Finite Element Analysis of Patient-Specific 3D-Printed Cranial Implant Manufactured with PMMA and PEEK: A Mechanical Comparative Study.

This article reports on a patient who required a cranial protection system. Using additive manufacturing techniques and surgical planning with the help of bio-models, a patient-specific bone implant solution was proposed that allows aesthetic restoration of the affected area and provides an adequate level of protection. In addition, through a comparative analysis with finite elements, the mechanical response to external actions of the medical device, printed with two materials: polymethylmethacrylate (PMMA) and polyether-ether-ketone (PEEK), is simulated. The tested materials have recognized biocompatibility properties, but their costs on the market differ significantly. The results obtained demonstrate the similarities in the responses of both materials. It offers the possibility that low-income people can access these devices, guaranteeing adequate biomechanical safety, considering that PMMA is a much cheaper material than PEEK.

Spheroids derived from the stromal vascular fraction of adipose tissue self-organize in complex adipose organoids and secrete leptin.

BACKGROUND: Adipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation. METHODS: SVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses. RESULTS: Cultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater Pparg, Plin1, and Lep expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix. CONCLUSION: Newborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation.

Disseminated intravascular coagulation phenotype is regulated by the TRPM7 channel during sepsis.

BACKGROUND: Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel-mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. RESULTS: The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. CONCLUSIONS: Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases.

Sepsis-Induced Coagulopathy Phenotype Induced by Oxidized High-Density Lipoprotein Associated with Increased Mortality in Septic-Shock Patients.

Sepsis syndrome is a highly lethal uncontrolled response to an infection, which is characterized by sepsis-induced coagulopathy (SIC). High-density lipoprotein (HDL) exhibits antithrombotic activity, regulating coagulation in vascular endothelial cells. Sepsis induces the release of several proinflammatory molecules, including reactive oxygen species, which lead to an increase in oxidative stress in blood vessels. Thus, circulating lipoproteins, such as HDL, are oxidized to oxHDL, which promotes hemostatic dysfunction, acquiring prothrombotic properties linked to the severity of organ failure in septic-shock patients (SSP). However, a rigorous and comprehensive investigation demonstrating that oxHDL is associated with a coagulopathy-associated deleterious outcome of SSP, has not been reported. Thus, we investigated the participation of plasma oxHDL in coagulopathy-associated sepsis pathogenesis and elucidated the underlying molecular mechanism. A prospective study was conducted on 42 patients admitted to intensive care units, (26 SSP and 16 non-SSP) and 39 healthy volunteers. We found that an increased plasma oxHDL level in SSP was associated with a prothrombotic phenotype, increased mortality and elevated risk of death, which predicts mortality in SSP. The underlying mechanism indicates that oxHDL triggers an endothelial protein expression reprogramming of coagulation factors and procoagulant adhesion proteins, to produce a prothrombotic environment, mainly mediated by the endothelial LOX-1 receptor. Our study demonstrates that an increased plasma oxHDL level is associated with coagulopathy in SSP through a mechanism involving the endothelial LOX-1 receptor and endothelial protein expression regulation. Therefore, the plasma oxHDL level plays a role in the molecular mechanism associated with increased mortality in SSP.

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Beta-coronaviruses exploit cellular stress responses by modulating TFEB and TFE3 activity.

Beta-coronaviruses have emerged as a severe threat to global health. Undercovering the interplay between host and beta-coronaviruses is essential for understanding disease pathogenesis and developing efficient treatments. Here we report that the transcription factors TFEB and TFE3 translocate from the cytosol to the nucleus in response to beta-coronavirus infection by a mechanism that requires activation of calcineurin phosphatase. In the nucleus, TFEB and TFE3 bind to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators is significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 is sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 results in increased cellular survival of infected cells but also in reduced lysosomal exocytosis and decreased viral infectivity. These results suggest a central role of TFEB and TFE3 in cellular response to beta-coronavirus infection.

Procoagulant phenotype induced by oxidized high-density lipoprotein associates with acute kidney injury and death.

BACKGROUND: Oxidative stress derived from severe systemic inflammation promotes conversion from high-density lipoprotein HDL to oxidized HDL (oxHDL), which interacts with vascular endothelial cells (ECs). OxHDL acquires procoagulant features playing a role in modulating coagulation, which has been linked with organ failure in ICU patients. However, whether oxHDL elicits a ECs-mediated procoagulant phenotype generating organ failure and death, and the underlying molecular mechanism is not known. Therefore, we studied whether oxHDL-treated rats and high-oxHDL ICU patients exhibit a procoagulant phenotype and its association with kidney injury and mortality and the endothelial underlying molecular mechanism. METHODS: Human ECs, oxHDL-treated rats and ICU patients were subjected to several cellular and molecular studies, coagulation analyses, kidney injury assessment and mortality determination. RESULTS: OxHDL-treated ECs showed a procoagulant protein expression reprograming characterized by increased E-/P-selectin and vWF mRNA expression through specific signaling pathways. OxHDL-treated rats exhibited a procoagulant phenotype and modified E-/P-selectin, vWF, TF and t-PA mRNA expression correlating with plasma TF, t-PA and D-dimer. Also, showed increased death events and the relative risk of death, and increased creatinine, urea, BUN/creatinine ratio, KIM-1, NGAL, ß2M, and decreased eGFR, all concordant with kidney injury, correlated with plasma TF, t-PA and D-dimer. ICU patients showed correlation between plasma oxHDL and increased creatinine, cystatin, BUN, BUN/creatinine ratio, KIM-1, NGAL, ß2M, and decreased GFR. Notably, ICU high-oxHDL patients showed decreased survival. Interestingly, altered coagulation factors TF, t-PA and D-dimer correlated with both increased oxHDL levels and kidney injury markers, indicating a connection between these factors. CONCLUSION: Increased circulating oxHDL generates an endothelial-dependent procoagulant phenotype that associates with acute kidney injury and increased risk of death.

Are birds more afraid in urban parks or cemeteries? A Latin American study contrasts with results from Europe.

The escape behaviour, measured as flight initiation distance (FID; the distance at which individuals take flight when approached by a potential predator, usually a human in the study systems), is a measure widely used to study fearfulness and risk-taking in animals. Previous studies have shown significant differences in the escape behaviour of birds inhabiting cemeteries and urban parks in European cities, where birds seem to be shyer in the latter. We collected a regional dataset of the FID of birds inhabiting cemeteries and parks across Latin America in peri-urban, suburban and urban parks and cemeteries. FIDs were recorded for eighty-one bird species. Mean species-specific FIDs ranged from 1.9 to 19.7 m for species with at least two observations (fifty-seven species). Using Bayesian regression modelling and controlling for the phylogenetic relatedness of the FID among bird species and city and country, we found that, in contrast to a recent publication from Europe, birds escape earlier in cemeteries than parks in the studied Latin American cities. FIDs were also significantly shorter in urban areas than in peri-urban areas and in areas with higher human density. Our results indicate that some idiosyncratic patterns in animal fearfulness towards humans may emerge among different geographic regions, highlighting difficulties with scaling up and application of regional findings to other ecosystems and world regions. Such differences could be associated with intrinsic differences between the pool of bird species from temperate European and mostly tropical Latin American cities, characterized by different evolutionary histories, but also with differences in the historical process of urbanization.

Disseminated intravascular coagulation phenotype is regulated by the TRPM7 channel during sepsis

BACKGROUND: Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. RESULTS: The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. CONCLUSIONS: Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases.

Oxidized High-Density Lipoprotein Induces Endothelial Fibrosis Promoting Hyperpermeability, Hypotension, and Increased Mortality.

During systemic inflammation, reactive oxygen species (ROS) are generated in the bloodstream, producing large amounts of oxidized HDL (oxHDL). OxHDL loses the vascular protective features of native HDL, acquiring detrimental actions. Systemic inflammation promotes endothelial fibrosis, characterized by adhesion protein downregulation and fibrotic-specific gene upregulation, disrupting endothelial monolayer integrity. Severe systemic inflammatory conditions, as found in critically ill patients in the intensive care unit (ICU), exhibit endothelial hyperpermeability, hypotension, and organ hypoperfusion, promoting organ dysfunction and increased mortality. Because endothelial fibrosis disturbs the endothelium, it is proposed that it is the cellular and molecular origin of endothelial hyperpermeability and the subsequent deleterious consequences. However, whether oxHDL is involved in this process is unknown. The aim of this study was to investigate the fibrotic effect of oxHDL on the endothelium, to elucidate the underlying molecular and cellular mechanism, and to determine its effects on vascular permeability, blood pressure, and mortality. The results showed that oxHDL induces endothelial fibrosis through the LOX-1/NOX-2/ROS/NF-κB pathway, TGF-ß secretion, and ALK-5/Smad activation. OxHDL-treated rats showed endothelial hyperpermeability, hypotension, and an enhanced risk of death and mortality, which was prevented using an ALK-5 inhibitor and antioxidant diet consumption. Additionally, the ICU patients showed fibrotic endothelial cells, and the resuscitation fluid volume administered correlated with the plasma oxHDL levels associated with an elevated risk of death and mortality. We conclude that oxHDL generates endothelial fibrosis, impacting blood pressure regulation and survival.

Surgical planning and finite element analysis for the neurocraneal protection in cranioplasty with PMMA: A case study.

New developments in terms of additive manufacturing, computational tools and mathematical simulation techniques have favored the development of successful methodologies for the restoration or restitution of bone structures in the human body. Likewise, achievements in Materials Science have allowed the development of biocompatible composites capable of achieving mechanical characteristics and biological similarities comparable to those of natural bone. Without considering the advantages and disadvantages of some biomaterials with respect to others, this research aims to evaluate the surgical planning, the design process, the impact resistance and the critical deflection of a customized cranial implant manufactured from polymethylmethacrylate (PMMA). With the support of finite element methods (FEM), the level of neurocranial protection offered by the implant is assessed.

Key Role of Astrocytes in Postnatal Brain and Retinal Angiogenesis.

Angiogenesis is a key process in various physiological and pathological conditions in the nervous system and in the retina during postnatal life. Although an increasing number of studies have addressed the role of endothelial cells in this event, the astrocytes contribution in angiogenesis has received less attention. This review is focused on the role of astrocytes as a scaffold and in the stabilization of the new blood vessels, through different molecules release, which can modulate the angiogenesis process in the brain and in the retina. Further, differences in the astrocytes phenotype are addressed in glioblastoma, one of the most devastating types of brain cancer, in order to provide potential targets involved in the cross signaling between endothelial cells, astrocytes and glioma cells, that mediate tumor progression and pathological angiogenesis. Given the relevance of astrocytes in angiogenesis in physiological and pathological conditions, future studies are required to better understand the interrelation between endothelial and astrocyte signaling pathways during this process.

Polyphenolic Composition and Antioxidant Activity (ORAC, EPR and Cellular) of Different Extracts of Argylia radiata Vitroplants and Natural Roots.

Plant biochemistry studies have increased in recent years due to their potential to improve human health. Argylia radiata is an extremophile plant with an interesting polyphenolic profile. However, its biomass is scarce and occasionally available. Argylia in vitro biomass was obtained from tissue culture and compared with in vivo roots regarding its polyphenolic and flavonoid content. Different solvents were used to prepare extracts from the in vitro tissue of callus and aerial plant organs and in vivo roots. UPLC-MS/MS was used to assess the chemical composition of each extract. ORAC-FL and scavenging of free radicals (DPPH and OH) methods were used to determine the antioxidant capacity of extracts. Furthermore, the biological activity of the extracts was established using the cellular antioxidant activity method. The vitroplants were a good source of polyphenols (25-68 mg GAE/100 g tissue FW), and methanol was the most efficient solvent. Eight polyphenolic compounds were identified, and their antioxidant properties were investigated by different chemical methods with EPR demonstrating its specific scavenging activity against free radicals. All extracts showed cellular dose-dependent antioxidant activity. The methanolic extract of vitroplants showed the highest cellular antioxidant activity (44.6% and 51%) at 1 and 10 µg/mL of extract, respectively. Vitroplants of A. radiata are proposed as a biotechnological product as a source of antioxidant compounds with multiple applications.

Decreased caveolae in AGPAT2 lacking adipocytes is independent of changes in cholesterol or sphingolipid levels: A whole cell and plasma membrane lipidomic analysis of adipogenesis.

BACKGROUND: Adipocytes from lipodystrophic Agpat2-/- mice have impaired adipogenesis and fewer caveolae. Herein, we examined whether these defects are associated with changes in lipid composition or abnormal levels of caveolae-associated proteins. Lipidome changes were quantified in differentiated Agpat2-/- adipocytes to identify lipids with potential adipogenic roles. METHODS: Agpat2-/- and wild type brown preadipocytes were differentiated in vitro. Plasma membrane was purified by ultracentrifugation. Number of caveolae and caveolae-associated proteins, as well as sterol, sphingolipid, and phospholipid lipidome were determined across differentiation. RESULTS: Differentiated Agpat2-/- adipocytes had decreased caveolae number but conserved insulin signaling. Caveolin-1 and cavin-1 levels were equivalent between Agpat2-/- and wild type adipocytes. No differences in PM cholesterol and sphingolipids abundance were detected between genotypes. Levels of phosphatidylserine at day 10 of differentiation were increased in Agpat2-/- adipocytes. Wild type adipocytes had increased whole cell triglyceride, diacylglycerol, phosphatidylglycerol, phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, and trihexosyl ceramide, and decreased 24,25-dihydrolanosterol and sitosterol, as a result of adipogenic differentiation. By contrast, adipogenesis did not modify whole cell neutral lipids but increased lysophosphatidylcholine, sphingomyelin, and trihexosyl ceramide levels in Agpat2-/- adipocytes. Unexpectedly, adipogenesis decreased PM levels of main phospholipids in both genotypes. CONCLUSION: In Agpat2-/- adipocytes, decreased caveolae is not associated with changes in PM cholesterol nor sphingolipid levels; however, increased PM phosphatidylserine content may be implicated. Abnormal lipid composition is associated with the adipogenic abnormalities of Agpat2 -/- adipocytes but does not prevent insulin signaling.

Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in ß-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

Polyphenolic distribution in organs of Argylia radiata, an extremophile plant from Chilean Atacama desert.

The polyphenolic distribution on different organs of Argylia radiata, an extremophile plant from the Atacama "Flowering Desert", is presented herein for the first time. For this purpose, the total phenolic content and antioxidant capacity of ethanolic extracts from leaves, tuberous root and flowers of different colors were evaluated. Orange and red flowers showed the highest polyphenolic and flavonoid content. The maximum anthocyanin concentration was found in red flowers and the antioxidant activity (FRAP) of extracts changed according to the organ. The UPLC-MS/MS analysis of the extracts allowed to identify 10 new polyphenols belonging to different families. Rutin was identified as the most abundant polyphenol in all plant organs, followed by quercetin and coumaric acid. Their role in plant response to abiotic and biotic stress, as well as their potential biotechnological application are discussed.

c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder.

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models.

Absence of AGPAT2 impairs brown adipogenesis, increases IFN stimulated gene expression and alters mitochondrial morphology.

BACKGROUND: Biallelic loss of function variants in AGPAT2, encoding 1-acylglycerol-3-phosphate O-acyltransferase 2, cause congenital generalized lipodystrophy type 1, a disease characterized by near total loss of white adipose tissue and metabolic complications. Agpat2 deficient (Agpat2-/-) mice completely lacks both white and interscapular brown adipose tissue (iBAT). The objective of the present study was to characterize the effects of AGPAT2 deficiency in brown adipocyte differentiation. METHODS: Preadipocytes obtained from newborn (P0.5) Agpat2-/- and wild type mice iBAT were differentiated into brown adipocytes, compared by RNA microarray, RT-qPCR, High-Content Screening (HCS), western blotting and electron microscopy. RESULTS: 1) Differentiated Agpat2-/- brown adipocytes have fewer lipid-laden cells and lower abundance of Pparγ, Pparα, C/ebpα and Pgc1α, both at the mRNA and protein levels, compared those to wild type cells. Prmd16 levels were equivalent in both, Agpat2-/- and wild type, while Ucp1 was only induced in wild type cells, 2) These differences were not due to lower abundance of preadipocytes, 3) Differentiated Agpat2-/- brown adipocytes are enriched in the mRNA abundance of genes participating in interferon (IFN) type I response, whereas genes involved in mitochondrial homeostasis were decreased, 4) Mitochondria in differentiated Agpat2-/- brown adipocytes had altered morphology and lower mass and contacting sites with lipid droplets concomitant with lower levels of Mitofusin 2 and Perlipin 5. CONCLUSION: AGPAT2 is necessary for normal brown adipose differentiation. Its absence results in a lower proportion of lipid-laden cells, increased expression of interferon-stimulated genes (ISGs) and alterations in mitochondrial morphology, mass and fewer mitochondria to lipid droplets contacting sites in differentiated brown adipocytes.