we had detected a relevant reduction of mitochondrial functionality in the skeletal muscle as shown by the reduced rate of phosphocreatine recovery from exercise, and in the occipital lobes by reduced [phosphocreatine] and a high [ADP] and [Pi]. After CoQ all brain variables were remarkably improved. Treatment with CoQ also improved the rate of muscle phosphocreatine recovery from exercise. Our in vivo findings support the hypothesis that the concentration of CoQ rather than the rate of its lateral diffusion in the mitochondrial membrane controls the efficiency of oxidative phosphorylation. Other experiments were undertaken to clarify the functional relationship between cytosolic free [Mg2+] and cell bioenergetics in the intact human brain. In the same group of patients with mitochondrial cytopathies we found decreased delta G of ATP hydrolysis and low cytosolic free [Mg2+]. Treatment with CoQ resulted in improved brain bioenergetics and increased free [Mg2+]. These findings strongly indicate that decreased free magnesium was secondary to defective mitochondrial respiration, and support the hypothesis that cytosolic free [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.

Deficit of brain and skeletal muscle bioenergetics and low brain magnesium in juvenile migraine: an in vivo 31P magnetic resonance spectroscopy interictal study.

We used phosphorus magnetic resonance spectroscopy (31P MRS) to investigate in vivo the brain and skeletal muscle energy metabolism of 15 children with migraine with aura in interictal periods. Brain 31P MRS disclosed low phosphocreatine and high inorganic phosphate contents, and high intracellular pH in all patients. Calculated [ADP] and the relative rate of mitochondrial oxidation were higher in the brain of patients than in control subjects, whereas the phosphorylation potential was lower. Brain intracellular free Mg2+ concentration was reduced by 25% in patients. Abnormal skeletal muscle mitochondrial respiration was also disclosed in 7 of 15 patients as shown by the slow rate of phosphocreatine postexercise recovery. The multisystem bioenergetic failure found in patients with juvenile migraine is comparable to that found in adults with different types of migraine.