Vitamin B12 Deficiency and the Nervous System: Beyond Metabolic Decompensation-Comparing Biological Models and Gaining New Insights into Molecular and Cellular Mechanisms.

Vitamin B12 (VitB12) is a micronutrient and acts as a cofactor for fundamental biochemical reactions: the synthesis of succinyl-CoA from methylmalonyl-CoA and biotin, and the synthesis of methionine from folic acid and homocysteine. VitB12 deficiency can determine a wide range of diseases, including nervous system impairments. Although clinical evidence shows a direct role of VitB12 in neuronal homeostasis, the molecular mechanisms are yet to be characterized in depth. Earlier investigations focused on exploring the biochemical shifts resulting from a deficiency in the function of VitB12 as a coenzyme, while more recent studies propose a broader mechanism, encompassing changes at the molecular/cellular levels. Here, we explore existing study models employed to investigate the role of VitB12 in the nervous system, including the challenges inherent in replicating deficiency/supplementation in experimental settings. Moreover, we discuss the potential biochemical alterations and ensuing mechanisms that might be modified at the molecular/cellular level (such as epigenetic modifications or changes in lysosomal activity). We also address the role of VitB12 deficiency in initiating processes that contribute to nervous system deterioration, including ROS accumulation, inflammation, and demyelination. Consequently, a complex biological landscape emerges, requiring further investigative efforts to grasp the intricacies involved and identify potential therapeutic targets.

Altered vitamin B12 metabolism in the central nervous system is associated with the modification of ribosomal gene expression: new insights from comparative RNA dataset analysis.

Recent studies have confirmed the direct role of vitamin B12 (VitB12) in the central nervous system (CNS) homeostasis; nevertheless, the detailed mechanisms are poorly understood. By analyzing RNA-Seq and microarray datasets obtained from databanks, this study aims to identify possible basic mechanisms, related to the brain, involved in altering the gene expression under VitB12 deficiency mimicking conditions. The database inquiry returned datasets generated from distinctly heterogeneous experimental sets and considering the quality and relevance requirements, two datasets from mouse and one from rat models were selected. The analyses of individual datasets highlighted a change in ribosomal gene expression in VitB12 deficiency mimicking conditions within each system. Specifically, a divergent regulation was observed depending on the animal model: mice showed a down regulation of the ribosomal gene expression, while rats an upregulation. Interestingly, E2f1 was significantly upregulated under VitB12 deficiency mimicking conditions in the animal models, with a greater upregulation in rats. The rat model also revealed putative E2F1 Transcription Factor Binding Sites (TFBSs) in the promoter of the differently regulated genes involved in ribosomal gene expression. This suggested the possibility that E2F1, being greater expressed in rats, could activate the ribosomal genes having E2F1 TFBSs, thus giving a plausible explication to the divergent regulation observed in animal models. Despite the great diversity of the experimental sets used to generate the datasets considered, a common alteration of the ribosomes exists, thereby indicating a possible basic and conserved response to VitB12 deficiency. Moreover, these findings could provide new insights on E2F1 and its association with CNS homeostasis and VitB12 deficiency.

Therapeutic Effect of Polymeric Nanomicelles Formulation of LY2157299-Galunisertib on CCl4-Induced Liver Fibrosis in Rats.

Hepatic fibrosis (HF) is a major cause of liver-related disorders and together with cancer-associated fibroblasts can favor liver cancer development by modulating the tumor microenvironment. Advanced HF, characterized by an excess of extracellular matrix (ECM), is mediated by TGF- ß1, that activates hepatic stellate cells (HSCs) and fibroblasts. A TGF-ß1 receptor inhibitor, LY2157299 or Galunisertib (GLY), has shown promising results against chronic liver progression in animal models, and we show that it can be further improved by enhancing GLYs bioavailability through encapsulation in polymeric polygalacturonic-polyacrylic acid nanomicelles (GLY-NMs). GLY-NMs reduced HF in an in vivo rat model of liver fibrosis induced by intraperitoneal injection of CCl4 as shown by the morphological, biochemical, and molecular biology parameters of normal and fibrotic livers. Moreover, GLY-NM was able to induce recovery from HF better than free GLY. Indeed, the encapsulated drug reduces collagen deposition, hepatic stellate cells (HSCs) activation, prevents fatty degeneration and restores the correct lobular architecture of the liver as well as normalizes the serum parameters and expression of the genes involved in the onset of HF. In summary, GLY-NM improved the pharmacological activity of the free TGF- ß1 inhibitor in the in vivo HF treatment and thus is a candidate as a novel therapeutic strategy.

Proinflammatory and Cancer-Promoting Pathobiont Fusobacterium nucleatum Directly Targets Colorectal Cancer Stem Cells.

Intestinal bacterial communities participate in gut homeostasis and are recognized as crucial in bowel inflammation and colorectal cancer (CRC). Fusobacterium nucleatum (Fn), a pathobiont of the oral microflora, has recently emerged as a CRC-associated microbe linked to disease progression, metastasis, and a poor clinical outcome; however, the primary cellular and/or microenvironmental targets of this agent remain elusive. We report here that Fn directly targets putative colorectal cancer stem cells (CR-CSCs), a tumor cell subset endowed with cancer re-initiating capacity after surgery and chemotherapy. A patient-derived CSC line, highly enriched (70%) for the stem marker CD133, was expanded as tumor spheroids, dissociated, and exposed in vitro to varying amounts (range 100-500 MOI) of Fn. We found that Fn stably adheres to CSCs, likely by multiple interactions involving the tumor-associated Gal-GalNac disaccharide and the Fn-docking protein CEA-family cell adhesion molecule 1 (CEACAM-1), robustly expressed on CSCs. Importantly, Fn elicited innate immune responses in CSCs and triggered a growth factor-like, protein tyrosine phosphorylation cascade largely dependent on CEACAM-1 and culminating in the activation of p42/44 MAP kinase. Thus, the direct stimulation of CSCs by Fn may contribute to microbiota-driven colorectal carcinogenesis and represent a target for innovative therapies.

Amino-functionalized mesoporous silica nanoparticles (NH2-MSiNPs) impair the embryonic development of the sea urchin Paracentrotus lividus.

Nanoparticles have found use in a wide range of applications, mainly as carriers of active biomolecules. It is thus necessary to assess their toxicity for human health, as well as for the environment, on which there is still a gap of knowledge. In this work, sea urchin Paracentrotus lividus, a widely used model for embryotoxicity and spermiotoxicity, has been used to assess potential detrimental effects of amino-functionalized mesoporous silica nanoparticles (NH2-MSiNPs) on embryonic development. Specifically, gametes quality, embryogenesis morphological and timing alterations, and cellular stress markers, such as mitochondrial functionality, were assessed in presence of different concentrations of NH2-MSiNPs in filtered seawater (FSW). Furthermore, dorsal-ventral axis development and skeletogenesis were characterized by microscopy imaging and gene expression analysis. NH2-MSiNPs determined a strong reduction in the egg fertilization rate. Consequently, the presence of NH2-MSiNPs resulted detrimental in P. lividus embryonic development, with severe morphological alterations correlated with an increased embryos mortality. Finally, NH2-MSiNPs treatment was responsible for other toxic effects, such as reduced mitochondrial function and skeletogenesis alterations, according to the reduced mineralization sites in the endoskeleton formation and the related genes altered expression. Taken together, these results suggest the potential toxic effects of NH2-MSiNPs on the marine ecosystem, with consequences for the development and reproduction of its organisms. Despite their promising potential as carriers of biomolecules, it is pivotal to consider that their uncontrolled use may result harmful to the environment and, consequently, to living organisms.

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. RESULTS: In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration. CONCLUSION: The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. LAY SUMMARY: Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

A TLR/CD44 axis regulates T cell trafficking in experimental and human multiple sclerosis.

In the pathogenesis of autoimmune disorders, the modulation of leukocytes' trafficking plays a central role, still poorly understood. Here, we focused on the effect of TLR2 ligands in trafficking of T helper cells through reshuffling of CD44 isoforms repertoire. Concurrently, strain background and TLR2 haplotype affected Wnt/ß-catenin signaling pathway and expression of splicing factors. During EAE, mCD44 v9- v 10 was specifically enriched in the forebrain and showed an increased ability to bind stably to osteopontin. Similarly, we observed that hCD44 v7 was highly enriched in cells of cerebrospinal fluid from MS patients with active lesions. Moreover, TLRs engagement modulated the composition of CD44 variants also in human T helper cells, supporting the hypothesis that pathogens or commensals, through TLRs, in turn modulate the repertoire of CD44 isoforms, thereby controlling the distribution of lesions in the CNS. The interference with this mechanism(s) represents a potential tool for prevention and treatment of autoimmune relapses and exacerbations.

Oleoylethanolamide Reduces Hepatic Oxidative Stress and Endoplasmic Reticulum Stress in High-Fat Diet-Fed Rats.

Long-term high-fat diet (HFD) consumption can cause weight gain and obesity, two conditions often associated with hepatic non-alcoholic fatty liver and oxidative stress. Oleoylethanolamide (OEA), a lipid compound produced by the intestine from oleic acid, has been associated with different beneficial effects in diet-induced obesity and hepatic steatosis. However, the role of OEA on hepatic oxidative stress has not been fully elucidated. In this study, we used a model of diet-induced obesity to study the possible antioxidant effect of OEA in the liver. In this model rats with free access to an HFD for 77 days developed obesity, steatosis, and hepatic oxidative stress, as compared to rats consuming a low-fat diet for the same period. Several parameters associated with oxidative stress were then measured after two weeks of OEA administration to diet-induced obese rats. We showed that OEA reduced, compared to HFD-fed rats, obesity, steatosis, and the plasma level of triacylglycerols and transaminases. Moreover, OEA decreased the amount of malondialdehyde and carbonylated proteins and restored the activity of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, which decreased in the liver of HFD-fed rats. OEA had also an improving effect on parameters linked to endoplasmic reticulum stress, thus demonstrating a role in the homeostatic control of protein folding. Finally, we reported that OEA differently regulated the expression of two transcription factors involved in the control of lipid metabolism and antioxidant genes, namely nuclear factor erythroid-derived 2-related factor 1 (Nrf1) and Nrf2, thus suggesting, for the first time, new targets of the protective effect of OEA in the liver.

Current Nanocarrier Strategies Improve Vitamin B12 Pharmacokinetics, Ameliorate Patients' Lives, and Reduce Costs.

Vitamin B12 (VitB12) is a naturally occurring compound produced by microorganisms and an essential nutrient for humans. Several papers highlight the role of VitB12 deficiency in bone and heart health, depression, memory performance, fertility, embryo development, and cancer, while VitB12 treatment is crucial for survival in inborn errors of VitB12 metabolism. VitB12 is administrated through intramuscular injection, thus impacting the patients' lifestyle, although it is known that oral administration may meet the specific requirement even in the case of malabsorption. Furthermore, the high-dose injection of VitB12 does not ensure a constant dosage, while the oral route allows only 1.2% of the vitamin to be absorbed in human beings. Nanocarriers are promising nanotechnology that can enable therapies to be improved, reducing side effects. Today, nanocarrier strategies applied at VitB12 delivery are at the initial phase and aim to simplify administration, reduce costs, improve pharmacokinetics, and ameliorate the quality of patients' lives. The safety of nanotechnologies is still under investigation and few treatments involving nanocarriers have been approved, so far. Here, we highlight the role of VitB12 in human metabolism and diseases, and the issues linked to its molecule properties, and discuss how nanocarriers can improve the therapy and supplementation of the vitamin and reduce possible side effects and limits.

Micro and Nanoplastics Identification: Classic Methods and Innovative Detection Techniques.

Micro and nanoplastics are fragments with dimensions less than a millimeter invading all terrestrial and marine environments. They have become a major global environmental issue in recent decades and, indeed, recent scientific studies have highlighted the presence of these fragments all over the world even in environments that were thought to be unspoiled. Analysis of micro/nanoplastics in isolated samples from abiotic and biotic environmental matrices has become increasingly common. Hence, the need to find valid techniques to identify these micro and nano-sized particles. In this review, we discuss the current and potential identification methods used in microplastic analyses along with their advantages and limitations. We discuss the most suitable techniques currently available, from physical to chemical ones, as well as the challenges to enhance the existing methods and develop new ones. Microscopical techniques (i.e., dissect, polarized, fluorescence, scanning electron, and atomic force microscopy) are one of the most used identification methods for micro/nanoplastics, but they have the limitation to produce incomplete results in analyses of small particles. At present, the combination with chemical analysis (i.e., spectroscopy) overcome this limit together with recently introduced alternative approaches. For example, holographic imaging in microscope configuration images microplastics directly in unfiltered water, thus discriminating microplastics from diatoms and differentiates different sizes, shapes, and plastic types. The development of new analytical instruments coupled with each other or with conventional and innovative microscopy could solve the current problems in the identification of micro/nanoplastics.

Nutrients and neurogenesis: the emerging role of autophagy and gut microbiota.

Adult neurogenesis, the generation of mature functional neurons from neural stem cells in specific regions of the adult mammalian brain, is implicated in brain physiology, neurodegeneration and mood disorders. Among the many intrinsic and extrinsic factors that modulate neurogenic activity, the role of nutrients, energy metabolism, and gut microbiota has recently emerged. It is increasingly evident that excessive calorie intake accelerates the age-dependent decline of neurogenesis, while calorie restriction and physical exercise have the opposite effect. Mechanistically, nutrient availability could affect neurogenesis by modulating autophagy, a cell-rejuvenating process, in neural stem cells. In parallel, diet can alter the composition of gut microbiota thus impacting the intestine-neurogenic niche communication. These exciting breakthroughs are here concisely reviewed.

The Promoter-Associated Noncoding RNA pncCCND1_B Assembles a Protein-RNA Complex to Regulate Cyclin D1 Transcription in Ewing Sarcoma.

Most Ewing sarcomas are characterized by the in-frame chromosomal translocation t(11;22) generating the EWS-FLI1 oncogene. EWS-FLI1 protein interacts with the RNA helicase DHX9 and affects transcription and processing of genes involved in neoplastic transformation, including CCND1 (the cyclin D1 gene), which contributes to cell-cycle dysregulation in cancer. In this study, we found that CCND1 expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the pncCCND1_B RNA, a previously uncharacterized CCND1 promoter-associated noncoding (pnc) transcript, is expressed in Ewing sarcoma cells. PncCCND1_B interacted with the RNA-binding protein Sam68 and repressed CCND1 expression. Notably, knockdown of Sam68 affected pncCCND1_B subcellular localization and cyclin D1 expression. Pharmacologic impairment of DHX9/EWS-FLI1 interaction promoted RNA-dependent association of Sam68 with DHX9 and recruitment of Sam68 to the CCND1 promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF1 impaired Sam68/DHX9 interaction and positively regulated CCND1 expression. These studies uncover a fine-tuned modulation of the proto-oncogene CCND1 in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS-FLI1 or pncCCND1_B-Sam68. SIGNIFICANCE: A pncRNA-based mechanism represses expression of CCND1 through the formation of a protein-RNA complex and provides new therapeutic opportunities for patients with Ewing sarcoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/14/3570/F1.large.jpg.

hnRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells.

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPM motifs were significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment.

Nutrients, neurogenesis and brain ageing: From disease mechanisms to therapeutic opportunities.

Appreciation of the physiological relevance of mammalian adult neurogenesis has in recent years rapidly expanded from a phenomenon of homeostatic cell replacement and brain repair to the current view of a complex process involved in high order cognitive functions. In parallel, an array of endogenous or exogenous triggers of neurogenesis has also been identified, among which metabolic and nutritional cues have drawn significant attention. Converging evidence from animal and in vitro studies points to nutrient sensing and energy metabolism as major physiological determinants of neural stem cell fate, and modulators of the whole neurogenic process. While the cellular and molecular circuitries underlying metabolic regulation of neurogenesis are still incompletely understood, the key role of mitochondrial activity and dynamics, and the importance of autophagy have begun to be fully appreciated; moreover, nutrient-sensitive pathways and transducers such as the insulin-IGF cascade, the AMPK/mTOR axis and the transcription regulators CREB and Sirt-1 have been included, beside more established "developmental" signals like Notch and Wnt, in the molecular networks that dictate neural-stem-cell self-renewal, migration and differentiation in response to local and systemic inputs. Many of these nutrient-related cascades are deregulated in the contest of metabolic diseases and in ageing, and may contribute to impaired neurogenesis and thus to cognition defects observed in these conditions. Importantly, accumulating knowledge on the metabolic control of neurogenesis provides a theoretical framework for the trial of new or repurposed drugs capable of interfering with nutrient sensing as enhancers of neurogenesis in the context of neurodegeneration and brain senescence.

Neural Stem Cells and Nutrients: Poised Between Quiescence and Exhaustion.

Adult neurogenesis initiated by neural stem cells (NSCs) contributes to brain homeostasis, damage repair, and cognition. Energy metabolism plays a pivotal role in neurogenic cell fate decisions regarding self-renewal, expansion and multilineage differentiation. NSCs need to fine-tune quiescence and proliferation/commitment to guarantee lifelong neurogenesis and avoid premature exhaustion. Accumulating evidence supports a model whereby calorie restriction or increased energy expenditure reinforce NSC quiescence and promote self-renewal. Conversely, growth/proliferation inputs and anabolic signals, although necessary for neurogenesis, deplete the NSCs pool in the long run. This framework incorporates the emerging neurogenic roles of nutrient-sensing signaling pathways, providing a rationale for the alarming connection between nutritional imbalances, metabolic disorders and accelerated brain aging.

The RNA helicase A in malignant transformation.

The RNA helicase A (RHA) is involved in several steps of RNA metabolism, such as RNA processing, cellular transit of viral molecules, ribosome assembly, regulation of transcription and translation of specific mRNAs. RHA is a multifunctional protein whose roles depend on the specific interaction with different molecular partners, which have been extensively characterized in physiological situations. More recently, the functional implication of RHA in human cancer has emerged. Interestingly, RHA was shown to cooperate with both tumor suppressors and oncoproteins in different tumours, indicating that its specific role in cancer is strongly influenced by the cellular context. For instance, silencing of RHA and/or disruption of its interaction with the oncoprotein EWS-FLI1 rendered Ewing sarcoma cells more sensitive to genotoxic stresses and affected tumor growth and maintenance, suggesting possible therapeutic implications. Herein, we review the recent advances in the cellular functions of RHA and discuss its implication in oncogenesis, providing a perspective for future studies and potential translational opportunities in human cancer.

Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9.

Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability.