Activation of the hypoxia-inducible factor pathway protects against acute ischemic stroke by reprogramming central carbon metabolism.

Cell metabolism reprogramming to sustain energy production, while reducing oxygen and energy consuming processes is crucially important for the adaptation to hypoxia/ischemia. Adaptive metabolic rewiring is controlled by hypoxia-inducible factors (HIFs). Accumulating experimental evidence indicates that timely activation of HIF in brain-resident cells improves the outcome from acute ischemic stroke. However, the underlying molecular mechanisms are still incompletely understood. Thus, we investigated whether HIF-dependent metabolic reprogramming affects the vulnerability of brain-resident cells towards ischemic stress. Methods: We used genetic and pharmacological approaches to activate HIF in the murine brain in vivo and in primary neurons and astrocytes in vitro. Numerous metabolomic approaches and molecular biological techniques were applied to elucidate potential HIF-dependent effects on the central carbon metabolism of brain cells. In animal and cell models of ischemic stroke, we analysed whether HIF-dependent metabolic reprogramming influences the susceptibility to ischemic injury. Results: Neuron-specific gene ablation of prolyl-4-hydroxylase domain 2 (PHD2) protein, negatively regulating the protein stability of HIF-α in an oxygen dependent manner, reduced brain injury and functional impairment of mice after acute stroke in a HIF-dependent manner. Accordingly, PHD2 deficient neurons showed an improved tolerance towards ischemic stress in vitro, which was accompanied by enhanced HIF-1-mediated glycolytic lactate production through pyruvate dehydrogenase kinase-mediated inhibition of the pyruvate dehydrogenase. Systemic treatment of mice with roxadustat, a low-molecular weight pan-PHD inhibitor, not only increased the abundance of numerous metabolites of the central carbon and amino acid metabolism in murine brain, but also ameliorated cerebral tissue damage and sensorimotor dysfunction after acute ischemic stroke. In neurons and astrocytes roxadustat provoked a HIF-1-dependent glucose metabolism reprogramming including elevation of glucose uptake, glycogen synthesis, glycolytic capacity, lactate production and lactate release, which enhanced the ischemic tolerance of astrocytes, but not neurons. We found that strong activation of HIF-1 in neurons by non-selective inhibition of all PHD isoenzymes caused a HIF-1-dependent upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 redirecting glucose-6-phosphate from pentose phosphate pathway (PPP) to the glycolysis pathway. This was accompanied by a reduction of NADPH production in the PPP, which further decreased the low intrinsic antioxidant reserve of neurons, making them more susceptible to ischemic stress. Nonetheless, in organotypic hippocampal cultures with preserved neuronal-glial interactions roxadustat decreased the neuronal susceptibility to ischemic stress, which was largely prevented by restricting glycolytic energy production through lactate transport blockade. Conclusion: Collectively, our results indicate that HIF-1-mediated metabolic reprogramming alleviates the intrinsic vulnerability of brain-resident cells to ischemic stress.

SUMOylation Modulates Reactive Oxygen Species (ROS) Levels and Acts as a Protective Mechanism in the Type 2 Model of Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is the prevalent type of peripheral neuropathy; it primarily impacts extremity nerves. Its multifaceted nature makes the molecular mechanisms of diabetic neuropathy intricate and incompletely elucidated. Several types of post-translational modifications (PTMs) have been implicated in the development and progression of DPN, including phosphorylation, glycation, acetylation and SUMOylation. SUMOylation involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, and it plays a role in various cellular processes, including protein localization, stability, and function. While the specific relationship between high blood glucose and SUMOylation is not extensively studied, recent evidence implies its involvement in the development of DPN in type 1 diabetes. In this study, we investigated the impact of SUMOylation on the onset and progression of DPN in a type 2 diabetes model using genetically modified mutant mice lacking SUMOylation, specifically in peripheral sensory neurons (SNS-Ubc9-/-). Behavioural measurement for evoked pain, morphological analyses of nerve fibre loss in the epidermis, measurement of reactive oxygen species (ROS) levels, and antioxidant molecules were analysed over several months in SUMOylation-deficient and control mice. Our longitudinal analysis at 30 weeks post-high-fat diet revealed that SNS-Ubc9-/- mice exhibited earlier and more pronounced thermal and mechanical sensation loss and accelerated intraepidermal nerve fibre loss compared to control mice. Mechanistically, these changes are associated with increased levels of ROS both in sensory neuronal soma and in peripheral axonal nerve endings in SNS-Ubc9-/- mice. In addition, we observed compromised detoxifying potential, impaired respiratory chain complexes, and reduced levels of protective lipids in sensory neurons upon deletion of SUMOylation in diabetic mice. Importantly, we also identified mitochondrial malate dehydrogenase (MDH2) as a SUMOylation target, the activity of which is negatively regulated by SUMOylation. Our results indicate that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the deletion of which causes oxidative stress and an impaired respiratory chain, resulting in energy depletion and subsequent damage to sensory neurons.

The Shear Stress-Regulated Expression of Glypican-4 in Endothelial Dysfunction In Vitro and Its Clinical Significance in Atherosclerosis.

Retention of circulating lipoproteins by their interaction with extracellular matrix molecules has been suggested as an underlying mechanism for atherosclerosis. We investigated the role of glypican-4 (GPC4), a heparan sulfate (HS) proteoglycan, in the development of endothelial dysfunction and plaque progression; Expression of GPC4 and HS was investigated in human umbilical vein/artery endothelial cells (HUVECs/HUAECs) using flow cytometry, qPCR, and immunofluorescent staining. Leukocyte adhesion was determined in HUVECs in bifurcation chamber slides under dynamic flow. The association between the degree of inflammation and GPC4, HS, and syndecan-4 expressions was analyzed in human carotid plaques; GPC4 was expressed in HUVECs/HUAECs. In HUVECs, GPC4 protein expression was higher in laminar than in non-uniform shear stress regions after a 1-day or 10-day flow (p < 0.01 each). The HS expression was higher under laminar flow after a 1 day (p < 0.001). Monocytic THP-1 cell adhesion to HUVECs was facilitated by GPC4 knock-down (p < 0.001) without affecting adhesion molecule expression. GPC4 and HS expression was lower in more-inflamed than in less-inflamed plaque shoulders (p < 0.05, each), especially in vulnerable plaque sections; Reduced expression of GPC4 was associated with atherogenic conditions, suggesting the involvement of GPC4 in both early and advanced stages of atherosclerosis.

A Bibliometric Analysis of Publications on Tinnitus: A Study Based on Web of Science Data From 1980 to 2020.

BACKGROUND: The incidence of tinnitus has been increasing together with its patient impact and societal costs. Much research has been conducted in the field of tinnitus, especially on treatment modalities because there still is no cure. This study aims to analyze the evolutions and developments in the scientific output relating to tinnitus. METHODS: We analyzed the Science Citation Index Expanded featured articles in the Web of Science Core Collection relating to tinnitus from 1980 to 2020. The publications were analyzed by characteristics such as the countries and institutions, journals, the most cited articles and references, and the most frequently used words in the abstracts and keywords. RESULTS: In total, 8282 articles relating to tinnitus were identified in the Web of Science. The number of publications has been significantly increasing after the 1990s. Of the 8282 articles, a major part originated from the American and European institutions. Most articles originated from high-impact journals, which consequently also covered the most cited papers. A major interest was seen in areas about treatment and pathogenic mechanisms. CONCLUSION: This bibliometric analysis here indicated an increasing trend in tinnitus research from 1980 to 2020, particularly with the increase in tinnitus burden and the societal costs by it. Specific interest has been seen in the specific tinnitus pathophysiological mechanisms and treatment. Individual researchers and institutions will gain a new perspective on their future studies based on the bibliometric data in our paper.

The Effects of Single and Combined Stressors on Daphnids-Enzyme Markers of Physiology and Metabolomics Validate the Impact of Pollution.

The continuous global increase in population and consumption of resources due to human activities has had a significant impact on the environment. Therefore, assessment of environmental exposure to toxic chemicals as well as their impact on biological systems is of significant importance. Freshwater systems are currently under threat and monitored; however, current methods for pollution assessment can neither provide mechanistic insight nor predict adverse effects from complex pollution. Using daphnids as a bioindicator, we assessed the impact in acute exposures of eight individual chemicals and specifically two metals, four pharmaceuticals, a pesticide and a stimulant, and their composite mixture combining phenotypic, biochemical and metabolic markers of physiology. Toxicity levels were in the same order of magnitude and significantly enhanced in the composite mixture. Results from individual chemicals showed distinct biochemical responses for key enzyme activities such as phosphatases, lipase, peptidase, ß-galactosidase and glutathione-S-transferase. Following this, a more realistic mixture scenario was assessed with the aforementioned enzyme markers and a metabolomic approach. A clear dose-dependent effect for the composite mixture was validated with enzyme markers of physiology, and the metabolomic analysis verified the effects observed, thus providing a sensitive metrics in metabolite perturbations. Our study highlights that sensitive enzyme markers can be used in advance on the design of metabolic and holistic assays to guide the selection of chemicals and the trajectory of the study, while providing mechanistic insight. In the future this could prove to become a useful tool for understanding and predicting freshwater pollution.

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

NFAT5/TonEBP Limits Pulmonary Vascular Resistance in the Hypoxic Lung by Controlling Mitochondrial Reactive Oxygen Species Generation in Arterial Smooth Muscle Cells.

Chronic hypoxia increases the resistance of pulmonary arteries by stimulating their contraction and augmenting their coverage by smooth muscle cells (SMCs). While these responses require adjustment of the vascular SMC transcriptome, regulatory elements are not well defined in this context. Here, we explored the functional role of the transcription factor nuclear factor of activated T-cells 5 (NFAT5/TonEBP) in the hypoxic lung. Regulatory functions of NFAT5 were investigated in cultured artery SMCs and lungs from control (Nfat5fl/fl) and SMC-specific Nfat5-deficient (Nfat5(SMC)-/-) mice. Exposure to hypoxia promoted the expression of genes associated with metabolism and mitochondrial oxidative phosphorylation (OXPHOS) in Nfat5(SMC)-/- versus Nfat5fl/fl lungs. In vitro, hypoxia-exposed Nfat5-deficient pulmonary artery SMCs elevated the level of OXPHOS-related transcripts, mitochondrial respiration, and production of reactive oxygen species (ROS). Right ventricular functions were impaired while pulmonary right ventricular systolic pressure (RVSP) was amplified in hypoxia-exposed Nfat5(SMC)-/- versus Nfat5fl/fl mice. Scavenging of mitochondrial ROS normalized the raise in RVSP. Our findings suggest a critical role for NFAT5 as a suppressor of OXPHOS-associated gene expression, mitochondrial respiration, and ROS production in pulmonary artery SMCs that is vital to limit ROS-dependent arterial resistance in a hypoxic environment.

Dysregulated paired related homeobox 1 impacts on hepatocellular carcinoma phenotypes.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of cancer-related death. Paired related homeobox 1 (PRRX1) is a transcription factor that regulates cell growth and differentiation, but its importance in HCC is unclear. METHODS: We examined the expression pattern of PRRX1 in nine microarray datasets of human HCC tumour samples (n > 1100) and analyzed its function in HCC cell lines. In addition, we performed gene set enrichment, Kaplan-Meier overall survival analysis, metabolomics and functional assays. RESULTS: PRRX1 is frequently upregulated in human HCC. Pathway enrichment analysis predicted a direct correlation between PRRX1 and focal adhesion and epithelial-mesenchymal transition. High expression of PRRX1 and low ZEB1 or high ZEB2 significantly predicted better overall survival in HCC patients. In contrast, metabolic processes correlated inversely and transcriptional analyses revealed that glycolysis, TCA cycle and amino acid metabolism were affected. These findings were confirmed by metabolomics analysis. At the phenotypic level, PRRX1 knockdown accelerated proliferation and clonogenicity in HCC cell lines. CONCLUSIONS: Our results suggest that PRRX1 controls metabolism, has a tumour suppressive role, and may function in cooperation with ZEB1/2. These findings have functional relevance in HCC, including in understanding transcriptional control of distinct cancer hallmarks.

Response of Downy Oak (Quercus pubescens Willd.) to Climate Change: Transcriptome Assembly, Differential Gene Analysis and Targeted Metabolomics.

Global change scenarios in the Mediterranean basin predict a precipitation reduction within the coming hundred years. Therefore, increased drought will affect forests both in terms of adaptive ecology and ecosystemic services. However, how vegetation might adapt to drought is poorly understood. In this report, four years of climate change was simulated by excluding 35% of precipitation above a downy oak forest. RNASeq data allowed us to assemble a genome-guided transcriptome. This led to the identification of differentially expressed features, which was supported by the characterization of target metabolites using a metabolomics approach. We provided 2.5 Tb of RNASeq data and the assembly of the first genome guided transcriptome of Quercus pubescens. Up to 5724 differentially expressed transcripts were obtained; 42 involved in plant response to drought. Transcript set enrichment analysis showed that drought induces an increase in oxidative pressure that is mitigated by the upregulation of ubiquitin-like protein protease, ferrochelatase, oxaloacetate decarboxylase and oxo-acid-lyase activities. Furthermore, the downregulation of auxin biosynthesis and transport, carbohydrate storage metabolism were observed as well as the concomitant accumulation of metabolites, such as oxalic acid, malate and isocitrate. Our data suggest that early metabolic changes in the resistance of Q. pubescens to drought involve a tricarboxylic acid (TCA) cycle shunt through the glyoxylate pathway, galactose metabolism by reducing carbohydrate storage and increased proteolytic activity.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

The combination of loss of glyoxalase1 and obesity results in hyperglycemia.

The increased formation of methylglyoxal (MG) under hyperglycemia is associated with the development of microvascular complications in patients with diabetes mellitus; however, the effects of elevated MG levels in vivo are poorly understood. In zebrafish, a transient knockdown of glyoxalase 1, the main MG detoxifying system, led to the elevation of endogenous MG levels and blood vessel alterations. To evaluate effects of a permanent knockout of glyoxalase 1 in vivo, glo1-/- zebrafish mutants were generated using CRISPR/Cas9. In addition, a diet-induced-obesity zebrafish model was used to analyze glo1-/- zebrafish under high nutrient intake. Glo1-/- zebrafish survived until adulthood without growth deficit and showed increased tissue MG concentrations. Impaired glucose tolerance developed in adult glo1-/- zebrafish and was indicated by increased postprandial blood glucose levels and postprandial S6 kinase activation. Challenged by an overfeeding period, fasting blood glucose levels in glo1-/- zebrafish were increased which translated into retinal blood vessel alterations. Thus, the data have identified a defective MG detoxification as a metabolic prerequisite and glyoxalase 1 alterations as a genetic susceptibility to the development of type 2 diabetes mellitus under high nutrition intake.

CNDP1 knockout in zebrafish alters the amino acid metabolism, restrains weight gain, but does not protect from diabetic complications.

The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1-/- zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1-/- zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala-His and Ser-His. Under diabetic conditions increased carnosine levels in CNDP1-/- embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications.

Pyrophosphate modulates plant stress responses via SUMOylation.

Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is correlated with reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance.

Activation of pro-survival metabolic networks by 1,25(OH)2D3 does not hamper the sensitivity of breast cancer cells to chemotherapeutics.

BACKGROUND: We have previously identified 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the bioactive form of vitamin D3, as a potent regulator of energy-utilization and nutrient-sensing pathways in prostate cancer cells. In the current study, we investigated the effects of 1,25(OH)2D3 on breast cancer (BCa) cell metabolism using cell lines representing distinct molecular subtypes, luminal (MCF-7 and T-47D), and triple-negative BCa (MDA-MB-231, MDA-MB-468, and HCC-1143). METHODS: 1,25(OH)2D3's effect on BCa cell metabolism was evaluated by employing a combination of real-time measurements of glycolysis/oxygen consumption rates using a biosensor chip system, GC/MS-based metabolomics, gene expression analysis, and assessment of overall energy levels. The influence of treatment on energy-related signaling molecules was investigated by immunoblotting. RESULTS: We show that 1,25(OH)2D3 significantly induces the expression and activity of the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (G6PD) in all BCa cell lines, however differentially influences glycolytic and respiratory rates in the same cells. Although 1,25(OH)2D3 treatment was found to induce seemingly anti-oxidant responses in MCF-7 cells, such as increased intracellular serine levels, and reduce the expression of its putative target gene thioredoxin-interacting protein (TXNIP), intracellular reactive oxygen species levels were found to be elevated. Serine accumulation in 1,25(OH)2D3-treated cells was not found to hamper the efficacy of chemotherapeutics, including 5-fluorouracil. Detailed analyses of the nature of TXNIP's regulation by 1,25(OH)2D3 included genetic and pharmacological inhibition of signaling molecules and metabolic enzymes including AMP-activated protein kinase and G6PD, as well as by studying the ITCH (E3 ubiquitin ligase)-TXNIP interaction. While these investigations demonstrated minimal involvement of such pathways in the observed non-canonical regulation of TXNIP, inhibition of estrogen receptor (ER) signaling by tamoxifen mirrored the reduction of TXNIP levels by 1,25(OH)2D3, demonstrating that the latter's negative regulation of ER expression is a potential mechanism of TXNIP modulation. CONCLUSIONS: Altogether, we propose that regulation of energy metabolism contributes to 1,25(OH)2D3's anti-cancer effects and that combining 1,25(OH)2D3 with drugs targeting metabolic networks in tumor cells may lead to synergistic effects.

1,25(OH)2D3 disrupts glucose metabolism in prostate cancer cells leading to a truncation of the TCA cycle and inhibition of TXNIP expression.

Prostate cell metabolism exhibits distinct profiles pre- and post-malignancy. The malignant metabolic shift converts prostate cells from "citrate-producing" to "citrate-oxidizing" cells, thereby enhancing glucose metabolism, a phenotype that contrasts classical tumoral Warburg metabolism. An on-line biosensor chip system (BIONAS 2500) was used to monitor metabolic changes (glycolysis and respiration) in response to the putative anti-cancer nutraceutical 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in different prostate cancer (PCa) cell lines (LNCaP, VCaP, DU145 and PC3). LNCaP cells exhibited profound metabolic responsiveness to the treatment and thus extensive analysis of metabolism-modulating effects of 1,25(OH)2D3 were performed, including mRNA expression analysis of key metabolic genes (e.g. GLUT1 and PDHK1), analysis of TCA cycle metabolites, glucose uptake/consumption measurements, ATP production, and mitochondrial biogenesis/activity. Altogether, data demonstrate a vivid disruption of glucose metabolism by 1,25(OH)2D3, illustrated by a decreased glucose uptake and an accumulation of citrate/isocitrate due to TCA cycle truncation. Depletion of glycolytic intermediates led to a consistent decrease in TXNIP expression in response to 1,25(OH)2D3, an effect that coincided with the activation of AMPK signaling and a reduction in c-MYC expression. Reduction in TXNIP levels in response to 1,25(OH)2D3 was rescued by an AMPK signaling inhibitor and mimicked by a MYC inhibitor highlighting the possible involvement of both pathways in mediating 1,25(OH)2D3's metabolic effects in PCa cells. Furthermore, pharmacological and genetic modulation of the androgen receptor showed similar and disparate effects on metabolic parameters compared to 1,25(OH)2D3 treatment, highlighting the AR-independent nature of 1,25(OH)2D3's metabolism-modulating effects.

Orbital Wall Reconstruction with Two-Piece Puzzle 3D Printed Implants: Technical Note.

The purpose of this article is to describe a technique for secondary reconstruction of traumatic orbital wall defects using titanium implants that act as three-dimensional (3D) puzzle pieces. We present three cases of large defect reconstruction using implants produced by Xilloc Medical B.V. (Maastricht, the Netherlands) with a 3D printer manufactured by LayerWise (3D Systems; Heverlee, Belgium), and designed using the biomedical engineering software programs ProPlan and 3-Matic (Materialise, Heverlee, Belgium). The smaller size of the implants allowed sequential implantation for the reconstruction of extensive two-wall defects via a limited transconjunctival incision. The precise fit of the implants with regard to the surrounding ledges and each other was confirmed by intraoperative 3D imaging (Mobile C-arm Systems B.V. Pulsera, Philips Medical Systems, Eindhoven, the Netherlands). The patients showed near-complete restoration of orbital volume and ocular motility. However, challenges remain, including traumatic fat atrophy and fibrosis.

A systematic review on soft-to-hard tissue ratios in orthognathic surgery part II: Chin procedures.

PURPOSE: Precise soft-to-hard tissue ratios in orthofacial chin procedures are not well established. The aim of this study was to determine useful soft-to-hard tissue ratios for planning the magnitude of sliding genioplasty (chin osteotomy), osseous chin recontouring and alloplastic chin augmentation. MATERIAL AND METHODS: A systematic review of English and non-English articles using PubMed central, ProQuest Dissertations and Theses, Science Citation Index, Elsevier Science Direct Complete, Highwire Press, Springer Standard Collection, SAGE premier 2011, DOAJ Directory of Open Access Journals, Sweetswise, Free E-Journals, Ovid Lippincott Williams & Wilkins total Access Collection, Wiley Online Library Journals, and Cochrane Plus databases from their onset until July 2014. Additional studies were identified by searching the references. Search terms included soft tissue, ratios, genioplasty, mentoplasty, chin, genial AND advancement, augmentation, setback, retrusion, impaction, reduction, vertical deficit, widening, narrowing, and expansion. Study selection criteria were as follows: only academic publications; human patients; no reviews; systematic reviews or meta-analyses; no cadavers; no syndromic patients; no pathology at the chin or mandible region; only articles of level of evidence from I to IV; number of patients must be cited in the articles; hard-to-soft tissue ratios must be cited in the articles or at least are able to be calculated with the quantitative data available in the article; if all patients of one article have had bilateral sagittal split osteotomy (BSSO) performed along with chin osteotomy, there should be an independent group evaluation of the data concerning to the chin; and no restriction regarding the size of the group. Independent extraction of articles by two authors using predefined data fields, including study quality indicators (level of evidence). RESULTS: The search identified 22 articles. Eleven additional articles were found in their reference sections. Of these, two were evidence level IIIb, three were evidence level IIb, and the rest were evidence level IV. Three studies were prospective in nature. A high variability of soft-to-hard tissue ratios regarding genioplasty seemed to disappear if data were stratified according to confounding factors. With the available data, a soft-to-hard pogonion ratio of 0.9:1 and 0.55:1 could be used for chin advancement and chin setback surgery, respectively. CONCLUSION: Advancement and extrusion movements of the chin segment show respectively a 0.9:1 of sPg:Pg horizontally and 0.95:1 of sMe:Me vertically. Setback and impaction movements show respectively a -0.52:1 of sPg:Pg horizontally and -0.43:1 of sMe:Me vertically. Prospective studies are needed that stratify by confounding factors such as type of osteotomy technique, magnitude of the movement, age, sex, race/ethnicity, and quantity and quality of the soft tissues. More specifically, studies are needed regarding soft-to-hard tissue changes after chin extrusion ("downgrafting"), intrusion ("impaction"), and widening and narrowing surgery.

[Limited mouth opening - what now?]. / Eingeschränkte Mundöffnung – was nun?

This case report illustrates a rare complex of symptoms leading to limited mouth opening in a young woman. The 28-year old suffered from a progressively limited mouth opening over several years that finally resulted in restricted alimentation and made dental treatment impossible. Clinical findings suggest a structural alteration, including a hyperplastic mandibular angle and marked hypertrophy of the masseter muscle. Further radiologic investigations reveal a thickened aponeurosis of the masticatory muscles and hyperplastic coronoid processes that are not interfering with the zygomatic bone. Primary therapeutic options for such conditions are mainly surgical, including reduction of the masseter muscles volume and aponeurorectomy as well as bony reductions, such as coronoidectomy and mandibular angle reduction. With this treatment, the outcome and prognosis are good. Long-term results depend on concomitant physical therapy. A uniform nomenclature for this condition is yet lacking and propositions such as “masticatory muscle tendon-aponeurosis hyperplasia” have been made. However, knowledge of this condition and its typical clinical signs can make the diagnosis and treatment straightforward, thus leading to an improved quality of life of affected patients.