Identifikation von Interaktionspartnern des humanen DNA-Reparaturproteins MLH1 mit einem bakteriellen Zweihybrid-System

Ein erblich bedingter Funktionsverlust der DNA Mismatch Reparatur (MMR) Proteine hMSH2 und hMLH1 führt zu genetischer Instabilität und frühzeitiger Ausbildung von Krebserkrankungen, insbesonders des Kolons und des Endometriums. Diese Erkrankung wird nach ihrem Erstbeschreiber als Lynch Syndrom oder im Falle eines kolorektalen Karzinoms auch als erbliches nichtpolypöses kolorektales Karzinom (hereditary non polyposis colorectal cancer, HNPCC) bezeichnet. Neben der DNA Reparatur sind MMR Proteine auch an weiteren regulatorischen Prozessen wie genetischer Rekombination, Antikörperbildung sowie Zellzyklus Regulation und Apoptose beteiligt. Aus onkologischer Sicht ist besonders der letzte Punkt von Interesse, da man davon ausgeht, dass MMR Proteine bei exzessiven DNA-Schäden den Zellzyklus anhalten und entweder die DNA reparieren oder die Apoptose einleiten. Dies ist von klinischer Bedeutung, da viele Chemotherapeutika künstliche DNA-Läsionen hervorrufen, die in MMR defizienten Zellen weder zu einem Zellzyklus Stop noch zur Apoptose führen, so dass die Wirksamkeit bestimmter Substanzen vermindert ist. Aus diesem Grund wurde im Rahmen der vorgelegten Dissertation ein Screening zur Identifikation von Interaktionspartnern von hMLH1 durchgeführt. hMLH1 ist hauptsächlich im Zellkern lokalisiert, über Interaktionspartner im Zytoplasma ist bisher wenig bekannt. Im Rahmen dieser Arbeit wurde ein neues bakterielles Zweihybrid-System etabliert. In diesem System besitzen die Wirtszellen keinen Zellkern, so dass die detektierten Interaktionen unabhängig von der intrazellulären Lokalisation stattfinden. Die Klone einer cDNA-Datenbank wurden mit hMLH1 in Bakterienzellen koexprimiert und die Interaktionen durch positives Zellwachstum auf Minimalnährböden angezeigt. Die positiven Klone wurden anschließend sequenziert und die Sequenz mit Hilfe der BLAST-Funktion mit der PubMed Online-Datenbank abgeglichen. Insgesamt wurden 108 bisher unbekannte Interaktionspartner von hMLH1 detektiert. Von diesen konnten zahlreiche Proteine den bekannten Funktionen der MMR zugeordnet werden. Von besonderem Interesse sind einige Interaktionspartner aus den Bereichen Zellzyklus Regulation (ANXA6, LPP2, NDRG1, PTP4A2/RH, RAPGEFL1, SPTLC2 und ARAF1), Apoptose (CTSB, CAPN5, DDX47, DES und Atmungskettenproteine) und ribosomale Proteine (EIF2A, EEF2, RPS2, RPS5 und RPS16). Weitere Untersuchungen müssen zeigen, welche dieser Interaktionspartner sich möglicherweise als prognostische bzw. prädiktive Biomarker nutzen lassen. Weiterhin könnten sich durch fundierte Kenntnis der Interaktion von MMR und Zellzyklus Regulation bzw. Apoptose neue Ansatzpunkte bei der Entwicklung zukünftiger therapeutischer Substanzen ergeben.Hereditary functional impairment of the DNA Mismatch Repair (MMR) proteins hMSH2 and hMLH1 lead to genetic instability and early onset of various malignancies, especially of colon and endometrium. This hereditary cancer syndrome has been firstly described by H.T. Lynch and is thereby called “Lynch Syndrome” or in case of colon carcinoma “Hereditary nonpolyposis colorectal cancer (HNPCC)”. Additionally to DNA repair, MMR proteins have been shown to be involved in other critical cellular processes such as genetic recombination, antibody generation, cell-cycle regulation and apoptosis. Especially those last two functions are of vital interest in the field of oncology, as MMR proteins are supposed to be key players in the recognition of DNA lesions and the initiation of cell-cycle arrest and/or apoptosis in case of excessive DNA damage. As many chemotherapeutic agents are causing artificial DNA lesions, which cannot initiate cell-cycle arrest in MMR deficient cells, this is of vital clinical importance. As hMLH1 is mostly located in the cellular nucleus, lots of information has been gathered about nuclear functions and interaction partners, though there is little known about functions of hMLH1 in the cytoplasm. Therefore, this screening for undiscovered interaction partners of hMLH1 has been performed by a new established bacterial two-hybrid-system. Here, host cells are prokaryotes and potential interaction partners can thereby interact quite independent of their original cellular compartment. The clones of a normal-colon tissue databank were coexpressed with hMLH1 in bacterial host cells and their interaction has been visualized by growth on minimal plates with different selection markers. Positive clones were sequenced and the encoded proteins identified by the PubMed BLAST online tool. 108 clones have been identified as new interaction partners of hMLH1 and most of them could be attributed two known functions of hMLH1. Here, proteins involved in cell-cycle control (ANXA6, LPP2, NDRG1, PTP4A2/RH, RAPGEFL1, SPTLC2 and ARAF1), apoptosis (CTSB, CAPN5, DDX47, DES and mitochondrial proteins) and ribosomal proteins (EIF2A, EEF2, RPS2, RPS5 und RPS16) are of special interest. Further experiments must show, which of these newly identified interaction partners of hMLH1 can possibly be used as future prognostical or predictive biomarkers. Additionally, new insights into the interaction mechanisms of MMR and cell-cycle/apoptosis could lead to new pharmacological approaches in the future

Prognostic assessment in patients operated for brain metastasis from systemic tumors

Background: Established models for prognostic assessment in patients with brain metastasis do not stratify for prior surgery. Here we tested the prognostic accuracy of the Graded Prognostic Assessment (GPA) score model in patients operated for BM and explored further prognostic factors. Methods: We included 285 patients operated for brain metastasis at the University Hospital Zurich in the analysis. Information on patient characteristics, imaging, staging, peri- and postoperative complications and survival were extracted from the files and integrated into a multivariate Cox hazard model. Results: The GPA score showed an association with outcome. We further identified residual tumor after surgery (p = 0.007, hazard ratio (HR) 1.6, 95% confidence interval (CI) 1.1-2.3) steroid use (p = 0.021, HR 1.7, 95% CI 1.1-2.6) and number of extracranial metastasis sites (p = 0.009, HR 1.4, 95% CI 1.1-1.6) at the time of surgery as independent prognostic factors. A trend was observed for postoperative infection of the subarachnoid space (p = 0.102, HR 3.5, 95% CI 0.8-15.7). Conclusions: We confirm the prognostic capacity of the GPA score in a cohort of operated patients with brain metastasis. However, extent of resection and steroid use provide additional aid for the prognostic assessment in these patients

Case Report Funicular Myelosis in a Butcher: It Was the Cream Cans

Background. Funicular myelosis is a known consequence of exposure to nitrous oxide. Nevertheless, there are only a few clinical trials assessing its long-term effects and there is no literature about the role of nutritional vitamin B12 supplementation in the context of nitrous oxide abuse. Case Descriptions. We diagnosed funicular myelosis in a young butcher, who consumed high amounts of meat regularly. Since the diagnostic process did not reveal any metabolic causes, reinterrogation of the patient uncovered recreational abuse of nitrous oxide out of whipped cream can gas cartridges. After stopping abuse and supplementation of vitamin B12, the patient recovered almost completely. Conclusions. In our case, even high nutritional vitamin B12 uptake could not compensate the noxious effects of nitrous oxide. Since there are emerging reports of increasing misuse, this should be considered in the diagnostic and therapeutic care of patients with nitrous oxide abuse. Furthermore, our case emphasizes that patients with vitamin B12 deficiency should be assessed for nitrous oxide abuse. Case Presentation The 27-year-old patient, a butcher, presented with a 4-week history of ascending symmetric numbness in the limbs, as well as tingling in the feet and fingers. He felt clumsy while writing and unsecure during walking, with report of several dropping events. The neurological examination revealed a diminished position and vibration sense and hypalgesia and hypaesthesia of the upper and lower limbs, compatible with distal symmetric polyneuropathy. However, the Romberg test was negative and the remainder of the examination was normal; in particular, there were no weaknesses or neuropsychological symptoms. The MRI of the brain was normal. The spinal MRI showed T2-hyperintense lesions in the dorsal columns of the cervical spine (C1-C6) without contrast enhancement as typical in patients with funicular myelosis [1] Discussion Vitamin B12 deficiency, subsequent hyperhomocysteinaemia, and funicular myelosis have been observed in patients after exposure to nitrous oxide, and the underlying biochemical pathomechanism has been revealed. It indicates an irreversible inhibition of the active cobalt centre of vitamin B12, leading to decreased activity of 5-methyltetrahydrofolatehomocysteine methyltransferase (MTR), a vitamin B12 dependent enzyme converting homocysteine to methionine In addition to several case reports, a small study revealed that long-term nitrous oxide exposure in operating theatres may lead to decreased vitamin B12 serum levels Besides this abuse of nitrous oxide and the resulting degenerative effects (myelopathy and peripheral neuropathy), Cousaert et al. We consider this report to be worth publishing, as we found a unique constellation, where even high nutritional Case Reports in Neurological Medicine 3 vitamin B12 uptake with a diet rich in meat was not able to compensate the noxious effects of N 2 O. To our knowledge, there are no controlled studies available that concern the influence of nutritional supply of vitamin B12; therefore, the present case suggests that alimentary increased vitamin B12 uptake may not prevent onset of funicular myelosis upon nitrous oxide exposure. This may be of clinical importance when assessing risks for patients with abuse of or other reasons of long-term exposure to nitrous oxide. Furthermore, the case emphasizes that patients (and their next kin) should be asked for nitrous oxide exposure or abuse in cases of vitamin B12 deficiency of unknown origin. Consent The authors have received permission to cite any personal communications. Disclosure All data were obtained in the course of the diagnostic process in the mentioned institutions. No financial disclosure was reported by any of the authors. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Authors' Contribution Fabian Wolpert and Roland Renzel were in charge for the diagnostic and therapeutic care during the hospitalization. Krisztina Baráth provided MRI diagnostics and figures. Andreas R. Gantenbein was the clinical supervisor during the hospitalization phase and is the senior author who reviewed the paper during the writing process. Michael Linnebank was providing fundamental and clinical advice during the diagnostic process and while reviewing the paper. Janis Brakowski contributed his expertise as experienced psychiatric clinician through reviewing the paper

Postoperative progression of brain metastasis is associated with seizures

Seizures in patients with brain metastases have an impact on morbidity and quality of life. The influence of tumor growth on the risk of seizures in these patients is not well defined. In this cohort study, we evaluated adult patients from the University Hospital of Zurich following resection of brain metastases from solid tumors, with or without preoperative seizures, at 3, 6, 9, and 12 months postoperatively. Brain magnetic resonance imaging was assessed for tumor progression using the Response Assessment in Neuro-Oncology criteria. The quarterly risk of unprovoked seizures was modeled with mixed effects logistic regression. We analyzed 444 time frames in 220 patients. Progression of brain metastases was independently associated with seizures during the respective quarterly follow-up period (odds ratio = 3.9, 95% confidence interval = 1.3-11.3, p = .014). Complete resection of brain metastases was associated with a lower risk of seizures (odds ratio = .2, 95% confidence interval = .04-.7, p = .015). Postoperative progression of brain metastases quadrupled the risk of seizures; therefore, vigorous follow-up may be useful to identify tumor progression and gauge the risk of seizures. The identification of patients at high seizure risk may have implications for treatment decisions and influence aspects of daily life. Breakthrough seizures may indicate brain metastases progression

Algorithm Portfolios for Noisy Optimization: Compare Solvers Early

International audienceNoisy optimization is the optimization of objective functions corrupted by noise. A portfolio of algorithms is a set of algorithms equipped with an algorithm selection tool for distributing the compu- tational power among them. We study portfolios of noisy optimization solvers, show that different settings lead to dramatically different perfor- mances, obtain mathematically proved adaptivity by an ad hoc selection algorithm dedicated to noisy optimization. A somehow surprising result is that it is better to compare solvers with some lag; i.e., recommend the current recommendation of the best solver, selected from a comparison based on their recommendations earlier in the run

Response assessment and outcome of combining immunotherapy and radiosurgery for brain metastasis from malignant melanoma

Background The optimal sequence of stereotactic radiotherapy (SRT) and immune checkpoint inhibition (ICI) and assessment of response in patients with brain metastases from melanoma remain challenging. Methods We reviewed clinical and neuroimaging data of 62 patients with melanoma, including 26 patients with BRAF-mutant tumours, with newly diagnosed brain metastases treated with ICI alone (n=10, group 1), SRT alone or in combination with other systemic therapies (n=20, group 2) or ICI plus SRT (n=32, group 3). Response was assessed retrospectively using response evaluation criteria in solid tumours (RECIST) V.1.1, response assessment in neuro-oncology (RANO) and immunotherapy RANO (iRANO) criteria. MRI follow-up from 43 patients was available for central review. Results Patients treated with ICI alone showed no objective responses and had worse outcome than patients treated with SRT without or with ICI. RECIST, RANO and iRANO criteria were concordant for complete response (CR) and partial response (PR). RANO called progression earlier than RECIST for clinical deterioration without MRI progression in some patients. Progression was called later when using iRANO criteria because of the need for a confirmatory scan. Pseudoprogression was documented in seven patients: three patients in group 2 and four patients in group 3. Radionecrosis was documented in seven patients: two patients in group 2 and five patients in group 3. Regression of non-irradiated lesions was seen neither in two patients treated with SRT alone nor in five patients treated with SRT plus ICI, providing no evidence for rare abscopal effects. Conclusions Pseudoprogression is uncommon with ICI alone, suggesting that growing lesions in such patients should trigger an intervention. Pseudoprogression rates were similar after SRT alone or SRT in combination with ICI. Abscopal effects are rare or do not exist. Response assessment criteria should be considered carefully when designing clinical studies for patients with brain metastases who receive SRT

Funicular myelosis in a butcher: it was the cream cans

Background. Funicular myelosis is a known consequence of exposure to nitrous oxide. Nevertheless, there are only a few clinical trials assessing its long-term effects and there is no literature about the role of nutritional vitamin B12 supplementation in the context of nitrous oxide abuse. Case Descriptions. We diagnosed funicular myelosis in a young butcher, who consumed high amounts of meat regularly. Since the diagnostic process did not reveal any metabolic causes, reinterrogation of the patient uncovered recreational abuse of nitrous oxide out of whipped cream can gas cartridges. After stopping abuse and supplementation of vitamin B12, the patient recovered almost completely. Conclusions. In our case, even high nutritional vitamin B12 uptake could not compensate the noxious effects of nitrous oxide. Since there are emerging reports of increasing misuse, this should be considered in the diagnostic and therapeutic care of patients with nitrous oxide abuse. Furthermore, our case emphasizes that patients with vitamin B12 deficiency should be assessed for nitrous oxide abuse