Molecular electron microscopy approaches to elucidating the mechanisms of protein fibrillogenesis

Electron microscopy (EM) has played a central role in our current understanding of the mechanisms underlying the pathogenesis of several amyloid diseases, including Alzheimer's disease, Parkinson's disease, and prion diseases. In this chapter, we discuss the application of various EM techniques to monitor and characterize quaternary structural changes during amyloid fibril formation in vitro and the potential of extending some of these techniques to characterizing ex vivo material. In particular, we would like to bring to the attention of the reader two very powerful molecular EM techniques that remain under utilized by researchers in the amyloid community, namely scanning transmission electron microscopy and single particle molecular averaging EM. An overview of the strength and limitations of these techniques as tools for elucidating the structural basis of amyloid fibril formation will be presented

The Location of the Carboxy-Terminal Region of γ Chains in Fibrinogen and Fibrin D Domains

Elongated fibrinogen molecules are comprised of two outer “D” domains, each connected through a “coiled-coil” region to the central “E” domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of γ chains in each D domain (the γXL site) by incorporating intermolecular ɛ-(γ-glutamyl)lysine bonds between amine donor γ406 lysine of one γ chain and a glutamine acceptor at γ398 or γ399 of another. Several lines of evidence show that crosslinked γ chains extend “transversely” between the strands of each fibril, but other data suggest instead that crosslinked γ chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the γXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled γ398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal γ chains in fibrinogen or fibrin are oriented toward the central domain and indicate that γXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking

A retrospective analysis of 20,178 adult neurological infection admissions to United Kingdom critical care units from 2001 to 2020

BACKGROUND: Neurological infection is an important cause of critical illness, yet little is known on the epidemiology of neurological infections requiring critical care. METHODS: We analysed data on all adults with proven or probable neurological infection admitted to UK (NHS) critical care units between 2001 and 2020 reported to the Intensive Care National Audit and Research Centre. Diagnoses, physiological variables, organ support and clinical outcomes were analysed over the whole period, and for consecutive 5-year intervals within it. Predictors of in-hospital mortality were identified using a backward stepwise regression model. RESULTS: We identified 20,178 critical care admissions for neurological infection. Encephalitis was the most frequent presentation to critical care, comprising 6725 (33.3%) of 20,178 cases. Meningitis- bacterial, viral or unspecified cases - accounted for 10,056 (49.8%) of cases. In-hospital mortality was high, at 3945/19,765 (20.0%) overall. Over the four consecutive 5-year periods, there were trends towards higher Glasgow Coma Scale scores on admission, longer critical care admissions (from median 4 [IQR 2-8] to 5 days [IQR 2-10]), and reduced in-hospital mortality (from 24.9 to 18.1%). We identified 12 independent predictors of in-hospital death which when used together showed good discrimination between patients who die and those who survive (AUC = 0.79). CONCLUSIONS: Admissions with neurological infection to UK critical care services are increasing and the mortality, although improving, remains high. To further improve outcomes from severe neurological infection, novel approaches to the evaluation of risk stratification, monitoring and management strategies are required

Evidence for a Second Type of Fibril Branch Point in Fibrin Polymer Networks, the Trimolecular Junction

Fibrin molecules polymerize to double-stranded fibrils by intermolecular end-to-middle domain pairing of complementary polymerization sites, accompanied by fibril branching to form a clot network. Mass/length measurements on scanning transmission electron microscopic images of fibrils comprising branch points showed two types of junctions. Tetramolecular junctions occur when two fibrils converge, creating a third branch with twice the mass/length of its constituents. Newly recognized trimolecular junctions have three fibril branches of equal mass/length, and occur when an extraneous fibrin molecule initiates branching in a propagating fibril by bridging across two unpaired complementary polymerization sites. When trimolecular junctions predominate, clots exhibit nearly perfect elasticity

The Rescue of Fannie Mae and Freddie Mac

Staff Report including the following:- Describes and evaluates the measures taken by the U.S. government to rescue Fannie Mae and Freddie Mac in September 2008. - Outlines the business model of these two firms and their role in the U.S. housing finance system. - The sources of financial distress that the firms experienced and the events that ultimately led the government to take action. - Describes the various resolution options available to policymakers. - Evaluates the success of the choice of conservatorship and other actions taken

Exposure to war and conflict: The individual and inherited epigenetic effects on health, with a focus on post-traumatic stress disorder

War and conflict are global phenomena, identified as stress-inducing triggers for epigenetic modifications. In this state-of-the-science narrative review based on systematic principles, we summarise existing data to explore the outcomes of these exposures especially in veterans and show that they may result in an increased likelihood of developing gastrointestinal, auditory, metabolic and circadian issues, as well as post-traumatic stress disorder (PTSD). We also note that, despite a potential "healthy soldier effect", both veterans and civilians with PTSD exhibit the altered DNA methylation status in hypothalamic-pituitary-adrenal (HPA) axis regulatory genes such as NR3C1. Genes associated with sleep (PAX8; LHX1) are seen to be differentially methylated in veterans. A limited number of studies also revealed hereditary effects of war exposure across groups: decreased cortisol levels and a heightened (sex-linked) mortality risk in offspring. Future large-scale studies further identifying the heritable risks of war, as well as any potential differences between military and civilian populations, would be valuable to inform future healthcare directives