The reactive pyruvate metabolite dimethylglyoxal mediates neurological consequences of diabetes.

Complications of diabetes are often attributed to glucose and reactive dicarbonyl metabolites derived from glycolysis or gluconeogenesis, such as methylglyoxal. However, in the CNS, neurons and endothelial cells use lactate as energy source in addition to glucose, which does not lead to the formation of methylglyoxal and has previously been considered a safer route of energy consumption than glycolysis. Nevertheless, neurons and endothelial cells are hotspots for the cellular pathology underlying neurological complications in diabetes, suggesting a cause that is distinct from other diabetes complications and independent of methylglyoxal. Here, we show that in clinical and experimental diabetes plasma concentrations of dimethylglyoxal are increased. In a mouse model of diabetes, ilvb acetolactate-synthase-like (ILVBL, HACL2) is the enzyme involved in formation of increased amounts of dimethylglyoxal from lactate-derived pyruvate. Dimethylglyoxal reacts with lysine residues, forms Nε-3-hydroxy-2-butanonelysine (HBL) as an adduct, induces oxidative stress more strongly than other dicarbonyls, causes blood-brain barrier disruption, and can mimic mild cognitive impairment in experimental diabetes. These data suggest dimethylglyoxal formation as a pathway leading to neurological complications in diabetes that is distinct from other complications. Importantly, dimethylglyoxal formation can be reduced using genetic, pharmacological and dietary interventions, offering new strategies for preventing CNS dysfunction in diabetes.

Midpalatal miniscrew insertion: The accuracy of digital planning and surgical placement.

INTRODUCTION: The objective of this study was to investigate the accuracy of palatal miniscrew insertion, evaluating the effect of guide fabrication and surgical placement. METHODS: Guided insertion of bilateral paramedian palatal miniscrews was undertaken using Appliance Designer software (3Shape, Copenhagen, Denmark). A resin surgical guide (P Pro Surgical Guide; Straumann AG, Basel, Switzerland) was used. Superimposition of the miniscrew position relative to the digital design was undertaken using bespoke software (Inspect 3D module, OnyxCeph; Image Instruments GmbH, Chemnitz, Germany) to assess surgical inaccuracy. Miniscrew position relative to the surgical guide was also assessed to isolate the effect of planning inaccuracies. Both horizontal and vertical discrepancies were evaluated at both implant locations. RESULTS: Twenty-seven patients having bilateral palatal insertions were examined. Mean discrepancies were <0.5 mm, both in the horizontal and vertical planes. The mean overall horizontal and vertical discrepancy between the digital design and final miniscrew position on the left side was 0.32 ± 0.15 mm and 0.34 ± 0.17 mm, respectively. The maximum horizontal discrepancy observed was 0.72 mm. No significant differences were observed in relation to the accuracy of mini-implant positioning on the basis of sidedness, either for horizontal (P = 0.29) or vertical (P = 0.86) discrepancy. CONCLUSIONS: High levels of accuracy associated with guided insertion of paramedian palatal implants were recorded with mean discrepancies of less than 0.5 mm both in the horizontal and vertical planes. No difference in accuracy was noted between the left and right sides. Very minor levels of inaccuracy associated both with surgical techniques and surgical guide fabrication were recorded.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells.

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

Formation and Investigation of Cell-Derived Nanovesicles as Potential Therapeutics against Chronic Liver Disease.

A new therapeutic approach using cell-derived nanovesicles (cdNVs) is offered here to overcome the lack of effective treatments for liver fibrosis, a reversible chronic liver disease. To achieve this goal the formation and purification of cdNVs from untreated, quiescent-like, or activated LX-2 cells, an immortalized human hepatic stellate cell (HSC) line with key features of transdifferentiated HSCs are established. Analysis of the genotype and phenotype of naïve and transdifferentiated LX-2 cells activated through transforming growth factor beta 1, following treatment with cdNVs, reveals a concentration-dependent fibrosis regression. The beneficial fibrosis-resolving effects of cdNVs are linked to their biomolecular corona. Liposomes generated using lipids extracted from cdNVs exhibit a reduced antifibrotic response in perpetuated LX-2 cells and show a reduced cellular uptake. However, incubation with soluble factors collected during purification results in a new corona, thereby restoring fibrosis regression activity. Overall, cdNVs display encouraging therapeutic properties, making them a promising candidate for the development of liver fibrosis resolving therapeutics.

Effect of high-dose glucocorticoid treatment on human brown adipose tissue activity: a randomised, double-blinded, placebo-controlled cross-over trial in healthy men.

BACKGROUND: Glucocorticoids (GCs) are widely applied anti-inflammatory drugs that are associated with adverse metabolic effects including insulin resistance and weight gain. Previous research indicates that GCs may negatively impact brown adipose tissue (BAT) activity in rodents and humans. METHODS: We performed a randomised, double-blinded cross-over trial in 16 healthy men (clinicaltrials.govNCT03269747). Participants received 40 mg of prednisone per day for one week or placebo. After a washout period of four weeks, participants crossed-over to the other treatment arm. Primary endpoint was the increase in resting energy expenditure (EE) in response to a mild-cold stimulus (cold-induced thermogenesis, CIT). Secondary outcomes comprised mean 18F-FDG uptake into supraclavicular BAT (SUVmean) as determined by FDG-PET/CT, volume of the BAT depot as well as fat content determined by MRI. The plasma metabolome and the transcriptome of supraclavicular BAT and of skeletal muscle biopsies after each treatment period were analysed. FINDINGS: Sixteen participants were recruited to the trial and completed it successfully per protocol. After prednisone treatment resting EE was higher both during warm and cold conditions. However, CIT was similar, 153 kcal/24 h (95% CI 40-266 kcal/24 h) after placebo and 186 kcal/24 h (95% CI 94-277 kcal/24 h, p = 0.38) after prednisone. SUVmean of BAT after cold exposure was not significantly affected by prednisone (3.36 g/ml, 95% CI 2.69-4.02 g/ml, vs 3.07 g/ml, 95% CI 2.52-3.62 g/ml, p = 0.28). Results of plasma metabolomics and BAT transcriptomics corroborated these findings. RNA sequencing of muscle biopsies revealed higher expression of genes involved in calcium cycling. No serious adverse events were reported and adverse events were evenly distributed between the two treatments. INTERPRETATION: Prednisone increased EE in healthy men possibly by altering skeletal muscle calcium cycling. Cold-induced BAT activity was not affected by GC treatment, which indicates that the unfavourable metabolic effects of GCs are independent from thermogenic adipocytes. FUNDING: Grants from Swiss National Science Foundation (PZ00P3_167823), Bangerter-Rhyner Foundation and from Nora van der Meeuwen-Häfliger Foundation to MJB. A fellowship-grant from the Swiss National Science Foundation (SNF211053) to WS. Grants from German Research Foundation (project number: 314061271-TRR 205) and Else Kröner-Fresenius (grant support 2012_A103 and 2015_A228) to MR.

Hepatocyte ABCA1 deficiency is associated with reduced HDL sphingolipids.

ATP binding cassette transporter A1 (ABCA1) limits the formation of high density lipoproteins (HDL) as genetic loss of ABCA1 function causes virtual HDL deficiency in patients with Tangier disease. Mice with a hepatocyte-specific ABCA1 knockout (Abca1 HSKO) have 20% of wild type (WT) plasma HDL-cholesterol levels, suggesting a major contribution of hepatic ABCA1 to the HDL phenotype. Whether plasma sphingolipids are reduced in Tangier disease and to what extent hepatic ABCA1 contributes to plasma sphingolipid (SL) levels is unknown. Here, we report a drastic reduction of total SL levels in plasma of a Tangier patient with compound heterozygosity for mutations in ABCA1. Compared to mutation-free controls, heterozygous mutations in ABCA1 had no significant effect on total SLs in plasma; however, apoB-depleted plasma showed a reduction in total SL also in het carriers. Similarly, liver specific Abca1 KO mice (Abca1 HSKO) showed reduced total sphingolipids in plasma and liver. In parallel, apoM and sphingosine-1-phosphate (S1P) levels were reduced in plasma of Abca1 HSKO mice. Primary hepatocytes from Abca1 HSKO mice showed a modest, but significant reduction in total SLs concentration compared to WT hepatocytes, although SL de novo synthesis and secretion were slightly increased in Abca1 HSKO hepatocytes. We conclude that hepatic ABCA1 is a signficant contributor to maintaining total plasma pool of HDL sphingolipids, including sphingomyelins and S1P.

Structure of the connexin-43 gap junction channel in a putative closed state.

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

Glyoxal in hyperglycaemic ischemic stroke - a cohort study.

BACKGROUND: Hyperglycaemia is frequent in acute ischemic stroke and denotes a bad prognosis, even in the absence of pre-existing diabetes. However, in clinical trials treatment of elevated glucose levels with insulin did not improve stroke outcome, suggesting that collateral effects rather than hyperglycaemia itself aggravate ischemic brain damage. As reactive glucose metabolites, glyoxal and methylglyoxal are candidates for mediating the deleterious effects of hyperglycaemia in acute stroke. METHODS: In 135 patients with acute stroke, we used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to measure glyoxal, methylglyoxal and several of their glycated amino acid derivatives in serum. Results were verified in a second cohort of 61 stroke patients. The association of serum concentrations with standard stroke outcome scales (NIHSS, mRS) was tested. RESULTS: Glucose, glyoxal, methylglyoxal, and the glyoxal-derived glycated amino acid Nδ-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) were positively correlated with a bad stroke outcome at 3 months as measured by mRS90, at least in one of the two cohorts. However, the glycated amino acids Nε-carboxyethyllysine (CEL) and in one cohort pyrraline showed an inverse correlation with stroke outcome probably reflecting lower food intake in severe stroke. Patients with a poor outcome had higher serum concentrations of glyoxal and methylglyoxal. CONCLUSIONS: The glucose-derived α-dicarbonyl glyoxal and glycated amino acids arising from a reaction with glyoxal are associated with a poor outcome in ischemic stroke. Thus, lowering α-dicarbonyls or counteracting their action could be a therapeutic strategy for hyperglycaemic stroke.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

Altered Distribution of Unesterified Cholesterol among Lipoprotein Subfractions of Patients with Diabetes Mellitus Type 2.

Biomarkers are important tools to improve the early detection of patients at high risk for developing diabetes as well as the stratification of diabetic patients towards risks of complications. In addition to clinical variables, we analyzed 155 metabolic parameters in plasma samples of 51 healthy volunteers and 66 patients with diabetes using nuclear magnetic resonance (NMR) spectrometry. Upon elastic net analysis with lasso regression, we confirmed the independent associations of diabetes with branched-chain amino acids and lactate (both positive) as well as linoleic acid in plasma and HDL diameter (both inverse). In addition, we found the presence of diabetes independently associated with lower concentrations of free cholesterol in plasma but higher concentrations of free cholesterol in small HDL. Compared to plasmas of non-diabetic controls, plasmas of diabetic subjects contained lower absolute and relative concentrations of free cholesterol in all LDL and HDL subclasses except small HDL but higher absolute and relative concentrations of free cholesterol in all VLDL subclasses (except very small VLDL). These disbalances may reflect disturbances in the transfer of free cholesterol from VLDL to HDL during lipolysis and in the transfer of cell-derived cholesterol from small HDL via larger HDL to LDL.

Basal re-esterification finetunes mitochondrial fatty acid utilization.

OBJECTIVE: Emerging evidence suggest the existence of constant basal lipolysis and re-esterification of a substantial fraction of thus liberated fatty acids. In stimulated lipolysis, the re-esterification is proposed to be a protective mechanism against lipotoxicity; however, the role of the lipolysis coupled to re-esterification under basal conditions has not been deciphered. METHODS: We used adipocytes (in vitro differentiated brown and white adipocytes derived from a cell line or primary SVF culture) to study the effect of inhibition of re-esterification by pharmacological DGAT1 and DGAT2 inhibitors alone or in combination. We then evaluated cellular energetics, lipolysis flux, and lipidomic parameters along with mitochondrial properties and fuel utilization. RESULTS: In adipocytes, DGAT1 and 2 mediated re-esterification is a moderator of fatty acid oxidation. Combined inhibition of both DGATs (D1+2i) increases oxygen consumption, which is largely due to enhanced mitochondrial respiration by lipolysis-derived fatty acids (FAs). Acute D1+2i selectively affects mitochondrial respiration without affecting the transcriptional homeostasis of genes relevant to mitochondrial health and lipid metabolism. D1+2i enhances the mitochondrial import of pyruvate and activates AMP Kinase to counteract CPT1 antagonism, thus facilitating the mitochondrial import of fatty acyl-CoA. CONCLUSIONS: These data implicate the process of re-esterification in the regulation of mitochondrial FA usage and uncover a mechanism of FAO regulation via crosstalk with FA re-esterification.

A functional analysis of 180 cancer cell lines reveals conserved intrinsic metabolic programs.

Cancer cells reprogram their metabolism to support growth and invasion. While previous work has highlighted how single altered reactions and pathways can drive tumorigenesis, it remains unclear how individual changes propagate at the network level and eventually determine global metabolic activity. To characterize the metabolic lifestyle of cancer cells across pathways and genotypes, we profiled the intracellular metabolome of 180 pan-cancer cell lines grown in identical conditions. For each cell line, we estimated activity for 49 pathways spanning the entirety of the metabolic network. Upon clustering, we discovered a convergence into only two major metabolic types. These were functionally confirmed by 13 C-flux analysis, lipidomics, and analysis of sensitivity to perturbations. They revealed that the major differences in cancers are associated with lipid, TCA cycle, and carbohydrate metabolism. Thorough integration of these types with multiomics highlighted little association with genetic alterations but a strong association with markers of epithelial-mesenchymal transition. Our analysis indicates that in absence of variations imposed by the microenvironment, cancer cells adopt distinct metabolic programs which serve as vulnerabilities for therapy.

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Variations in COVID-19 Vaccine Attitudes and Acceptance among Refugees and Lebanese Nationals Pre- and Post-Vaccine Rollout in Lebanon.

Vaccine hesitancy among displaced populations is associated with inequitable access to services and mistrust of authorities, among other factors. This study evaluated variations in attitudes toward COVID-19 vaccines and factors associated with vaccine acceptance among refugees and Lebanese nationals accessing 60 International Medical Corps-supported health facilities through two cross-sectional surveys pre- (n = 3927; Survey 1) and post- (n = 4174; Survey 2) vaccine rollout. Logistic regression was used to assess predictors of vaccine acceptance using the health beliefs model. Refugees comprised 52.9% (Survey 1) and 54.2% (Survey 2) of respondents. Vaccine acceptance was low among both groups in Survey 1 (25.9% refugees vs. 23.1% Lebanese nationals), but higher in Survey 2 in Lebanese (57.6%) versus refugees (32.9%). Participants reported greater perceived benefits of vaccination, higher perceived COVID-19 susceptibility, and lower perceived vaccination barriers in Survey 2 versus Survey 1. Post-vaccine rollout, refugees had lower odds of vaccine acceptance compared to Lebanese (OR 0.50, 95%CI 0.41-0.60), while older age (OR 1.37, 95%CI 1.06-1.78, ≥51 years vs. 18-30 years) was associated with greater vaccine acceptance. Health beliefs model variables were associated with vaccine acceptance in both surveys. Tailored strategies to respond dynamically to changes in vaccine attitudes among vulnerable groups in Lebanon are essential for equitable vaccine uptake.

The AT1 Receptor Blocker Telmisartan Reduces Intestinal Mucus Thickness in Obese Mice.

The angiotensin II (type 1) (AT1) receptor blocker telmisartan (TEL) is beneficial for the treatment of individuals suffering from metabolic syndrome. As we have shown that TEL has an impact on gut microbiota, we investigated here whether TEL influences gut barrier function. C57BL/6N mice were fed with chow or high-fat diet (HFD) and treated with vehicle or TEL (8 mg/kg/day). Mucus thickness was determined by immunohistochemistry. Periodic Acid-Schiff staining allowed the number of goblet cells to be counted. Using western blots, qPCR, and immunohistochemistry, factors related to mucus biosynthesis (Muc2, St6galnac), proliferation (Ki-67), or necroptosis (Rip3) were measured. The influence on cell viability was determined in vitro by using losartan, as the water solubility of TEL was too low for in vitro experiments. Upon HFD, mice developed obesity as well as leptin and insulin resistance, which were prevented by TEL. Mucus thickness upon HFD-feeding was diminished. Independent of feeding, TEL additionally reduced mucus thickness. Numbers of goblet cells were not affected by HFD-feeding and TEL. St6galnac expression was increased by TEL. Rip3 was increased in TEL-treated and HFD-fed mice, while Ki-67 decreased. Cell viability was diminished by using >1 mM losartan. The anti-obese effect of TEL was associated with a decrease in mucus thickness, which was likely not related to a lower expression of Muc2 and goblet cells. A decrease in Ki-67 and increase in Rip3 indicates lower cell proliferation and increased necroptosis upon TEL. However, direct cell toxic effects are ruled out, as in vivo concentrations are lower than 1 mM.

Liposome-based nanocapsules for the controlled release of dietary curcumin: PDDA and silica nanoparticle-coated DMPC liposomes enhance the fluorescence efficiency and anticancer activity of curcumin.

Nanosystems with various compositions and biological properties are being extensively investigated for drug and gene delivery applications. Many nanotechnology methods use novel nanocarriers, such as liposomes, in therapeutically targeted drug delivery systems. However, liposome matrices suffer from several limitations, including drug leakage and instability. Therefore, the surface modification of liposomes by coating them or adding polymers has advanced their application in drug delivery. Hence, the prevention of drug release from the liposome bilayers was the main focus of this work. For this purpose, liposomes were synthesized according to a thin film hydration method by applying various surface modifications. Three different nanocapsules, N1, N2, and N3, were prepared using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), poly(diallyldimethylammonium)chloride (PDAA) polymer, and silica nanoparticles. PDDA and silica nanoparticles were coated on the surface of liposomes using a layer-by-layer assembly method, completely encapsulating curcumin into the core of the liposome. Fluorescence spectroscopy, TGA, DLS, XRD, SEM, and zeta potential methods were used to characterize the prepared nanocapsules. Interestingly, the fluorescence of curcumin showed a blue shift and the fluorescence efficiency was extraordinarily enhanced ∼25-, ∼54-, and ∼62-fold in the N1, N2, and N3 nanocapsules, respectively. Similarly, encapsulation efficiency, drug loading, and the anticancer activity of dietary curcumin were investigated for the different types of DMPC nanocapsules. The drug efficiencies of the liposomes were established according to the release of curcumin from the liposomes. The results showed that the release of curcumin from the nanocapsules decreased as the number of layers at the surface of the liposomes increased. The release of curcumin follows the Higuchi model; thus, a slow rate of diffusion is observed when a number of layers is added. The better encapsulation and higher anti-cancer activity of curcumin were also observed when more layers were added, which is due to electrostatic interactions inhibiting curcumin from being released.

Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome.

BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.

Postendodontic pain in asymptomatic necrotic teeth prepared with different rotary instrumentation techniques.

OBJECTIVES: to investigate the postendodontic pain in asymptomatic necrotic teeth prepared with different rotary instrumentation techniques after single-visit root canal treatment. MATERIALS AND METHODS: A total of 60 single-rooted teeth with single root canal were treated endodontically. Teeth were divided randomly into four equaled groups (n = 15) according to instrumentation systems as follows: group I were shaped using ProTaper Universal (control group) (Dentsply/Maillefer, Ballaigues), group 2 were shaped with 2Shape (Micro-Mega) till TS2 (25.06), group 3 were shaped with XP-endo Shaper file (FKG Dentaire) till #30.04, and group 4 were shaped with Reciproc blue (VDW) till R25 (25.08). All groups were prepared according to manufacturer's instructions and obturated with lateral condensation technique. Pain levels were assessed by visual analog scale (VAS) and verbal evaluation of pain questionnaire after 6, 12, 24, 48 h, and 7 days of canal obturation. Data were then analyzed using Kruskal-Wallis and Mann-Whitney U tests at P value of 0.05. RESULTS: Postendodontic pain started after 6 h of treatment with highest values and then decreased gradually until almost vanished after 1 week of treatment, with no significant differences in VAS among studied groups (ProTaper, 2 shape, XP endo Shaper, and Reciproc Blue) after 6,12, and 48 h of treatment. On the other hand, XP endo Shaper group, showed the lowest pain values after 24 h of treatment, and the highest pain values were found in 2 shape group after 1 week with significance (P < 0.05). CONCLUSION: Root canals prepared with XP endo Shaper resulted in the lowest pain levels after 24 h of treatment. Root canal preparation with 2Sshape rotary system resulted in the highest pain levels after 1 week of treatment.