Identification and manipulation of chromatin barriers in transcriptional reprogramming

Master transcription factors are cell fate determining factors that can force new identities onto cells by evoking distinct gene transcription patterns. This potential can be detrimental for an organism when activated erroneously, which is why genes of master transcription factors have to be regulated tightly. Chromatin features like DNA methylation have been implied in transcriptional regulation, the direct causalities and the underlying mechanisms are however still largely unclear. Here I investigated the role of the neurogenic transcription factor Sexdetermining-region-y-box 1 (Sox1) in directing neural stem cell identity. I employed a targeted trans-activating domain (dCas9-VP64) to induce Sox1 expression in vitro in neural progenitor cells (NPCs) and characterized the invoked phenotypic changes. Inducing Sox1 expression in NPCs restored their neuronal differentiation potential, and transcriptome analysis revealed a shift in cell identity towards neural stem cells (NSCs), underlining the role of Sox1 as cell fate determining factor. Analysis on single cell basis however revealed that only a small subset of NPCs responded to the targeted gene induction with Sox1 upregulation. Using a GFP knock in as reporter, I separated responsive from unresponsive cells and investigated differences in chromatin features at the Sox1 promoter. I identified DNA methylation as a strong barrier against trans-activation by combining transcriptional engineering and epigenome editing via dCas9-Tet1. Furthermore, I found similar barriers at the promoters of other master transcription factor genes, including Oct4 and Nkx2-2. Lastly, I employed a screening approach to identify potential regulatory regions distal of the Sox1 gene. By transducing NPCs with a gRNA library of high complexity, I was able to identify targeting sites for dCas9-VP64 in the locus of Sox1 that have the potency to induce gene transcription even outside of the promoter. In conclusion, I have confirmed Sox1 as a neurogenic master transcription factor and identified mechanisms that control expression of this gene. These findings could serve to optimize future trans-activation approaches and underline the importance of chromatin features in the regulation of cell fate determining factors

On Soft Limits of Inflationary Correlation Functions

Soft limits of inflationary correlation functions are both observationally relevant and theoretically robust. Various theorems can be proven about them that are insensitive to detailed model-building assumptions. In this paper, we re-derive several of these theorems in a universal way. Our method makes manifest why soft limits are such an interesting probe of the spectrum of additional light fields during inflation. We illustrate these abstract results with a detailed case study of the soft limits of quasi-single-field inflation.Comment: 26 pages, 5 figures; V2: references added + pedagogical improvements of Sec. 2 and App.

Experimental implementation of a silicon wafer tandem solar cell

We combine aluminum back surface field (Al-BSF) solar cell precursors with an additional rear side infrared active floating emitter in a tandem cell configuration. This emitter is implemented area selectively by fs-laser hyperdoping in a sulfurous atmosphere. Its design as a floating emitter conceals losses induced by the laser process as long as n-doping occurs. All processes are adapted and supplemented by just a single new process step

In vivo expression and purification of aptamer-tagged small RNA regulators

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria

Correction to: Two years later: Is the SARS-CoV-2 pandemic still having an impact on emergency surgery? An international cross-sectional survey among WSES members

Background: The SARS-CoV-2 pandemic is still ongoing and a major challenge for health care services worldwide. In the first WSES COVID-19 emergency surgery survey, a strong negative impact on emergency surgery (ES) had been described already early in the pandemic situation. However, the knowledge is limited about current effects of the pandemic on patient flow through emergency rooms, daily routine and decision making in ES as well as their changes over time during the last two pandemic years. This second WSES COVID-19 emergency surgery survey investigates the impact of the SARS-CoV-2 pandemic on ES during the course of the pandemic. Methods: A web survey had been distributed to medical specialists in ES during a four-week period from January 2022, investigating the impact of the pandemic on patients and septic diseases both requiring ES, structural problems due to the pandemic and time-to-intervention in ES routine. Results: 367 collaborators from 59 countries responded to the survey. The majority indicated that the pandemic still significantly impacts on treatment and outcome of surgical emergency patients (83.1% and 78.5%, respectively). As reasons, the collaborators reported decreased case load in ES (44.7%), but patients presenting with more prolonged and severe diseases, especially concerning perforated appendicitis (62.1%) and diverticulitis (57.5%). Otherwise, approximately 50% of the participants still observe a delay in time-to-intervention in ES compared with the situation before the pandemic. Relevant causes leading to enlarged time-to-intervention in ES during the pandemic are persistent problems with in-hospital logistics, lacks in medical staff as well as operating room and intensive care capacities during the pandemic. This leads not only to the need for triage or transferring of ES patients to other hospitals, reported by 64.0% and 48.8% of the collaborators, respectively, but also to paradigm shifts in treatment modalities to non-operative approaches reported by 67.3% of the participants, especially in uncomplicated appendicitis, cholecystitis and multiple-recurrent diverticulitis. Conclusions: The SARS-CoV-2 pandemic still significantly impacts on care and outcome of patients in ES. Well-known problems with in-hospital logistics are not sufficiently resolved by now; however, medical staff shortages and reduced capacities have been dramatically aggravated over last two pandemic years