Search

SummaryAltering ubiquitination by disruption of individual deubiquitinating enzymes (DUBs) has proven to affect hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. To accomplish this goal, we systematically inactivated DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNAs (shRNAs) screens. We found that multiple DUBs may be individually required for hematopoiesis and that the ubiquitin-specific protease 15 (USP15) is particularly important for the maintenance of murine hematopoietic stem and progenitor cells in vitro and in vivo. Consistently, Usp15 knockout mice exhibited a reduced HSC pool. The defect was intrinsic to HSCs, as demonstrated by competitive repopulation assays. Importantly, USP15 is highly expressed in normal human hematopoietic cells and leukemias, and USP15 depletion in murine early progenitors and myeloid leukemia cells impaired in vitro expansion and increased genotoxic stress. Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSC and in leukemia cells.

One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or in the 2',3'-O-position (i.e., 3a-7a and 3c), and tested their cytostatic/cytotoxic activities using HT-29 human colon carcinoma cells, in comparison with, e.g., 5-FU (1) and 5-FUrd (2a). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5'-O-labelled Atto 425 derivatives were incorporated by the human HT-29 cells and accumulated in their cytoplasm. Moreover, after 24-h treatment of HT-29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80 μM) revealed a significant (14-23 or 33-45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80 μM) led to a significant (77-95 or 89-96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63-72% or ca. 75%, respectively more effective than 5-FU (1; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80 μM, respectively in comparison with the (negative) control. Some synthesized 5-FUrd derivatives turned out to be more effective than 5-FU (1) or 5-FUrd (2a).

Sulcus-deepening trochleoplasty restores the trochlear groove in patients with patellofemoral instability and underlying trochlear dysplasia. There are types of dysplasia both with (B and D) and without (A and C) a supratrochlear spur. The aim of this study was to identify influencing factors for the clinical outcome following trochleoplasty.Forty-four knees in 38 patients who underwent trochleoplasty for instability (type A in 9, B in 15, C in 9 and D in 11 knees) were assessed clinically with the Kujala score and radiologically with radiographs and MRI. The median follow-up was 4 (2-7.8) years.At follow-up, the median Kujala score had improved from 68 (29-84) to 90 (42-100) points (P 0.001). Instability (P 0.001) and pain (P = 0.027) decreased significantly, but in 3 knees, pain was worse postoperatively. Twenty-seven knees were ranked as excellent, 10 as good, 2 as fair and 5 as poor. Overall, dysplasia types B and D benefited more from surgery than types A and C. The postoperative MRI revealed no chondrolysis or subchondral necrosis, but deterioration of cartilage on the lateral trochlear facet was identified.Trochleoplasty is a useful and reliable surgical technique to improve patellofemoral instability in patients with a dysplastic trochlea. While improved stability is predictable, pain is less predictable and may even increase following surgery. The overall results were directly dependent on the type of the dysplasia, with a significantly better clinical outcome in type B and D. The clinical relevance of this study is that severe dysplasia can successfully be treated with trochleoplasty.III.

In our former studies a diagnostic approach for the detection of transmissible spongiform encephalopaties (TSE) based on FT-IR spectroscopy in combination with artificial neural networks was described, based on a controlled animal study with terminally ill Syrian hamsters and control animals. As a consequence of the bovine spongiform encephalopathy (BSE) crisis in Europe, the development of a disgnostic ante mortem test for cattle has become a matter of great scientific importance and public interest. Since 1986 more than 180,000 clinical cases of BSE have been observed in the UK alone. Most of these cases were confirmed by post mortem examination of brain tissue. However, BSE-related risk assessment and risk-management would greatly benefit from ante mortem testing on living animals. For example, a serum-based test could allow for screening of the cattle population, thus, even a BSE eradication program would be conceivable. Here we report on a novel method for ante mortem BSE testing, which combines infrared spectroscopy of serum samples with multivariate pattern recognition analysis. A classification algorithm was trained using infrared spectra of sera from more than 800 animals from a field study (including BSE positive, healthy controls and animals suffering from viral or bacterial infections). In two validation studies sensitivities of 85% and 87% and specificities of 84% and 91% were achieved, respectively. The combination of classification algorithms increased sensitivity and specificity to 96% and 92%, respectively.

Since 1986, more than 180 000 clinical cases of bovine spongiform encephalopathy (BSE) have been observed in the U.K. alone. Most of these cases were confirmed by postmortem examination of brain tissue. However, BSE-related risk assessment and risk management would greatly benefit from antemortem testing on living animals. A serum-based test could allow for screening of the cattle population; thus, even a BSE eradication program would be conceivable. Here we report on a novel method for antemortem BSE testing, which combines infrared spectroscopy of serum samples with multivariate pattern recognition analysis. A classification algorithm was trained using infrared spectra of bovine sera from more than 800 animals (including BSE-positive, healthy controls and animals suffering from classical viral or bacterial infections). In two validation studies, sensitivities of 85 and 84% and specificities of 86 and 91% were achieved, respectively. The combination of classification algorithms increased the sensitivity and specificity of BSE detection to 96 and 92%, respectively.

Staphylococcus aureus has been well known as a major pathogen for many years, causing a variety of nosocomial infections. The rapid and reliable detection of methicillin-resistant S . aureus (MRSA) organisms is of utmost importance to prevent the spread of infection. Species identification is

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. Significance: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses. This article is highlighted in the In This Issue feature, p. 521