The Varkud Satellite RNA contains a self-cleaving ribozyme that has been shown to function independently of its surroundings. This 160 nucleotide ribozyme adopts a catalytically active tertiary ...structure that includes a kissing hairpin complex formed by stem-loop I and stem-loop V (SLV). The five-nucleotide 5′-rUGACU loop of the isolated SLV has been shown to adopt a Mg2+-dependent U-turn structure by solution NMR. This U-turn hairpin is examined here by molecular dynamics simulations in the presence of monovalent and divalent ions. Simulations confirm on an all-atom level the hypotheses for the role of the Mg2+ ions in stabilizing the loop, as well as the role of the solvent exposed U700 base. Additionally, these simulations suggest the Mg2+-free stem-loop adopts a wide range of structures, including energetically favorable structures similar to the Mg2+-bound loop structure. We propose this structure is a “gatekeeper” or precursor to Mg2+ binding when those ions are present.
Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole-cell proteomics of genome-edited cells, we show that the oxidative stress ...response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid-peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative-stress-induced cell death, demonstrating a crucial role for this organelle in cellular homeostasis and wound response.
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•Released CAVIN1 inactivates NRF2 under oxidative stress•Membrane lipid peroxidation triggers CAVIN1 release under oxidative stress•Cavin1 mediates ROS accumulation and tissue regeneration in zebrafish•Cancer cells lacking CAVIN1 are resistant to ferroptosis
Wu et al. define a conserved pathway involving cell surface pits called caveolae, NRF2, and lipid peroxidation in sensing and responding to oxidative stress. They show the crucial role of this pathway in maintaining homeostatic cellular balance by promoting the elimination of severely damaged or malignant cells.
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both ...development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.
Self‐assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient ...conditions. This report demonstrates how charged side‐chain chemistry affects the hierarchical co‐assembly of a family of charge‐complementary β‐sheet‐forming peptide pairs known as CATCH(X+/Y−) at physiologic pH and ionic strength in water. In a concentration‐dependent manner, the CATCH(6K+) (Ac‐KQKFKFKFKQK‐Am) and CATCH(6D−) (Ac‐DQDFDFDFDQD‐Am) pair formed either β‐sheet‐rich microspheres or β‐sheet‐rich gels with a micron‐scale plate‐like morphology, which were not observed with other CATCH(X+/Y−) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N‐ and C‐terminal amino acids in CATCH(6K+) and CATCH(6D−) to Qs, increased observed co‐assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)‐green fluorescent protein fusions could be incorporated into the β‐sheet plates and microspheres formed by the CATCH(6K+/6D−) pair, demonstrating the potential to endow functionality.
Co‐assembly of charge‐complementary peptides affords opportunities to create fibrillar biomaterials with molecular‐level features that cannot be achieved in single‐component systems. However, our ability to predict fibrillar network architecture based on peptide pair sequence characteristics is lacking. This report shows how the type of charged amino acid affects supramolecular organization in charge‐complementary β‐sheet peptide co‐assemblies.
Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is ...progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural ...localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.
Healthcare workers (HCWs) found the 2003 outbreak of severe acute respiratory syndrome (SARS) to be stressful, but the long-term impact is not known. From 13 to 26 months after the SARS outbreak, 769 ...HCWs at 9 Toronto hospitals that treated SARS patients and 4 Hamilton hospitals that did not treat SARS patients completed a survey of several adverse outcomes. Toronto HCWs reported significantly higher levels of burnout (p = 0.019), psychological distress (p<0.001), and posttraumatic stress (p<0.001). Toronto workers were more likely to have reduced patient contact and work hours and to report behavioral consequences of stress. Variance in adverse outcomes was explained by a protective effect of the perceived adequacy of training and support and by a provocative effect of maladaptive coping style and other individual factors. The results reinforce the value of effective staff support and training in preparation for future outbreaks.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Telomere length has been implicated in the organismal response to stress, but the underlying mechanisms are unknown.
Here we examine the impact of telomere length changes on the responses to ...three contrasting abiotic environments in Arabidopsis, and measure 32 fitness, developmental, physiological and leaf‐level anatomical traits.
We report that telomere length in wild‐type and short‐telomere mutants is resistant to abiotic stress, while the elongated telomeres in ku70 mutants are more plastic. We detected significant pleiotropic effects of telomere length on flowering time and key leaf physiological and anatomical traits. Furthermore, our data reveal a significant genotype by environment (G × E) interaction for reproductive fitness, with the benefits and costs to performance depending on the growth conditions.
These results imply that life‐history trade‐offs between flowering time and reproductive fitness are impacted by telomere length variation. We postulate that telomere length in plants is subject to natural selection imposed by different environments.
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