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FragIdent – Automatic identification and characterisation of cDNA-fragments
Dominik Seelow, Heike Goehler, Katrin Hoffmann
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-95
Abstract: Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available.Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs) within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones.We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at http://compbio.charite.de/genetik/FragIdent/ webcite.cDNA clones or the cDNA contained in them are frequently used in yeast two-hybrid assays [1] and hybridisation studies [2]. Although whole clones are employed in some hybridisation studies [3], usually only the insert or a fragment of it is used as a probe. The DNA can be obtained either by amplification of the corresponding plasmid, by insert or vector specific PCR reactions.While the contents of clones experimentally derived from complex mRNA samples are necessarily unknown, even clones from characterised libraries are in many cases wrongly annotated [4] or might have been mixed up
Evanescent field Sensors Based on Tantalum Pentoxide Waveguides – A Review
Katrin Schmitt,Kerstin Oehse,Gerd Sulz,Christian Hoffmann
Sensors , 2008, DOI: 10.3390/s8020711
Abstract: Evanescent field sensors based on waveguide surfaces play an important rolewhere high sensitivity is required. Particularly tantalum pentoxide (Ta2O5) is a suitablematerial for thin-film waveguides due to its high refractive index and low attenuation.Many label-free biosensor systems such as grating couplers and interferometric sensors aswell as fluorescence-based systems benefit from this waveguide material leading toextremely high sensitivity. Some biosensor systems based on Ta2O5 waveguides alreadytook the step into commercialization. This report reviews the various detection systems interms of limit of detection, the applications, and the suitable surface chemistry.
Evanescent field Sensors Based on Tantalum Pentoxide Waveguides ¢ € “ A Review
Katrin Schmitt,Kerstin Oehse,Gerd Sulz,Christian Hoffmann
Sensors , 2008,
Abstract: Evanescent field sensors based on waveguide surfaces play an important rolewhere high sensitivity is required. Particularly tantalum pentoxide (Ta2O5) is a suitablematerial for thin-film waveguides due to its high refractive index and low attenuation.Many label-free biosensor systems such as grating couplers and interferometric sensors aswell as fluorescence-based systems benefit from this waveguide material leading toextremely high sensitivity. Some biosensor systems based on Ta2O5 waveguides alreadytook the step into commercialization. This report reviews the various detection systems interms of limit of detection, the applications, and the suitable surface chemistry.
The HIV-1 Rev Protein Enhances Encapsidation of Unspliced and Spliced, RRE-Containing Lentiviral Vector RNA
Bastian Grewe, Katrin Ehrhardt, Bianca Hoffmann, Maik Blissenbach, Sabine Brandt, Klaus überla
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0048688
Abstract: Background During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein. Findings Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly. Conclusions Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.
Translating microarray data for diagnostic testing in childhood leukaemia
Katrin Hoffmann, Martin J Firth, Alex H Beesley, Nicholas H de Klerk, Ursula R Kees
BMC Cancer , 2006, DOI: 10.1186/1471-2407-6-229
Abstract: We examined published microarray data from 104 ALL patients specimens, that represent six different subgroups defined by cytogenetic features and immunophenotypes. Using the decision-tree based supervised learning algorithm Random Forest (RF), we determined a small set of genes for optimal subgroup distinction and subsequently validated their predictive power in an independent patient cohort.We achieved very high overall ALL subgroup prediction accuracies of about 98%, and were able to verify the robustness of these genes in an independent panel of 68 specimens obtained from a different institution and processed in a different laboratory. Our study established that the selection of discriminating genes is strongly dependent on the analysis method. This may have profound implications for clinical use, particularly when the classifier is reduced to a small set of genes. We have demonstrated that as few as 26 genes yield accurate class prediction and importantly, almost 70% of these genes have not been previously identified as essential for class distinction of the six ALL subgroups.Our finding supports the feasibility of qRT-PCR technology for standardized diagnostic testing in paediatric ALL and should, in conjunction with conventional cytogenetics lead to a more accurate classification of the disease. In addition, we have demonstrated that microarray findings from one study can be confirmed in an independent study, using an entirely independent patient cohort and with microarray experiments being performed by a different research team.Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease characterized by the presence of several subtypes that are of prognostic relevance. These subtypes can be distinguished based on immunophenotype, differentiation status, as well as chromosomal and molecular abnormalities. The identification of different ALL subtypes, the characterization of prognostic features, and the finding that ALL subtypes differ in their response to t
Involvement of the epidermal growth factor receptor in the modulation of multidrug resistance in human hepatocellular carcinoma cells in vitro
Katrin Hoffmann, Zhi Xiao, Clemens Franz, Elvira Mohr, Susanne Serba, Markus W Büchler, Peter Schemmer
Cancer Cell International , 2011, DOI: 10.1186/1475-2867-11-40
Abstract: Chemotherapeutic treatment induces multidrug resistance and significantly increases ABC-transport protein expression and function in a time- and dose-dependent manner in HCC cells. Furthermore, cytostatic treatment increases the mRNA expression of tyrosine kinases and induces the phosphorylation of ERK. EGF activation of the tyrosine kinase pathway up-regulated the ABC-transport protein mRNA expression and enhanced the survival of resistant HCC cells. Consistent with these effects, inhibition of the EGFR using siRNA decreased the ABC-transport protein mRNA expression and inhibited the proliferation of resistant cells. Additional treatment with Gefitinib, a clinically approved EGFR inhibitor, caused a dose-dependent reversal of resistance to conventional chemotherapy.The present study demonstrates that the multidrug resistance of HCC is modulated through the EGF-activated tyrosine kinase cascade. Consequentially, the restoration of chemosensitivity by EGFR inhibition may lead towards new tailored therapies in patients with highly resistant tumors.Hepatocellular carcinoma (HCC) is a major health problem worldwide. Its incidence is increasing continuously in the Western world. In the United States and Europe the diagnosis of HCC has almost doubled during the last two decades [1]. Despite recent improvements in surveillance programs and diagnostic tools, only 30-40% of HCC patients are eligible for liver resection or transplantation, the only curative treatment options to date [2]. The tyrosine kinase inhibitor sorafenib is the current standard of care for palliative treatment; the partial response rate, however, is only about 10% [3]. Conventional systemic chemotherapy has shown only minor effectiveness with response rates far below 10% [4]. A substantial resistance against structurally and functionally unrelated cytostatic drugs develops through the destruction of vulnerable and negative chemoresistant tumor cell populations during hepatocarcinogenesis. An increased c
Major combined electrolyte deficiency during therapy with low-dose Cisplatin, 5-Fluorouracil and Interferon alpha: report on several cases and review of the literature [ISRCTN62866759]
Katrin Hoffmann, Angela Marten, Katja Lindel, Stefan Fritz, Dirk Jager, Markus W Buchler, Jan Schmidt
BMC Cancer , 2006, DOI: 10.1186/1471-2407-6-128
Abstract: A patient with resected adenocarcinoma of the pancreas was treated with adjuvant radio-chemo-immunotherapy using a combination of low-dose Cisplatin, 5-Fluorouracil and Interferon alpha together with external beam radiation. Severe hypocalcaemia without signs of acute renal failure or electrolyte disturbance occurred within 2 days at the 4th week of treatment and required intensive care treatment.Combination of biological and cytotoxic therapies may increase the incidence of severe hypocalcaemia in pancreatic cancer. Oncologists should remain attentive of this problem as more highly active regimes become available.Hypocalcaemia is a known side-effect in high-dose Cisplatin chemotherapy of solid tumors [1,2]. In the classic case, hypocalcaemia is caused by excessive urinary loss and decreased renal up-take during high-dose Cisplatin treatment. Proximal tubular damage leads to decreased reabsorption of cations. Acute nephrotoxicity presents with increased creatinine and persistent protein and electrolyte losses. Chronic nephrotoxicity is characterized by a decrease of glomerular filtration rate and a slightly elevated but persistent magnesium, potassium and calcium excretion [3]. Hypomagnaesemic induced hypocalcaemia is caused by inhibition of parathyroid hormone secretion, impaired calcium release from the bones and low tissue responses to PTH due to low magnesium levels. Cisplatin associated hypocalcaemia and hypercalciuria can not be influenced by calcium supplementation. Correction of magnesium blood levels usually should improve the hypocalcaemia [4]. Hypocalcaemia may be associated with tetany, depression, carpopedal spasm, neuromuscular excitability, cardiac arrythmias with prolonged Q-T interval and sudden death, making it a true oncological emergency [5].Outside cytotoxic therapy hypocalcaemia has been observed after thyroid and parathyroid surgery, chronic renal failure, acute rhabdomyolysis and pancreatitis [6-9]. Cisplatin is often used in combinations wit
Investigating a training supporting shared decision making (IT'S SDM 2011): study protocol for a randomized controlled trial
Friedemann Geiger, Katrin Liethmann, Frauke Hoffmann, Jutta Paschedag, Jürgen Kasper
Trials , 2011, DOI: 10.1186/1745-6215-12-232
Abstract: In a multi-center randomized controlled trial with a waiting control group, 40 physicians from 7 medical fields are enrolled. Each physician contributes a sequence of four medical consultations including a diagnostic or treatment decision.The intervention consists of two condensed video-based individual coaching sessions (15min.) supported by a manual and a DVD. The interventions alternate with three measurement points plus follow up (6 months).Realized patient involvement is measured using the coefficient SDMMASS drawn from the Multifocal Approach to the Sharing in SDM (MAPPIN'SDM) which includes objective involvement, involvement as perceived by the patient, and the doctor-patient concordance regarding their judges of the involvement. For validation purposes, all three components of SDMMASS are supplemented by similar measures, the OPTION observer scale, the Shared Decision Making Questionnaire (SDM-Q) and the dyadic application of the Decisional Conflict Scale (DCS). Training effects are analyzed using t-tests. Spearman correlation coefficients are used to determine convergent validities, the influence of involvement (SDMMASS) on the perceived decision quality (DCS) and on the elaboration of the decision. The latter is operationalised by the ELAB coefficient from the UP24 (Uncertainty Profile, 24 items version).Due to the rigorous blinded randomized controlled design, the current trial promises valid and reliable results. On the one hand, we expect this condensed time-saving training to be adopted in clinical routine more likely than previous trainings. On the other hand, the exhaustivity of the MAPPIN'SDM measurement system qualifies it as a reference measure for simpler instruments and to deepen understanding of decision-making processes.Current Controlled Trials ISRCTN78716079Good clinical practice is characterized by valuable interactions between an informed and activated patient and a proficient, proactive health care team [1]. Such interactions require a pa
Outcome Results of Treatment with Selective Internal Radiation Therapy (SIRT) in Patients with Hepatocellular Carcinoma: A Single Center Experience  [PDF]
Jan Pfeiffenberger, Tatjana Zimmermann, Daniel N. Gotthardt, Christoph Springfeld, Wolfgang Stremmel, Peter Schirmacher, Henning Schulze-Bergkamen, Arianeb Mehrabi, Christoph W. Michalski, Katrin Hoffmann, Nikolas Kortes, Boris Radeleff, Uwe Haberkorn, Clemens Kratochwil, Karl Heinz Weiss, Carsten Grüllich
Journal of Cancer Therapy (JCT) , 2017, DOI: 10.4236/jct.2017.84031
Abstract: Background: Hepatocellular carcinoma (HCC) has a poor prognosis. Selective internal radiation therapy (SIRT) with microspheres is a treatment option for HCC. This study aimed to assess safety and survival (OS) in patients with HCC treated with SIRT, to stratify patients with tumor vascularization and analyze the impact of sequential sorafenib treatment. Methods: Thirty-nine patients who received SIRT for HCC between 2010 and 2013 at our center were included in this retrospective analysis. Tumor vascularization was assessed using a combination of MRI, MAA-scintigraphy and angiography. Tumor vascularization was correlated with survival. Subgroups are treated with two commercially available 90Y-labeled products SIR-Spheres (n = 16) and TheraSpheres (n = 23) and sequential therapy with sorafenib compared to SIRT only was analyzed. Results: Adverse events occurred in 49% of patients with only four grade 3 and no grade 4 event. Median survival for all patients was 12.5 months (95% CI: 8.7 - 16.3). No significant differences were detectable between Thera Spheres or SIR Spheres. Survival was shorter in patients with low tumor vascularization score (OS: 3.8 months (95% CI 0 - 15.0), p = 0.043). Survival was longer with sorafenib upon progression after SIRT (n=16) with an OS of 17.4 months (95% CI: 12.1 – 22.7) compared to no sorafenib (n = 13; 9.1 months; 95% CI: 3.0 - 15.1) or progression upon sorafenib before SIRT (n = 10; 8.6 months; 95% CI: 5.5 - 11.7). Conclusions: SIRT is safe in HCC patients. Tumor vascularization by radiography and scintigraphy may predict survival benefit. Sorafenib is active after SIRT and significantly prolongs survival.
MEK inhibition induced downregulation of MRP1 and MRP3 expression in experimental hepatocellular carcinoma
Shibo Lin, Katrin Hoffmann, Zhi Xiao, Nan Jin, Uwe Galli, Elvira Mohr, Markus W Büchler, Peter Schemmer
Cancer Cell International , 2013, DOI: 10.1186/1475-2867-13-3
Abstract: The Raf1 kinase inhibitor (GW5074) and different MEK inhibitors (U0126 and AZD6244) were used to treat HCC cells to identify their effects on HCC cell growth and ABC proteins expression in vitro. Cell viability tests were performed after the treatment of MAPK pathway inhibitors and in combination with gemcitabine or doxorubicin. Western blot was applied to assess the changes of MAPK pathway and protein expression of MRP1 and MRP3. Flow cytometry was used to measure intracellular doxorubicin accumulation after the treatment of MEK inhibitors.Both Raf1 inhibitor (GW5074) and MEK inhibitors (U0126 and AZD6244) suppressed HCC cell growth in a dose dependent manner. Pre-treatment of MEK inhibitor U0126 or AZD6244 sensitized HCC cells to gemcitabine or doxorubicin based chemotherapy. Raf1 inhibitor GW5074 had no effect on MRP1 and MRP3 protein expression. Treatment of gemcitabine or doxorubicin activated phosphorylated ERK and induced the upregulation of MRP1 and MRP3. MEK inhibitors U0126 and AZD6244 deactivated phosphorylated ERK, decreased endogenous MRP1 expression, reversed gemcitabine or doxorubicin induced MRP1 and MRP3 upregulation, and increased the intracellular doxorubicin accumulation.This study provides evidence that MEK inhibitors sensitize HCC cells to chemotherapy by increasing intracellular chemodrug accumulation. MEK inhibirors U0126 and AZD6244 reduced MRP1 as well as MRP3 expression, and may contribute partially to the sensitization. The combination of MEK inhibitor and conventional chemotherapy may offer new therapeutic option for the treatment of resistant HCC.Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality and causes more than half a million deaths annually worldwide [1,2]. The number of new cases of primary liver cancer increases globally and HCC accounts for 70% to 85% of them [3]. Potentially curative treatment, including liver resection, transplantation and local ablation, could provide promising 5-year-survival
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