Dynamic changes in Sox2 spatio-temporal expression promote the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos.

Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerize on adjacent cis regulatory motifs, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In the present study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy. We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast and primitive endoderm populations within the inner cell mass of the developing rodent blastocyst.

Fluorescence-Guided versus Conventional Surgical Resection of High Grade Glioma: A Single-Centre, 7-Year, Comparative Effectiveness Study.

BACKGROUND: High grade gliomas (HGGs) are locally invasive brain tumours that carry a dismal prognosis. Although complete resection increases median survival, the difficulty in reliably demonstrating the tumour border intraoperatively is a norm. The Department of Neurosurgery, Hospital Sungai Buloh is the first public hospital in Malaysia to overcome this problem by adopting fluorescence-guided (FG) surgery using 5-aminolevulinic acid (5-ALA). METHODS: A total of 74 patients with histologically proven HGGs treated between January 2008 and December 2014, who fulfilled the inclusion criteria, were enrolled. Kaplan-Meier survival estimates and Cox proportional hazard regression were used. RESULTS: Significant longer survival time (months) was observed in the FG group compared with the conventional group (12 months versus 8 months, P < 0.020). Even without adjuvant therapy, HGG patients from FG group survived longer than those from the conventional group (8 months versus 3 months, P = 0.006). No significant differences were seen in postoperative Karnofsky performance scale (KPS) between the groups at 6 weeks and 6 months after surgery compared to pre-operative KPS. Cox proportional hazard regression identified four independent predictors of survival: KPS > 80 (P = 0.010), histology (P < 0.001), surgical method (P < 0.001) and adjuvant therapy (P < 0.001). CONCLUSION: This study showed a significant clinical benefit for HGG patients in terms of overall survival using FG surgery as it did not result in worsening of post-operative function outcome when compared with the conventional surgical method. We advocate a further multicentered, randomised controlled trial to support these findings before FG surgery can be implemented as a standard surgical adjunct in local practice for the benefit of HGG patients.

Knotting and unknotting of a protein in single molecule experiments.

Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.

Selective influence of Sox2 on POU transcription factor binding in embryonic and neural stem cells.

Embryonic stem cell (ESC) identity is orchestrated by co-operativity between the transcription factors (TFs) Sox2 and the class V POU-TF Oct4 at composite Sox/Oct motifs. Neural stem cells (NSCs) lack Oct4 but express Sox2 and class III POU-TFs Oct6, Brn1 and Brn2. This raises the question of how Sox2 interacts with POU-TFs to transcriptionally specify ESCs versus NSCs. Here, we show that Oct4 alone binds the Sox/Oct motif and the octamer-containing palindromic MORE equally well. Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif. In contrast, Oct6 binds preferentially to MORE and is unaffected by Sox2. ChIP-Seq in NSCs shows the MORE to be the most enriched motif for class III POU-TFs, including MORE subtypes, and that the Sox/Oct motif is not enriched. These results suggest that in NSCs, co-operativity between Sox2 and class III POU-TFs may not occur and that POU-TF-driven transcription uses predominantly the MORE cis architecture. Thus, distinct interactions between Sox2 and POU-TF subclasses distinguish pluripotent ESCs from multipotent NSCs, providing molecular insight into how Oct4 alone can convert NSCs to pluripotency.