Towards a Medically Approved Technology for Large-Scale Stem Cell Banks: Tools and Method

The importance, of the development of stem cell cryobanking has increased recently with an augmentation of stem cell research and its therapeutic applications. The development of therapies is, among other things, limited by high sensitivity of stem cells to freezingthawing procedures. Thus, new approaches are needed for preservation and related evaluation methods, as well as new technologies for long term storage of large numbers of stem cells. Here we present selected recent improvements of stem cell cryopreservation, e.g. for freezing of adherent human embryonic stem cells using gel-like matrices. We report the application and performance of novel microsystem-based cryosubstrates and devices and describe new evaluation methods and the results of a thermal stress cycle study.В настоящее время возросла важность развития криобанков стволовых клеток в связи с их расширенным изучением и терапевтическим применением. Однако, наряду с другими факторами, вышеуказанная терапия ограничена высокой чувствительностью стволовых клеток к процедурам замораживания-оттаивания. Необходимы как новые подходы к криоконсервированию и связанным с ним методам оценки, так и новые технологии для долгосрочного хранения большого количества стволовых клеток. В настоящей работе мы представляем некоторые улучшенные методы криоконсервирования стволовых клеток, например замораживание эмбриональных стволовых клеток человека с использованием гелеобразного матрикса. Мы представляем результаты применения разработанных на базе микросистемной техники новых криосубстратов и устройств, а также описываем новые методы оценки и результаты изучения циклов температурного стресса.Наразі зросла важливість розвитку кріобанків стовбурових клітин у зв’язку з їх розширеним вивченням і терапевтичним застосуванням. Але водночас з іншими факторами вищезгадана терапія обмежена високою чутливістю стовбурових клітин до процедур заморожування-відтавання. Необхідні як нові підходи до кріоконсервування та повязаних з ним методам оцінки, так і нові технології для довгострокового зберігання великої кількості стовбурових клітин. В цій роботі ми представляємо деякі покращені методи кріоконсервування стовбурових клітин, наприклад заморожування ембріональних стовбурових клітин людини з використанням гелеподібного матриксу. Ми представляємо результати застосування розроблених на базі мікросистемної техніки нових кріосубстратів та приладів, а також описуємо нові методи оцінки і результати вивчення циклів температурного стресу

Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis

Functional analysis of human foamy virus accessory reading frames.

Foamy viruses belong to the retroviruses which possess a complex genome structure. The human foamy virus (HFV) isolate bears three open reading frames (the so-called bel genes) in the 3' region of the genome which have been reported to give rise to possibly six different proteins via alternative splicing (W. Muranyi and R. M. Flügel, J. Virol. 65:727-735, 1991). In order to analyze the requirements of these proteins for HFV replication in vitro, we constructed a set of single and combinatory bel gene mutants of an infectious molecular clone of HFV. The mutant which lacked the transacting activator, bel-1, was found to be replication incompetent. All other mutants replicated equally well and gave rise to comparable titers of infectious cell-free virus. When HFV proviruses were put under the control of a heterologous promoter (simian virus 40), none of the accessory gene products was found to be required for expression of structural (gag) proteins. There was no evidence for a posttranscriptional regulatory protein that is present in other complex retroviruses

The transcriptional transactivator of human foamy virus maps to the bel 1 genomic region.

The human foamy virus (HFV) genome possesses three open reading frames (bel 1, 2, and 3) located between env and the 3' long terminal repeat. By analogy to other human retroviruses this region was selected as the most likely candidate to encode the viral transactivator. Results presented here confirmed this and showed further that a deletion introduced only into the bel 1 open reading frame of a plasmid derived from an infectious molecular clone of HFV abolished transactivation. In contrast, deletions in bel 2 and bel 3 had only minor effects on the ability to transactivate. The role of the bel 1 genomic region as a transactivator was further investigated by eukaryotic expression of a genome fragment of HFV spanning the bel 1 open reading frame. A construct expressing bel 1 under control of a heterologous promoter was found to transactivate the HFV long terminal repeat in a dose-dependent fashion. Furthermore, it is shown that the U3 region of the HFV long terminal repeat is sufficient to respond to the HFV transactivator

Publishing in Gender and Sexualities Journals

Panel discussion held at Southern Sociological Society’s Committee on Gender and Sexuality at Southern Sociological Society Annual Meeting. Progra