PPARG activation promotes the proliferation of colorectal cancer cell lines and enhances the antiproliferative effect of 5-fluorouracil.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARG) is a member of the nuclear receptor family. It is involved in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, vascular homeostasis and inflammation. In addition, PPARG agonists, known as thiazolidinediones, are well established in the treatment of type 2 diabetes mellitus. PPARGs role in cancer is a matter of debate, as pro- and anti-tumour properties have been described in various tumour entities. Currently, the specific role of PPARG in patients with colorectal cancer (CRC) is not fully understood. MATERIAL AND METHODS: The prognostic impact of PPARG expression was investigated by immunohistochemistry in a case-control study using a matched pair selection of CRC tumours (n = 246) with either distant metastases to the liver (n = 82), lung (n = 82) or without distant metastases (n = 82). Its effect on proliferation as well as the sensitivity to the chemotherapeutic drug 5-fluorouracil (5-FU) was examined after activation, inhibition, and transient gene knockdown of PPARG in the CRC cell lines SW403 and HT29. RESULTS: High PPARG expression was significantly associated with pulmonary metastasis (p = 0.019). Patients without distant metastases had a significantly longer overall survival with low PPARG expression in their tumours compared to patients with high PPARG expression (p = 0.045). In the pulmonary metastasis cohort instead, a trend towards longer survival was observed for patients with high PPARG expression in their tumour (p = 0.059). Activation of PPARG by pioglitazone and rosiglitazone resulted in a significant dose-dependent increase in proliferation of CRC cell lines. Inhibition of PPARG by its specific inhibitor GW9662 and siRNA-mediated knockdown of PPARG significantly decreased proliferation. Activating PPARG significantly increased the CRC cell lines sensitivity to 5-FU while its inhibition decreased it. CONCLUSION: The prognostic effect of PPARG expression depends on the metastasis localization in advanced CRC patients. Activation of PPARG increased malignancy associated traits such as proliferation in CRC cell lines but also increases sensitivity towards the chemotherapeutic agent 5-FU. Based on this finding, a combination therapy of PPARG agonists and 5-FU-based chemotherapy constitutes a promising strategy which should be further investigated.

Training doctoral students in critical thinking and experimental design using problem-based learning.

BACKGROUND: Traditionally, doctoral student education in the biomedical sciences relies on didactic coursework to build a foundation of scientific knowledge and an apprenticeship model of training in the laboratory of an established investigator. Recent recommendations for revision of graduate training include the utilization of graduate student competencies to assess progress and the introduction of novel curricula focused on development of skills, rather than accumulation of facts. Evidence demonstrates that active learning approaches are effective. Several facets of active learning are components of problem-based learning (PBL), which is a teaching modality where student learning is self-directed toward solving problems in a relevant context. These concepts were combined and incorporated in creating a new introductory graduate course designed to develop scientific skills (student competencies) in matriculating doctoral students using a PBL format. METHODS: Evaluation of course effectiveness was measured using the principals of the Kirkpatrick Four Level Model of Evaluation. At the end of each course offering, students completed evaluation surveys on the course and instructors to assess their perceptions of training effectiveness. Pre- and post-tests assessing students' proficiency in experimental design were used to measure student learning. RESULTS: The analysis of the outcomes of the course suggests the training is effective in improving experimental design. The course was well received by the students as measured by student evaluations (Kirkpatrick Model Level 1). Improved scores on post-tests indicate that the students learned from the experience (Kirkpatrick Model Level 2). A template is provided for the implementation of similar courses at other institutions. CONCLUSIONS: This problem-based learning course appears effective in training newly matriculated graduate students in the required skills for designing experiments to test specific hypotheses, enhancing student preparation prior to initiation of their dissertation research.

Muscle active force-length curve explained by an electrophysical model of interfilament spacing.

The active isometric force-length relation (FLR) of striated muscle sarcomeres is central to understanding and modeling muscle function. The mechanistic basis of the descending arm of the FLR is well explained by the decreasing thin:thick filament overlap that occurs at long sarcomere lengths. The mechanistic basis of the ascending arm of the FLR (the decrease in force that occurs at short sarcomere lengths), alternatively, has never been well explained. Because muscle is a constant-volume system, interfilament lattice distances must increase as sarcomere length shortens. This increase would decrease thin and thick-filament electrostatic interactions independently of thin:thick filament overlap. To examine this effect, we present here a fundamental, physics-based model of the sarcomere that includes filament molecular properties, calcium binding, sarcomere geometry including both thin:thick filament overlap and interfilament radial distance, and electrostatics. The model gives extremely good fits to existing FLR data from a large number of different muscles across their entire range of measured activity levels, with the optimized parameter values in all cases lying within anatomically and physically reasonable ranges. A local first-order sensitivity analysis (varying individual parameters while holding the values of all others constant) shows that model output is most sensitive to a subset of model parameters, most of which are related to sarcomere geometry, with model output being most sensitive to interfilament radial distance. This conclusion is supported by re-running the fits with only this parameter subset being allowed to vary, which increases fit errors only moderately. These results show that the model well reproduces existing experimental data, and indicate that changes in interfilament spacing play as central a role as changes in filament overlap in determining the FLR, particularly on its ascending arm.

Bcl-xL as prognostic marker and potential therapeutic target in cholangiocarcinoma.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-xL and Mcl-1. Expression of Bcl-xL , Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-xL and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl-xL (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-xL and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-xL as a key protein in cell death resistance of CCA and may pave the way for clinical application.

The short term influence of right ventricular pacing burden on echocardiographic and spiroergometric parameters in patients with preserved left ventricular ejection fraction.

BACKGROUND: The incidence of worsened clinical outcome due to high right ventricular (RV) pacing burden in patients with preserved left ventricular function remains controversial. OBJECTIVE: To investigate the impact of RV pacing on several echocardiographic and spiroergometric parameters. METHODS: In 60 pacemaker patients with preserved left ventricular ejection fraction (LVEF) serial echocardiographies and spiroergometries were performed over a time course of 12 months. Additionally, in 48 patients retrospective echocardiographic analyses of the LV- and RV function were carried out up to 24 months after pacemaker implantation. RESULTS: The patients were divided into two groups: The high RV pacing burden group (hRVP: ≥ 40%) and the low RV pacing group (lRVP < 40%) according to the definitions in previous randomized MOST and DAVID trials. After a period of 12-month pacemaker therapy no changes to left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), LVEF, E/A-ratio; E/E'-ratio and tricuspid annular plane systolic excursion (TAPSE) could be revealed, independently of the RV pacing burden. Additionally, after 24-month long term follow-up there were no differences in LVEF and TAPSE in both groups. Accordingly, no relevant changes of peak exercise capacity, ventilatory anaerobic threshold or maximal oxygen consumption could be demonstrated independently of the RV pacing. CONCLUSIONS: In pacemaker patients with preserved LVEF the burden of RV pacing has no adverse influence on several echocardiographic and spiroergometric surrogate parameters of pacemaker-induced cardiomyopathy after a follow-up of 12 to 24 month. Despite this, screening for pacemaker induced cardiomyopathy should be performed especially in the presence of new heart failure symptoms.

Rules of nature's Formula Run: Muscle mechanics during late stance is the key to explaining maximum running speed.

The maximum running speed of legged animals is one evident factor for evolutionary selection-for predators and prey. Therefore, it has been studied across the entire size range of animals, from the smallest mites to the largest elephants, and even beyond to extinct dinosaurs. A recent analysis of the relation between animal mass (size) and maximum running speed showed that there seems to be an optimal range of body masses in which the highest terrestrial running speeds occur. However, the conclusion drawn from that analysis-namely, that maximum speed is limited by the fatigue of white muscle fibres in the acceleration of the body mass to some theoretically possible maximum speed-was based on coarse reasoning on metabolic grounds, which neglected important biomechanical factors and basic muscle-metabolic parameters. Here, we propose a generic biomechanical model to investigate the allometry of the maximum speed of legged running. The model incorporates biomechanically important concepts: the ground reaction force being counteracted by air drag, the leg with its gearing of both a muscle into a leg length change and the muscle into the ground reaction force, as well as the maximum muscle contraction velocity, which includes muscle-tendon dynamics, and the muscle inertia-with all of them scaling with body mass. Put together, these concepts' characteristics and their interactions provide a mechanistic explanation for the allometry of maximum legged running speed. This accompanies the offering of an explanation for the empirically found, overall maximum in speed: In animals bigger than a cheetah or pronghorn, the time that any leg-extending muscle needs to settle, starting from being isometric at about midstance, at the concentric contraction speed required for running at highest speeds becomes too long to be attainable within the time period of a leg moving from midstance to lift-off. Based on our biomechanical model, we, thus, suggest considering the overall speed maximum to indicate muscle inertia being functionally significant in animal locomotion. Furthermore, the model renders possible insights into biological design principles such as differences in the leg concept between cats and spiders, and the relevance of multi-leg (mammals: four, insects: six, spiders: eight) body designs and emerging gaits. Moreover, we expose a completely new consideration regarding the muscles' metabolic energy consumption, both during acceleration to maximum speed and in steady-state locomotion.

A geometry- and muscle-based control architecture for synthesising biological movement.

A key problem for biological motor control is to establish a link between an idea of a movement and the generation of a set of muscle-stimulating signals that lead to the movement execution. The number of signals to generate is thereby larger than the body's mechanical degrees of freedom in which the idea of the movement may be easily expressed, as the movement is actually executed in this space. A mathematical formulation that provides a solving link is presented in this paper in the form of a layered, hierarchical control architecture. It is meant to synthesise a wide range of complex three-dimensional muscle-driven movements. The control architecture consists of a 'conceptional layer', where the movement is planned, a 'structural layer', where the muscles are stimulated, and between both an additional 'transformational layer', where the muscle-joint redundancy is resolved. We demonstrate the operativeness by simulating human stance and squatting in a three-dimensional digital human model (DHM). The DHM considers 20 angular DoFs and 36 Hill-type muscle-tendon units (MTUs) and is exposed to gravity, while its feet contact the ground via reversible stick-slip interactions. The control architecture continuously stimulates all MTUs ('structural layer') based on a high-level, torque-based task formulation within its 'conceptional layer'. Desired states of joint angles (postural plan) are fed to two mid-level joint controllers in the 'transformational layer'. The 'transformational layer' communicates with the biophysical structures in the 'structural layer' by providing direct MTU stimulation contributions and further input signals for low-level MTU controllers. Thereby, the redundancy of the MTU stimulations with respect to the joint angles is resolved, i.e. a link between plan and execution is established, by exploiting some properties of the biophysical structures modelled. The resulting joint torques generated by the MTUs via their moment arms are fed back to the conceptional layer, closing the high-level control loop. Within our mathematical formulations of the Jacobian matrix-based layer transformations, we identify the crucial information for the redundancy solution to be the muscle moment arms, the stiffness relations of muscle and tendon tissue within the muscle model, and the length-stimulation relation of the muscle activation dynamics. The present control architecture allows the straightforward feeding of conceptional movement task formulations to MTUs. With this approach, the problem of movement planning is eased, as solely the mechanical system has to be considered in the conceptional plan.

Enantioselective Construction of Modular and Asymmetric Baskets.

The precise positioning of functional groups about the inner space of abiotic hosts is a challenging task and of interest for developing more effective receptors and catalysts akin to those found in nature. To address it, we herein report a synthetic methodology for preparing basket-like cavitands comprised of three different aromatics as side arms with orthogonal esters at the rim for further functionalization. First, enantioenriched A (borochloronorbornene), B (iodobromonorbornene), and C (boronorbornene) building blocks were obtained by stereoselective syntheses. Second, consecutive A-to-B and then AB-to-C Suzuki-Miyaura (SM) couplings were optimized to give enantioenriched ABC cavitand as the principal product. The robust synthetic protocol allowed us to prepare (a) an enantioenriched basket with three benzene sides and each holding either tBu, Et, or Me esters, (b) both enantiomers of a so-called "spiral staircase" basket with benzene, naphthalene, and anthracene groups surrounding the inner space, and (c) a photo-responsive basket bearing one anthracene and two benzene arms.

Twist-Turn-Twist Motif Chaperoned Inside Molecular Baskets.

Two molecular baskets 16-, each with three (S)-glutamic acids at its rim, were found (NMR, ITC) to complex diammonium alkanes 22+-52+ (+H3N(CH2)nNH3+, n = 7-10) giving ternary [2-5⊂12]10- assemblies (K = 107-109 M-2). From the magnetic perturbation of proton nuclei (1H NMR, NICS), we deduced that each guest assumed a U shape within the binary complex, [2-5⊂1]4-. Two ammonium groups were bound in the "anionic nest" at the top of 16-, while the hydrocarbon chain resided in its nonpolar cavity. From detailed ITC analyses, we showed that the binary complex [2-5⊂1]4- forms first and then another 16- capped [2-5⊂1]4- to give [2-5⊂12]10-, via interactions of the carboxylates at the rims with ammoniums on the guest. Long-range NOEs revealed that U-shaped 22+ and 52+ coiled into highly strained twist-turn-twist formations, for the first time observed within an abiotic host while curiously resembling helix-turn-helix motif found in DNA binding proteins.

Light-Triggered Transformation of Molecular Baskets into Organic Nanoparticles.

Discovering novel and functional photoresponsive materials is of interest for improving controlled release of molecules and scavenging toxic compounds for cleaning our environment or designing chemosensors. In this study, we report on the photoinduced decarboxylation of basket 16- , containing three glutamic acids at its rim. This concave compound is, in an aqueous environment (30 mm phosphate buffer at pH 7.0), monomeric (1 H NMR DOSY, DLS) with glutamic acid residues randomly oriented about its rim (1 H NMR and MM-OPLS3). The irradiation (300 nm) of 16- leads to the exclusive removal of its α-carboxylates to give amphiphilic 23- possessing γ-carboxylates. The photochemical transformation is a consecutive reaction with mono- and bis-decarboxylated products observed with 1 H NMR spectroscopy and ESI mass spectrometry. Amphiphilic 23- is a preorganized molecule (MM-OPLS3) that, in water, aggregates into organic nanoparticles (ca. 50-200 nm in diameter; DLS, TEM and cryo-TEM) having a critical aggregation concentration of 12 µm (UV/Vis). As the transition of monomeric 16- into nanoparticulate 23- is triggered with light, we reasoned that stimuli-responsive formation of the soft material lends itself to nanotechnology applications such as controlled release or scavenging of targeted compounds.

Stereo- and Regioselective Synthesis of Molecular Baskets.

We describe a stereoselective method for obtaining multigram quantities of molecular basket 1 syn in overall 11% yield, using inexpensive cyclopentadiene and diethyl fumarate as starting materials. First, an asymmetric synthesis of enantioenriched bromo(trimethylstannyl)alkene (-)-8 was accomplished by the stereoselective bromination of dibromonorbornene (+)-4 guided by anchimeric assistance and subsequent syn- exo-elimination of tetrabromonorbornane (-)-5a as the key steps. Subsequent Cu(I)-catalyzed cyclotrimerization of (-)-8 was optimized to give 1 syn/ anti in 85% yield and 1:1 ratio of diastereomers. Importantly, the results of our mechanistic experiments were in line with the cyclotrimerization occurring in a chain-type fashion with racemization of a Cu(I) homochiral dimeric intermediate, reducing the stereoselectivity of the transformation. Enabled by more facile access to molecular baskets of type 1 syn, a range of recognition studies can now be completed for producing novel supramolecular catalysts, organophosphorus scavengers, and nanostructured materials.

A Three-Dimensional Musculoskeletal Model of the Western Lowland Gorilla Foot: Examining Muscle Torques and Function.

Due to difficulty of obtaining accurate quantitative data on foot muscles, relatively little has been done to study foot muscle function in non-human apes. Gorilla feet are known to be similar in bony proportions and mechanics to those of humans, hence are key to understanding human foot evolution and its ecological context. We present the first 3D musculoskeletal computer model of a western lowland gorilla foot, giving muscle torques about the tarsometatarsal, metatarsophalangeal and interphalangeal joints of digits 2-5. Peak flexor torque around the fifth metatarsophalangeal joint occurs at a highly flexed position, suggesting an ability to maintain flexed postures around lateral metatarsophalangeal joints, useful for grasping vertical supports. For distal interphalangeal joints, flexor torques peaked the more medial the digit at relatively flexed postures. We report, for the first time, interossei acting upon proximal and distal interphalangeal joints. All these facilitate maintenance of flexed positions around distal interphalangeal joints, likely used for grasping of small supports/objects. Humans lack these features, suggesting that semi-arboreal early hominins made less use of the peripheral canopy than gorillines. Information here could be used in gorilla enclosure design to encourage wild-type locomotor repertoires in captivity.

Inter-filament spacing mediates calcium binding to troponin: A simple geometric-mechanistic model explains the shift of force-length maxima with muscle activation.

The maximum of a muscle fiber's force-length curve (FLC) shifts to shorter lengths as muscle activation increases. State-of-the-art muscle models cannot explain the mechanistic basis for this shift, which is therefore either omitted or added ad hoc in a descriptive manner. A more theoretical approach developed by Hatze, who had particularly modeled the process of muscle activation, does predict this shift but can be shown to consist of multiple mathematical attempts that are all inconsistent with their common assertion: to represent local volume constancy. What mechanism may underlie the experimentally well-known shift has thus remained unclear. We work out here that the simple assumption of sarcomere volume constancy can, first of all, indeed explain the shift in the activity-Ca2+ relation as a function of sarcomere length by the enforcement of a decrease in inter-filament spacing that must occur as sarcomere length increases. We show that physiological data of this shift are consistent with a simply linear dependency of troponin (volumetric) density on sarcomere length. Further incorporating filament overlap as a second, independent mechanism, we can moreover reproduce, by means of a single master equation, an entire set of measured FLCs from literature, which testify shifts in their maxima at different levels of activation. We conclude that both phenomena, the shift in activity-Ca2+ relations with length and the shift in the maxima of FLCs with Ca2+, can be explained by the superposition of two mechanisms immediately connected to the same sarcomere state variable length: filament overlap and inter-filament spacing.

The basic mechanical structure of the skeletal muscle machinery: One model for linking microscopic and macroscopic scales.

Measuring, analysing, and modelling muscle contraction has a long history. In consequence, some signature characteristics of skeletal muscle contraction have been found. On a microscopic level, these are the typical non-steady-state responses of the cross-bridge bindings to steps in force and length. On a macroscopic level, the force-velocity, enthalpy-velocity, and efficiency-velocity relations for concentric steady-state contractions are crucial characteristics. As these characteristics were repeatedly confirmed across animal species and sizes, they are expected to pinpoint basic physical properties of the mechanical structure that embodies the skeletal muscle machinery. The approach presented in this article explains, for the first time, these characteristics at both the microscopic and the macroscopic scale with one model and one set of parameters. According to expectation, this model is solely built on the basic mechanical structure of the muscular, contractile machinery. Its four mechanical elements represent the source of work, the serial elasticity, damping due to mechanical deformation, and damping due to the biochemical ATP hydrolysis in the energy conversion process. For explaining all mentioned non-steady-state and steady-state characteristics at once, the model requires, at maximum, ten parameters of which only three parameters representing damping properties plus one representing muscle-internal steady-state kinematics were free to be chosen. All other parameters were already fixed by literature knowledge of the geometrical structure and force characteristics of one cross-bridge. Amongst other results, we found that (i) the most reduced variant of the model is mathematically equivalent to a former version and (ii) the curvature parameter of the Hill relation can be interpreted as the ratio of strengths of the two modelled damping processes. This model approach not only unifies microscopic and macroscopic experimental findings, but further allows to interpret findings of molecular damping and elasticity and scaling of muscle properties, as discussed in this article.

Experience With the Wearable Cardioverter-Defibrillator in Patients at High Risk for Sudden Cardiac Death.

BACKGROUND: This study evaluated the wearable cardioverter-defibrillator (WCD) for use and effectiveness in preventing sudden death caused by ventricular tachyarrhythmia or fibrillation. METHODS: From April 2010 through October 2013, 6043 German WCD patients (median age, 57 years; male, 78.5%) were recruited from 404 German centers. Deidentified German patient data were used for a retrospective, nonrandomized analysis. RESULTS: Ninety-four patients (1.6%) were treated by the WCD in response to ventricular tachyarrhythmia/fibrillation. The incidence rate was 8.4 (95% confidence interval, 6.8-10.2) per 100 patient-years. Patients with implantable cardioverter-defibrillator explantation had an incidence rate of 19.3 (95% confidence interval, 12.2-29.0) per 100 patient-years. In contrast, an incidence rate of 8.2 (95% confidence interval, 6.4-10.3) was observed in the remaining cardiac diagnosis groups, including dilated cardiomyopathy, myocarditis, and ischemic and nonischemic cardiomyopathies. Among 120 shocked patients, 112 (93%) survived 24 hours after treatment, whereas asystole was observed in 2 patients (0.03%) with 1 resulting death. CONCLUSIONS: This large cohort represents the first nationwide evaluation of WCD use in patients outside the US healthcare system and confirms the overall value of the WCD in German treatment pathways.

A 3D musculoskeletal model of the western lowland gorilla hind limb: moment arms and torque of the hip, knee and ankle.

Three-dimensional musculoskeletal models have become increasingly common for investigating muscle moment arms in studies of vertebrate locomotion. In this study we present the first musculoskeletal model of a western lowland gorilla hind limb. Moment arms of individual muscles around the hip, knee and ankle were compared with previously published data derived from the experimental tendon travel method. Considerable differences were found which we attribute to the different methodologies in this specific case. In this instance, we argue that our 3D model provides more accurate and reliable moment arm data than previously published data on the gorilla because our model incorporates more detailed consideration of the 3D geometry of muscles and the geometric constraints that exist on their lines-of-action about limb joints. Our new data have led us to revaluate the previous conclusion that muscle moment arms in the gorilla hind limb are optimised for locomotion with crouched or flexed limb postures. Furthermore, we found that bipedalism and terrestrial quadrupedalism coincided more regularly with higher moment arms and torque around the hip, knee and ankle than did vertical climbing. This indicates that the ability of a gorilla to walk bipedally is not restricted by musculoskeletal adaptations for quadrupedalism and vertical climbing, at least in terms of moment arms and torque about hind limb joints.

The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids.

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.