Skeletal muscle transcriptomics dissects the pathogenesis of Friedreich's ataxia.

OBJECTIVE: In Friedreich's ataxia (FRDA), the most affected tissues are not accessible to sampling and available transcriptomic findings originate from blood-derived cells and animal models. Herein, we aimed at dissecting for the first time the pathophysiology of FRDA by means of RNA-sequencing in an affected tissue sampled in vivo. METHODS: Skeletal muscle biopsies were collected from seven FRDA patients before and after treatment with recombinant human Erythropoietin (rhuEPO) within a clinical trial. Total RNA extraction, 3'-mRNA library preparation and sequencing were performed according to standard procedures. We tested for differential gene expression with DESeq2 and performed gene set enrichment analysis with respect to control subjects. RESULTS: FRDA transcriptomes showed 1873 genes differentially expressed from controls. Two main signatures emerged: (1) a global downregulation of the mitochondrial transcriptome as well as of ribosome/translational machinery and (2) an upregulation of genes related to transcription and chromatin regulation, especially of repressor terms. Downregulation of the mitochondrial transcriptome was more profound than previously shown in other cellular systems. Furthermore, we observed in FRDA patients a marked upregulation of leptin, the master regulator of energy homeostasis. RhuEPO treatment further enhanced leptin expression. INTERPRETATION: Our findings reflect a double hit in the pathophysiology of FRDA: a transcriptional/translational issue and a profound mitochondrial failure downstream. Leptin upregulation in the skeletal muscle in FRDA may represent a compensatory mechanism of mitochondrial dysfunction, which is amenable to pharmacological boosting. Skeletal muscle transcriptomics is a valuable biomarker to monitor therapeutic interventions in FRDA.

Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies.

Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli.

Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.

Mobile phone induced EMF stress is reversed upon the use of protective devices: results from two experiments testing different boundary conditions.

This work examines (a) the impact of electromagnetic fields (EMF) on heart rate variability (HRV), saliva cortisol, arterial blood oxygenation, and tympanic temperature, and (b) the potential effect of protective devices developed to counter EMF-induced stress. In a pilot study, recordings were taken during a 15-min mobile phone call emitting a high burden of EMF (electric, magnetic, high frequency) after a baseline measurement at rest with very low EMF. In a second visit, this was repeated with participants using three protective devices (insoles, pendant, mobile phone chip). In the main study, four experimental arms were employed, two of which replicated the experimental setup of the pilot study, and two of which examined the effect of only one mobile phone chip in an open-hidden-paradigm. In both experiments, exposure to EMF decreased HRV and increased salivary cortisol. In the protective experimental condition, HRV increased above and cortisol decreased below the level of the baseline measures. All differences were large and specific and not modulated by non-specific effects like placebo effects.

Efficient In Silico Saturation Mutagenesis of a Member of the Caspase Protease Family.

Rational-design methods have proven to be a valuable toolkit in the field of protein design. Numerical approaches such as free-energy calculations or QM/MM methods are fit to widen the understanding of a protein-sequence space but require large amounts of computational time and power. Here, we apply an efficient method for free-energy calculations that combines the one-step perturbation (OSP) with the third-power-fitting (TPF) approach. It is fit to calculate full free energies of binding from three different end states only. The nonpolar contribution to the free energies are calculated for a set of chosen amino acids from a single simulation of a judiciously chosen reference state. The electrostatic contributions, on the other hand, are predicted from simulations of the neutral and charged end states of the individual amino acids. We used this method to perform in silico saturation mutagenesis of two sites in human Caspase-2. We calculated relative binding free energies toward two different substrates that differ in their P1' site and in their affinity toward the unmutated protease. Although being approximate, our approach showed very good agreement upon validation against experimental data. 76% of the predicted relative free energies of amino acid mutations was found to be true positives or true negatives. We observed that this method is fit to discriminate amino acid mutations because the rate of false negatives is very low (<1.5%). The approach works better for a substrate with medium/low affinity with a Matthews correlation coefficient (MCC) of 0.63, whereas for a substrate with very low affinity, the MCC was 0.38. In all cases, the combined TPF + OSP approach outperformed the linear interaction energy method.

The increase in urinary serotonin and decrease in salivary cortisol concentrations following direct inhalations of concentrated essential oils is not induced by non-specific effects.

Objectives. The effectiveness of exogenously triggered serotonin (e.g., dietary supplements, drugs) increase is varied. However, since urinary serotonin concentrations were found to correlate with those in the cerebrospinal fluid, the olfactory system might be an efficient and testable pathway to quickly elevate serotonin levels due to its fast-acting central neurophysiological and peripheral pathways. However, little research has been devoted to investigate this assumption. This paper extends previous findings of parasympathetic activation of a specially designed essential oil inhaler (AromaStick® Balance) by experimentally testing its impact on urine serotonin and saliva cortisol excretion. Method. Two experiments involving healthy individuals were conducted to test the efficacy of essential oil application to the nose by employing different inhalation protocols and control conditions. Results. In the pilot study (n=8), serotonin urine excretion was increased after six inhalations (effect size Cohen's d=0.7). In the second experiment (n=80), inhalations proved superior to both the natural control condition and the pseudo placebo condition after three and six inhalation cycles (0.6

Enhancing the promiscuity of a member of the Caspase protease family by rational design.

The N-terminal cleavage of fusion tags to restore the native N-terminus of recombinant proteins is a challenging task and up to today, protocols need to be optimized for different proteins individually. Within this work, we present a novel protease that was designed in-silico to yield enhanced promiscuity toward different N-terminal amino acids. Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease. These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations. The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results. Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids. We believe that the created mutants constitute an important step toward generalized procedures to restore original N-termini of recombinant fusion proteins.

KinCon: Cell-based recording of full-length kinase conformations.

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation-oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient-mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho-transfer. Such non-catalytic kinase functions are related to transitions of full-length kinase conformations. Recent findings underline that cell-based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live-cell recording of kinase states. KinCon is a genetically encoded bioluminescence-based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full-length kinase-targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

Essential oil inhaler (AromaStick®) improves heat tolerance in the Hot Immersion Test (HIT). Results from two randomized, controlled experiments.

BACKGROUND: A recent review article on an aromatherapeutic inhaler demonstrated clinical effects on a number of bodily systems, like the cardiovascular system, the respiratory system, the nervous system and the endocrine system. OBJECTIVE: This paper extends these findings and investigates whether specially designed essential oils inhalers are capable to counter experimentally induced stressful heat sensations. METHOD: Two prospective, randomized, controlled experiments using the Hot Immersion Test Paradigm (HIT) were conducted to investigate whether deep odor inhalations increase heat tolerance. RESULTS: In both experiments, the inhaler strongly prolonged pain tolerance and increased blood oxygenation (1 < d < 1.3). In the second experiment, the inhaler also increased heart rate variability (d = 1.3) as a mechanism to cope with heat stress. CONCLUSION: The ability to resist a stressful thermal stimulus can be exogenously improved by short and deep inhalations of essential scents directly delivered to the olfactory system.

Opportunities in Interventional and Diagnostic Imaging by Using High-Performance Low-Field-Strength MRI.

Background Commercial low-field-strength MRI systems are generally not equipped with state-of-the-art MRI hardware, and are not suitable for demanding imaging techniques. An MRI system was developed that combines low field strength (0.55 T) with high-performance imaging technology. Purpose To evaluate applications of a high-performance low-field-strength MRI system, specifically MRI-guided cardiovascular catheterizations with metallic devices, diagnostic imaging in high-susceptibility regions, and efficient image acquisition strategies. Materials and Methods A commercial 1.5-T MRI system was modified to operate at 0.55 T while maintaining high-performance hardware, shielded gradients (45 mT/m; 200 T/m/sec), and advanced imaging methods. MRI was performed between January 2018 and April 2019. T1, T2, and T2* were measured at 0.55 T; relaxivity of exogenous contrast agents was measured; and clinical applications advantageous at low field were evaluated. Results There were 83 0.55-T MRI examinations performed in study participants (45 women; mean age, 34 years ± 13). On average, T1 was 32% shorter, T2 was 26% longer, and T2* was 40% longer at 0.55 T compared with 1.5 T. Nine metallic interventional devices were found to be intrinsically safe at 0.55 T (<1°C heating) and MRI-guided right heart catheterization was performed in seven study participants with commercial metallic guidewires. Compared with 1.5 T, reduced image distortion was shown in lungs, upper airway, cranial sinuses, and intestines because of improved field homogeneity. Oxygen inhalation generated lung signal enhancement of 19% ± 11 (standard deviation) at 0.55 T compared with 7.6% ± 6.3 at 1.5 T (P = .02; five participants) because of the increased T1 relaxivity of oxygen (4.7e-4 mmHg-1sec-1). Efficient spiral image acquisitions were amenable to low field strength and generated increased signal-to-noise ratio compared with Cartesian acquisitions (P < .02). Representative imaging of the brain, spine, abdomen, and heart generated good image quality with this system. Conclusion This initial study suggests that high-performance low-field-strength MRI offers advantages for MRI-guided catheterizations with metal devices, MRI in high-susceptibility regions, and efficient imaging. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Grist in this issue.

Evaluation of a real-time magnetic resonance imaging-guided electrophysiology system for structural and electrophysiological ventricular tachycardia substrate assessment.

AIMS: Potential advantages of real-time magnetic resonance imaging (MRI)-guided electrophysiology (MR-EP) include contemporaneous three-dimensional substrate assessment at the time of intervention, improved procedural guidance, and ablation lesion assessment. We evaluated a novel real-time MR-EP system to perform endocardial voltage mapping and assessment of delayed conduction in a porcine ischaemia-reperfusion model. METHODS AND RESULTS: Sites of low voltage and slow conduction identified using the system were registered and compared to regions of late gadolinium enhancement (LGE) on MRI. The Sorensen-Dice similarity coefficient (DSC) between LGE scar maps and voltage maps was computed on a nodal basis. A total of 445 electrograms were recorded in sinus rhythm (range: 30-186) using the MR-EP system including 138 electrograms from LGE regions. Pacing captured at 103 sites; 47 (45.6%) sites had a stimulus-to-QRS (S-QRS) delay of ≥40 ms. Using conventional (0.5-1.5 mV) bipolar voltage thresholds, the sensitivity and specificity of voltage mapping using the MR-EP system to identify MR-derived LGE was 57% and 96%, respectively. Voltage mapping had a better predictive ability in detecting LGE compared to S-QRS measurements using this system (area under curve: 0.907 vs. 0.840). Using an electrical threshold of 1.5 mV to define abnormal myocardium, the total DSC, scar DSC, and normal myocardium DSC between voltage maps and LGE scar maps was 79.0 ± 6.0%, 35.0 ± 10.1%, and 90.4 ± 8.6%, respectively. CONCLUSION: Low-voltage zones and regions of delayed conduction determined using a real-time MR-EP system are moderately associated with LGE areas identified on MRI.

Medical aromatherapy revisited-Basic mechanisms, critique, and a new development.

OBJECTIVE: According to a series of recent meta-analyses and systematic reviews, aromatherapy has shown to be effective in treating patients with different medical conditions. However, many of the clinical studies are of rather low methodological quality. Moreover, there is much conceptual ambiguity with regard to what aromatherapy actually constitutes. METHOD: In this paper, we discuss the conditions under which aromatherapy is most likely to be of medical value by outlining the workings of the olfactory system and the necessary requirements of odors to be therapeutic. We then introduce an aromatherapeutic inhaler that was tested in a series of studies involving 465 participants. RESULTS: This inhaler (AromaStick®) produced large to very large effects across a variety of physiological target systems (e.g., cardiovascular, endocrine, blood oxygenation, and pain), both short term and long term. DISCUSSION: Inhalation of volatile compounds from essential oils yields almost immediate, large, and clinically relevant effects as long as the scents are delivered highly concentrated from an appropriate device. The changes caused in the body seem side effect-free and can be sustained when inhalation is repeated.

Epicardial electroanatomical mapping, radiofrequency ablation, and lesion imaging in the porcine left ventricle under real-time magnetic resonance imaging guidance-an in vivo feasibility study.

Aims: Magnetic resonance imaging (MRI) is the gold standard for defining myocardial substrate in 3D and can be used to guide ventricular tachycardia ablation. We describe the feasibility of using a prototype magnetic resonance-guided electrophysiology (MR-EP) system in a pre-clinical model to perform real-time MRI-guided epicardial mapping, ablation, and lesion imaging with active catheter tracking. Methods and results: Experiments were performed in vivo in pigs (n = 6) using an MR-EP guidance system research prototype (Siemens Healthcare) with an irrigated ablation catheter (Vision-MR, Imricor) and a dedicated electrophysiology recording system (Advantage-MR, Imricor). Following epicardial access, local activation and voltage maps were acquired, and targeted radiofrequency (RF) ablation lesions were delivered. Ablation lesions were visualized in real time during RF delivery using MR-thermometry and dosimetry. Hyper-acute and acute assessment of ablation lesions was also performed using native T1 mapping and late-gadolinium enhancement (LGE), respectively. High-quality epicardial bipolar electrograms were recorded with a signal-to-noise ratio of greater than 10:1 for a signal of 1.5 mV. During epicardial ablation, localized temperature elevation could be visualized with a maximum temperature rise of 35 °C within 2 mm of the catheter tip relative to remote myocardium. Decreased native T1 times were observed (882 ± 107 ms) in the lesion core 3-5 min after lesion delivery and relative location of lesions matched well to LGE. There was a good correlation between ablation lesion site on the iCMR platform and autopsy. Conclusion: The MR-EP system was able to successfully acquire epicardial voltage and activation maps in swine, deliver, and visualize ablation lesions, demonstrating feasibility for intraprocedural guidance and real-time assessment of ablation injury.

Feasibility of real-time MR thermal dose mapping for predicting radiofrequency ablation outcome in the myocardium in vivo.

BACKGROUND: Clinical treatment of cardiac arrhythmia by radiofrequency ablation (RFA) currently lacks quantitative and precise visualization of lesion formation in the myocardium during the procedure. This study aims at evaluating thermal dose (TD) imaging obtained from real-time magnetic resonance (MR) thermometry on the heart as a relevant indicator of the thermal lesion extent. METHODS: MR temperature mapping based on the Proton Resonance Frequency Shift (PRFS) method was performed at 1.5 T on the heart, with 4 to 5 slices acquired per heartbeat. Respiratory motion was compensated using navigator-based slice tracking. Residual in-plane motion and related magnetic susceptibility artifacts were corrected online. The standard deviation of temperature was measured on healthy volunteers (N = 5) in both ventricles. On animals, the MR-compatible catheter was positioned and visualized in the left ventricle (LV) using a bSSFP pulse sequence with active catheter tracking. Twelve MR-guided RFA were performed on three sheep in vivo at various locations in left ventricle (LV). The dimensions of the thermal lesions measured on thermal dose images, on 3D T1-weighted (T1-w) images acquired immediately after the ablation and at gross pathology were correlated. RESULTS: MR thermometry uncertainty was 1.5 °C on average over more than 96% of the pixels covering the left and right ventricles, on each volunteer. On animals, catheter repositioning in the LV with active slice tracking was successfully performed and each ablation could be monitored in real-time by MR thermometry and thermal dosimetry. Thermal lesion dimensions on TD maps were found to be highly correlated with those observed on post-ablation T1-w images (R = 0.87) that also correlated (R = 0.89) with measurements at gross pathology. CONCLUSIONS: Quantitative TD mapping from real-time rapid CMR thermometry during catheter-based RFA is feasible. It provides a direct assessment of the lesion extent in the myocardium with precision in the range of one millimeter. Real-time MR thermometry and thermal dosimetry may improve safety and efficacy of the RFA procedure by offering a reliable indicator of therapy outcome during the procedure.

The E3 ubiquitin ligase MID1 catalyzes ubiquitination and cleavage of Fu.

SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control.

Automated slice-specific simultaneous z-shim method for reducing B1 inhomogeneity and susceptibility-induced signal loss with parallel transmission at 3T.

PURPOSE: Through-plane susceptibility-induced signal loss in gradient recalled echo (GRE)-based sequences can considerably impair both the clinical diagnosis and functional analysis of certain brain areas. In this work, a fully automated simultaneous z-shim approach is proposed on the basis of parallel transmit (pTX) to reduce those signal dropouts at 3T. THEORY AND METHODS: The approach uses coil-specific time-delayed excitations to impose a z-shim phase. It was extended toward B1 inhomogeneity mitigation and adequate slice-specific signal-dephasing cancellation on the basis of the prevailing B0 and B1 spatial information. Local signal recovery level and image quality preservation were analyzed using multi-slice FLASH experiments in humans and compared to the standard excitation. Spatial blood-oxygen-level-dependent (BOLD) activation coverage was further compared in breath-hold functional MRI. RESULTS: The pTX z-shim approach recovered approximately 47% of brain areas affected by signal loss in standard excitation images across all subjects. At the same time, B1 shading effects could be substantially reduced. In these areas, BOLD activation coverage could be also increased by approximately 57%. CONCLUSION: The proposed fully automated pTX z-shim method enables time-efficient and robust signal recovery in GRE-based sequences on a clinical scanner using two standard whole-body transmit coils.

A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling.

BACKGROUND: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. METHODS: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. RESULTS: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. CONCLUSION: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.

Shaped saturation with inherent radiofrequency-power-efficient trajectory design in parallel transmission.

PURPOSE: A target-pattern-driven (TD) trajectory design is introduced in combination with parallel transmit (pTX) radiofrequency (RF) pulses to provide localized suppression of unwanted signals. The design incorporates target-pattern and B1+ information to adjust denser sampling and coverage in k-space regions where the main pattern information lies. Based on this approach, two-dimensional RF spiral saturation pulses sensitive to RF power limits were applied in vivo for the first time. THEORY AND METHODS: The TD method was compared with two state-of-the-art spiral design methods. Simulations at different spatial fidelities, acceleration factors and anatomical regions were carried out for an eight-channel pTX 3 Tesla (T) coil. Human in vivo experiments were performed on a two-channel pTX 3T scanner saturating shaped patterns in the brain, heart, and thoracic spine. RESULTS: Using the TD trajectory, RF pulse power can be substantially reduced by up to 34% compared with other trajectory designs with the same spatial accuracy. Local and global specific absorption rates are decreased in most cases. CONCLUSION: The TD trajectory design uses available a priori information to enhance RF power efficiency and spatial response of the RF pulses. Shaped saturation pulses show improved spatial accuracy and saturation performance. Thus, RF pulses can be designed more efficiently and can be further accelerated.

B0-informed variable density trajectory design for enhanced correction of off-resonance effects in parallel transmission.

PURPOSE: To improve B1 and B0 inhomogeneity mitigation performance of spatially selective radio-frequency (RF) pulses in parallel transmission while decreasing RF pulse power. Further enhancement of off-resonance correction for rectilinear spoke-trajectory-based RF pulses with known residual geometric distortions after optimization. METHODS: The appropriate definition of the target magnetization pattern is discussed regarding the maximum physical excitation resolution. Furthermore, a novel variable-density trajectory design is introduced, which subsamples accrued B0 phase error elevations in k-space. A simulation study (echo-planar and spiral 2DRF) at different off-resonance levels and pulse acceleration factors was pursued using data from a whole-body 2-channel parallel transmit 3T MRI system. The new trajectory design for echo-planar 2DRF was validated in human in-vivo experiments. RESULTS: Proper target pattern definition can require spatial filtering, such that RF pulse optimization is prevented from lower excitation performance with significant higher RF power level. The new trajectory design proposed can considerably improve off-resonance compensation, while further reducing the RF power, e.g., 43% less RMSE with 79% less RF power for spoke based pulses. CONCLUSION: The proposed methods offer significant improvements of the excitation performance (homogeneity and acceleration), while significantly decreasing the RF power. Furthermore, single-channel transmit RF pulse performance can be similarly improved.