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.

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.

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.

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.

Multifocal sclerosing angiomatoid nodular transformation of the spleen in a patient with simultaneous metachronous liver metastasis after colon cancer surgery: a first case report.

Sclerosing angiomatoid nodular transformation of the spleen (SANT) is a benign, extremely rare vascular lesion of the spleen with unknown pathogenesis. SANT is often discovered incidentally, and can sometimes be found in patients with a history of cancer. Based on absent definitive radiological signs and varying growth patterns, distinction from malignant processes such as metastasis can be very difficult. Therefore, surgical resection of the spleen is indicated in most cases of patients with history of cancer. We report a case of a bifocal manifestation of SANT in the spleen in a patient with history of colon cancer and newly-diagnosed metachronous liver metastases.

Evolutionary-guided de novo structure prediction of self-associated transmembrane helical proteins with near-atomic accuracy.

How specific protein associations regulate the function of membrane receptors remains poorly understood. Conformational flexibility currently hinders the structure determination of several classes of membrane receptors and associated oligomers. Here we develop EFDOCK-TM, a general method to predict self-associated transmembrane protein helical (TMH) structures from sequence guided by co-evolutionary information. We show that accurate intermolecular contacts can be identified using a combination of protein sequence covariation and TMH binding surfaces predicted from sequence. When applied to diverse TMH oligomers, including receptors characterized in multiple conformational and functional states, the method reaches unprecedented near-atomic accuracy for most targets. Blind predictions of structurally uncharacterized receptor tyrosine kinase TMH oligomers provide a plausible hypothesis on the molecular mechanisms of disease-associated point mutations and binding surfaces for the rational design of selective inhibitors. The method sets the stage for uncovering novel determinants of molecular recognition and signalling in single-spanning eukaryotic membrane receptors.

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/.

[Surgical management of a retrorectal tumor with consideration of a rare differential diagnosis]. / Management eines retrorektalen Tumors unter Berücksichtigung einer seltenen Differenzialdiagnose.

We present a case of a retrorectal space occupying lesion diagnosed as an adenocarcinoma of unknown primary origin by preoperative histopathology. Localization slightly above the anal sphincter would have required extirpation of the rectum. Rectal palpation, endosonography and radiological imaging, however, suggested a retrorectal tumor or a metastasis of an adenocarcinoma. Both entities would have required local resection. We applied a surgical algorithm including frozen biopsy allowing a stepwise choice of operative procedure from the spectrum in question. The operation performed was thus tailored to the entity of the tumor.

[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.

[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.

Analysis of clinically suspected orbital cavernomas.

BACKGROUND: Orbital cavernomas are low-flow vascular malformations that are the most common benign neoplasms of the orbit in adults, typically becoming symptomatic in the middle age. METHODS: The medical records of six patients with clinically suspected orbital cavernomas receiving elective surgical excision were analysed concerning symptoms, physical findings, treatment results and visual outcome. The pathologic slides were evaluated, and additional immunohistochemical stains were done if necessary to obtain diagnosis. RESULTS: Histologic evaluation revealed three of six cases not being cavernomas, although the clinical and macroscopic findings were consistent with orbital cavernomas. Two of them were haemorrhagic lymphangiomas, and one was a solitary fibrous tumour. CONCLUSIONS: Haemorrhagic lymphangiomas and other vascular tumours may mimic orbital cavernomas regarding anamnesis, radiologic and intraoperative findings and gross examination. Therefore, exact histologic evaluation is necessary to get the correct diagnosis.

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.

Clear cell follicular adenoma of the thyroid--a challenge in intra-operative diagnostics.

Pure clear cell lesions of the thyroid gland are rare and prone to pose severe diagnostic challenges. We report the case of a 61-year-old man with a clear cell adenoma of the thyroid. The patient presented with a hypoechoic thyroid nodule. Fine needle aspiration cytology rendered the primary diagnosis of a follicular neoplasia, and right thyroid lobectomy was performed. By intra-operative frozen section, the diagnosis given was clear cell lesion of unknown malignant potential. Based on the light microscopic findings and the immunohistochemical profile, the lesion was diagnosed as clear cell follicular adenoma of the thyroid. A follicular thyroid lesion presenting with clear cell changes in fine needle aspiration cytology or intra-operative frozen section consultation constitutes a diagnostic challenge to every surgical pathologist. As immunohistochemistry of cytologic specimens is hampered by several methodological problems, any thyroid lesion with clear cell features warrants further histologic assessment to render the correct diagnosis.

Prospective study on neck dissection after primary chemoradiation therapy in stage IV pharyngeal cancer.

BACKGROUND: Definitive chemoradiation is a well-established option in the treatment of locally advanced squamous cell carcinoma of the head and neck. The intention of this study was to evaluate its efficacy on cervical lymph node metastases in a prospective study after a standardized protocol for chemoradiation (CRT) and histopathological evaluation, respectively. PATIENTS AND METHODS: The data of 25 patients (10 oropharynx, 15 hypopharynx) who received planned neck dissection after definitive chemoradiation for UICC stage IV carcinomas of the pharynx were analyzed. All patients were sonomorphologically staged positive for lymph nodes (3 patients: N1; 2 patients N2a; 7 patients N2b; 9 patients N2c and 4 patients N3). A neck dissection was carried out 8.9+/-1.5 weeks (range 6-13) post treatment. The specimens obtained from the different neck levels were histologically examined for viable tumour cells. RESULTS: Local control was achieved in 100% of all patients on endoscopy 9 weeks after the chemoradiation. In 14/25 patients (56%), still viable tumour tissue was found in the neck dissection (ND) specimen. Only one of these 14 patients (7%) was deemed suspicious for residual lymphadenopathy from clinical and diagnostic findings at re-staging after chemoradiation, the others were staged yN0. Postsurgical complications occurred in six patients (24%) such as bleeding and prolonged wound healing in one patient each and functional deficits in an additional four patients. One patient developed a scar recurrence seven months after surgery. CONCLUSION: Based on these findings, the ultimate efficacy of primary CRT should not be judged 8-10 weeks after the treatment. Therefore planned neck dissection should be performed no earlier than 12 weeks after primary CRT.

["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.