Intermolecular Structure Determination of Amyloid Fibrils with 2 Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range [superscript 13]C–[superscript 13]C correlation spectra and the third based on the identification of intermolecular interactions in [superscript 13]C–[superscript 15]N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient [superscript 13]C–[superscript 13]C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The [superscript 13]C–[superscript 13]C experiments are corroborated by [superscript 15]N–[superscript 13]C correlation spectra obtained from a mixed [[superscript 15]N,[superscript 12]C]/[[superscript 14]N,[superscript 13]C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular [superscript 15]N–[superscript 13]C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.National Institutes of Health (U.S.) (Grant EB-003151)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026

B Cell apoptosis in the central nervous system in experimental autoimmune Encephalomyelitis: Roles of B Cell CD95, CD95L and Bcl-2 expression

The role and fate of B cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) are unknown. Using enzyme-linked immunospot assays we now show that B cells reactive to myelin basic protein (MBP) accumulate in the CNS of Lewis rats with acute EAE induced by immunization with MBP and adjuvants. We also report that B cells are eliminated from the CNS by apoptosis during spontaneous recovery from this disease. Apoptotic B cells were identified by flow cytometry of inflammatory cells extracted from the spinal cord and by histological sections of the spinal cord using light and electron microscopic immunocytochemistry. B cell apoptosis occurred preferentially in the CNS rather than in the peripheral lymphoid organs and was maximal just prior to the onset of spontaneous clinical recovery. Three colour flow cytometry indicated that B cells expressing CD95 (Fas) or CD95 ligand (CD95L) were highly vulnerable to apoptosis, whereas B cells expressing Bcl-2 were relatively protected from apoptosis. We propose that B cells are eliminated from the CNS by the interaction of CD95L and CD95 on the same B cell and that this contributes to the spontaneous resolution of CNS inflammation and clinical recovery in acute EAE

Interventions to improve emergency department use for mental health reasons:Protocol for a mixed-methods systematic review

BACKGROUND: Healthcare resources are limited and unnecessary, and inappropriate emergency department use is now a highly visible healthcare priority. Individuals visiting the emergency department for mental health-related reasons are often amongst the most frequent presenters. In response, researchers and clinicians have created interventions to streamline emergency department use and several primary studies describe the effects of these interventions. Yet, no consensus exists on the optimal approach, and information on the quality of development, effectiveness, acceptability, and economic considerations is hard to find. The purpose of this study is to systematically review interventions designed to improve appropriate use of the emergency department for mental health reasons. METHOD: A mixed-method systematic review using Joanna Briggs Methodology. Search combining electronic databases (EMBASE, MEDLINE, PsycINFO, CINAHL, HealthSTAR, PROQUEST, Cumulative Index to Nursing and Allied Health) and secondary searches (grey literature and hand search with consultation). Two independent reviewers will screen titles and abstracts using predetermined eligibility criteria and a third reviewer will resolve conflicts. Full texts will also be screened by two independent reviews and conflicts resolved in a consensus meeting with a third reviewer. A pilot-tested data extraction form will be used to retrieve data relevant to the study objectives. We will assess the quality and of all included studies. Data describing interventions will be summarized using logic models and reported narratively. Quality of development will be assessed using the Oxford Implementation Index. For data on intervention effectiveness, we will assess statistical heterogeneity and conduct a meta-analysis using a random effects method, if appropriate. For interventions that cannot be pooled, we will report outcomes narratively and descriptively. Qualitative data on acceptability will be synthesized using meta-aggregation and an economic evaluation of interventions will be done. The reporting of this protocol follows the PRISMA-P statement. DISCUSSION: Using a combined systematic review methodology and integrated knowledge translation plan, the project will provide decision makers with concrete evidence to support the implementation and evaluation of interventions to improve emergency department use for mental health reasons. These interventions reflect widespread priorities in the area of mental health care. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42018087430

Amyloid as a Depot for the Formulation of Long-Acting Drugs

Amyloids are highly organized protein aggregates that are associated with both neurodegenerative diseases such as Alzheimer disease and benign functions like skin pigmentation. Amyloids self-polymerize in a nucleation-dependent manner by recruiting their soluble protein/peptide counterpart and are stable against harsh physical, chemical, and biochemical conditions. These extraordinary properties make amyloids attractive for applications in nanotechnology. Here, we suggest the use of amyloids in the formulation of long-acting drugs. It is our rationale that amyloids have the properties required of a long-acting drug because they are stable depots that guarantee a controlled release of the active peptide drug from the amyloid termini. This concept is tested with a family of short- and long-acting analogs of gonadotropin-releasing hormone (GnRH), and it is shown that amyloids thereof can act as a source for the sustained release of biologically active peptides

Low-penetrance alleles predisposing to sporadic colorectal cancers: a French case-controlled genetic association study

<p>Abstract</p> <p>Background</p> <p>Sporadic colorectal cancers (CRC) are multifactorial diseases resulting from the combined effects of numerous genetic, environmental and behavioral risk factors. Genetic association studies have suggested low-penetrance alleles of extremely varied genes to be involved in susceptibility to CRC in Caucasian populations.</p> <p>Methods</p> <p>Through a large genetic association study based on 1023 patients with sporadic CRC and 1121 controls, we tested a panel of these low-penetrance alleles to find out whether they could determine "genotypic profiles" at risk for CRC among individuals of the French population. We examined 52 polymorphisms of 35 genes – drawn from inflammation, xenobiotic detoxification, one-carbon, insulin signaling, and DNA repair pathways – for their possible contribution to colorectal carcinogenesis. The risk of cancer associated with these polymorphisms was assessed by calculation of odds ratios (OR) using multivariate analyses and logistic regression.</p> <p>Results</p> <p>Whereas all these polymorphisms had previously been found to be associated with CRC risk, especially in Caucasian populations, we were able to replicate the association for only five of them. Three SNPs were shown to increase CRC risk: <it>PTGS1 </it>c.639C>A (p.Gly213Gly), <it>IL8 </it>c.-352T>A, and <it>MTHFR </it>c.1286A>C (p.Ala429Glu). On the contrary, two other SNPs, <it>PLA2G2A </it>c.435+230C>T and <it>PPARG </it>c.1431C>T (p.His477His), were associated with a decrease in CRC risk. Further analyses highlighted genotypic combinations having a greater predisposing effect on CRC (OR 1.97, 95%CI 1.31–2.97, p = 0.0009) than the allelic variants that were examined separately.</p> <p>Conclusion</p> <p>The identification of CRC-predisposing combinations, composed of alleles <it>PTGS1 </it>c.639A, <it>PLA2G2A </it>c.435+230C, <it>PPARG </it>c.1431C, <it>IL8 </it>c.-352A, and <it>MTHFR </it>c.1286C, highlights the importance of inflammatory processes in susceptibility to sporadic CRC, as well as a possible crosstalk between inflammation and one-carbon pathways.</p

Alveolar Hemorrhage in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: Results of an International Randomized Controlled Trial (PEXIVAS)

Rationale: Diffuse alveolar hemorrhage (DAH) is a life-threatening manifestation of antineutrophil cytoplasmic antibody–associated vasculitis (AAV). The PEXIVAS (Plasma Exchange and Glucocorticoids in Severe Antineutrophil Cytoplasmic Antibody–Associated Vasculitis) (NCT00987389) trial was the largest in AAV and the first to enroll participants with DAH requiring mechanical ventilation. Objectives: Evaluate characteristics, treatment effects, and outcomes for patients with AAV with and without DAH. Methods: PEXIVAS randomized 704 participants to plasma exchange (PLEX) or no-PLEX and reduced or standard-dose glucocorticoids (GC). DAH status was defined at enrollment as no-DAH, nonsevere, or severe (room air oxygen saturation of ⩽ 85% as measured by pulse oximetry, or use of mechanical ventilation). Measurements and Main Results: At enrollment, 191 (27.1%) participants had DAH (61 severe, including 29 ventilated) and were younger, more frequently relapsing, PR3 (proteinase 3)-ANCA positive, and had lower serum creatinine but were more frequently dialyzed than participants without DAH (n = 513; 72.9%). Among those with DAH, 8/95 (8.4%) receiving PLEX died within 1 year versus 15/96 (15.6%) with no-PLEX (hazard ratio, 0.52; confidence interval [CI], 0.21–1.24), whereas 13/96 (13.5%) receiving reduced GC died versus 10/95 (10.5%) with standard GC (hazard ratio, 1.33; CI, 0.57–3.13). When ventilated, ventilator-free days were similar with PLEX versus no-PLEX (medians, 25; interquartile range [IQR], 22–26 vs. 22–27) and fewer with reduced GC (median, 23; IQR, 20–25) versus standard GC (median, 26; IQR, 25–28). Treatment effects on mortality did not vary by presence or severity of DAH. Overall, 23/191 (12.0%) with DAH died within 1 year versus 34/513 (6.6%) without DAH. End-stage kidney disease and serious infections did not differ by DAH status or treatments. Conclusions: Patients with AAV and DAH differ from those without DAH in multiple ways. Further data are required to confirm or refute a benefit of PLEX or GC dosing on mortality.publishedVersio

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Contemporary enclosures and resistance : women in struggle in Ireland, Scotland and Brazilian Amazon

This paper traces a discussion between women to the fore of struggles against contemporary enclosures in Ireland, Scotland and Brazil. The forms of resource capitalisation; wildcat and corporate mining, 'rewilding' schemes and 'green' energy infrastructures, lead to different material effects and subjective experiences for those who inhabit the zones of intrusion, expansion and encroachment. They lead also to differentiated motivations for -and forms of- resistance by individuals and communities whose histories, cultures and livelihoods interdepend with the territories in which new movements are born or reborn. A discussion with women taking and remaking collective actions in their particular places reveals distinctive challenges, strategies and successes in the face of acute power inequalities, and highlight capital-labour antagonisms that lie outside of conventional readings of industrial and worker organising. The interconnectedness of the experiences across disparate territories that are shared here helps to makes visible the current wave of capitalist enclosure and the effects on rural, island and forest communities. Furthermore, the dialogue unveils the effective and positioned methods by which women make and remake communities of resistance on the grounds on which they tread, and create recognition, affection, solidarity and commitment with others across territories in ways that, as the paper concludes, point to revitalisation of a much needed global solidarity, from below

Dynamic Context-Aware Representation for Semantic Alignment in Large Language Models

The capacity of modern neural networks to generate human-like text is accompanied by ongoing challenges in maintaining semantic coherence, particularly across dynamically evolving contexts in long-form text generation. Dynamic Context-Aware Representation (DCAR) addresses this limitation through a novel mechanism that enables continuous recalibration of context vectors, ensuring more accurate semantic alignment throughout the generation process. Through the integration of a dynamic adjustment layer within a state-of-the-art transformer-based LLM, significant improvements were observed in perplexity, BLEU score, and semantic coherence, especially in cases where traditional static embeddings fall short. Experimental results validated the effectiveness of DCAR in managing context shifts fluidly, with minimal computational overhead, providing a flexible yet powerful solution for enhancing the performance of LLMs in handling complex, multi-turn conversations and extended text. The findings suggest that the application of DCAR offers a substantial leap in both the accuracy and adaptability of LLM architectures, enabling more precise and consistent generation of language across a variety of domains. These advancements position DCAR as a transformative step in overcoming the inherent limitations of static context representations in language models, pushing the boundaries of contextual comprehension and generation in neural networks