Cryogenic electron tomography (cryo-ET), a method that enables the viewing of biomolecules in near-native environments at high resolution, is rising in accessibility and applicability. Over the past ...several years, once slow sample preparation and data collection procedures have seen innovations which enable rapid collection of the large datasets required for attaining high resolution structures. Increased data availability has provided a driving force for exciting improvements in cryo-ET data processing methodologies throughout the entire processing pipeline and the development of accessible graphical user interfaces (GUIs) that enable individuals inexperienced in computational fields to convert raw tilt series into 3D structures. These advances in data processing are enabling cryo-ET to attain higher resolution and extending its applicability to more complex samples.
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•Advances in cryo-ET data processing streamline the analysis of cellular tomograms.•Constrained single-particle tomography improves the resolution of in situ structures.•Availability of end-to-end pipelines for analysis of tilt-series increases access to cryo-ET.•AI-based structure prediction combined with cryo-ET enables de novo structure determination.
Abstract Silicon may be important for bone and connective tissue health. Higher concentrations of silicon are suggested to be associated with bone and the connective tissues, compared with the ...non-connective soft tissues. Moreover, in connective tissues it has been suggested that silicon levels may decrease with age based upon analyses of human aorta. These claims, however, have not been tested under controlled conditions. Here connective and non-connective tissues were collected and analysed for silicon levels from female Sprague–Dawley rats of different ages (namely, 3, 5, 8, 12, 26 and 43 weeks; n = 8–10 per age group), all maintained on the same feed source and drinking water, and kept in the same environment from weaning to adulthood. Tissues (696 samples) were digested in nitric acid and analysed by inductively coupled plasma optical emission spectrometry for total silicon content. Fasting serum samples were also collected, diluted and analysed for silicon. Higher concentrations of silicon (up to 50-fold) were found associated with bone and the connective tissues compared with the non-connective tissues. Although total silicon content increased with age in all tissues, the highest connective tissue silicon concentrations (up to 9.98 μg/g wet weight) were found in young weanling rats, decreasing thereafter with age (by 2–6 fold). Fasting serum silicon concentrations reflected the pattern of connective tissue silicon concentrations and, both measures, when compared to collagen data from a prior experiment in Sprague–Dawley rats, mirrored type I collagen turnover with age. Our findings confirm the link between silicon and connective tissues and would imply that young growing rats have proportionally higher requirements for dietary silicon than mature adults, for bone and connective tissue development, although this was not formally investigated here. However, estimation of total body silicon content suggested that actual Si requirements may be substantially lower than previously estimated which could explain why absolute silicon deficiency is difficult to achieve but, when it is achieved in young growing animals, it results in stunted growth and abnormal development of bone and other connective tissues.
This paper proposes an approach for including deeply uncertain factors directly into a multi-objective search procedure, to aid in incorporating divergent quantitative scenarios within the ...model-based decision support process. Specifically, we extend Many Objective Robust Decision Making (MORDM), a framework for finding and evaluating planning solutions under multiple objectives, to include techniques from robust optimization. Traditional MORDM first optimized a problem under a baseline scenario, then evaluated candidate solutions under an ensemble of uncertain conditions, and finally discovered scenarios under which solutions are vulnerable. In this analysis, we perform multiple multi-objective search trials that directly incorporate these discovered scenarios within the search. Through the analysis, we have created multiple problem formulations to show how methodological choices of severe scenarios affect the resulting candidate planning solutions. We demonstrate the approach through a water planning portfolio example in the Lower Rio Grande Valley of Texas.
•A multi-scenario optimization technique for deep uncertainty is proposed.•Including severe scenarios within the optimization yields more robust solutions.•Parallel coordinates effectively show solution performance in alternate scenarios.
Background and Aims
In nonalcoholic fatty liver disease (NAFLD), fibrosis is the most important factor contributing to NAFLD‐associated morbidity and mortality. Prevention of progression and ...reduction in fibrosis are the main aims of treatment. Even in early stages of NAFLD, hepatic and systemic hyperammonemia is evident. This is due to reduced urea synthesis; and as ammonia is known to activate hepatic stellate cells, we hypothesized that ammonia may be involved in the progression of fibrosis in NAFLD.
Approach and Results
In a high‐fat, high‐cholesterol diet–induced rodent model of NAFLD, we observed a progressive stepwise reduction in the expression and activity of urea cycle enzymes resulting in hyperammonemia, evidence of hepatic stellate cell activation, and progressive fibrosis. In primary, cultured hepatocytes and precision‐cut liver slices we demonstrated increased gene expression of profibrogenic markers after lipid and/or ammonia exposure. Lowering of ammonia with the ammonia scavenger ornithine phenylacetate prevented hepatocyte cell death and significantly reduced the development of fibrosis both in vitro in the liver slices and in vivo in a rodent model. The prevention of fibrosis in the rodent model was associated with restoration of urea cycle enzyme activity and function, reduced hepatic ammonia, and markers of inflammation.
Conclusions
The results of this study suggest that hepatic steatosis results in hyperammonemia, which is associated with progression of hepatic fibrosis. Reduction of ammonia levels prevented progression of fibrosis, providing a potential treatment for NAFLD.
The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of ...integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α5β1, which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell–cell fusion. Our results showed that α5β1 does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell–cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α5β1 inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α5 cytoplasmic tail (CT). We detected a direct interaction between α5β1 and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α5β1 homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1β. These effects can be attenuated by the loss of α5 expression or inhibition of the α5 CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α5 CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α5β1 and suggest potential targets for antiviral treatment.
The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of ...integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α
β
, which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell-cell fusion. Our results showed that α
β
does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell-cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α
β
inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α
cytoplasmic tail (CT). We detected a direct interaction between α
β
and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α
β
homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1β. These effects can be attenuated by the loss of α
expression or inhibition of the α
CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α
CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α
β
and suggest potential targets for antiviral treatment.
The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in ...subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-β-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc may be a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, suggesting a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.
Precision cut liver slices (PCLSs) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two‐dimensional mono‐ or ...co‐cultures. The aim of this study was to develop a bioreactor system to increase the healthy life span of PCLSs and model fibrogenesis. PCLSs were generated from normal rat or human liver, or fibrotic rat liver, and cultured in our bioreactor. PCLS function was quantified by albumin enzyme‐linked immunosorbent assay (ELISA). Fibrosis was induced in PCLSs by transforming growth factor beta 1 (TGFβ1) and platelet‐derived growth factor (PDGFββ) stimulation ± therapy. Fibrosis was assessed by gene expression, picrosirius red, and α‐smooth muscle actin staining, hydroxyproline assay, and soluble ELISAs. Bioreactor‐cultured PCLSs are viable, maintaining tissue structure, metabolic activity, and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell‐cultured PCLSs rapidly deteriorate, and albumin secretion is significantly impaired by 48 hours. TGFβ1/PDGFββ stimulation of rat or human PCLSs induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts, and histological fibrosis. Fibrogenesis slowly progresses over 6 days in cultured fibrotic rat PCLSs without exogenous challenge. Activin receptor‐like kinase 5 (Alk5) inhibitor (Alk5i), nintedanib, and obeticholic acid therapy limited fibrogenesis in TGFβ1/PDGFββ‐stimulated PCLSs, and Alk5i blunted progression of fibrosis in fibrotic PCLS. Conclusion: We describe a bioreactor technology that maintains functional PCLS cultures for 6 days. Bioreactor‐cultured PCLSs can be successfully used to model fibrogenesis and demonstrate efficacy of antifibrotic therapies.
Silicon and boron share many similarities, both chemically and biochemically, including having similar effects on bone, although their mechanisms of action are not known. Here we compared the loading ...of silicon and boron into bone, their localization and how they are influenced by age (growth & development), to obtain further clues as to the biological effects of these elements and, especially, to see if they behave the same or not. Bone samples were obtained from two different studies where female Sprague Dawley rats had been maintained on a normal maintenance diet for up to 43 weeks. Total bone elemental levels were determined by ICP-OES following microwave assisted acid digestion. Silicon and boron levels in the decalcified bones (i.e. the collagen fraction) were also investigated. Silicon and boron showed marked differences in loading and in their localization in bone. Highest silicon and lowest boron concentrations were found in the under-mineralized bone of younger rats and lowest silicon and highest boron concentrations were found in the fully mineralized bone of the adult rat. Overall, however total bone silicon content increased with age, as did boron content, the latter mirroring the increase in calcium (mineral) content of bone. However, whereas silicon showed equal distribution in the collagen and mineral fractions of bone, boron was exclusively localized in the mineral fraction. These findings confirm the reported association between silicon and collagen, especially at the early stages of bone mineralization, and show that boron is associated with the bone mineral but not connective tissues. These data suggest that silicon and boron have different biological roles and that one is unlikely, therefore, to substitute for the other, or at least boron would not substitute for Si in the connective tissues. Finally, we noted that silicon levels in the mineral fraction varied greatly between the two studies, suggesting that one or more nutritional factor(s) may influence the loading of Si into the mineral fraction of bone. This and the nature of the interaction between Si and collagen deserve further attention.
This investigation is focused on studying the relationship between channel entrenchment and subparallel salt diapir movement in the Levant Basin, located off the western coast of Lebanon. The main ...objective was to determine the chronological order of events and the specific causes that led to their formation. This study utilized 3D seismic data collected along the eastern margin of the basin, enabling a detailed evaluation of individual salt and channel structures. Extensive faulting oriented in a north-south direction, resulting from gravity sliding, was observed and identified as the primary mechanism for salt growth. However, the close spatial correlation between the placement of turbidite channels and diapirs necessitated a comprehensive understanding of these systems in conjunction.The findings may suggest that certain diapirs (referred to as D2, D3, and D4) in the area differed from others and began rising not as a response to extensional faulting, but rather due to the displacement of sediment caused by channel entrenchment, leading to differential loading. In these instances, the channels formed first, and the upward movement of salt occurred as a direct response to the erosion and sediment displacement caused by the channels. Further research and analysis of seismic data are required to fully validate this theory.
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