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Background and Aims: The risk for infection in alcohol-related liver disease (ALD) has rarely been investigated at a population level, nor if the underlying liver histopathology is associated with infection risk. We examined the rate of hospital-based infections in a nationwide cohort of biopsy-proven ALD, and the subsequent risk of death. Methods: Population-based cohort study in Sweden comparing 4028 individuals with an international classification of disease (ICD) code for ALD and a liver biopsy from 1969 to 2017 with 19,296 matched general population individuals. Swedish national registers were used to ascertain incident infections in secondary or tertiary care and subsequent mortality until 2019. We used Cox regression, adjusted for sex, age, education, country of birth, diabetes, and number of hospitalizations in the year preceding liver biopsy date, to estimate hazard ratios (HRs) in ALD and histopathological subgroups compared to reference individuals. Results: Median age at ALD diagnosis was 59 years, 65% were men and 59% had cirrhosis at baseline. Infections were more common in patients with ALD (84 cases/1000 person-years [PY]) compared to reference individuals (29/1000 PYs; adjusted hazard ratio [aHR] 3.06, 95% CI = 2.85–3.29). This excess risk corresponded to one additional infection per 18 ALD patients each year. The rate of infections was particularly high in individuals with cirrhosis (aHR = 3.46) and in those with decompensation (aHR = 5.20). Restricting our data to those with an infection, ALD (aHR = 3.63, 95%CI = 3.36–3.93), and especially ALD cirrhosis (aHR = 4.31, 95%CI = 3.89–4.78) were linked to subsequent death. Conclusions: Individuals with biopsy-proven ALD have a three-fold increased rate of infections compared with the general population. The risk of death after an infection is also considerably higher in individuals with ALD.

Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full-length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter-species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model.

There are around 30 human diseases associated with protein misfolding and amyloid formation, each one caused by a certain protein or peptide. Many of these diseases are lethal and together they pose an enormous burden to society. The prion protein has attracted particular interest as being shown to be the pathogenic agent in transmissible diseases such as kuru, Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Whether similar transmission could occur also in other amyloidoses such as Alzheimer's disease, Parkinson's disease and serum amyloid A amyloidosis is a matter of intense research and debate. Furthermore, it has been suggested that novel biomaterials such as artificial spider silk are potentially amyloidogenic. Here, we provide a brief introduction to amyloid, prions and other proteins involved in amyloid disease and review recent evidence for their potential transmission. We discuss the similarities and differences between amyloid and silk, as well as the potential hazards associated with protein-based biomaterials.

The transmembrane protease complex γ-secretase is a key enzyme in Alzheimer disease pathogenesis as it liberates the neurotoxic amyloid β-peptide (Aβ); however, the mechanism of regulation of its activity in various cell types and subcellular compartments is largely unknown. Several γ-secretase inhibitors have been developed, but none have been released due to side-effects that appear to arise from reduced processing of Notch, one of many γ-secretase substrates. Hence, it is desirable to specifically inhibit Aβ production. In our previous studies, we have identified several γ-secretase-associated proteins (GSAPs) from brain, which affect Aβ production without having any major effects on Notch processing. In the present study using detergent-resistant membranes prepared from brain, we have identified four GSAPs that affect Aβ production to a greater extent than Notch processing. We evaluated the interaction between GSAPs and γ-secretase in various cell types and their mRNA expression in various human organs. Using an in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably greater interaction with γ-secretase in neurons than in human embryonic kidney cells stably over-expressing APP, and showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein-protein interactions in relevant cell types, and suggests that reducing Aβ production by interfering with brain- or neuron-specific γ-secretase/GSAP interactions may reduce the risk of unwanted side-effects associated with treatment of Alzheimer disease.

It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post-mortem brains by laser capture microdissection. By using 18O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat-shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron-specific analysis. Hence, we conclude that cell-specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis.

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ(40) and Aβ(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ(42) accompanied by increased intracellular Aβ(42) /Aβ(40) ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ(42) /Aβ(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ(42) /Aβ(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.

γ-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. Little is known about how the substrates are selected by γ-secretase, but it has been suggested that γ-secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ-secretase and regulates the processing of APP-derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ-secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ-secretase inhibitor coupled to biotin via a long linker and a S-S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC-MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ-secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid beta-peptide (Abeta) in senile plaques. Two major forms of Abeta are produced, 40 (Abeta40) and 42 (Abeta42) residues long. The most abundant form of Abeta is Abeta40, while Abeta42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C-terminal seems to be very important for the oligomerization and neurotoxicity of the Abeta peptide. Here we investigated which Abeta species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Abeta standard peptides. Cyanogen bromide was used to generate C-terminal Abeta fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Abeta43, to be more frequent than Abeta40. No variants longer than Abeta43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Abeta1-43 polymerizes rapidly and we suggest that this variant may be of importance for AD.

A regular nest spacing of Golden Eagle pairs was found in every carefully searched area within the species range in Sweden. The spacing regularity of nests used by pairs in forests (mostly tree-nesting) was similar to that in mountain areas (cliff-nesting), indicating that regular nest spacing is affected more by spacing behaviour than by shortage of nest sites. Distances to nearest neighbours were shorter in the mountain region (mean distance 102 km) than in the forests (170) of northern Sweden. Densities of the most important prey species for Golden Eagle were apparently higher in the mountains than in the forests. Differences in topography and habitat diversity between the two regions may also have contributed to the difference in breeding density.

Several lines of evidence suggest that polymerization of the amyloid β-peptide (Aβ) into amyloid plaques is a pathogenic event in Alzheimer’s disease (AD). Aβ is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by β-secretase and γ-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. γ-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that γ-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied γ-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in γ-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the γ-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the γ-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in γ-secretase activity, suggesting that DRMs from fresh rat brain may be useful for γ-secretase activity studies. Finally, confocal microscopy showed co-localization of γ-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active γ-secretase complex is localized to lipid rafts in human brain.

Summary The missense mutation of cysteine 2362 to a phenylalanine in von Willebrand factor (VWF) has been detected in several Italian families with autosomal recessive, severe von Willebrand disease. We investigated how this amino acid change in VWF may lead to a predominantly quantitative defect. This mutation was studied in vitro by transient expression of the full-length mutant VWF-C2362F protein and in vivo by analysis of plasma VWF after infusion of 1-deamino-8-d-arginine vasopressin (DDAVP) in a patient homozygous for this mutation. Single transfections of pSVHVWF-C2362F and cotransfections of mutant and wild-type constructs resulted in 8% and 50% VWF antigen, respectively, in conditioned medium. These reduced levels are in accordance with observations in homozygous and heterozygous carriers of the mutation. In addition, VWF-C2362F was retained intracellularly. Similar results were obtained for C2362F and C2362A. After infusion of DDAVP in a homozygous patient, a twofold decrease in half-life of plasma VWF-C2362F was observed. This was not explained by increased susceptibility of recombinant VWF-C2362F to ADAMTS13. It was concluded that VWF-C2362F causes reduced VWF plasma levels due to impaired secretion and intracellular retention. Furthermore, it is the loss of cysteine 2362 rather than the introduction of the bulky amino acid side chain that causes these effects.

Recently, a novel plaque-associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimer's disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC-P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimer's disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid β-peptide (Aβ). Here, we examined the effects of CLAC on Aβ fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Aβ fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Aβ was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Aβ fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T-binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Aβ fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Aβ fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.

Background:Alzheimer’s disease (AD) is characterized by the presence of amyloid beta (Ab) plaques and neurofibrillary tangles (NFT). SPPL2b is a novel enzyme that can process TNFa and BRI2, substrates involved in Ab plaque and NFT formation. Since changes in BRI2 and TNFa were previously identified in early stages of AD, we have now extensively characterized SPPL2b in human brain tissue from controls and AD patients at different stages of the disease. We also investigated the relation of SPPL2b with important pathological AD hallmarks including amyloid plaques, NFTs, ER stress and ubiquitin. Methods: SPPL2b expression levels were quantified in post-mortem human homogenates from AD patients (n1⁄414) and controls (n1⁄413) by western blot. SPPL2b immunostaining was evaluated on paraffin sections from AD patients (n 1⁄4 12) and age-matched controls (n1⁄415). Results: SPPL2b levels were 10-fold increased (p < 0.0001) in AD hippocampus compared to non-demented controls. SPPL2b was strongly correlated (r 1⁄4 0.785, p < 0.0001) to Braak stages showing a significant increased from Braak III to IV, stages in which cognitive impairment starts. Strong SPPL2b immunoreactivity in the hippocampus was observed in early AD stages (p

A variety of synchrotron radiation techniques like soft X-ray magnetic circular dichroism, near-edge as well as extended X-ray absorption fine structure, and synchrotron radiation X-ray diffraction have been used to characterise the crystal and electronic structure as well as the magnetic properties of superparamagnetic nanocrystalline samples with the nominal composition γ-Fe2O3. The results are compared with data from bulk reference samples like Fe3O4, Fe3—δO4, α-Fe2O3, and FeO.

We have used two-dimensional electrophoresis (2-DE) to analyze changes in protein expression profiles during a microbial cultivation process on an industrial scale. An Escherichia coli strain W31 10 containing the gene for recombinant human growth hormone production was used. Samples were taken at time intervals ranging from fast to slow growth rate (late growth phase at high cell density/starvation) and 2-DE analysis combined with image analysis using the PDQuest software showed significant alterations in expression levels of a number of proteins. Twenty-four protein spots were identified using a combination of matching with SWISS-2DPAGE E. coli map, N-terminal sequence analysis and mass spectrometry matrix-assisted laser desorption/ionization (MALDI). Two of the most abundant proteins expressed at late growth phase (pI 5.4/28 kDa and pI 5.5/28 kDa) were subjected to N-terminal sequence analysis after electrotransfer of the proteins from a preparative 2-DE gel to polyvinylidene difluoride (PVDF) membrane. Sequence tags of five amino acids in combination with approximate pI and Mr identified both proteins as deoxyribose phosphate aldolase (gene name deoC). In addition, both spots were subjected to tryptic in-gel digestion and analyzed using MALDI. Peptide mass fingerprints from both spots showed similar MALDI spectra and 10 of 10 tryptic fragments confirmed the identity as deoC. The identification of the acidic variant of deoC on 2-DE gels and the observation of this variant as induced during late growth phase is novel.