The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoterspecific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA- DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and sigma 3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex

Learning curve and period of experience required for the competent diagnosis of acute appendicitis using abdominal computed tomography: a prospective observational study

Objective To assess the learning curve of novice residents in diagnosing acute appendicitis using abdominal computed tomography (CT) scans. Methods This prospective observational study was conducted within a 4-month period from March 1 to June 30, 2015. After CT scans for right lower quadrant pain or similar acute abdomen were evaluated, postgraduate year 1 (PGY-1) residents completed an interpretation checklist. The primary outcome was evaluation of the learning curve for competent CT scan interpretation under suspicion of acute appendicitis. Secondary outcomes were cumulative numbers of accurate abdominal CT interpretations regardless of initial clinical impression and training period. Results PGY-1 residents recorded a total of 230 interpretation checklists. There were 53, 51, 46, 44, and 36 checklists recorded by individual residents and 92, 92, 91, 91, and 61 respective training days in the emergency department, excluding rotation periods in other departments. After 16 to 20 interpretations of abdominal CT scans performed under suspicion of acute appendicitis, the residents could diagnose acute appendicitis with more than 95% accuracy. Overall, the sensitivity and specificity for diagnosing acute appendicitis were 97% (95% confidence interval, 94 to 100) and 83% (95% confidence interval, 80 to 87), respectively. After 61 to 80 abdominal CT interpretations regardless of suspicion of acute appendicitis and after 41 to 50 days in training, PGY-1 emergency department residents could diagnose acute appendicitis with more than 95% accuracy. Conclusion PGY-1 residents require 16 to 20 checklist interpretations to acquire acceptable abdominal CT interpretation. After performing 61 to 80 CT scans regardless of suspicion of acute appendicitis, they could diagnose acute appendicitis with acceptable accuracy

The dynamic landscape of transcription initiation in yeast mitochondria

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription. Conformational dynamics during the early stage of transcription is crucial to understanding the regulation of transcription efficiency and fidelity. Here the authors, by single-molecule fluorescence resonance energy transfer approaches, examine the conformational dynamics of the two-component transcription system of yeast mitochondria with single-base resolution

Anti-apoptotic effects of silk fibroin hydrolysate in RIN5F cell on high glucose condition

Hyperglycemia-induced pancreatic β-cell apoptosis causes serious health complications in diabetic patients. Recently, studies demonstrated that silk and silk-related materials have anti-diabetic effects. We previously reported that silk fibroin hydrolysate (SFH) has anti-diabetic effects through increased pancreatic β-cell mass in type 2 diabetic animals (C57BL/KsJdb/db). However, it is not known whether SFH has anti-apoptotic effects in hyperglycemic conditions. The present study investigates the anti-apoptotic effects of SFH on high glucoseinduced apoptosis using RIN5F cells, a rat pancreatic β-cell line. Hyperglycemic conditions decreased RIN5F cell viability in a concentration-dependent manner. High glucose treatment of 33 mM or higher caused a significant decrease in RIN5F cell viability. However, addition of SFH significantly recovered cell viability in the presence of high glucose. Flow cytometry analysis showed that high glucose treatment significantly increased early stage apoptosis in RIN5F cells. This was inhibited by SFH treatment, which significantly decreased not only early stage apoptosis but also decreased the production of nitrite. Additionally, SFH protected RIN5F cells from oxidative stress-induced apoptosis. These results suggest that SFH has anti-apoptotic effects by protecting pancreatic β-cell from high glucose and/or oxidative stress. Our results support in vivo anti-diabetic effects of SFH and validation of the traditional use of silkworm and silkworm materials in the treatment of diabetes. © 2015 Korean Society for Integrative Biology1111sciescopuskc

Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore

The higher-order structural organization and dynamics of the chromosomes play a central role in gene regulation. To explore this structure-function relationship, it is necessary to directly visualize genomic elements in living cells. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. This feature enables the application of CRISPR-based imaging to loci throughout the genome and opens up new possibilities for the study of nuclear processes in living cells

A Universal Perovskite Nanocrystal Ink for High-Performance Optoelectronic Devices

Semiconducting lead halide perovskite nanocrystals (PNCs) are regarded as promising candidates for next-generation optoelectronic devices due to their solution processability and outstanding optoelectronic properties. While the field of light-emitting diodes (LEDs) and photovoltaics (PVs), two prime examples of optoelectronic devices, has recently seen a multitude of efforts toward high-performance PNC-based devices, realizing both devices with high efficiencies and stabilities through a single PNC processing strategy has remained a challenge. In this work, diphenylpropylammonium (DPAI) surface ligands, found through a judicious ab-initio-based ligand search, are shown to provide a solution to this problem. The universal PNC ink with DPAI ligands presented here, prepared through a solution-phase ligand-exchange process, simultaneously allows single-step processed LED and PV devices with peak electroluminescence external quantum efficiency of 17.00% and power conversion efficiency of 14.92% (stabilized output 14.00%), respectively. It is revealed that a careful design of the aromatic rings such as in DPAI is the decisive factor in bestowing such high performances, ease of solution processing, and improved phase stability up to 120 days. This work illustrates the power of ligand design in producing PNC ink formulations for high-throughput production of optoelectronic devices; it also paves a path for "dual-mode" devices with both PV and LED functionalities

Additional file 1 of Augmented interpretation of HER2, ER, and PR in breast cancer by artificial intelligence analyzer: enhancing interobserver agreement through a reader study of 201 cases

Additional file 1. Supplementary Method, Supplementary Results, Figures S1–6; Table S1–7

Assembly of the Five-Way Junction in the Ribosomal Small Subunit Using Hybrid MD-Go Simulations

Assembly of the bacterial ribosomal small subunit (SSU) begins with the folding of the five-way junction upon interaction with the primary binding protein S4. This complex contains the largest contiguous molecular signature, which is a conserved feature in all bacterial 16S rRNAs. In a previous study, we used all-atom molecular dynamics simulations to demonstrate that the co-evolving signature in the N-terminus of S4 is intrinsically disordered and capable of accelerating the binding process through a fly casting mechanism. In this paper, comparisons between the all-atom MD simulations and FRET experiments identify multiple metastable conformations of the naked five-way junction without the presence of S4. Furthermore, we capture the simultaneous folding and binding of the five-way junction and r-protein S4 by use of a structure-based Gō potential implemented within the framework of the all-atom molecular dynamics CHARMM force field. Different folding pathways are observed for the refolding of the five-way junction upon partial binding of S4. Our simulations illustrate the complex nature of RNA folding in the presence of a protein binding partner and provide insight into the role of population shift and the induced fit mechanisms in the protein:RNA folding and binding process.close10