Radical-free hyperpolarized MRI using endogenously-occurring pyruvate analogues and UV-induced nonpersistent radicals

It was demonstrated that nonpersistent radicals can be generated in frozen solutions of metabolites such as pyruvate by irradiation with ultraviolet (UV) light, enabling radical-free dissolution DNP. Although pyruvate is endogenous, an excess of additional pyruvate may perturb metabolic processes, making it potentially unsuitable as a polarizing agent when studying fatty acids or carbohydrate metabolism. Therefore, the aim of the study was to characterize solutions containing endogenously-occurring alternatives to pyruvate as UV-induced nonpersistent radical precursors for in vivo hyperpolarized MRI. The metabolites alpha-ketovalerate (AKV) and alpha-ketobutyrate (AKB) are analogues of pyruvate and were chosen as potential radical precursors. Sample formulations containing AKV and AKB were studied with UV-visible spectroscopy, irradiated with UV light, and their nonpersistent radical yields were quantified with ESR and compared to pyruvate. The addition of 13C labeled substrates to the sample matrix altered the radical yield of the precursors. Using AKB increased the 13C-labeled glucose liquid state polarization to 16.3 +/- 1.3% compared with 13.3 +/- 1.5% obtained with pyruvate, and 8.9 +/- 2.1% with AKV. For [1-13C]butyric acid, polarization levels of 12.1 +/- 1.1% for AKV and 12.9 +/- 1.7% for AKB were achieved. Hyperpolarized [1-13C]butyrate metabolism in the heart revealed label incorporation into [1-13C]acetylcarnitine, [1-13C]acetoacetate, [1-13C]butyrylcarnitine, [5-13C]glutamate and [5-13C]citrate. This study demonstrates the potential of AKV and AKB as endogenous polarizing agents for in vivo radical-free hyperpolarized MRI. UV-induced, nonpersistent radicals generated in endogenous metabolites enable high polarization without requiring radical filtration, thus simplifying the quality-control tests in clinical applications.Comment: 38 pages, 5 Tables, 8 Figures, Submitted to NMR in Biomedicin

The polo-like kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia

CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56+ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56+ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML

SARS-CoV-2 vaccination and multiple sclerosis: a large multicentric study on relapse risk after the third booster dose

Background: COVID-19 vaccines have been recommended to people with multiple sclerosis (pwMS) and, to ensure durable immunity, a third booster dose has been administered in several countries. Data about potential risks associated with the third booster dose in pwMS, such as vaccine-triggered disease exacerbations, are still scarce. Objective: To investigate whether the administration of a third booster dose of mRNA COVID-19 vaccines was associated with an increased risk of short-term disease reactivation in a large cohort of pwMS. Methods: We retrospectively selected 1265 pwMS who received a third booster dose of an mRNA COVID-19 vaccine. Demographic and clinical data were collected, including the presence, number and characteristics of relapses in the 60 days prior to and after the third booster dose. Results: In the selected cohort, the relapse rate in the two months after administration of the third booster dose of mRNA COVID-19 vaccines did not increase when compared with the prior two months. Indeed, the percentage of pwMS experiencing relapses in the 60 days following the administration of the third booster dose was 2.1%, similar to the percentage recorded in 60 days prior to vaccination, which was 1.9%. Conclusions: The third booster dose of mRNA COVID-19 vaccines appeared to be safe for pwMS

Mechanistic studies of DNP and applications of hyperpolarized probes to study renal physiology and metabolism

Dissolution dynamic nuclear polarization (dDNP) is a powerful technique that enhances the magnetic resonance signal of nuclear spins by several orders of magnitude. DNP relies on the principle of cross-relaxation by electron spins driven out of equilibrium to enhance nuclear polarization. When coupled to magnetic resonance spectroscopy/imaging (MRS/I), infusion of 13C-labeled tracers hyperpolarized via DNP in vivo provides real-time estimates of metabolic fluxes, physiological parameters and/or tissue perfusion. The present work focuses on fundamental aspects of DNP, in particular how sample composition affects the DNP of low-gamma nuclei, as well as on in vivo dDNP applications targeting renal metabolism and physiology. The DNP properties of the SA-BDPA radical were studied in a sample of 13C-urea. SA-BDPA, a water-soluble derivative of BDPA, is characterized by a small g-anisotropy and unresolved hyperfine coupling to protons. As a result, at 6.7 T, 1.1 K its ESR linewidth is much smaller than the 13C Larmor frequency, which enabled the observation for the first time of 13C DNP via the solid effect and pure thermal mixing, the latter defined as the process where the electron non-Zeeman reservoir alone provides the energy required for triple-spin flips. The effect of solvent deuteration on 6Li DNP and radical ESR properties was studied in 6LiCl water:glycerol solutions doped with either the nitroxide radical TEMPOL or the trityl radical OX063. Unexpectedly, the TEMPOL-doped samples polarized better than the trityl-doped ones. Across all samples, the relationship between the degree of solvent deuteration with the buildup time constant and polarization level was notably different from what has been reported for 13C. This behavior is indicative of DNP via a combination of the cross effect and thermal mixing mechanisms. The uptake and metabolism of hyperpolarized L-[1-13C]alaninamide was investigated in the rat kidney in vivo. This probe of aminopeptidase N, which can play a role in tumor growth, was previously studied in vitro. Our study showed that alanine production from alaninamide also occurs in vivo, however, with spectral overlap of substrate and product. Alaninamide, having a pKa of 7.9, proved to be sensitive to local pH. Three spectral peaks, corresponding to at least three environments with different pH values, could be observed in the kidney. The two peaks at higher pH were assigned to the blood extra- and (partially) intracellular compartments, while the third one was mainly in the inner part of the kidney. Finally, alaninamide was shown to also be sensitive to dissolved CO2, with the rapid formation of a carbamate adduct following infusion. The renal metabolism of D-[1-13C]alanine by D-amino acid oxidase (DAO) was also studied. Conversion of hyperpolarized D-alanine to pyruvate and further metabolism to lactate and bicarbonate was observed in the kidney only when DAO was not inhibited. DAO activity could also be detected in blood, where leukocytes express the enzyme, but not in the brain and liver, in line with their lower DAO activity. Overall, this thesis provides additional insight into how the experimental conditions can favor a particular DNP mechanism over another. It also significantly expands the scope of in vivo dDNP applications, showing that additional enzyme-catalyzed processes can be detected, along with the potential of amino-acid based hyperpolarized 13C sensors for physiological studies

Detection of D-amino acid oxidase using hyperpolarized molecular probes

D-amino acid oxidase (DAO) is an enzyme that catalyzes the degradation of D-amino acids in the body. Here, we explored the possibility of detecting D-amino acid oxidase activity by monitoring its metabolism in the rat kidney after a bolus injection of hyperpolarized D-[1-13C]alanine. Our data show that D-alanine is readily converted to lactate only when the DAO enzyme is not inhibited, indicating that the observed metabolism is that of DAO

In vivo detection of d-amino acid oxidase with hyperpolarized d-[1-C-13]alanine

d-amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d-amino acids to their corresponding alpha-keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d-amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N-methyl-d-aspartate (NMDA) receptor hypo/hyper-function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d-[1-C-13]alanine. Following a bolus of hyperpolarized d-alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate-to-d-alanine ratio was comparable to the values measured when the l-enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d-[1-C-13]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate-to-d-alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1-C-13]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d-[1-C-13]alanine as a probe of d-amino acid metabolism

The Impact of Peri-Implantitis on Systemic Diseases and Conditions: A Review of the Literature

While periodontitis has been proven to have an impact on systemic conditions, such as cardiovascular diseases, pregnancy complications, or poor glycemic control in diabetic patients, the influence of peri-implantitis on systemic health has not been adequately explored in the literature as yet. The existing evidence suggests that peri-implant lesions lead to more intense inflammatory response than periodontitis. Given the analogies between periodontal diseases and peri-implantitis, the aim of the present paper was to review the scientific evidence about the potential correlation between peri-implantitis and systemic diseases and conditions. Two clinical trials on animals reported that experimental peri-implantitis determined an alteration in hematological and biological parameters. One human study explored the risk indicators for cardiovascular diseases and found that patients with peri-implantitis had significantly higher levels of triglyceride, uric acid, and white blood cells and lower levels of vitamin D. It was described in the literature that periodontitis affects cardiovascular health through a number of mechanisms, including the increase in systemic mediators of inflammation, which also has a role in the worsening of glycemic control in diabetic patients. Similarly, peri-implantitis may influence the systemic status through inflammatory cytokines such as IL-1, IL-6, and IL-10 and matrix metalloproteinases. One microbiological mechanism, based on the systemic dissemination of periodontal bacteria, has been hypothesized for cardiovascular diseases and pregnancy complications. Again, it is plausible that the same could occur in peri-implantitis. In conclusion, only few studies explored the systemic impact of peri-implantitis. Although changes in hematological parameters, biochemical parameters, and inflammatory markers have been reported in peri-implantitis, further studies are needed to investigate this correlation

Probing renal pH using hyperpolarized [1-13C]alaninamide

Hyperpolarized molecular probes can be effectively used as pH markers. To date, the only two probes reported in vivo as extracellular pH sensors are 13C-bicarbonate, and [1,5-13C2]zymonic acid. Alaninamide is a derivative of alanine which is found to be sensitive to variations of pH in the physiological range. The aim of the present study was to assess the feasibility of using alaninamide as a pH probe in vivo. The alaninamide titration curve was determined by performing 13C NMR measurements at 9.4 T, 37° on a set of 500 mM Ala-NH2∙HCl samples of varying pH referenced to 13C urea. [1-13C]Alaninamide was polarized at 1 K in a 7 T polarizer, then rapidly dissolved in a buffered solution and injected IV into a Sprague Dawley rat (n=6) located in a 9.4 T animal scanner. 13C FIDs were acquired with 30° BIR4 pulses using a single loop 1H / quadrature 13C surface coil placed over the left kidney. The pH was perturbed by injecting acetazolamide IV (10 mg/kg) one hour prior to infusion. The alaninamide titration curve shows a 13C1 chemical shift change of ≈ 8.4 ppm, and a pKa of 7.9. The pH sensitivity of 13C1 results in three distinct alaninamide spectral peaks, corresponding to three different extracellular pH compartments within the kidney (pH = 7.46, pH = 7.22, pH = 6.58) that can be tentatively assigned to the cortex/blood, medulla and calyx/ureter. With acetazolamide treatment, the pH in the first compartment follows the change in pH of the blood, while the pH in the third compartment does not reflect the urine pH and shifts during the brief experiment. No change is observed in the pH value of the second compartment

¹³C Dynamic Nuclear Polarization using SA-BDPA at 6.7 T and 1.1 K:Coexistence of Pure Thermal Mixing and Well-Resolved Solid Effect

SA-BDPA is a water-soluble, narrow-line width radical previously used for dynamic nuclear polarization (DNP) signal enhancement in solid-state magic angle spinning NMR spectroscopy. Here, we report the first study using SA-BDPA under dissolution DNP conditions (6.7 T and 1.15 K). Longitudinal-detected (LOD)-electron spin resonance (ESR) and C-13 DNP measurements were performed on samples containing 8.4 M [C-13]urea dissolved in 50:50 water:glycerol (v/v) doped with either 60 or 120 mM SA-BDPA. Two distinct DNP mechanisms, both "pure" thermal mixing and a well-resolved solid effect could clearly be identified. The radical's ESR line width (30-40 MHz), broadened predominantly by dipolar coupling, excluded any contribution from the cross effect. Microwave frequency modulation increased the enhancement by DNP at the lower radical concentration but not at the higher radical concentration. These results are compared to data acquired with trityl radical AH111501, highlighting the unusual C-13 DNP properties of SA-BDPA

Benefícios flexíveis: estudo de caso em uma indústria de autopeças

As empresas estão investindo em diversas estratégias para atrair e reter talentos. Uma delas é a implantação de um plano de benefícios flexíveis, que possibilita o funcionário escolher entre uma série de benefícios. O objetivo geral deste artigo é investigar o que pensam os colaboradores quanto a implantação de um plano flexível de benefícios. O estudo é fundamentado com base no referencial teórico de alguns autores, como, Boog e Boog (2002), Maximiano (2012), Robbins (2005), Marras (2011) e outros. A metodologia utilizada é estudo de caso, com caráter exploratório-descritivo e abordagem qualiquantitativa. Analisando o resultado do questionário aplicado, considera-se viável a implantação de um programa de benefícios flexíveis na empresa, pois 72% dos respondentes entendem que os benefícios flexíveis são fator importante para retenção de talentos e motivação de pessoal e 69% não estão satisfeitos com os benefícios oferecidos pela empresa