Self-assembly of tissue transglutaminase into amyloid-like fibrils using physiological concentration of Ca 2+

Tissue transglutaminase (tTG or TG2) is a member of the transglutaminase family that catalyzes calcium dependent formation of isopeptide bonds. It has been shown that the expression of TG2 is elevated in neurodegenerative diseases such as Parkinson's, Huntington's, and Alzheimer's. We have investigated the self-assembly of TG2 in vitro. First, using software, hot spots, which are prone for aggregation, were identified in domain 2 of the enzyme. Next we expressed and purified recombinant TG2 and its truncated version that contains only the catalytic domain, and examined their amyloidogenic behavior in various conditions including different temperatures and pHs, in the presence of metal ions and Guanosine triphosphate (GTP).To analyze various stages leading to TG2 fibrillation, we employed various techniques including Thioflavin T (ThT) binding assay, Congo-Red, birefringence, Circular Dichroism (CD), 8-anilino-1-naphthalene sulfonic acid (ANS) binding, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). Our results indicated that using low concentrations of Ca 2+, TG2 self-assembled into amyloid-like fibrils; this self-assembly occurred at the physiological temperature (37 °C) and at a higher temperature (57 °C). The truncated version of TG2 (domain 2) also forms amyloid-like fibrils only in the presence of Ca 2+. Because amyloid formation has occurred with domain 2 alone where no enzymatic activity was shown, self-cross-linking by the enzyme was ruled out as a mechanism of amyloid induction. The self-assembly of TG2 was not significant with magnesium and zinc ions, indicating specificity of the self-assembly for calcium ions. The calcium role in self-assembly of TG2 into amyloid may be extended to other proteins with similar biophysical properties to produce novel biomaterials. © 2011 American Chemical Society

Rosetta Brains: A Strategy for Molecularly-Annotated Connectomics

We propose a neural connectomics strategy called Fluorescent In-Situ Sequencing of Barcoded Individual Neuronal Connections (FISSEQ-BOINC), leveraging fluorescent in situ nucleic acid sequencing in fixed tissue (FISSEQ). FISSEQ-BOINC exhibits different properties from BOINC, which relies on bulk nucleic acid sequencing. FISSEQ-BOINC could become a scalable approach for mapping whole-mammalian-brain connectomes with rich molecular annotations

The effects of combined low level laser therapy and mesenchymal stem cells on bone regeneration in rabbit calvarial defects

Abstract: This study evaluated the effect of Low Level Laser Therapy (LLLT) and Mesenchymal Stem Cells (MSCs) on bone regeneration. Background data: Although several studies evaluated the effects of MSCs and LLLT, there is little information available regarding in vivo application of LLLT in conjunction with MSCs. Methods: Forty-eight circular bone defects (6 mm in diameter) were prepared in the calvaria of 12 New- Zealand white rabbits. The defects of each animal were randomly assigned to 4 groups: (C) no treatment; (L) applying LLLT; (SC) filled with MSCs; (SCL) application of both MSCs and LLLT. LLL was applied on alternate days at wavelength of 810 nm, power density of 0.2 W/cm2 and a fluency of 4 J/cm2 using a Gallium–Aluminum–Arsenide (GaAlAs) diode laser. The animals were sacrificed after 3 weeks and then histological samples were evaluated to determine the amount of new bone formation and the remaining scaffold and inflammation. Results: The histological evaluation showed a statistically significant increase in new bone formation of LLLT group relative to the control and the other two experimental groups (p < 0.05). There was no significant difference in bone formation of the control group compared to experimental groups filled with MSCs. Laser irradiation had no significant effect on resorption of the scaffold material. In addition, inflammation was significantly reduced in LLLT group compared to the control defects and the other two experimental groups. Conclusion: Low level laser therapy could be effective in bone regeneration but there is no evidence of a synergistic effect when applied in conjunction with MSCs

Nurses� perception of patient safety culture and its relationship with adverse events: a national questionnaire survey in Iran

Background: Patient safety culture is an important factor in determining hospitals� ability to address and reduce the occurrence of adverse events (AEs). However, few studies have reported on the impact of nurses� perceptions of patient safety culture on the occurrence of AEs. Our study aimed to assess the association between nurses� perception of patient safety culture and their perceived proportion of adverse events. Methods: A cross-sectional survey was carried out among 2295 nurses employed in thirty-two teaching hospitals in Iran. Nurses completed the Persian version of the hospital survey of patients� safety culture between October 2018 and September 2019. Results: Positive Response Rates of overall patient safety culture was 34.1 and dimensions of patient safety culture varied from 20.9 to 43.8. Also, nurses estimated that the occurrence of six adverse events varied from 51.2�63.0 in the past year. The higher nurses� perceptions of �Staffing�, �Hospital handoffs and transitions�, �Frequency of event reporting�, �Non-punitive response to error�, �Supervisor expectation and actions promoting safety�, �Communication openness�, �Organizational learning continuous improvement�, �Teamwork within units�, and �Hospital management support patient safety� were significantly related to lower the perceived occurrence at least two out of six AEs (OR = 0.69 to 1.46). Conclusions: Our findings demonstrated that nurses� perception regarding patient safety culture was low and the perceived occurrence of adverse events was high. The research has also shown that the higher level of nurses� perception of patient safety culture was associated with lowered occurrence of AEs. Hence, managers could provide prerequisites to improve patient safety culture and reduce adverse events through different strategies, such as encouraging adverse events� reporting and holding training courses for nurses. However, further research is needed to assess how interventions addressing patient safety culture might reduce the occurrence of adverse events. © 2021, The Author(s)

Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture (3C) assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single-cell Hi-C, combined with genome-wide statistical analysis and structural modelling of single-copy X chromosomes, to show that individual chromosomes maintain domain organization at the megabase scale, but show variable cell-to-cell chromosome structures at larger scales. Despite this structural stochasticity, localization of active gene domains to boundaries of chromosome territories is a hallmark of chromosomal conformation. Single-cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organization underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns

Highly multiplexed subcellular RNA sequencing in situ

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ