Opticool: Cutting-edge transgenic optical tools.

Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

A critical role of retinoic acid concentration for the induction of a fully human-like atrial action potential phenotype in hiPSC-CM.

Retinoic acid (RA) induces an atrial phenotype in human induced pluripotent stem cells (hiPSCs), but expression of atrium-selective currents such as the ultrarapid (IKur) and acetylcholine-stimulated K+ current is variable and less than in the adult human atrium. We suspected methodological issues and systematically investigated the concentration dependency of RA. RA treatment increased IKur concentration dependently from 1.1 ± 0.54 pA/pF (0 RA) to 3.8 ± 1.1, 5.8 ± 2.5, and 12.2 ± 4.3 at 0.01, 0.1, and 1 µM, respectively. Only 1 µM RA induced enough IKur to fully reproduce human atrial action potential (AP) shape and a robust shortening of APs upon carbachol. We found that sterile filtration caused substantial loss of RA. We conclude that 1 µM RA seems to be necessary and sufficient to induce a full atrial AP shape in hiPSC-CM in EHT format. RA concentrations are prone to methodological issues and may profoundly impact the success of atrial differentiation.

An arrhythmogenic metabolite in atrial fibrillation.

BACKGROUND: Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. METHODS AND RESULTS: Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. CONCLUSION: Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.

A genome-wide genetic screen identifies CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis.

Opsonin-independent phagocytosis mediated by human carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) has evolved to control a subset of human-restricted bacterial pathogens. CEACAM3 engagement triggers rapid GTP-loading of the small GTPase Rac as a master regulator of cytoskeletal rearrangements and lamellipodia-driven internalization. To identify components of the CEACAM3-initiated signaling cascade, we performed a genome-wide CRISPR/Cas9-based screen in human myeloid cells. Following infection with fluorescently labeled bacteria, cells exhibiting elevated phagocytosis (gain-of-function) as well as cells showing reduced phagocytosis (loss-of-function) were sorted and enrichment of individual single-guide RNAs (sgRNAs) was determined by next generation sequencing. Concentrating on genes whose targeting by three distinct sgRNAs consistently resulted in a gain-of-function phenotype, we identified the Rac-GTP-sequestering protein CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis. Clonal HL-60 cell lines with CYRI-B knockout showed enhanced CEACAM3-downstream signaling, such as Rac GTP loading and phosphorylation of PAK kinases, leading to increased phagocytosis of bacteria. Complementation of the CYRI-B knockout cells reverted the knockout phenotype. Our results unravel components of CEACAM3-initiated opsonin-independent phagocytosis on a genome-wide level and highlight CYRI-B as a negative regulator of CEACAM3-initiated signaling in myeloid cells.

Human-Engineered Atrial Tissue for Studying Atrial Fibrillation.

This chapter details the generation of atrial fibrin-based engineered heart tissue (EHT) in standard 24-well format as a 3D model for the human atrium. Compared to 2D cultivation, human-induced pluripotent stem cells (hiPSCs)-derived atrial cardiomyocytes demonstrated a higher degree of maturation in 3D format. Furthermore, we have demonstrated in previous work that the model displayed atrial characteristics in terms of contraction and gene expression patterns, electrophysiology, and pharmacological response. Here, we describe how to embed atrial cardiomyocytes differentiated from hiPSCs in a fibrin hydrogel to form atrial EHT attached to elastic silicone posts, allowing auxotonic contraction. In addition, we describe how force and other contractility parameters can be derived from these beating atrial EHTs by video-optical monitoring. The presented atrial EHT model is suitable to study chamber-specific mechanisms, drug effects and to serve for disease modeling.

Tissue-specific multi-omics analysis of atrial fibrillation.

Genome-wide association studies (GWAS) for atrial fibrillation (AF) have uncovered numerous disease-associated variants. Their underlying molecular mechanisms, especially consequences for mRNA and protein expression remain largely elusive. Thus, refined multi-omics approaches are needed for deciphering the underlying molecular networks. Here, we integrate genomics, transcriptomics, and proteomics of human atrial tissue in a cross-sectional study to identify widespread effects of genetic variants on both transcript (cis-eQTL) and protein (cis-pQTL) abundance. We further establish a novel targeted trans-QTL approach based on polygenic risk scores to determine candidates for AF core genes. Using this approach, we identify two trans-eQTLs and five trans-pQTLs for AF GWAS hits, and elucidate the role of the transcription factor NKX2-5 as a link between the GWAS SNP rs9481842 and AF. Altogether, we present an integrative multi-omics method to uncover trans-acting networks in small datasets and provide a rich resource of atrial tissue-specific regulatory variants for transcript and protein levels for cardiovascular disease gene prioritization.

Long-Chain Acyl-Carnitines Interfere with Mitochondrial ATP Production Leading to Cardiac Dysfunction in Zebrafish.

In the human heart, the energy supplied by the production of ATP is predominately accomplished by ß-oxidation in mitochondria, using fatty acids (FAs) as the primary fuel. Long-chain acylcarnitines (LCACs) are intermediate forms of FA transport that are essential for FA delivery from the cytosol into mitochondria. Here, we analyzed the impact of the LCACs C18 and C18:1 on mitochondrial function and, subsequently, on heart functionality in the in vivo vertebrate model system of zebrafish (Danio rerio). Since LCACs are formed and metabolized in mitochondria, we assessed mitochondrial morphology, structure and density in C18- and C18:1-treated zebrafish and found no mitochondrial alterations compared to control-treated (short-chain acylcarnitine, C3) zebrafish embryos. However, mitochondrial function and subsequently ATP production was severely impaired in C18- and C18:1-treated zebrafish embryos. Furthermore, we found that C18 and C18:1 treatment of zebrafish embryos led to significantly impaired cardiac contractile function, accompanied by reduced heart rate and diminished atrial and ventricular fractional shortening, without interfering with cardiomyocyte differentiation, specification and growth. In summary, our findings provide insights into the direct role of long-chain acylcarnitines on vertebrate heart function by interfering with regular mitochondrial function and thereby energy allocation in cardiomyocytes.

Long-chain acylcarnitine 18:1 acutely increases human atrial myocardial contractility and arrhythmia susceptibility.

Long-chain acylcarnitines (LCACs) are known to directly alter cardiac contractility and electrophysiology. However, the acute effect of LCACs on human cardiac function is unknown. We aimed to determine the effect of LCAC 18:1, which has been associated with cardiovascular disease, on the contractility and arrhythmia susceptibility of human atrial myocardium. Additionally, we aimed to assess how LCAC 18:1 alters Ca2+ influx and spontaneous Ca2+ release in vitro. Human right atrial trabeculae (n = 32) stimulated at 1 Hz were treated with LCAC 18:1 at a range of concentrations (1-25 µM) for a 45-min period. Exposure to the LCAC induced a dose-dependent positive inotropic effect on myocardial contractility (maximal 1.5-fold increase vs. control). At the 25 µM dose (n = 8), this was paralleled by an enhanced propensity for spontaneous contractions (50% increase). Furthermore, all LCAC 18:1 effects on myocardial function were reversed following LCAC 18:1 washout. In fluo-4-AM-loaded HEK293 cells, LCAC 18:1 dose dependently increased cytosolic Ca2+ influx relative to vehicle controls and the short-chain acylcarnitine C3. In HEK293 cells expressing ryanodine receptor (RyR2), this increased Ca2+ influx was linked to an increased propensity for RyR2-mediated spontaneous Ca2+ release events. Our study is the first to show that LCAC 18:1 directly and acutely alters human myocardial function and in vitro Ca2+ handling. The metabolite promotes proarrhythmic muscle contractions and increases contractility. The exploratory findings in vitro suggest that LCAC 18:1 increases proarrhythmic RyR2-mediated spontaneous Ca2+ release propensity. The direct effects of metabolites on human myocardial function are essential to understand cardiometabolic dysfunction.NEW & NOTEWORTHY For the first time, the fatty acid metabolite, long-chain acylcarnitine 18:1, is shown to acutely increase the arrhythmia susceptibility and contractility of human atrial myocardium. In vitro, this was linked to an influx of Ca2+ and an enhanced propensity for spontaneous RyR2-mediated Ca2+ release.

Hypertrophic signaling compensates for contractile and metabolic consequences of DNA methyltransferase 3A loss in human cardiomyocytes.

The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using engineered heart tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 µM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease.

[Is the Bavarian Population Open for Teledermatology? A Cross-Sectional Study in Rural and Urban Regions of Bavaria, Germany]. / Wie offen ist die bayerische Bevölkerung für Teledermatologie? Eine Querschnittsstudie in ländlichen und städtischen Regionen Bayerns.

AIM: Teledermatology has a great potential to improve dermatologic care in rural regions. The aim of this study was to assess the willingness to use teledermatology in the rural population of Bavaria, Southern Germany, and to explore major concerns regarding teledermatology. METHODS: The data of this cross-sectional study were collected both as part of a health survey conducted in the Bavarian forest region in Q1/2017 and as part of a follow-up survey (Q1/2018) of a running cohort study recruited at a Bavarian agricultural festival. Study participants were asked in a standardized questionnaire whether they would send pictures of skin rashes or other skin changes to their dermatologist via the internet, and if "no", why not. RESULTS: Data on 1,116 participants living in Bavaria were analysed (mean age 50.2 years, 57.3% female, 80.4% living in rural regions). Of the whole sample, 36.6% were willing to use teledermatology. Women, older participants and participants living in rural regions were less open to the use of teledermatology. Major concerns regarding teledermatology were impersonality, doubts about the quality of the service and data safety and privacy concerns. DISCUSSION: The willingness to use teledermatology in the rural population of Bavaria is still rather low. Before teledermatology can realise its full potential for improving health care in rural regions, the population needs to be educated about the functioning and the advantages of teledermatology, and data safety concerns need to be addressed.

Long-Chain Acylcarnitines and Cardiac Excitation-Contraction Coupling: Links to Arrhythmias.

A growing number of metabolomic studies have associated high circulating levels of the amphiphilic fatty acid metabolites, long-chain acylcarnitines (LCACs), with cardiovascular disease (CVD) risk. These studies show that plasma LCAC levels can be correlated with the stage and severity of CVD and with indices of cardiac hypertrophy and ventricular function. Complementing these recent clinical associations is an extensive body of basic research that stems mostly from the twentieth century. These works, performed in cardiomyocyte and multicellular preparations from animal and cell models, highlight stereotypical derangements in cardiac electrophysiology induced by exogenous LCAC treatment that promote arrhythmic muscle behavior. In many cases, this is coupled with acute inotropic modulation; however, whether LCACs increase or decrease contractility is inconclusive. Linked to the electromechanical alterations induced by LCAC exposure is an array of effects on cardiac excitation-contraction coupling mechanisms that overload the cardiomyocyte cytosol with Na+ and Ca2+ ions. The aim of this review is to revisit this age-old literature and collate it with recent findings to provide a pathophysiological context for the growing body of metabolomic association studies that link circulating LCACs with CVD.

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility.

BACKGROUND: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. METHODS: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. RESULTS: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. CONCLUSION: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

[Primary and secondary prevention of skin cancer in rural areas : A cross-sectional study in the Bavarian Forest]. / Primär- und Sekundärprävention von Hautkrebs in ländlichen Regionen : Querschnittstudie im Bayerischen Wald.

BACKGROUND: Skin cancer is the most common malignancy of the fair-skinned population worldwide. To reduce skin cancer's burden primary and secondary prevention are critical. However, various studies indicate an inadequate prevention behavior among rural populations. OBJECTIVE: To examine the risk and prevention behavior with respect to skin cancer and to identify subgroups in rural areas with specific need for prevention efforts. MATERIALS AND METHODS: In a cross-sectional study carried out in the first quarter of 2017, patients and their accompanying persons (≥18 years) were interviewed on the subject of primary and secondary prevention in waiting rooms of nondermatological medical practices in the Bavarian Forest, Germany. Data were collected using paper-based questionnaires. Associations were calculated using logistic regression models. RESULTS: In all, 880 persons (57.7% women, mean age = 49.5 years) were included in the analysis, of whom 53.6% had undergone a skin cancer screening at least once before. Sunscreen was the most frequently used sun protection measure. Male sex and being 18-34 years of age were significantly associated with not using prevention measures (depending on the measure: odds ratio [OR]: 1.4-2.4 and 1.8-3.7, respectively). In addition, not using skin cancer screening was associated with UV exposure more than 6 h daily in summer (OR: 1.8, 95%-CI [confidence interval]: 1.14-2.97). CONCLUSION: Future prevention strategies should increasingly focus on young adults, on men and people with high solar UV exposition particularly, to reduce the burden of skin cancer in rural areas.

A model-based analysis of the reactive transport behaviour of 37 trace organic compounds during field-scale bank filtration.

Though bank filtration diminishes the loads of many trace organic compounds (TOrCs) present in the source water, still there is a wide uncertainty on the influence of local environmental conditions on biodegradation processes. This research addresses the fate and transport behaviour of 37 trace organic compounds at a bank filtration site in Germany over a relatively long-time span of six years. Using two-dimensional heat and reactive transport modelling in FEFLOW, TOrCs are classified according to their occurrence in bank filtration wells with a residence time of up to 4 months. We identify 12 persistent compounds, 20 reactive compounds and 5 transformation products formed during aquifer passage. Estimates of first-order biodegradation rate constants are given for six reactive compounds. Minimum biodegradation rate constants (i.e. maximum half-lives) are approximated for eight compounds only present in the surface water. For some compounds, a simple first-order degradation model did not yield satisfactory results and the behaviour appears to be more complex. Processes like sorption, redox- and/or temperature-dependent biodegradation and temperature-dependent desorption are suspected but incorporating these into the model was beyond the scope of this paper that provides an overview for many compounds. Results highlight the ability of the sub-surface to improve the water quality during bank filtration, yet at the same time show the persistence of several compounds in the aquifer.

Prevalence and treatment of allergies in rural areas of Bavaria, Germany: a cross-sectional study.

BACKGROUND: There is a high prevalence of allergies in Germany, with approximately 20% of the population having at least one allergy and only about 10% of these being treated adequately. We conducted a cross-sectional study in a rural area of Bavaria (Southern Germany) to evaluate the prevalence of allergies and their treatment, because research regarding this topic is currently limited. METHODS: Data were collected in 10 offices of non-dermatological doctors using a self-filled questionnaire to ask participants about allergies and treatment. RESULTS: A total of 641 questionnaires were appropriate for analysis. The prevalence of allergies in the Bavarian countryside was higher than that reported for Germany (37.3% vs. 20.0%). Furthermore, almost a third (30.4%) of allergies were not treated at all. The most frequently consulted therapist was found to be a general practitioner. CONCLUSIONS: Based on the study results, there is a need for prevention programs and establishment of treatments for certain allergies to minimize long-term health effects. Moreover, more studies are needed to analyze the prevalence of allergies in farmers who had a higher prevalence of allergies compared to previously reported prevalence in literature reviews. TRIAL REGISTRATIONS: The study was approved by the ethical review committee of the Technical University Munich (EC number 548/16S).

Rat atrial engineered heart tissue: a new in vitro model to study atrial biology.

Engineered heart tissue (EHT) from rat cells is a useful tool to study ventricular biology and cardiac drug safety. Since atrial and ventricular cells differ significantly, EHT and other 3D cell culture formats generated from ventricular cells have been of limited value to study atrial biology. To date, reliable in vitro models that reflect atrial physiology are lacking. Therefore, we established a novel EHT model using rat atrial cells (atrial EHT, aEHT) to assess atrial physiology, contractility and drug response. The tissue constructs were characterized with regard to gene expression, histology, electrophysiology, and the response to atrial-specific drugs. We observed typical functional properties of atrial tissue in our model such as more regular spontaneous beating with lower force, shorter action potential duration, and faster contraction and relaxation compared to ventricular EHT (vEHT). The expression of atrial-specific genes and proteins was high, whereas ventricle-specific transcripts were virtually absent. The atrial-selective drug carbachol had a strong negative inotropic and chronotropic effect on aEHT only. Taken together, the results demonstrate the feasibility of aEHT as a novel atrial 3D model and as a benchmark for tissue engineering with human induced pluripotent stem cell-derived atrial-like cardiomyocytes. Atrial EHT faithfully recapitulates atrial physiology and shall be useful to study atrial molecular physiology in health and disease as well as drug response.

Novel DNA Methylation Sites Influence GPR15 Expression in Relation to Smoking.

Smoking is a major risk factor for cardiovascular diseases and has been implicated in the regulation of the G protein-coupled receptor 15 (GPR15) by affecting CpG methylation. The G protein-coupled receptor 15 is involved in angiogenesis and inflammation. An effect on GPR15 gene regulation has been shown for the CpG site CpG3.98251294. We aimed to analyze the effect of smoking on GPR15 expression and methylation sites spanning the GPR15 locus. DNA methylation of nine GPR15 CpG sites was measured in leukocytes from 1291 population-based individuals using the EpiTYPER. Monocytic GPR15 expression was measured by qPCR at baseline and five-years follow up. GPR15 gene expression was upregulated in smokers (beta (ß) = -2.699, p-value (p) = 1.02 × 10-77) and strongly correlated with smoking exposure (ß = -0.063, p = 2.95 × 10-34). Smoking cessation within five years reduced GPR15 expression about 19% (p = 9.65 × 10-5) with decreasing GPR15 expression over time (ß = 0.031, p = 3.81 × 10-6). Additionally, three novel CpG sites within GPR15 affected by smoking were identified. For CpG3.98251047, DNA methylation increased steadily after smoking cessation (ß = 0.123, p = 1.67 × 10-3) and strongly correlated with changes in GPR15 expression (ß = 0.036, p = 4.86 × 10-5). Three novel GPR15 CpG sites were identified in relation to smoking and GPR15 expression. Our results provide novel insights in the regulation of GPR15, which possibly linked smoking to inflammation and disease progression.

[Terms for the feeling of itching in the Bavarian dialect]. / Wenn's "ameiselt" ­ Bezeichnungen für das Empfinden von Juckreiz im bairischen Dialekt.

BACKGROUND: Pruritus can have many different causes and is a very subjective sensation. As dialects show a greater diversity of linguistic expressions than standard languages, the description of the sensation of pruritus in a dialect might carry additional information about the quality of pruritus. METHOD: This cross-sectional study was carried out in Q1/2016 in the rural Bavarian Forest region in Bavaria, Germany. Participants were recruited in the waiting rooms of local doctors. They were asked to complete four sentences composed in the Bavarian dialect in which one word per sentence that signified the sensation of pruritus had been replaced by a blank space. RESULTS: In total, the 1,007 participants (mean age 49.97 years, SD = 15.76; 58.2% female) named 2,870 expressions, 144 excluding duplicates. 98.7% of the expressions could be matched to 13 underlying terms. The most frequent ones were "jucken" ('to itch'', 59.5%), "brennen" ('to burn', 26.5%), "kribbeln" (no English equivalent, 1.1%), "kratzen" ('to scratch', 0.7%) and "beißen" ('to bite', 0.6%). The use of the different expressions was strongly situational: In 75% of the cases "brennen" ('to burn') was used in the context of contact to a stinging nettle (in German 'Brennnessel'). Two of the 13 expressions ("kribbeln" and "bitzeln", no English equivalents) were even used only in this scenario. DISCUSSION: In the Bavarian dialect several different expressions for the sensation of pruritus exist. Although "jucken" ('to itch') is the most common expression for the sensation of pruritus, the German medical terms "Pruritus" ('pruritus') and "Juckreiz" ('itch') don't do justice to the situational use of expressions in the dialect.

Immediate release pellets with lipid binders obtained by solvent-free cold extrusion.

Lipid-based drug delivery systems have spread in their use in pharmaceutical drug development. This work focuses on the use of lipid binders as alternative non-toxic extrusion aid for pellet formulations. The preparation of immediate release pellets with solid lipid binders through a solvent-free cold extrusion/spheronisation process was investigated in this study. Various binary, ternary and quaternary mixtures of powdered lipids and the model drug sodium benzoate were investigated and compared to well-known wet extrusion binders like microcrystalline cellulose and kappa-carrageenan. The cold lipid extrusion process offers multiple advantages as it is suitable for thermal sensitive as well as for hygroscopic drugs, furthermore no drying process to evaporate the solvent is needed and the process is feasible for different extruder types. Some of the developed pellets showed favourable properties like spherical shape, narrow size distribution, a high drug load of 80% sodium benzoate and a drug release of more than 90% within 40 min. The stability of drug release, which can be problematic when using lipid excipients, was sufficient for some mixtures, as storage under elevated temperatures changed the release profiles only slightly and no formulation released less than 80% within the first 60 min. A formulation with a mixture of hard fat, glycerol distearate and glycerol trimyristate showed the best results, as pellets with a low aspect ratio, narrow size distribution and complete drug release were obtained. Using appropriate mixtures of acylglycerides it becomes possible to produce pharmaceutical pellets with immediate release characteristics by cold extrusion and subsequent spheronisation. Thus, lipids are very promising alternatives to commonly used extrusion/spheronisation binders.