Non-polyadenylated transcription in embryonic stem cells reveals novel non-coding RNA related to pluripotency and differentiation

The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of whole-genome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs

NucTools: analysis of chromatin feature occupancy profiles from high-throughput sequencing data

Background: Biomedical applications of high-throughput sequencing methods generate a vast amount of data in which numerous chromatin features are mapped along the genome. The results are frequently analysed by creating binary data sets that link the presence/absence of a given feature to specific genomic loci. However, the nucleosome occupancy or chromatin accessibility landscape is essentially continuous. It is currently a challenge in the field to cope with continuous distributions of deep sequencing chromatin readouts and to integrate the different types of discrete chromatin features to reveal linkages between them. Results: Here we introduce the NucTools suite of Perl scripts as well as MATLAB- and R-based visualization programs for a nucleosome-centred downstream analysis of deep sequencing data. NucTools accounts for the continuous distribution of nucleosome occupancy. It allows calculations of nucleosome occupancy profiles averaged over several replicates, comparisons of nucleosome occupancy landscapes between different experimental conditions, and the estimation of the changes of integral chromatin properties such as the nucleosome repeat length. Furthermore, NucTools facilitates the annotation of nucleosome occupancy with other chromatin features like binding of transcription factors or architectural proteins, and epigenetic marks like histone modifications or DNA methylation. The applications of NucTools are demonstrated for the comparison of several datasets for nucleosome occupancy in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). Conclusions: The typical workflows of data processing and integrative analysis with NucTools reveal information on the interplay of nucleosome positioning with other features such as for example binding of a transcription factor CTCF, regions with stable and unstable nucleosomes, and domains of large organized chromatin K9me2 modifications (LOCKs). As potential limitations and problems we discuss how inter-replicate variability of MNase-seq experiments can be addressed

Intra-operative fiducial-based CT/fluoroscope image registration framework for image-guided robot-assisted joint fracture surgery

Purpose Joint fractures must be accurately reduced minimising soft tissue damages to avoid negative surgical outcomes. To this regard, we have developed the RAFS surgical system, which allows the percutaneous reduction of intra-articular fractures and provides intra-operative real-time 3D image guidance to the surgeon. Earlier experiments showed the effectiveness of the RAFS system on phantoms, but also key issues which precluded its use in a clinical application. This work proposes a redesign of the RAFS’s navigation system overcoming the earlier version’s issues, aiming to move the RAFS system into a surgical environment. Methods The navigation system is improved through an image registration framework allowing the intra-operative registration between pre-operative CT images and intra-operative fluoroscopic images of a fractured bone using a custom-made fiducial marker. The objective of the registration is to estimate the relative pose between a bone fragment and an orthopaedic manipulation pin inserted into it intra-operatively. The actual pose of the bone fragment can be updated in real time using an optical tracker, enabling the image guidance. Results Experiments on phantom and cadavers demonstrated the accuracy and reliability of the registration framework, showing a reduction accuracy (sTRE) of about 0.88 ±0.2mm (phantom) and 1.15±0.8mm (cadavers). Four distal femur fractures were successfully reduced in cadaveric specimens using the improved navigation system and the RAFS system following the new clinical workflow (reduction error 1.2±0.3mm, 2±1∘). Conclusion Experiments showed the feasibility of the image registration framework. It was successfully integrated into the navigation system, allowing the use of the RAFS system in a realistic surgical application

A Destruction Layer from the Hellenistic Period at Tel Shiloh

Excavations at Tel Shiloh revealed a building from the end of the third century BCE. The building was destroyed violently in the middle of the second century BCE. Based on the archaeological context, its relationship with later buildings and other finds from the period in its vicinity, it can be determined that its occupants were gentiles. Shiloh is located between Judea and Samaria, in an area included in different administrative districts at different times during the Second Temple period. In the Persian and Early Hellenistic periods, this area was outside the Land of Judea, but at the end of the Second Temple period it was within the Province of Judea. Historical sources indicate that the population on the eve of the Hasmonean Revolt was heterogeneous; during the revolt there were conflicts between the various ethnic groups. Edomites are mentioned by several sources as being in the area, and Jewish localities are also implied. Discharged Seleucid soldiers may have settled in the area as well, as they did in western Samaria. In any case, it is clear that the foreign settlement in Shiloh was destroyed in a violent confrontation. Based on its dating, the destruction must be attributed to the conquests of Jonathan and Simeon as part of the expansion of the borders of the Hasmonean state.</jats:p