Transcriptome analysis of differentiating trypanosomes reveals the existence of multiple post-transcriptional regulons

<p>Abstract</p> <p>Background</p> <p>Trypanosome gene expression is regulated almost exclusively at the post-transcriptional level, with mRNA degradation playing a decisive role. When trypanosomes are transferred from the blood of a mammal to the midgut of a Tsetse fly, they transform to procyclic forms: gene expression is reprogrammed, changing the cell surface and switching the mode of energy metabolism. Within the blood, trypanosomes can pre-adapt for Tsetse transmission, becoming growth-arrested stumpy forms. We describe here the transitions in gene expression that occur during differentiation of <it>in-vitro </it>cultured bloodstream forms to procyclic forms.</p> <p>Results</p> <p>Some mRNAs showed changes within 30 min of <it>cis-</it>aconitate addition, whereas others responded 12-24 hours later. For the first 12 h after addition of <it>cis</it>-aconitate, cells accumulated at the G1 phase of the cell cycle, and showed decreases in mRNAs required for proliferation, mimicking the changes seen in stumpy forms: many mRNAs needed for ribosomal and flagellar biogenesis showed striking co-regulation. Other mRNAs encoding components of signal transduction pathways and potential regulators were specifically induced only during differentiation. Messenger RNAs encoding proteins required for individual metabolic pathways were often co-regulated.</p> <p>Conclusion</p> <p>Trypanosome genes form post-transcriptional regulons in which mRNAs with functions in particular pathways, or encoding components of protein complexes, show almost identical patterns of regulation.</p

Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma

Background: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. Methods: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. Results: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). Conclusion: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma

Use of Complex DNA and Antibody Microarrays as Tools in Functional Analyses

While the deciphering of basic sequence information on a genomic scale is yielding complete genomic sequences in ever-shorter intervals, experimental procedures for elucidating the cellular effects and consequences of the DNA-encoded information become critical for further analyses. In recent years, DNA microarray technology has emerged as a prime candidate for the performance of many such functional assays. Technically, array technology has come a long way since its conception some 15 years ago, initially designed as a means for large-scale mapping and sequencing

Primary osteoblasts, osteoblast precursor cells or osteoblast‐like cell lines: Which human cell types are (most) suitable for characterizing 45S5‐bioactive glass?

Abstract The question how bioactive glasses (BGs) influence the viability and osteogenic differentiation of human osteogenic cells has already been addressed by several studies. However, a literature review revealed great differences in the type of cells used for these experiments. Primary human osteoblasts (hOBs) represent the desired standard, but possess the limitation of patient variability and time‐consuming isolation protocols. Therefore, several alternative cell types have been used including primary mesenchymal stromal cells (BMSCs) and the “osteoblast‐like” cell lines MG‐63, Saos‐2, HOS, and U2OS. The aim of our study was the identification of the cell type most suitable for tissue engineering projects involving BGs by comparative analysis of cell viability and osteogenic differentiation in response to crystallized 45S5‐BG. We observed that hOBs, BMSCs, and MG‐63 cells were resistant to 45S5‐BG induced cytotoxicity, while the viability of Saos‐2, HOS, and U2OS cells was significantly reduced. In addition, we detected alkaline phosphatase activity, except in U2OS cells, that increased upon 45S5‐BG cocultivation, demonstrating the induction of osteogenic differentiation. Our data and the fact that the donor‐dependent variations can be avoided when using MG‐63 cells suggest that these are a promising alternative to primary cells and remain an important cell line for future BG related studies

Surgical therapy of benign and low-grade malignant intramedullary chondroid lesions of the distal femur: intralesional resection and bone cement filling with or without osteosynthesis

Surgical treatment of benign and low-grade malignant intramedullary chondroid lesions at the distal femur is not well analyzed compared to higher-grade chondrosarcomas. Localization at the distal femur offers high biomechanical risks requiring sophisticated treatment strategy, but scientific guidelines are missing. We therefore wanted to analyze a series of equally treated patients with intralesional resection and bone cement filling with and without additional osteosynthesis. Twenty-two consecutive patients could be included with intralesional excision and filling with polymethylmethacrylate bone cement alone (n = 10) or with compound bone cement osteosynthesis using a locking compression plate (n = 12). Clinical and radiological outcome was retrospectively evaluated including tumor recurrences, complications, satisfaction, pain, and function. Mean follow-up was 55 months (range 7–159 months). Complication rate was generally high with lesion-associated fractures both in the osteosynthesis group (n = 2) and in the non-osteosynthesis group (n = 2). All fractures occurred in lesions that reached the diaphysis. No fractures were found in meta-epiphyseal lesions. No tumor recurrence was found until final follow-up. Clinical outcome was good to excellent for both groups, but patients with additional osteosynthesis had significantly longer surgery time, more blood loss, longer postoperative stay in the hospital, more complications, more pain, less satisfaction, and worse functional outcome. Intralesional resection strategy was oncologically safe without local recurrences but revealed high risk of biomechanical complications if the lesion reached the diaphysis with an equal fracture rate no matter whether osteosynthesis was used or not. Additional osteosynthesis significantly worsened final clinical outcome and had more overall complications. This study may help guide surgeons to avoid overtreatment with additional osteosynthesis after curettage and bone cement filling of intramedullary lesions of the distal femur. Meta-epiphyseal lesions will need additional osteosynthesis rarely, contrary to diaphyseal lesions with considerable cortical thinning

Retrospective analysis of 51 intralesionally treated cases with progressed giant cell tumor of the bone: local adjuvant use of hydrogen peroxide reduces the risk for tumor recurrence

Background: Giant cell tumor of the bone (GCT) has high local recurrence rates and the prognosis is hard to predict. We therefore retrospectively analyzed clinical outcome and recurrences of 51 GCT cases focusing on the effects of adjuvant local use of hydrogen peroxide. Methods: The series enclosed 51 advanced GCT cases of the upper and lower extremities (n = 27 Campanacci grade III; n = 24 grade II; n = 39 surgery at our institution, n = 12 elsewhere). Mean follow-up was 88.3 (± 62.0) months. Surgical details, histology, metastases, recurrences, and interview-based data on satisfaction and function including the Musculoskeletal Tumor Society (MSTS) score were evaluated. It was investigated whether hydrogen peroxide was additionally used or not to clean the tumor cavity after curettage as we hypothesized influence on recurrences. To analyze the underlying mechanisms, GCT-derived stromal cell lines were cultured in vitro and tested for cell viability and apoptosis after treatment with hydrogen peroxide. Statistical analysis was performed with Student’s t tests, analysis of variance (ANOVA) with post hoc testing, Mann-Whitney U tests, chi-square tests, Kaplan-Meier analysis, and multivariate Cox regression analysis. Results: The whole series had 21 recurrences (41%). Eleven recurrences were found (28%) after surgery at our institution. Kaplan-Meier analysis of cumulative recurrence-free survival revealed at 2 years follow-up 69% (72%, only our institution) and at 10 years follow-up 54% (68%, only our institution). Intralesional resection was performed by vigorous curettage, burring, and defect filling with either polymethylmethacrylate bone cement (n = 45) or cancellous bone from the iliac crest (n = 6). Univariate chi-square analysis showed significantly lower recurrence rate after bone cement filling (2.3-fold, p = 0.024). Cleaning of the lesion cavity with hydrogen peroxide significantly reduced recurrence rate (whole collective 2.9-fold, p = 0.004; our institution 2.8-fold, p = 0.04) and significantly increased cumulative recurrence-free survival rate (whole collective at 10 years follow-up 74% versus 31%, p = 0.002; our institution 79% versus 48%, p = 0.02) compared to cases without hydrogen peroxide treatment. In multivariate analysis, significant risk factors for recurrence were pathological fracture (hazard ratio 3.7; p = 0.04), high mitosis rate (hazard ratio 15.6; p = 0.01), and lack of hydrogen peroxide use (hazard ratio 6.0; p = 0.02). In vitro cell culture analyses found apoptotic nature of hydrogen peroxide induced GCT cell death. Conclusions: The present series proved for the first time that additional cleaning of the tumor cavity with hydrogen peroxide before defect filling significantly reduced recurrence rate and significantly increased recurrence-free survival in advanced but intralesionally treated GCT cases

Overexpression of Inosine 5′-Monophosphate Dehydrogenase Type II Mediates Chemoresistance to Human Osteosarcoma Cells

overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance..IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression

Genotypic resistance testing in HIV by arrayed primer extension

The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere

Replication and Virus-Induced Transcriptome of HAdV-5 in Normal Host Cells versus Cancer Cells - Differences of Relevance for Adenoviral Oncolysis

Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication