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

Reactivity of primate sera to foamy virus Gag and Bet proteins

In order to establish criteria for the Serodiagnosis of foamy virus infections we investigated the extent to which sera from iofected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M$_r$ and the cytoplasmic 60K M$_r$ Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rahbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and Mrican green monkey origin. This was reßected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72 o/o) showed antiborlies against the Bet protein, indicating that Bet antigen is of value in sero1ogical screening for foamy virus infections

Reactivity of primate sera to foamy virus Gag and Bet

In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M r and the cytoplasmic 60K M r Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey an

One-Pot Chemoenzymatic Synthesis of Microviridin Analogs Containing Functional Tags

Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage‐like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest. While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags. To date – although highly desirable – no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides. In this study, a chemoenzymatic in vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners. This straightforward approach paves the way for customized protease inhibitors with built‐in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition