The atrophic effect of 1,25(Oh)2 vitamin d3 (calcitriol) on c2c12 myotubes depends on oxidative stress

Dysfunctional mitochondrial metabolism has been linked to skeletal muscle loss in several physio-pathological states. Although it has been reported that vitamin D (VD) supports cellular redox homeostasis by maintaining normal mitochondrial functions, and VD deficiency often occurs in conditions associated with skeletal muscle loss, the efficacy of VD supplementation to overcome muscle wasting is debated. Investigations on the direct effects of VD metabolites on skeletal muscle using C2C12 myotubes have revealed an unexpected pro-atrophic activity of calcitriol (1,25VD), while its upstream metabolites cholecalciferol (VD3) and calcidiol (25VD) have anti-atrophic effects. Here, we investigated if the atrophic effects of 1,25VD on myotubes depend on its activity on mitochondrial metabolism. The impact of 1,25VD and its upstream metabolites VD3 and 25VD on mitochondria dynamics and the activity of C2C12 myotubes was evaluated by measuring mitochondrial content, architecture, metabolism, and reactive oxygen species (ROS) production. We found that 1,25VD induces atrophy through protein kinase C (PKC)-mediated ROS production, mainly of extramito-chondrial origin. Consistent with this, cotreatment with the antioxidant N-acetylcysteine (NAC), but not with the mitochondria-specific antioxidant mitoTEMPO, was sufficient to blunt the atrophic activity of 1,25VD. In contrast, VD3 and 25VD have antioxidant properties, suggesting that the efficacy of VD supplementation might result from the balance between atrophic pro-oxidant (1,25VD) and protective antioxidant (VD3 and 25VD) metabolites

Effect of unacylated ghrelin on peripheral nerve regeneration

Ghrelin is a circulating peptide hormone released by enteroendocrine cells of the gastrointestinal tract as two forms, acylated and unacylated. Acylated ghrelin (AG) binds to the growth hormone secretagogue receptor 1a (GHSR1a), thus stimulating food intake, growth hormone release, and gastrointestinal motility. Conversely, unacylated GHR (UnAG), through binding to a yet unidentified receptor, protects the skeletal muscle from atrophy, stimulates muscle regeneration, and protects cardiomyocytes from ischemic damage. Recently, interest about ghrelin has raised also among neuroscientists because of its effect on the nervous system, especially the stimulation of neurogenesis in spinal cord, brain stem, and hippocampus. However, few information is still available about its effectiveness on peripheral nerve regeneration. To partially fill this gap, the aim of this study was to assess the effect of UnAG on peripheral nerve regeneration after median nerve crush injury and after nerve transection immediately repaired by means of an end-to-end suture. To this end, we exploited FVB1 Myh6/Ghrl transgenic mice in which overexpression of the ghrelin gene (Ghrl) results in selective up-regulation of circulating UnAG levels, but not of AG. Regeneration was assessed by both functional evaluation (grasping test) and morphometrical analysis of regenerated myelinated axons. Results obtained lead to conclude that UnAG could have a role in development of peripheral nerves and during more severe lesions

A novel method to optimize autologous adipose tissue recovery with extracellular matrix preservation

This work aims to characterize a new method to recover low-manipulated human adipose tissue, enriched with adipose tissue-derived mesenchymal stem cells (ATD-MSCs) for autologous use in regenerative medicine applications. Lipoaspirated fat collected from patients was processed through Lipocell, a Class II-a medical device for dialysis of adipose tissue, by varying filter sizes and washing solutions. ATD-MSC yield was measured with flow cytometry after stromal vascular fraction (SVF) isolation in fresh and cultured samples. Purification from oil and blood was measured after centrifugation with spectrophotometer analysis. Extracellular matrix preservation was assessed through hematoxylin and eosin (H&E) staining and biochemical assay for total collagen, type-2 collagen, and glycosaminoglycans (GAGs) quantification. Flow cytometry showed a two-fold increase of ATD-MSC yield in treated samples in comparison with untreated lipoaspirate; no differences where reported when varying filter size. The association of dialysis and washing thoroughly removed blood and oil from samples. Tissue architecture and extracellular matrix integrity were unaltered after Lipocell processing. Dialysis procedure associated with Ringer’s lactate preserves the proliferation ability of ATD-MSCs in cell culture. The characterization of the product showed that Lipocell is an efficient method for purifying the tissue from undesired byproducts and preserving ATD-MSC vitality and extracellular matrix (ECM) integrity, resulting in a promising tool for regenerative medicine applications

Enhancing resistance against African weevils through development of transgenic sweetpotato cultivars (Ipomoea batatas (L.) Lam.) expressing cry7Aa1, cry3Ca1 and ET33-34 genes.

Sweetpotato (Ipomoea batatas) is one of the most important food crops in tropical and subtropical countries. In Sub-Saharan Africa, sweetpotato is mainly produced for consumption and as a source of income by resource-poor farmers. However, their production is limited by severe damage caused by pests and diseases. The African weevils Cylas puncticollis and C. brunneus are the main biological constraints that may cause losses between 50 and 100%. Biotechnological approaches to control weevils include the introduction of genes encoding Cry proteins found to be active against these pests. To that end, several protocols for sweetpotato regeneration and transformation by organogenesis or somatic embryogenesis have been developed but their efficiency remains largely genotype-dependent and time-consuming. In this study, 31 African sweetpotato cultivars from CIP genebank were screened for regeneration and transformation efficiencies by organogenesis and somatic embryogenesis. Additionally, “Jewel” and “Jonathan” cultivars were used as organogenic and embryogenic controls, respectively. Regeneration by organogenesis was conducted using a two-step protocol including 2,4-D then thidiazuron, zeatin or kinetin while regeneration by embryogenesis was performed using a three-step protocol, each one using a different hormone (2,4,5-T, ABA and AG3). Higher than 40% regeneration efficiencies were obtained for 8 cultivars (Jewel, Imby, Kawogo, Luapula, Mafutha, CIP440163, Zambezi and Ukerewe) with an organogenesis protocol and 8 cultivars (Jonathan, Imby, K51/3251, Bwanjule, CIP440163, SPK004, New Kawogo and KSP 11) with an embryogenesis protocol. Genetic transformation of sweetpotato with Jewel by organogenesis and Imby, CIP440163 and Jonathan by somatic embryogenesis has been achieved using chimerical genes coding for three of the most active proteins (Cry7Aa1, ET33-34, and Cry3Ca1) against African weevils. Transgenic events have been confirmed by kanamycin resistant calli test, PCR and Southern blot. Transcriptional activity and Cry protein accumulation are being tested in leaves and storage roots by Real time PCR and DAS-ELISA respectively

A novel method to optimize autologous adipose tissue recovery with extracellular matrix preservation

This work aims to characterize a new method to recover low-manipulated human adipose tissue, enriched with adipose tissue-derived mesenchymal stem cells (ATD-MSCs) for autologous use in regenerative medicine applications. Lipoaspirated fat collected from patients was processed through Lipocell, a Class II-a medical device for dialysis of adipose tissue, by varying filter sizes and washing solutions. ATD-MSC yield was measured with flow cytometry after stromal vascular fraction (SVF) isolation in fresh and cultured samples. Purification from oil and blood was measured after centrifugationwith spectrophotometer analysis. Extracellularmatrix preservationwas assessed through hematoxylin and eosin (H&E) staining and biochemical assay for total collagen, type-2 collagen, and glycosaminoglycans (GAGs) quantification. Flow cytometry showed a two-fold increase of ATD-MSC yield in treated samples in comparisonwith untreated lipoaspirate; no differenceswhere reportedwhen varying filter size. The association of dialysis and washing thoroughly removed blood and oil from samples. Tissue architecture and extracellular matrix integrity were unaltered after Lipocell processing. Dialysis procedure associated with Ringer's lactate preserves the proliferation ability of ATD-MSCs in cell culture. The characterization of the product showed that Lipocell is an efficient method for purifying the tissue from undesired byproducts and preserving ATD-MSC vitality and extracellular matrix (ECM) integrity, resulting in a promising tool for regenerative medicine applications

Boosting intracellular sodium selectively kills hepatocarcinoma cells and induces hepatocellular carcinoma tumor shrinkage in mice

Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, Na-23-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O-2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.The ionophore monensin is shown to have cancer-selective cytotoxic action by selectively increasing the sodium content in cultured hepatocellular carcinoma cells (HCC) and allografts, highlighting the sensitivity of HCC cells to pharmacologically induced Na+ load

ACYLATED AND UNACYLATED GHRELIN IMPAIR SKELETAL MUSCLE ATROPHY IN MICE.

Cachexia is a wasting syndrome associated with cancer, AIDS, and multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1aindependent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3K\u3b2-, mTORC2-, and p38-mediated pathways in myotubes. Up-regulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsrdeficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner

Both ghrelin deletion and unacylated ghrelin overexpression preserve muscles in aging mice

Sarcopenia, the decline in muscle mass and functionality during aging, might arise from age-associated endocrine dysfunction. Ghrelin is a hormone circulating in both acylated (AG) and unacylated (UnAG) forms with antiatrophic activity on skeletal muscle. Here, we show that not only lifelong overexpression of UnAG (Tg) in mice, but also the deletion of ghrelin gene (Ghrl KO) attenuated the age-associated muscle atrophy and functionality decline, as well as systemic inflammation. Yet, the aging of Tg and Ghrl KO mice occurs with different dynamics: while old Tg mice seem to preserve the characteristics of young animals, Ghrl KO mice features deteriorate with aging. However, young Ghrl KO mice show more favorable traits compared to WT animals that result, on the whole, in better performances in aged Ghrl KO animals. Treatment with pharmacological doses of UnAG improved muscle performance in old mice without modifying the feeding behavior, body weight, and adipose tissue mass. The antiatrophic effect on muscle mass did not correlate with modifications of protein catabolism. However, UnAG treatment induced a strong shift towards oxidative metabolism in muscle. Altogether, these data confirmed and expanded some of the previously reported findings and advocate for the design of UnAG analogs to treat sarcopenia

Personalized at-home neurofeedback compared with long-acting methylphenidate in an european non-inferiority randomized trial in children with ADHD

Neurofeedback (NF) has gained increasing interest among non-pharmacological treatments for Attention Deficit Hyperactivity Disorder (ADHD). NF training aims to enhance self-regulation of brain activities. The goal of the NEWROFEED study is to assess the efficacy of a new personalized NF training device, using two different protocols according to each child's electroencephalographic pattern, and designed for use at home. This study is a non-inferiority trial comparing NF to methylphenidate. The study is a prospective, multicentre, randomized, reference drug-controlled trial. One hundred seventy-nine children with ADHD, aged 7 to 13 years will be recruited in 13 clinical centres from 5 European countries. Subjects will be randomized to two groups: NF group (Neurofeedback Training Group) and MPH group (Methylphenidate group). Outcome measures include clinicians, parents and teachers' assessments, attention measures and quantitative EEG (qEEG). Patients undergo eight visits over a three-month period: pre-inclusion visit, inclusion visit, 4 "discovery" (NF group) or titration visits (MPH group), an intermediate and a final visit. Patients will be randomized to either the MPH or NF group. Children in the NF group will undergo either an SMR or a Theta/Beta training protocol according to their baselineTheta/Beta Ratio obtained from the qEEG. This is the first non-inferiority study between a personalized NF device and pharmacological treatment. Innovative aspects of Mensia Koala™ include the personalization of the training protocol according to initial qEEG characteristics (SMR or Theta/Beta training protocols) and an improved accessibility of NF due to the opportunity to train at home with monitoring by the clinician through a dedicated web portal. NCT02778360 . Date registration (retrospectively registered): 5-12-2016. Registered May 19, 2016