Computational design of orthogonal membrane receptor-effector switches for rewiring signaling pathways.

Membrane receptors regulate numerous intracellular functions. However, the molecular underpinnings remain poorly understood because most receptors initiate multiple signaling pathways through distinct interaction interfaces that are structurally uncharacterized. We present an integrated computational and experimental approach to model and rationally engineer membrane receptor-intracellular protein systems signaling with novel pathway selectivity. We targeted the dopamine D2 receptor (D2), a G-protein-coupled receptor (GPCR), which primarily signals through Gi, but triggers also the Gq and beta-arrestin pathways. Using this approach, we designed orthogonal D2-Gi complexes, which coupled with high specificity and triggered exclusively the Gi-dependent signaling pathway. We also engineered an orthogonal chimeric D2-Gs/i complex that rewired D2 signaling from a Gi-mediated inhibitory into a Gs-dependent activating pathway. Reinterpreting the evolutionary history of GPCRs in light of the designed proteins, we uncovered an unforeseen hierarchical code of GPCR-G-protein coupling selectivity determinants. The results demonstrate that membrane receptor-cytosolic protein systems can be rationally engineered to regulate mammalian cellular functions. The method should prove useful for creating orthogonal molecular switches that redirect signals at the cell surface for cell-engineering applications.

Multicenter phase II trial to investigate safety and efficacy of gemcitabine combined with cetuximab as adjuvant therapy in pancreatic cancer (ATIP).

BACKGROUND: To investigate whether addition of cetuximab to standard adjuvant chemotherapy with gemcitabine improves outcome in pancreatic cancer, specifically whether the rate of disease-free survival (DFS) at 18 months (primary end point) exceeds the previously reported 35% of gemcitabine alone. PATIENTS AND METHODS: Prospective, open-label, multicenter, nonrandomized phase II study in 76 patients with R0- or R1-resected ductal adenocarcinoma of the pancreas included between October 2006 and November 2008. Gemcitabine and cetuximab were administered for 24 weeks. Secondary end points included overall survival (OS) and toxic effect. RESULTS: Seventy-three patients received cetuximab. Median DFS was 10.0 [95% confidence interval (CI) 8.9-13.6] months and the DFS rate at month 18 of 27.1% (16.7%-37.6%) was inferior to 35%. Median OS was 22.4 (18.2-27.9) months. Subgroup analyses revealed a nonsignificant increase in DFS for patients with versus without skin toxic effect ≥ grade 2 (median 14.7 versus 8.3 months, P = 0.073) and wild-type versus mutated K-Ras (median 11.5 versus 9.3 months, P = 0.57). Grade 3/4 toxic effects included neutropenia (11.0%), thrombopenia (7%), skin toxic effect (7%) and allergic reactions (7%). CONCLUSION: Addition of cetuximab to adjuvant gemcitabine does not seem to improve DFS or OS of unstratified pancreatic cancer patients. Trends for improved DFS in patients with wild-type K-Ras and skin toxic effect remain to be confirmed.

An optimized video system for augmented reality in endodontics: a feasibility study.

OBJECTIVES: We propose an augmented reality system for the reliable detection of root canals in video sequences based on a k-nearest neighbor color classification and introduce a simple geometric criterion for teeth. MATERIAL AND METHODS: The new software was implemented using C++, Qt, and the image processing library OpenCV. Teeth are detected in video images to restrict the segmentation of the root canal orifices by using a k-nearest neighbor algorithm. The location of the root canal orifices were determined using Euclidean distance-based image segmentation. A set of 126 human teeth with known and verified locations of the root canal orifices was used for evaluation. RESULTS: The software detects root canals orifices for automatic classification of the teeth in video images and stores location and size of the found structures. Overall 287 of 305 root canals were correctly detected. The overall sensitivity was about 94 %. Classification accuracy for molars ranged from 65.0 to 81.2 % and from 85.7 to 96.7 % for premolars. CONCLUSION: The realized software shows that observations made in anatomical studies can be exploited to automate real-time detection of root canal orifices and tooth classification with a software system. CLINICAL RELEVANCE: Automatic storage of location, size, and orientation of the found structures with this software can be used for future anatomical studies. Thus, statistical tables with canal locations will be derived, which can improve anatomical knowledge of the teeth to alleviate root canal detection in the future. For this purpose the software is freely available at: http://www.dental-imaging.zahnmedizin.uni-mainz.de/.

Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain.

Key cellular functions depend on the transduction of extracellular mechanical signals by specialized membrane receptors including adhesion G-protein coupled receptors (aGPCRs). While recently solved structures support aGPCR activation through shedding of the extracellular GAIN domain, the molecular mechanisms underpinning receptor mechanosensing remain poorly understood. When probed using single-molecule atomic force spectroscopy and molecular simulations, ADGRG1 GAIN dissociated from its tethered agonist at forces significantly higher than other reported signaling mechanoreceptors. Strong mechanical resistance was achieved through specific structural deformations and force propagation pathways under mechanical load. ADGRG1 GAIN variants computationally designed to lock the alpha and beta subdomains and rewire mechanically-induced structural deformations were found to modulate the GPS-Stachel rupture forces. Our study provides unprecedented insights into the molecular underpinnings of GAIN mechanical stability and paves the way for engineering mechanosensors, better understanding aGPCR function, and informing drug-discovery efforts targeting this important receptor class.

Prediction of membrane protein structures with complex topologies using limited constraints.

Reliable structure-prediction methods for membrane proteins are important because the experimental determination of high-resolution membrane protein structures remains very difficult, especially for eukaryotic proteins. However, membrane proteins are typically longer than 200 aa and represent a formidable challenge for structure prediction. We have developed a method for predicting the structures of large membrane proteins by constraining helix-helix packing arrangements at particular positions predicted from sequence or identified by experiments. We tested the method on 12 membrane proteins of diverse topologies and functions with lengths ranging between 190 and 300 residues. Enforcing a single constraint during the folding simulations enriched the population of near-native models for 9 proteins. In 4 of the cases in which the constraint was predicted from the sequence, 1 of the 5 lowest energy models was superimposable within 4 A on the native structure. Near-native structures could also be selected for heme-binding and pore-forming domains from simulations in which pairs of conserved histidine-chelating hemes and one experimentally determined salt bridge were constrained, respectively. These results suggest that models within 4 A of the native structure can be achieved for complex membrane proteins if even limited information on residue-residue interactions can be obtained from protein structure databases or experiments.

[Significance of autopsy in patients with head and neck cancer]. / Stellenwert der Autopsie bei Patienten mit Kopf-Halstumoren.

BACKGROUND: Nowadays, the morphological assessment of samples obtained from living patients has a greater importance than the scientific knowledge which is gained by autopsy. Therefore, the aim of the study was a retrospective analysis of causes of death in patients with head and neck cancer. MATERIAL AND METHODS: The autopsy rate, clinical parameters of oncologic patients as well as autopsy findings like lethal complications, distant metastases and second primary tumors were retrospectively analyzed. RESULTS: From 1968 to 2007 in 91 patients with malignant tumors of the head and neck an autopsy was performed. In these 39 years an autopsy was performed in 45.9% of dead oncologic patients. Autopsy findings revealed distant metastases in 46.2% and second primary tumors in 17.6% of the patients. 49.5% of the patients died from pneumonia, 20.9% from tumor bleeding and 10% from progressive cachexia. CONCLUSION: The study confirms the global trend of a decline in autopsy numbers in the last 3 decades. However, as an important instrument of quality assurance autopsies continue to play an essential and indispensable role in medical research.

CUX1: target of Akt signalling and mediator of resistance to apoptosis in pancreatic cancer.

BACKGROUND AND AIMS: The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer. METHODS: The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues. RESULTS: Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts. CONCLUSION: CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.

[Contrast enhanced sonographic patterns of hepatic candidiasis]. / Kontrastunterstützte sonografische Befunde bei hepatischer Candidiasis.

PURPOSE: B-mode ultrasound (US) of hepatic candidiasis (HC) shows an uncharacteristic pattern. The aim of this study is to display the pattern of HC by performing contrast-enhanced ultrasound (CEUS). PATIENTS AND METHODS: Between May 2006 and June 2008 HC was diagnosed in 12 patients (4 female, 8 male) by clinical and sonographic findings. The underlying diseases were acute leukemia (n = 10), aplastic anemia (n = 1), and testicular cancer (n = 1) in either the state of complete remission (n = 10) or relapse (n = 2). Due to neutropenic fever after chemotherapy all of the patients had received antifungal therapy. When HC was diagnosed all patients were afebrile and in a recovered hematological constitution. Additional diagnostic procedures were histological examination (n = 5), computed tomography (n = 8) and sonographic follow-up examinations (n = 12). All patients were examined with B-mode US and CEUS. RESULTS: In B-mode US the lesions were hypoechoic (n = 12), multiple (n = 10) and > 1 cm (n = 8) as well as > 2 cm (n = 4) in diameter. During CEUS no enhancement of contrast media in the centre of the lesions was seen in all cases during both phases. Additionally, in the arterial phase, the lesions showed no rim enhancement (n = 3) (type I), an isoechoic rim enhancement (n = 5) (type II), or a hyperechoic rim enhancement (n = 4) (type III). During sonographic follow-up a complete regression of the lesions (n = 9) or a stable disease (n = 2) was seen. One patient died due to a relapse. CONCLUSION: The CEUS pattern of HC is variable but characteristic. Therefore, CEUS should be performed in all patients with suspected HC.

Single dose escalation studies with inhaled POL6014, a potent novel selective reversible inhibitor of human neutrophil elastase, in healthy volunteers and subjects with cystic fibrosis.

BACKGROUND: POL6014 is a novel, orally inhaled neutrophil elastase (NE) inhibitor in development for cystic fibrosis (CF). METHODS: Two studies, one in healthy volunteers (HVs, doses 20 to 960 mg) and one in subjects with CF (doses 80 to 320 mg) were conducted to evaluate the safety, tolerability and pharmacokinetics (PK) of single ascending doses of inhaled POL6014 with a Pari eFlow® nebuliser. PK was evaluated over a period of 24 h. In addition, NE activity in CF sputum was measured. RESULTS: After single doses, POL6014 was safe and well tolerated up to 480 mg in HVs and at all doses in subjects with CF. POL6014 showed a dose-linear PK profile in both populations with Cmax between 0.2 and 2.5 µM in HVs and between 0.2 and 0.5 µM in subjects with CF. Tmax was reached at approximately 2-3 h. Mean POL6014 levels in CF sputum rapidly reached 1000 µM and were still above 10 µM at 24 h. >1-log reduction of active NE was observed at 3 h after dosing. CONCLUSION: Inhalation of POL6014 can safely lead to high concentrations within the lung and simultaneously low plasma concentrations, allowing for a clear inhibition of NE in the sputum of subjects with CF after single dosing. TRIAL REGISTRATION: European Medicines Agency EudraCT-Nr. 2015-001618-83 and 2016-000493-38.

["Malignant" ARDS]. / "Malignes" ARDS.

Acute respiratory failure and the "acute respiratory distress syndrome" (ARDS) are frequent medical conditions in critically ill patients. Various causes can potentially result in the development of ARDS. Two cases are presented, in which malignant diseases were identified as causes of the respiratory failure. The first patient was diagnosed with an acute myeloic leukemia M5 (FAB). In the second patient, lung histology revealed an adenocarcinoma of the lung. These case reports show that in addition to the classical causes of ARDS, specific disease entities can mimic this form of respiratory failure. Beside solid cancers and lymphomas, acute and progressive forms of inflammatory, parenchymal lung diseases (such as acute interstitial pneumonitis, acute eosinophilic pneumonia, diffuse alveolar hemorrhagia, and acute hypersensitivity pneumonitis) can manifest with this picture. As a consequence, the diagnostic workup of respiratory failure of unknown cause should include these entities.

Survival in SMA type I: a prospective analysis of 34 consecutive cases.

Thirty-four children with genetically proven SMA type I (age at onset <6 months, unable to sit during study period) were included in a 3-year prospective cohort study and neurologically followed-up until death or the end of the study. At the end of the study period 31/34 children had died. The median age at death was 176 days (95% Confidence Interval 150-214 days), the median survival from the time of diagnosis was 158 days (95% CI 137-232 days). The median survival after diagnosis did not differ significantly between children diagnosed at birth (median survival 137 days, 95% CI 111-232 days) and those diagnosed later (median survival 159 days, 95% CI 141-256), implying that SMA I cases with different ages of onset show the same progression rate of the disease. The number of SMN2 copies was not clearly correlated with survival duration, possibly because of lack of statistical power due to the small number of cases with 1 or 3 SMN2 copies. The three cases alive at the end of the study had either three or an unknown number of SMN2 copies, which is in agreement with previously described cases showing longer survival with increasing number of SMN2 copies. All deceased children died of respiratory insufficiency and/or an intercurrent lung infection, indicating that the susceptibility of the child with SMA type I to respiratory infections plays an important role in determining the survival.

Reprogramming G protein coupled receptor structure and function.

The prominence of G protein-coupled receptors (GPCRs) in human physiology and disease has resulted in their intense study in various fields of research ranging from neuroscience to structural biology. With over 800 members in the human genome and their involvement in a myriad of diseases, GPCRs are the single largest family of drug targets, and an ever-present interest exists in further drug discovery and structural characterization efforts. However, low GPCR expression and stability outside the natural lipid environments have challenged these efforts. In vivo functional studies of GPCR signaling are complicated not only by the need for specific spatiotemporal activation, but also by downstream effector promiscuity. In this review, we summarize the present and emerging GPCR engineering methods that have been employed to overcome the challenges involved in receptor characterization, and to better understand the functional role of these receptors.

Sequential MR imaging changes in nonketotic hyperglycinemia.

Serial diffusion-weighted (DWI) and diffusion tensor imaging (DTI) were performed in a patient with neonatal onset nonketotic hyperglycinemia (NKH). At 3 weeks areas that are normally myelinated at this time showed increased T2-signal intensity and restricted diffusion, consistent with vacuolating myelinopathy. At 3 months, these areas had increased in the topographic pattern of normal myelination, whereas fractional anisotropy was compatible with axonal sparing. At 17 months, diffusion restriction had disappeared, likely because of coalescence of myelin vacuoles. A decrease of fractional anisotropy was observed in the previously myelinated areas indicative of axonal loss. We conclude that DWI and DTI can be used to identify and characterize white matter tract abnormalities in patients with NKH.

The status of neck node metastases in breast cancer--loco-regional or distant?

Metastases to cervical lymph nodes do not exclusively derive from malignancies of the head and neck area. In the literature the region where distant metastases of breast carcinomas to the neck occur is exclusively named "supraclavicular". The system established by head and neck surgeons regarding neck node topography allows interdisciplinary management of patients with cervical lymph node metastases from breast cancer. Twelve patients suffering from breast cancer who presented with cervical masses have been examined. Most lymph node metastases were found in the posterior triangle of the neck and at the caudo-jugular level, but some metastases were even found in the upper jugular levels. The results presented show that neck node metastases of breast cancer are located superiorly to the supraclavicular region in more than 50% of the cases. According to the AJCC Staging System for Breast Cancer metastases located in the supraclavicular fossa are assessed as loco-regional metastases (N3c). Lymph node metastases situated above the supraclavicular region are not mentioned, but should be considered as distant metastases. This important question remains unanswered and deserves clarification in the current classification of the AJCC Staging System for Breast Cancer.

Colour Doppler ultrasound patterns and clinical follow-up of incidentally found hypoechoic, vascular tumours of the spleen: evidence for a benign tumour.

Between January 1990 and January 2005, incidental hypoechoic, vascular tumours of the spleen were identified in 13 patients using B-mode and colour Doppler ultrasound (CDS). All lesions found were well demarcated, intrasplenically located, and ranged in size between 1 cm and 4 cm. The increased vascular pattern on CDS was confirmed in 9 of the 13 cases by contrast enhanced ultrasound (CES), while two patients showed reduced vascularity on CES. In 10 patients, lesions were confirmed by contrast enhanced CT. Histological examination was performed in three patients with the diagnosis of capillary haemangioma (n = 2) and hamartoma (n = 1). In the remaining cases, ultrasound follow-up was performed (range 4 months to 13 years) and demonstrated no evidence of tumour growth in all but one patient. During a 4 year follow-up, one lesion increased in size from 1.0 cm to 1.5 cm and in the same patient an additional 0.5 cm sized hypoechoic increased vascular lesion was also found. In the spleen a hypoechoic lesion with an increased vascular pattern incidentally found by ultrasound most likely indicates a benign tumour with capillary haemangioma/hamartoma as the most likely diagnosis. However, it should be emphasised that in all cases a careful ultrasound follow-up is warranted.

POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker-Warburg syndrome.

BACKGROUND: Walker-Warburg syndrome (WWS) is an autosomal recessive condition characterised by congenital muscular dystrophy, structural brain defects, and eye malformations. Typical brain abnormalities are hydrocephalus, lissencephaly, agenesis of the corpus callosum, fusion of the hemispheres, cerebellar hypoplasia, and neuronal overmigration, which causes a cobblestone cortex. Ocular abnormalities include cataract, microphthalmia, buphthalmos, and Peters anomaly. WWS patients show defective O-glycosylation of alpha-dystroglycan (alpha-DG), which plays a key role in bridging the cytoskeleton of muscle and CNS cells with extracellular matrix proteins, important for muscle integrity and neuronal migration. In 20% of the WWS patients, hypoglycosylation results from mutations in either the protein O-mannosyltransferase 1 (POMT1), fukutin, or fukutin related protein (FKRP) genes. The other genes for this highly heterogeneous disorder remain to be identified. OBJECTIVE: To look for mutations in POMT2 as a cause of WWS, as both POMT1 and POMT2 are required to achieve protein O-mannosyltransferase activity. METHODS: A candidate gene approach combined with homozygosity mapping. RESULTS: Homozygosity was found for the POMT2 locus at 14q24.3 in four of 11 consanguineous WWS families. Homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Immunohistochemistry in muscle showed severely reduced levels of glycosylated alpha-DG, which is consistent with the postulated role for POMT2 in the O-mannosylation pathway. CONCLUSIONS: A fourth causative gene for WWS was uncovered. These genes account for approximately one third of the WWS cases. Several more genes are anticipated, which are likely to play a role in glycosylation of alpha-DG.

A 'specificity' pocket inferred from the crystal structures of the complexes of aldose reductase with the pharmaceutically important inhibitors tolrestat and sorbinil.

BACKGROUND: Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket. RESULTS: The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase. CONCLUSIONS: The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

Frequent abnormalities of the putative tumor suppressor gene FHIT at 3p14.2 in pancreatic carcinoma cell lines.

The FHIT gene is localized on chromosome 3p14, a region including a tumor cell-specific, commonly deleted region. To determine the role of the FHIT gene in pancreatic carcinogenesis, 14 pancreatic carcinoma cell lines were analyzed by reverse transcription-PCR and exon-specific PCR amplification of genomic DNA. The full-length FHIT transcript was lost in 70% of the pancreatic carcinoma cell lines analyzed, while 66% also revealed intragenic homozygous deletions of exons 3, 4, and 5. Truncated FHIT transcripts lacking a variable number of exons most likely represented alternative splicing products. Fhit protein expression was dependent on a full-length FHIT transcript. The results suggest that the FHIT gene may be a target tumor suppressor gene involved in pancreatic carcinogenesis.