Successful proteome analyses of highly dilute samples are strongly dependent on optimized workflows considering especially sample preparation prior to highly sensitive mass spectrometric analysis. ...Various methods are available for enrichment of proteome samples, each characterized by specific advantages and disadvantages limiting their general application as a method of choice. Here we suggest an optimized universal protocol ensuring reproducibility and effective enrichment of dilute samples by commercial affinity beads. By comparably assessing the performance of the new protocol with selected standard enrichment techniques, we show the seamless application of the enrichment in common mass spectrometry based proteomic workflows. Further, novel applications are suggested including a facile storage and shipping of desiccated, trapped proteome samples at ambient temperatures and usage of the affinity beads for gel-free proteomic approaches.
The recent SARS-CoV-2 pandemic and the vaccination campaign posed a challenge to patients with autoimmune disease, such as multiple sclerosis (MS). We aimed for investigating whether ...psychological/sociodemographic/clinical characteristics of MS patients are associated with SARS-CoV-2 vaccination status and self-reported vaccination side effects (SEs). We have asked patients with MS about their willingness to receive recommended standard vaccinations pre-pandemically since June 2019. Between 10/2021 and 01/2022, we surveyed 193 of these MS patients about their current SARS-CoV-2 vaccination status, their perception of vaccination-related SEs, and reasons for and against SARS-CoV-2 vaccination. 75.6% of the patients declared their willingness to receive standard vaccinations before the pandemic. 84.5%, 78.2%, and 13.0% of the patients had received the first, second, and third SARS-CoV-2 vaccination, respectively, until the follow-up survey. The most common reason for not getting vaccinated against SARS-CoV-2 was concern about possible side effects (82.1%), followed by the belief that the vaccines had not been adequately tested (64.3%). Vaccination-related SEs were reported by 52.8% of the patients. Younger age, higher education, lower degree of disability, relapsing disease course, shorter disease duration, not receiving a disease-modifying therapy and higher anxiety and depression levels were associated with the occurrence of certain vaccination-related SEs. Concerns about novel vaccines are widespread among MS patients and necessitate targeted education of the patients, especially to those with more severe psychopathological symptoms (anxiety or depression) and those who are generally skeptical of vaccination.
Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking ...are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO 2 . Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis , a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include ( i ) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; ( ii ) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; ( iii ) the potential use of hydrogen as an energy source; ( iv ) the strong expression of high-affinity uptake transporters; and ( v ) as yet undescribed energy-efficient steps in CO 2 fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O . algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.
Multiple sclerosis (MS) affects about three times more women than men. Due to variable MS courses, multiple therapies are necessary in clinical practice.
We aimed at conducting sex-specific analyses ...of MS patients regarding polypharmacy (≥ 5 drugs) and at identifying differences in the medication spectrum.
Clinico-demographic data were gathered from 306 patients using clinical examinations, structured patient interviews, and patient records. Statistical data analyses were performed to evaluate whether the same or different factors are associated with polypharmacy in both genders.
Women (N = 218) and men (N = 88) showed similar polypharmacy rates (56.0% vs. 58.0%; p = 0.799). For both genders, higher age, severe disability degrees, comorbidities, and inpatient treatment were significantly associated with a higher polypharmacy risk. Low educational levels were predictors of polypharmacy only in women. Fampridine (p < 0.021) and antispasmodics (p < 0.010) were used more often by men, while women took more frequently thyroid medications (p < 0.001) and contraceptives (p < 0.001). The age-related increase in medication use was much stronger in women (p < 0.001).
Male and female MS patients with older age, comorbidities, higher disability degree, and inpatient treatment are at greater risk of polypharmacy. Future studies should examine the occurrence of clinically relevant drug interactions in MS patients stratified by sex.
Telomeres are protective structures at the ends of linear chromosomes. Shortened telomere lengths (TL) are an indicator of premature biological aging and have been associated with a wide spectrum of ...disorders, including multiple sclerosis (MS). MS is a chronic inflammatory, demyelinating and neurodegenerative disease of the central nervous system. The exact cause of MS is still unclear. Here, we provide an overview of genetic, environmental and lifestyle factors that have been described to influence TL and to contribute to susceptibility to MS and possibly disease severity. We show that several early-life factors are linked to both reduced TL and higher risk of MS, e.g., adolescent obesity, lack of physical activity, smoking and vitamin D deficiency. This suggests that the mechanisms underlying the disease are connected to cellular aging and senescence promoted by increased inflammation and oxidative stress. Additional prospective research is needed to clearly define the extent to which lifestyle changes can slow down disease progression and prevent accelerated telomere loss in individual patients. It is also important to further elucidate the interactions between shared determinants of TL and MS. In future, cell type-specific studies and advanced TL measurement methods could help to better understand how telomeres may be causally involved in disease processes and to uncover novel opportunities for improved biomarkers and therapeutic interventions in MS.
Background
Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with ...increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS.
Methods
Our study focused on 10 bioinformatically prioritized SNP–gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays.
Results
We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from
GSDMB
and
SP140
. We also observed an increased
IL7R
exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 (
EFCAB13
), rs1131123 (
HLA-C
), rs10783847 (TSFM), and rs2014886 (
TSFM
) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of
TSFM
due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short
TSFM
transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed.
Conclusion
In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.
In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established ...a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MSE). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (>1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification.
The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of “protein costs” of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities.
Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution ...therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS.
The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy.
B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration.
Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction.
We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.
MicroRNAs (miRNAs) are small non-coding RNA molecules acting as post-transcriptional regulators of gene expression. They are involved in many biological processes, and their dysregulation is ...implicated in various diseases, including multiple sclerosis (MS). Interferon-beta (IFN-beta) is widely used as a first-line immunomodulatory treatment of MS patients. Here, we present the first longitudinal study on the miRNA expression changes in response to IFN-beta therapy. Peripheral blood mononuclear cells (PBMC) were obtained before treatment initiation as well as after two days, four days, and one month, from patients with clinically isolated syndrome (CIS) and patients with relapsing-remitting MS (RRMS). We measured the expression of 651 mature miRNAs and about 19,000 mRNAs in parallel using real-time PCR arrays and Affymetrix microarrays. We observed that the up-regulation of IFN-beta-responsive genes is accompanied by a down-regulation of several miRNAs, including members of the mir-29 family. These differentially expressed miRNAs were found to be associated with apoptotic processes and IFN feedback loops. A network of miRNA-mRNA target interactions was constructed by integrating the information from different databases. Our results suggest that miRNA-mediated regulation plays an important role in the mechanisms of action of IFN-beta, not only in the treatment of MS but also in normal immune responses. miRNA expression levels in the blood may serve as a biomarker of the biological effects of IFN-beta therapy that may predict individual disease activity and progression.
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