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In situ reverse transcription: the magic of strength and anonymity

In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2′-deoxyuridine into the growing cDNA strand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin, 5-bromo-2′-deoxyuridine is ‘invisible’ in the DNA–DNA duplex but easily detectable in the DNA–RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. We have also shown that the replacement of deoxythymidine by 5-bromo-2′-deoxyuridine considerably stabilizes the growing DNA–RNA duplex, thus enabling the one-step detection of polyadenylated RNA in structurally well-preserved cells. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed for the cell's preparation, the ratio is higher than 80

Building Change Detection in Airborne Laser Scanning and Dense Image Matching Point Clouds Using a Residual Neural Network

National Mapping Agencies (NMAs) acquire nation-wide point cloud data from Airborne Laser Scanning (ALS) sensors as well as using Dense Image Matching (DIM) on aerial images. As these datasets are often captured years apart, they contain implicit information about changes in the real world. While detecting changes within point clouds is not a new topic per se, detecting changes in point clouds from different sensors, which consequently have different point densities, point distributions and characteristics, is still an on-going problem. As such, we approach this task using a residual neural network, which detects building changes using height and class information on a raster level. In the experiments, we show that this approach is capable of detecting building changes automatically and reliably independent of the given point clouds and for various building sizes achieving mean F1-Scores of 80.5% and 79.8% for ALS-ALS and ALS-DIM point clouds on an object-level and F1-Scores of 91.1% and 86.3% on a raster-level, respectively

Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy. © 2006 by The American Society for Cell Biology

Copanlisib synergizes with conventional and targeted agents including venetoclax in B- And T-cell lymphoma models

Copanlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor with preferred activity toward PI3Ka and PI3Kd. Despite the clear overall clinical benefit, the number of patients achieving complete remissions with the single agent is relatively low, a problem shared by the vast majority of targeted agents. Here, we searched for novel copanlisib-based combinations. Copanlisib was tested as a single agent, in combination with an additional 17 drugs in 26 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and T-cell lymphomas. In vivo experiments, transcriptome analyses, and immunoblotting experiments were also performed. Copanlisib as a single agent showed in vitro dose-dependent antitumor activity in the vast majority of the models. Combination screening identified several compounds that synergized with copanlisib. The strongest combination was with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax. The benefit of the combination over single agents was also validated in an MZL xenograft model and in MCL primary cells, and was due to increased induction of apoptosis, an effect likely sustained by the reduction of the antiapoptotic proteins myeloid cell leukemia 1 (MCL1) and BCL-XL, observed in MCL and MZL cell lines, respectively. These data supported the rationale for the design of the Swiss Group for Clinical Cancer Research (SAKK) 66/18phase 1 study currently exploring the combination of copanlisib and venetoclax in relapsed/refractory lymphomas