Expression of a SOX1 overlapping transcript in neural differentiation and cancer models

SOX1 is a member of the SOXB1 subgroup of transcription factors involved in early embryogenesis, CNS development and maintenance of neural stem cells. The structure and regulation of the human SOX1 locus has been less studied than that of SOX2, another member of the SOXB1 subgroup for which an overlapping transcript has been reported. Here we report that the SOX1 locus harbours a SOX1 overlapping transcript (SOX1-OT), and describe expression, splicing variants and detection of SOX1-OT in different stem and cancer cells. RT-PCR and RACE experiments were performed to detect and characterize the structure of SOX1-OT in neuroprogenitor cultures and across different cancer cell lines. SOX1-OT was found to present a complex structure including several unannotated exons, different transcript variants, and at least two potential transcription start sites. SOX1-OT was found to be highly expressed in differentiated neural stem cells across different time points of differentiation, and its expression correlated with SOX1 gene expression. Concomitant expression of SOX1 and SOX1-OT was further observed in several cancer cell models. While the function of this transcript is unknown, the regulatory role reported for other lncRNAs strongly suggests a possible role for SOX1-OT in regulating SOX1 expression, as previously observed for SOX2. The elucidation of the genetic and regulatory context governing SOX1 expression will contribute to clarifying its role in stem cell differentiation and tumorigenesis

Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography

Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in-vitro. However, obtaining quantitative molecular information from the Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least square fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 256 mg/m, while proteins were distributed more uniformly and reaching concentrations as high as ~5012 mg/ml. The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman map of fixed cells (n=10), we found a linear relationship between the scores corresponding to the first component (PC1) and cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n=10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were obtained concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in-vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease

Differences in the pattern and regulation of mineral deposition in human cell lines of osteogenic and non-osteogenic origin

Bone marrow-derived mesenchymal stem cells (MSCs) are widely used as a cellular model of bone formation, and can mineralize in vitro in response to osteogenic medium (OM). It is unclear, however, whether this property is specific to cells of mesenchymal origin. We analysed the OM response in 3 non-osteogenic lines, HEK293, HeLa and NTera, compared to MSCs. Whereas HEK293 cells failed to respond to OM conditions, the 2 carcinoma-derived lines NTera and HeLa deposited a calcium phosphate mineral comparable to that present in MSC cultures. However, unlike MSCs, HeLa and NTera cultures did so in the absence of dexamethasone. This discrepancy was confirmed, as bone morphogenetic protein inhibition obliterated the OM response in MSCs but not in HeLa or NTera, indicating that these 2 models can deposit mineral through a mechanism independent of established dexamethasone or bone morphogenetic protein signalling

In Silico Identification of SOX1 Post-Translational Modifications Highlights a Shared Protein Motif

The transcription factor SOX1 is a key regulator of neural stem cell development, acting to keep neural stem cells (NSCs) in an undifferentiated state. Postnatal expression of Sox1 is typically confined to the central nervous system (CNS), however, its expression in non-neural tissues has recently been implicated in tumorigenesis. The mechanism through which SOX1 may exert its function is not fully understood, and studies have mainly focused on changes in SOX1 expression at a transcriptional level, while its post-translational regulation remains undetermined. To investigate this, data were extracted from different publicly available databases and analysed to search for putative SOX1 post-translational modifications (PTMs). Results were compared to PTMs associated with SOX2 in order to identify potentially key PTM motifs common to these SOXB1 proteins, and mapped on SOX1 domain structural models. This approach identified several putative acetylation, phosphorylation, glycosylation and sumoylation sites within known functional domains of SOX1. In particular, a novel SOXB1 motif (xKSExSxxP) was identified within the SOX1 protein, which was also found in other unrelated proteins, most of which were transcription factors. These results also highlighted potential phospho-sumoyl switches within this SOXB1 motif identified in SOX1, which could regulate its transcriptional activity. This analysis indicates different types of PTMs within SOX1, which may influence its regulatory role as a transcription factor, by bringing changes to its DNA binding capacities and its interactions with partner proteins. These results provide new research avenues for future investigations on the mechanisms regulating SOX1 activity, which could inform its roles in the contexts of neural stem cell development and cancer

Implementation of an innovative hands-on training to improve adherence to hygiene rules: A feasibility Study

Background: Hospital-acquired infections (HAI) still pose a major problem in inpatient care. The single most important measure for preventing HAIs is to improve adherence to hand hygiene among health care professionals. Objective: To assess the feasibility of an innovative hands-on training to improve adherence to hygiene rules under standardized and under real life conditions. Design: Before-after controlled cohort trial to assess the feasibility of implementing an innovative hands-on training to improve hand hygiene adherence. Setting: Large university hospital in Germany. Participants: Fifty trained nurses from three wards with an average age of 32 years (+/- 10.22 years) and an average vocational experience of 6.85 years (+/- 7.54 years). Methods: The intervention consisted of a hands-on training in the skills lab of the University of Cologne complemented by a 12-week observation period before and after the training on participating wards. The training comprised important skills with respect to hand hygiene, venipuncture, dressing changes of central venous catheters, preparation of IV infusions, and donning of gloves using sterile technique. A communication training was included to enable nurses to enforce hygiene rules in their collaboration with peers and physicians. The intervention was taught in small groups with a wide array of interactive teaching methods. It was evaluated using the objective structured clinical examination (OSCE) format. Observations were conducted by a trained infection control nurse. Results: Before (after) the intervention 622 (612) occasions of hand hygiene were documented. A highly significant improvement in hygiene compliance was observed pre- and post-intervention (64.3% vs. 79.2%; p <= 0.0001). The OSCE evaluation showed significant improvements in all subscales. Conclusion: The developed and conducted hands-on training seems feasible and is successful in significantly improving adherence to hygiene rules under standardized and real life conditions. Whether the effect is stable over time is subject to further investigation. (C) 2015 Elsevier Ltd. All rights reserved