In vitro glycoengineering using exoenzymes for specific modification is recognized as appropriate method to tailor sugar moieties of glycan structures during the recombinant production of monoclonal antibodies (mAbs). This report describes enhanced in vitro glycoengineering approaches using beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase to improve the efficiency of galactosylation and sialylation with the aim to implement in vitro glycoengineering into common mAb purification processes. Feasibility studies tested the potential of different in vitro glycoengineering protocols (two-step vs. one-step) to facilitate the overall procedure. Scalability of the reactions was demonstrated for mAb amounts ranging from 1 mg to 1 g. Additionally, the reactions of beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase were shown to work on column during affinity chromatography using Protein A or KappaSelect, the latter providing more efficient galactosylation and sialylation of IgG1 and IgG4 mAbs. Performing in vitro glycoengineering on column enabled the use of cell culture harvest that yielded results comparable to those of purified bulk. Based thereon, an optimized two-step mixed mode approach was found most appropriate to integrate in vitro glycoengineering of the IgG1 mAb into the overall manufacturing process. Using harvest for on-column reaction of beta 1,4-galactosyltransferase combined with in-solution reaction of alpha 2,6-sialyltransferase, this approach yielded 100% biantennary galactosylation and 61% biantennary sialylation. Moreover, the enzymes applied in in vitro glycoengineering could be separated, recycled and reused in further reactions to improve economic efficiency. Overall, the study provides a toolbox for in vitro glycoengineering and presents an optimized easy-to-handle workflow to implement this method into the downstream processing of industrial mAb production.

We previously generated a highly thermostable triple variant of Moloney murine leukemia virus reverse transcriptase, MM3 (E286R/E302K/L435R), by introducing positive charges by site-directed mutagenesis at positions that have been implicated in the interaction with template-primer (Yasukawa et al., (2010) J. Biotechnol., 150, 299-306). In this study, we attempted to further increase the thermostability of MM3. Twenty-nine mutations were newly designed, focusing on the number of surface charge, stabilization of hydrophobic core, and introduction of salt bridge. The corresponding 29 single variants were produced in Escherichia coli and characterized for activity and stability. Six mutations (A32V, L41D, L72R, I212R, L272E and W388R) were selected as the candidates for further stabilize MM3. Fifteen multiple variants were designed by combining two or more of the six mutations with the MM3 mutations, produced and characterized. The sextuple variant MM3.14 (A32V/L72R/E286R/E302K/W388R/L435R) exhibited higher thermostability than MM3.

The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT-qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT-qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT-qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the goal of personalized health care. (C) 2012 Elsevier Inc. All rights reserved.

La presente invention concerne des biomarqueurs, des methodes et des kits pour l'evaluation d'echantillons de sang total, ladite evaluation ciblant n'importe quel element faisant partie du groupe constitue par : cancer colorectal, adenome colorectal, et polype colorectal. Lesdits biomarqueurs sont des polynucleotides issus de loci genetiques choisis a partir de l'un quelconque des loci suivants : ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, et LRRN3.

Die kombinierte transgluteale N.-ischiadicus- und N.-femoralis-Blockade mit Kathetertechnik kann ein gutes Alternativverfahren zu einer Allgemeinanästhesie oder einer rückenmarknahen Technik bei Amputationen der unteren Extremität sein. Bei multimorbiden Hochrisikopatienten kann hiermit der operative Eingriff ohne kontrollierte Beatmung sowie mit geringerer Kreislaufdepression durchgeführt und noch über Tage eine vollständige Schmerzfreiheit erzielt werden. In der retrospektiven Analyse an 65 Patienten mit Beinamputationen konnte der operative Eingriff bei 58 Patienten (89%) in alleiniger Katheterregionalanästhesie durchgeführt werden; lediglich 7 Patienten (11%) benötigten zusätzlich eine Allgemeinanästhesie. Das postoperative schmerztherapeutische Management mit kontinuierlicher Katheteranalgesie erzielte eine hohe perioperative Patientenzufriedenheit. Voraussetzungen sind jedoch ausreichende personelle und strukturelle Ressourcen sowie regionalanästhesiologische Erfahrung durch Schulung und Praxis.

For amputations of the lower limbs the combined blockade of the sciatic nerve (via the transgluteal approach) and the femoral nerve (via the inguinal route) may be a suitable alternative to general or neuroaxial anaesthetic methods. In highly comorbid, high risk patients this catheter-linked regional anaesthesia combines the advantages of avoiding controlled ventilation with decreased cardiovascular depression, as conveyed by the use of general anaesthesia or neuraxial techniques. Furthermore, improved postoperative analgesia may be achieved for several days. In our retrospective study, 65 patients who were scheduled for amputation of the lower limbs were analysed. In 58 patients (89%), a regional anaesthesia technique employing only a catheter technique was performed and only 7 patients (11%) required additional general anaesthesia. For postoperative pain management, local anaesthetics were administered via an indwelling nerve block catheter and excellent pain scores were achieved in these patients. However, successful employment of this technique necessitates sufficiently trained personnel as well as on-going training in the performance of catheter-based local analgesia.

European Journal of PainVolume 10, Issue S1 p. S244a-S244 942 P.A.I.N. QUALITY — OUTCOME DRIVEN PAIN MANAGEMENT B.J. Morlion, B.J. Morlion Dept. for Pain Management, UZ Gasthuisberg, Leuven, BelgiumSearch for more papers by this authorH. Walch, H. Walch Dept. of Anaesthesiology, LKH Graz-West, Graz, AustriaSearch for more papers by this authorD.J. Vermeij, D.J. Vermeij Dirk Vermey Quality Management, Wijk bij Duurstede, The NetherlandsSearch for more papers by this authorM. Stanton-Hicks, M. Stanton-Hicks Pain Management Center C-25, The Cleveland Clinic Foundation, Cleveland OH, USASearch for more papers by this authorJ. de Andres, J. de Andres Multidisciplinary Pain Management Departments, Valencia University General Hospital, Valencia, SpainSearch for more papers by this authorG. Yihune, G. Yihune International Healthcare Management, Gruenenthal GmbH, Aachen, GermanySearch for more papers by this author B.J. Morlion, B.J. Morlion Dept. for Pain Management, UZ Gasthuisberg, Leuven, BelgiumSearch for more papers by this authorH. Walch, H. Walch Dept. of Anaesthesiology, LKH Graz-West, Graz, AustriaSearch for more papers by this authorD.J. Vermeij, D.J. Vermeij Dirk Vermey Quality Management, Wijk bij Duurstede, The NetherlandsSearch for more papers by this authorM. Stanton-Hicks, M. Stanton-Hicks Pain Management Center C-25, The Cleveland Clinic Foundation, Cleveland OH, USASearch for more papers by this authorJ. de Andres, J. de Andres Multidisciplinary Pain Management Departments, Valencia University General Hospital, Valencia, SpainSearch for more papers by this authorG. Yihune, G. Yihune International Healthcare Management, Gruenenthal GmbH, Aachen, GermanySearch for more papers by this author First published: 13 January 2012 https://doi.org/10.1016/S1090-3801(06)60945-2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume10, IssueS1September 2006Pages S244a-S244 RelatedInformation

Fruchtfliegen der Mutante VamKS74 zeigen eine semidominante Degeneration des Gehirns. Dabei entstehen bei adulten Fliegen altersabhangig histologisch nachweisbare Locher, bzw. Vakuolen in den optischen Neuropilen der Lamina und der Medulla. In dieser Arbeit wird gezeigt, das das Auftreten dieser Locher mit apoptotischem Zelltod von Laminamonopolarzellen im Laminakortex einhergeht. Zudem wird dargelegt, das eine Reduktion der X-Gal Farbeintensitat verschiedener lacZ Enhancer-Trap Linien auf eine transkriptionelle Repression des P-Element Promotors im genetischen Vam Hintergrund zuruckzufuhren ist. Zur Identifizierung der molekularen Ursachen der Degeneration wurde eine genetische Analyse der Vam-Mutante begonnen. Mehrere Gene, die in der fraglichen Region des X-Chromosoms liegen wurden molekular charakterisiert, wobei in einem der Gene in der Vam Mutante eine charakteristische Punktmutation identifiziert werden konnte. Dabei handelt es sich um ein vollig neuartiges Gen mit bisher unbekannter Funktion. Dieses Gen ist jedoch in hoheren Eukaryoten hoch konserviert und kann auch im menschlichen Genom nachgewiesen werden.