AGC1 Deficiency: Pathology and Molecular and Cellular Mechanisms of the Disease

AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named “early infantile epileptic encephalopathy 39” (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.     

Pyroptosis-Mediated Periodontal Disease

Pyroptosis is a caspase-dependent process relevant to the understanding of beneficial host responses and medical conditions for which inflammation is central to the pathophysiology of the disease. Pyroptosis has been recently suggested as one of the pathways of exacerbated inflammation of periodontal tissues. Hence, this focused review aims to discuss pyroptosis as a pathological mechanism in the cause of periodontitis. The included articles presented similarities regarding methods, type of cells applied, and cell stimulation, as the outcomes also point to the same direction considering the cellular events. The collected data indicate that virulence factors present in the diseased periodontal tissues initiate the inflammasome route of tissue destruction with caspase activation, cleavage of gasdermin D, and secretion of interleukins IL-1β and IL-18. Consequently, removing periopathogens’ virulence factors that trigger pyroptosis is a potential strategy to combat periodontal disease and regain tissue homeostasis.     

Endothelial ADAM17 Expression in the Progression of Kidney Injury in an Obese Mouse Model of Pre-Diabetes

Disintegrin and metalloproteinase domain 17 (ADAM17) activates inflammatory and fibrotic processes through the shedding of various molecules such as Tumor Necrosis Factor-α (TNF-α) or Transforming Growht Factor-α (TGF-α). There is a well-recognised link between TNF-α, obesity, inflammation, and diabetes. In physiological situations, ADAM17 is expressed mainly in the distal tubular cell while, in renal damage, its expression increases throughout the kidney including the endothelium. The aim of this study was to characterize, for the first time, an experimental mouse model fed a high-fat diet (HFD) with a specific deletion of Adam17 in endothelial cells and to analyse the effects on different renal structures. Endothelial Adam17 knockout male mice and their controls were fed a high-fat diet, to induce obesity, or standard rodent chow, for 22 weeks. Glucose tolerance, urinary albumin-to-creatinine ratio, renal histology, macrophage infiltration, and galectin-3 levels were evaluated. Results showed that obese mice presented higher blood glucose levels, dysregulated glucose homeostasis, and higher body weight compared to control mice. In addition, obese wild-type mice presented an increased albumin-to-creatinine ratio; greater glomerular size and mesangial matrix expansion; and tubular fibrosis with increased galectin-3 expression. Adam17 deletion decreased the albumin-to-creatinine ratio, glomerular mesangial index, and tubular galectin-3 expression. Moreover, macrophage infiltration in the glomeruli of obese Adam17 knockout mice was reduced as compared to obese wild-type mice. In conclusion, the expression of ADAM17 in endothelial cells impacted renal inflammation, modulating the renal function and histology in an obese pre-diabetic mouse model.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

Effects of composite viscosity and adhesive curing mode on marginal sealing of restorations

Objectives: Evaluate the influence of composite viscosity, adhesive systems curing mode and artificial aging on marginal sealing of composite resin restorations. Methods: 240 cavities were prepared in bovine incisors. Teeth were divided in two groups: GR – GrandioSO (Voco), conventional viscosity and GF – GrandioSO Heavy Flow (Voco), low viscosity. Each group was divided into 6 subgroups: FM – Futura Bond M (Voco), FDCL and FDCC – Futura Bond DC (Voco), light and chemical cured, CS – Clearfil S³ Bond (Kuraray), CDCL and CDCC – Cleafil DC (Kuraray), light and chemical cured. Half of all specimens were subjected to mechanical (300.000) and thermal cycling (1.000). Marginal gap was measured in an optical microscope. For microleakage, specimens were stained in 50% ammoniacal silver nitrate. Results: For marginal gap mean (μm), significant differences were shown for all factors (p = 0.000). Results for resin were GR: 28.1 and GF: 40.6; for adhesive CDCC: 28.67, CDCL: 32.25, CS: 34.12, FDCL: 36.10, FM: 36.50 and FDCC: 38.46; and for artificial aging WITHOUT: 29.05 and WITH: 39.65. For microleakage mean (mm), there were also differences for all factors (p = 0.000). Results for resin were GR: 2.68 and GF: 3.46; for adhesive FM: 2.85, CDCC: 3.00, FDCL: 3.08, CDCL: 3.15, CS: 3.16 and FDCC: 3.19; and for artificial aging WITHOUT: 2.66 and WITH: 3.48. Conclusion: Composite with conventional viscosity resulted in narrower marginal gaps and less microleakage. CDCC showed less marginal gap formation. The presence of thermo-mechanical cycling increased the values of gap and microleakage.     

Influence of nutritional status on the effectiveness of a dietary supplement of live lactobacillus to prevent and cure diarrhoea in children

The influence of undernutrition on the effectiveness of a dietary supplement of live lactobacillus regarding prevention and treatment of infantile diarrhoea was studied on 200 children 6 to 24m old. Children, undernourished (D) or controls (C), received for 90 days, in a blind experiment, a fermented milk providing L. Acidofilus y L. Casei (10(7)-10(8)/ml) (LB) or an equivalent amount of fluid milk (L). diarrhoea episodes were recorded and classified according duration: 1-4, 5-14 days, and protracted diarrhea (DP) those lasting beyond 14 days. Study requirements were fulfilled by 119 children: D-L: n=25; D-LB: n=32; C-L: n=27 y C-LB: n=35. Preventive aspects were evaluated through number of episodes and through their lasting the therapeutic ones. Episodes recorded were 134: 29 in D-L; 48 in D-LB; 37 in C-L and 20 in C-LB. In the D-LB group over 90% episodes were very short (1-4 days), percentage far higher to that observed in D-L and similar to C-LB. There were 12 episodes of DP, 9 D and 3 in C, all in the L groups. In conclusion, although the fermented milk prevent half episode in the controls but not in the undernourished, it was able to shorter episodes duration and prevent protracted diarrhoea irrespectively of nutritional status. Therefore, undernutrition impaired the ability of the lactobacillus supplement to prevent children diarrhoea, which implicates immune system, but not curative effects which are the result of local actions.     

Postprandial and short-term effects of dietary virgin olive oil on oxidant/antioxidant status

It is generally believed that virgin olive oil consumption has beneficial effects, but little is known about its effects postprandially on oxidant/antioxidant status. The aim of this study was to determine changes in oxidative stress biomarkers and lipid profile after a single dose of virgin olive oil and after 1 wk of daily consumption. Sixteen subjects (9 men, 7 women) ingested 50 mL of virgin olive oil in a single dose. Blood samples were collected from 0 to 24 h. Thereafter, 14 participants (8 men, 6 women) followed a 1-wk 25 mg/d virgin olive oil dietary intervention. Blood samples were collected at the end of this period. Serum TAG (P=0.016), plasma FA (P<0.001) and lipid peroxidation products in plasma (P<0.001) and VLDL (P=0.007) increased, reaching a peak at 4–6 h, and returning to baseline values at 24 h after oil ingestion. The opposite changes were observed in plasma glutathione peroxidase (P=0.001) and glutathione reductase (GR) (P=0.042). No changes in LDL lipid peroxidation or resistance to oxidation were observed postprandially. At 24 h, plasma oleic acid remained increased (P<0.05) and resistance of LDL to oxidation improved (P<0.05). After 1 wk of virgin olive oil consumption, plasma oleic acid (P=0.031), resistance of LDL to oxidation (P<0.05), and plasma GR activity (P=0.005) increased. These results indicate that changes in oxidant/antioxidant status occur after oral virgin olive oil. Virgin olive oil consumption could provide short-term benefits for LDL resistance to oxidation and in glutathione-related enzyme activities.     

Nucleobase-Derived Nitrones: Synthesis and Antioxidant and Neuroprotective Activities in an In Vitro Model of Ischemia–Reperfusion

Herein, we report the synthesis, antioxidant, and neuroprotective properties of some nucleobase-derived nitrones named 9a–i. The neuroprotective properties of nitrones, 9a–i, were measured against an oxygen-glucose-deprivation in vitro ischemia model using human neuroblastoma SH-SY5Y cells. Our results indicate that nitrones, 9a–i, have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN) and are similar to N-acetyl-L-cysteine (NAC), a well-known antioxidant and neuroprotective agent. The nitrones with the highest neuroprotective capacity were those containing purine nucleobases (nitrones 9f, g, B = adenine, theophylline), followed by nitrones with pyrimidine nucleobases with H or F substituents at the C5 position (nitrones 9a, c). All of these possess EC₅₀ values in the range of 1–6 μM and maximal activities higher than 100%. However, the introduction of a methyl substituent (nitrone 9b, B = thymine) or hard halogen substituents such as Br and Cl (nitrones 9d, e, B = 5-Br and 5-Cl uracil, respectively) worsens the neuroprotective activity of the nitrone with uracil as the nucleobase (9a). The effects on overall metabolic cell capacity were confirmed by results on the high anti-necrotic (EC_50′s ≈ 2–4 μM) and antioxidant (EC_50′s ≈ 0.4–3.5 μM) activities of these compounds on superoxide radical production. In general, all tested nitrones were excellent inhibitors of superoxide radical production in cultured neuroblastoma cells, as well as potent hydroxyl radical scavengers that inhibit in vitro lipid peroxidation, particularly, 9c, f, g, presenting the highest lipoxygenase inhibitory activity among the tested nitrones. Finally, the introduction of two nitrone groups at 9a and 9d (bis-nitronas 9g, i) did not show better neuroprotective effects than their precursor mono-nitrones. These results led us to propose nitrones containing purine (9f, g) and pyrimidine (9a, c) nucleobases as potential therapeutic agents for the treatment of cerebral ischemia and/or neurodegenerative diseases, leading us to further investigate their effects using in vivo models of these pathologies.     

Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function

Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.