The Bright and Dark Side of DNA Methylation: A Matter of Balance

DNA methylation controls several cellular processes, from early development to old age, including biological responses to endogenous or exogenous stimuli contributing to disease transition. As a result, minimal DNA methylation changes during developmental stages drive severe phenotypes, as observed in germ-line imprinting disorders, while genome-wide alterations occurring in somatic cells are linked to cancer onset and progression. By summarizing the molecular events governing DNA methylation, we focus on the methods that have facilitated mapping and understanding of this epigenetic mark in healthy conditions and diseases. Overall, we review the bright (health-related) and dark (disease-related) side of DNA methylation changes, outlining how bulk and single-cell genomic analyses are moving toward the identification of new molecular targets and driving the development of more specific and less toxic demethylating agents

Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases

The burst of reprogramming technology in recent years has revolutionized the field of stem cell biology, offering new opportunities for personalized, regenerative therapies. The direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has provided an invaluable tool to study and model a wide range of human diseases. Here, we review the transforming potential of such a strategy in research and in therapies applicable to the hematology field

Quantificazione della Nicotinamide Riboside e dei suoi precursori NMN e NAD nel latte mediante un nuovo saggio enzimatico fluorimetrico

QUANTIFICAZIONE DELLA NICOTINAMIDE RIBOSIDE E DEI SUOI PRECURSORI NMN E NAD NEL LATTE, MEDIANTE UN NUOVO SAGGIO ENZIMATICO FLUORIMETRICO. La Nicotinammide Riboside (NR), è una forma della vitamina B3 che può essere assunta come tale con la dieta o può derivare dalla digestione del NAD (Nicotinammide Adenin Dinucleotide) e/o dell’NMN (Nicotinammide Mononucleotide) ad opera degli enzimi della flora o della mucosa intestinale. Una volta assorbita, la vitamina viene convertita a NAD, che ne rappresenta la forma biologicamente attiva. Studi recenti hanno dimostrano che la somministrazione di NR e NMN a modelli murini determina un significativo aumento dei livelli intracellulari di NAD, con conseguente protezione nei confronti di disordini di varia natura, metabolici, mitocondriali e nervosi. La mancanza, ad oggi, di saggi adatti alla quantificazione di NR e NMN in matrici complesse ha limitato l’analisi di queste molecole negli alimenti. Qui presentiamo un saggio enzimatico accoppiato, sensibile, rapido ed economico, che consente la quantificazione simultanea di NR, NMN e NAD. Il saggio è stato applicato all’analisi dei tre analiti in latti di diverse specie animali, compreso l’uomo. I risultati hanno dimostrato che NR, sia come tale, sia sotto forma dei suoi precursori, contribuisce in maniera significativa al contenuto totale di vitamina B3. Il saggio ha anche permesso di valutare la stabilità della vitamina ai trattamenti termici HTST e UHT, rivelando che NR è resistente alla pastorizzazione, ma è completamente distrutta dal trattamento UHT. L’analisi del contenuto di NAD nel latte umano a diversi tempi di lattazione ha mostrato oscillazioni ritmiche dei livelli della molecola nelle 24 ore, in accordo con il ritmo circadiano di sintesi del coenzima. Il saggio è stato anche ottimizzato per la determinazione dei tre analiti in alimenti fermentati, come kefir e birra. Questo studio sarà strumentale alla comprensione della distribuzione di NR, NMN e NAD nei vari alimenti, anche al fine di impiegare i migliori processi tecnologici per preservare le proprietà della vitamina.QUANTITATION OF NICOTINAMIDE RIBOSIDE AND ITS PRECURSORS NMN AND NAD IN MILK, THROUGH A NOVEL ENZYMATIC FLUOROMETRIC ASSAY. Nicotinamide Riboside (NR) is a form of vitamin B3 that can be found as such in the diet or formed from dietary NAD (Nicotinamide Adenine Dinucleotide) and/or NMN (Nicotinamide Mononucleotide) by the combined action of enzymes from the intestinal mucosa and gut microbiota. Recent studies demonstrated that administration of NR and NMN to mice is able to boost intracellular NAD levels resulting in multiple health benefits, including protection against damage to nerves, hearing loss, age-related disorders, metabolic and mitochondrial diseases. The lack of suitable assays to accurately quantitate the amount of NR and NMN in complex matrices has limited so far the assessment of these molecules in food sources. Here, we introduce a novel, sensitive, rapid and economical enzyme-coupled assay for the simultaneous quantitation of NR, NMN and NAD. The assay was tested to measure the concentration of the three analytes in milk collected from different species including, buffalo, bovine, ewe, caprine, donkey, and human. The results showed that NR, both as such and in the form of dietary precursors, accounts for a significant fraction of the total vitamin B3 content. Furthermore, the assay allowed us to evaluate the thermal stability of the vitamin upon heating treatment, showing that NR is resistant to pasteurization, but it is fully destroyed by UHT processing. Interestingly, by studying the analytes’ content in human milk at different lactation times, we observed rhythmic fluctuations of NAD over the 24 h, which is in keeping with the circadian intracellular synthesis of the coenzyme. Finally, the assay was also optimized to measure the analytes in fermented foods, like kefir and beer. This study might pave the way to a better understanding of the distribution of NR, NMN and NAD among different food-types, and to design better processing technologies able to preserve the vitamin and its precursors

Quantificazione della Nicotinamide Riboside e dei suoi precursori NMN e NAD nel latte mediante un nuovo saggio enzimatico fluorimetrico

QUANTIFICAZIONE DELLA NICOTINAMIDE RIBOSIDE E DEI SUOI PRECURSORI NMN E NAD NEL LATTE, MEDIANTE UN NUOVO SAGGIO ENZIMATICO FLUORIMETRICO. La Nicotinammide Riboside (NR), è una forma della vitamina B3 che può essere assunta come tale con la dieta o può derivare dalla digestione del NAD (Nicotinammide Adenin Dinucleotide) e/o dell’NMN (Nicotinammide Mononucleotide) ad opera degli enzimi della flora o della mucosa intestinale. Una volta assorbita, la vitamina viene convertita a NAD, che ne rappresenta la forma biologicamente attiva. Studi recenti hanno dimostrano che la somministrazione di NR e NMN a modelli murini determina un significativo aumento dei livelli intracellulari di NAD, con conseguente protezione nei confronti di disordini di varia natura, metabolici, mitocondriali e nervosi. La mancanza, ad oggi, di saggi adatti alla quantificazione di NR e NMN in matrici complesse ha limitato l’analisi di queste molecole negli alimenti. Qui presentiamo un saggio enzimatico accoppiato, sensibile, rapido ed economico, che consente la quantificazione simultanea di NR, NMN e NAD. Il saggio è stato applicato all’analisi dei tre analiti in latti di diverse specie animali, compreso l’uomo. I risultati hanno dimostrato che NR, sia come tale, sia sotto forma dei suoi precursori, contribuisce in maniera significativa al contenuto totale di vitamina B3. Il saggio ha anche permesso di valutare la stabilità della vitamina ai trattamenti termici HTST e UHT, rivelando che NR è resistente alla pastorizzazione, ma è completamente distrutta dal trattamento UHT. L’analisi del contenuto di NAD nel latte umano a diversi tempi di lattazione ha mostrato oscillazioni ritmiche dei livelli della molecola nelle 24 ore, in accordo con il ritmo circadiano di sintesi del coenzima. Il saggio è stato anche ottimizzato per la determinazione dei tre analiti in alimenti fermentati, come kefir e birra. Questo studio sarà strumentale alla comprensione della distribuzione di NR, NMN e NAD nei vari alimenti, anche al fine di impiegare i migliori processi tecnologici per preservare le proprietà della vitamina.QUANTITATION OF NICOTINAMIDE RIBOSIDE AND ITS PRECURSORS NMN AND NAD IN MILK, THROUGH A NOVEL ENZYMATIC FLUOROMETRIC ASSAY. Nicotinamide Riboside (NR) is a form of vitamin B3 that can be found as such in the diet or formed from dietary NAD (Nicotinamide Adenine Dinucleotide) and/or NMN (Nicotinamide Mononucleotide) by the combined action of enzymes from the intestinal mucosa and gut microbiota. Recent studies demonstrated that administration of NR and NMN to mice is able to boost intracellular NAD levels resulting in multiple health benefits, including protection against damage to nerves, hearing loss, age-related disorders, metabolic and mitochondrial diseases. The lack of suitable assays to accurately quantitate the amount of NR and NMN in complex matrices has limited so far the assessment of these molecules in food sources. Here, we introduce a novel, sensitive, rapid and economical enzyme-coupled assay for the simultaneous quantitation of NR, NMN and NAD. The assay was tested to measure the concentration of the three analytes in milk collected from different species including, buffalo, bovine, ewe, caprine, donkey, and human. The results showed that NR, both as such and in the form of dietary precursors, accounts for a significant fraction of the total vitamin B3 content. Furthermore, the assay allowed us to evaluate the thermal stability of the vitamin upon heating treatment, showing that NR is resistant to pasteurization, but it is fully destroyed by UHT processing. Interestingly, by studying the analytes’ content in human milk at different lactation times, we observed rhythmic fluctuations of NAD over the 24 h, which is in keeping with the circadian intracellular synthesis of the coenzyme. Finally, the assay was also optimized to measure the analytes in fermented foods, like kefir and beer. This study might pave the way to a better understanding of the distribution of NR, NMN and NAD among different food-types, and to design better processing technologies able to preserve the vitamin and its precursors

The PARP Way to Epigenetic Changes

ADP-ribosylation, is a reversible post-translational modification implicated in major biological functions. Poly ADP-ribose polymerases (PARP) are specialized enzymes that catalyze the addition of ADP ribose units from “nicotinamide adenine dinucleotide-donor molecules” to their target substrates. This reaction known as PARylation modulates essential cellular processes including DNA damage response, chromatin remodeling, DNA methylation and gene expression. Herein, we discuss emerging roles of PARP1 in chromatin remodeling and epigenetic regulation, focusing on its therapeutic implications for cancer treatment and beyond

Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases

The burst of reprogramming technology in recent years has revolutionized the field of stem cell biology, offering new opportunities for personalized, regenerative therapies. The direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has provided an invaluable tool to study and model a wide range of human diseases. Here, we review the transforming potential of such a strategy in research and in therapies applicable to the hematology field

Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay

Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time

NAD modulates dna methylation and cell differentiation

10.3390/cells10112986Cells1011298

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+ /K+ ATPase

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca2+/calmodulin\u2010activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein\u2010expressing astrocytes (astroglial calcineurin KO [ACN\u2010KO]). Here, we report that postnatal and astrocyte\u2010specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K+ homeostasis. Indeed, blockade of Na+/K+\u2010ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN\u2010KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN\u2010KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center\u2010stage as an astrocytic Ca2+\u2010sensitive switch