An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids.     

Short arm dicentric Y chromosome with associated statural defects in a sterile man

Summary A short arm dicentric Y chromosome is described as the predominant cell line in a sterile man. The patient also presents with short stature. Tooth development appears normal. Only Sertoli cells are present in the seminiferous tubules. It is suggested that the function of the gene controlling spermatogenesis in Yq11 in man might be to prevent proliferation or migration of germ cells to the gonad of the early embryo.     

An enzymatic activity in uninfected cells that cleaves the linkage between poliovirion RNA and the 5' terminal protein.

The 5' terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of heLa cells and is activated by Mg++. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5' terminal protein. The role of this enzyme in the uninfected cell is not known.     

Reproducibility of nuclear morphometric measurements in benign human epithelial cells. Simulation of variations in routine tissue processing.

The value of nuclear morphometric measurements in diagnostic pathology is determined largely by the reproducibility of the measurements. Although a variety of factors have been shown to affect tissues during processing, the regulation of fixative type and the avoidance of air drying in particular have been shown to avoid significant variations in nuclear measurements. The current study simulated routine tissue processing in order to identify any factors that may introduce variability of nuclear morphometric values in day-to-day processing if air drying is avoided and fixative type and pH are regulated. Samples of benign endometrium were collected from three uteri, fixed in phosphate-buffered formalin (PBF) from 2 hours to 15 days and dehydrated in an automated tissue processor on four occasions. In addition, tissue from one case was cut at 4, 6 and 8 microns, simulating the potential variations in section thickness that may occur during routine processing. Mean nuclear areas and shape factors of epithelial cells were then determined using computed planimetry. By analysis of variance, no significant differences were found in nuclear morphometric values in relation to time of fixation, dehydration runs or tissue section thickness; coefficients of variation for all variables were less than 7%. This study suggested that routinely processed tissues are adequate for morphometric analysis, including retrospective analysis, provided that tissues are fixed in a pH-regulated fixative such as PBF and air drying is avoided.     

Multimodality Multiparametric Imaging of Early Tumor Response to a Novel Antiangiogenic Therapy Based on Anticalins

Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [¹⁸F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = −0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy.     

Chronobiological study of the pharmacological response of rats to combination ketamine-midazolam

Ketamine is commonly administered in combination with benzodiazepines to achieve surgical anaesthesia in rats. The aim of the present study was to analyze the pharmacological response of the combination ketamine-midazolam injected intraperitoneally at different times of day to rats. The study was conducted in July 2003, during the winter in the Southern hemisphere. Female prepuberal Sprague-Dawley rats synchronized to a 12h light:12h dark cycle (light, 07:00-19:00h) were used as experimental animals. A combination treatment of ketamine (40 mg/kg) and midazolam (2 mg/kg) was administered to five different clock-time groups of rats (n=7/group). Duration of the latency period, ataxia, loss-of-righting reflex (LRR), post-LRR ataxia, and total pharmacological response were assessed by visual assessment. Significant treatment-time differences were detected in the duration of LRR, post-LRR ataxia, and total pharmacological response duration. The longest pharmacological response occurred in rats injected during the light (rest) phase, and the shortest pharmacological response occurred in rats injected during the dark (activity) phase. Cosinor analysis documented circadian rhythmicity in the duration of post-LRR ataxia. The findings of the study indicate the duration of CNS-depression of the ketamine-midazolam combination exhibits treatment-time-dependent variation in the rat.     

Comparative Oncology: Evaluation of 2-Deoxy-2-[18F]fluoro-D-glucose (FDG) Positron Emission Tomography/Computed Tomography (PET/CT) for the Staging of Dogs with Malignant Tumors

Introduction

2-Deoxy-2-[18F]fluoro-D-glucose PET/CT is a well-established imaging method for staging, restaging and therapy-control in human medicine. In veterinary medicine, this imaging method could prove to be an attractive and innovative alternative to conventional imaging in order to improve staging and restaging. The aim of this study was both to evaluate the effectiveness of this image-guided method in canine patients with spontaneously occurring cancer as well as to illustrate the dog as a well-suited animal model for comparative oncology.

Methods

Ten dogs with various malignant tumors were included in the study and underwent a whole body FDG PET/CT. One patient has a second PET-CT 5 months after the first study. Patients were diagnosed with histiocytic sarcoma (n = 1), malignant lymphoma (n = 2), mammary carcinoma (n = 4), sertoli cell tumor (n = 1), gastrointestinal stromal tumor (GIST) (n = 1) and lung tumor (n = 1). PET/CT data were analyzed with the help of a 5-point scale in consideration of the patients’ medical histories.

Results

In seven of the ten dogs, the treatment protocol and prognosis were significantly changed due to the results of FDG PET/CT. In the patients with lymphoma (n = 2) tumor extent could be defined on PET/CT because of increased FDG uptake in multiple lymph nodes. This led to the recommendation for a therapeutic polychemotherapy as a treatment. In one of the dogs with mammary carcinoma (n = 4) and in the patient with the lung tumor (n = 1), surgery was cancelled due to the discovery of multiple metastasis. Consequently no treatment was recommended.

Conclusion

FDG PET/CT offers additional information in canine patients with malignant disease with a potential improvement of staging and restaging. The encouraging data of this clinical study highlights the possibility to further improve innovative diagnostic and staging methods with regard to comparative oncology. In the future, performing PET/CT not only for staging but also in therapy control could offer a significant improvement in the management of dogs with malignant tumors.     

DNA damage, somatic aneuploidy, and malignant sarcoma susceptibility in muscular dystrophies

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD-pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD-gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.     

A uniform system for microRNA annotation

MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. Guidelines are presented for the identification and annotation of new miRNAs from diverse organisms, particularly so that miRNAs can be reliably distinguished from other RNAs such as small interfering RNAs. We describe specific criteria for the experimental verification of miRNAs, and conventions for naming miRNAs and miRNA genes. Finally, an online clearinghouse for miRNA gene name assignments is provided by the Rfam database of RNA families.