Practice patterns and clinical outcomes in acute appendicitis differ in the elderly patient

Background: Appendicitis is the most frequent global abdominal surgical emergency. An ageing population, who often exhibit atypical symptoms and delayed presentations, challenge conventional diagnostic and treatment paradigms. Objectives: This study aims to delineate disparities in presentation, management, and outcomes between elderly patients and younger adults suffering from acute appendicitis. Methods: This subgroup analysis forms part of ESTES SnapAppy, a time-bound multi-center prospective, observational cohort study. It includes patients aged 15 years and above who underwent laparoscopic appendectomy during a defined 90-day observational period across multiple centers. Statistical comparisons were performed using appropriate tests with significance set at p < 0.05. Results: The study cohort comprised 521 elderly patients (≥65 years) and 4,092 younger adults (18–64 years). Elderly patients presented later (mean duration of symptoms: 7.88 vs. 3.56 days; p < 0.001) and frequently required computed tomography (CT) scans for diagnosis (86.1% vs. 54.0%; p < 0.001). The incidence of complicated appendicitis was higher in the elderly (46.7% vs. 20.7%; p < 0.001). Delays in surgical intervention were notable in the elderly (85.0% operated within 24 h vs. 88.7%; p = 0.018), with longer operative times (71.1 vs. 60.3 min; p < 0.001). Postoperative complications were significantly higher in the elderly (27.9% vs. 12.9%; p < 0.001), including severe complications (6.9% vs. 2.4%; p < 0.001) and prolonged hospital stays (7.9 vs. 3.6 days; p < 0.001). Conclusions: Our findings highlight significant differences in the clinical course and outcomes of acute appendicitis in the elderly compared to younger patients, suggesting a need for age-adapted diagnostic pathways and treatment strategies to improve outcomes in this vulnerable population

Evaluation of Three Principally Different Intact Protein Prefractionation Methods for Plasma Biomarker Discovery

The aim of this study was to evaluate three principally different top-down protein prefractionation methods for plasma: high-abundance protein depletion, size fractionation and peptide ligand affinity beads, focusing in particular on compatibility with downstream analysis, reproducibility and analytical depth. Our data clearly demonstrates the benefit of high-abundance protein depletion. However, MS/MS analysis of the proteins eluted from the high-abundance protein depletion column show that more proteins than aimed for are removed and, in addition, that the depletion efficacy varies between the different high-abundance proteins. Although a smaller number of proteins were identified per fraction using the peptide ligand affinity beads, this technique showed to be both robust and versatile. Size fractionation, as performed in this study, focusing on the low molecular weight proteome using a combination of gel filtration chromatography and molecular weight cutoff filters, showed limitations in the molecular weight cutoff precision leading detection of high molecular weight proteins and, in the case of the cutoff filters, high variability. GeLC-MS/MS analysis of the fractionation methods in combination with pathway analysis demonstrates that increased fractionation primarily leads to high proteome coverage of pathways related to biological functions of plasma, such as acute phase reaction, complement cascade and coagulation. Further, the prefractionation methods in this study induces limited effect on the proportion of tissue proteins detected, thereby highlighting the importance of extensive or targeted downstream fractionation

Abstract 18975: Increased Arterial Blood Pressure and Vascular Fibrosis/remodeling in Mice Lacking Salt-inducible Kinase 1 (sik1)

Arterial fibrosis is one of the main underlying causes of vascular stiffness and is increasingly recognized as an important prognostic factor in the development of high blood pressure (BP), yet its physiophatology is not fully known. Therefore, elucidating the precise mechanism leading to arterial fibrosis will help improving current anti-hypertensive therapies. We previously described in human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene, affecting the activity of the protein, which is associated with hypertension (lower SIK1 activity is associated with higher BP). Also, lower SIK1 expression induces the expression of transcription factors commonly involved in fibrosis. Therefore, we hypothesized that SIK1 can regulate BP by reducing vascular fibrosis and hence vascular stiffness. To test this, BP and aortic wall structure/composition was evaluated in sik1 -/- and sik1 +/+ mice challenged with a chronic high salt (HS) or normal salt diet (NS). The results showed that the s ik1 -/- mice challenged with HS developed high BP and presented signs of cardiac hypertrophy (increased left ventricle wall thickness and enhanced expression of hypertrophic genes) when compared to their counterparts on NS. On the contrary, BP and cardiac parameters remained unaffected in the sik1 +/+ mice under HS when compared to NS fed mice. Immunohistochemistry analysis revealed increased intima/media thickness and collagen deposition in the aorta of the sik1 -/- vs. sik1 +/+ mice under NS and before becoming hypertensive. Also, qPCR analysis of the aorta of the sik1 -/- mice showed increased expression of other extracellular matrix (ECM) components such as fibronectin and laminin-β1. In addition, the transient knock-down of the SIK1 gene in vascular smooth muscle cell (VSMC) cultures using siRNAs showed a similar increase in the expression of collagen as well as other ECM genes. The present study shows that SIK1 activity is necessary to prevent the development of hypertension, possibly through an anti-fibrotic mechanism regulating vascular wall structure and function. Therefore, SIK1 could become a novel therapeutic target in the treatment of hypertension and other vascular fibrosis-associated diseases

Gene Cluster of Rhodothermus marinus High-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, a caa(3)-Type Oxidase Belonging to the Superfamily of Heme-Copper Oxidases

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases

Study on stomach content of fish to update databases and analyse possible changes in diet or food web interactions

Fish stomach content is a compelling data source, as stomachs can provide information about diet of predators, distribution of prey fish, predator-prey preference or changes in diet over time. For instance, stomach samples have historically been used to inform the natural mortality of stock assessments in the Baltic and North Seas. Since stomach samples tend to vary substantially over time, due to differences in biomass of predator and prey, time series must be constructed to provide critical information on food web interactions in time and space. In this project, we aimed (1) to analyse new stomach samples, from the North and Baltic Seas; (2) to continue historical time series; (3) to update and create an online ICES database for the broader community to explore new and old data in conjunction; and (4) to perform preliminary analysis on the newly added data. In total, 10 087 new stomachs will be provided from the two case study areas. We analysed and uploaded 5 512 new stomach samples so far. Additionally, 27 744 historical Baltic cod stomach data have been uploaded. The analysis provided here show that the predation on saduria and sprat by cod in the Baltic Sea has changed over the last 30 years. In the North Sea, the new samples consisted primarily of unidentified matter, benthic food and crustaceans, whereas the historical samples contain a larger number of commercial fish

Macromolecular Crowding Fails To Fold a Globular Protein in Cells

Proteins perform their function in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules will stabilize globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells with a variant of protein L, a 7-kDa globular protein with seven lysine residues replaced by glutamic acids. Eighty-four percent of the variant molecules populate the denatured state in dilute buffer at room temperature, compared to 0.1% for the wild-type protein. We then used in-cell nuclear magnetic resonance spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (~1 kcal/mol) free energy deficit. The data are consistent with the idea that non-specific interactions between cytoplasmic components can overcome the excluded volume effect. Evidence for these interactions is provided by the observation that adding simple salts folds the variant in dilute solution, but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with other studies of protein stability in cells, and suggest that stabilizing excluded volume effects, which must be present under crowded conditions, can be ameliorated by non-specific interactions between cytoplasmic components

Intricate Role of Water in Proton Transport through Cytochrome c

Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O(2) to H(2)O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from R. sphaeroides. Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules half way up the D pathway, were replaced by more bulky hydrophobic residues (Ser200Ile, Ser200Val/Ser201Val and Ser200Val/Ser201Tyr) in order to query the effects of changing the local structure on enzyme activity as well as proton uptake, release and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcOs were lowered, both the Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P(R) → F and F → O transitions. Furthermore, the P(R) → F transition is shown to be essentially pH independent up to pH 12 (i.e. the apparent pK(a) of Glu286 is increased from 9.4. by at least 3 pK(a) units) in the S200V/S201V mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO