300 research outputs found
The quest for improved reproducibility in MALDI mass spectrometry
© 2016 Wiley Periodicals, Inc. Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat “hit and miss” with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217–228, 2018
'What did i do wrong?' An empirical evaluation of sample preparation methodologies in matrix-assisted laser desorption/ionization-mass spectrometry imaging
© 2019 MO Rourke. Aim: This guide aims to broaden the uptake of MALDI-MSI biomedical research by removing the initial 'lag phase' associated with empirical determination in sample preparation and data analysis. Methods: Samples from several tissue types were prepared for lipid, protein and peptide MSI analysis. Broadly, samples were cryo sectioned, mounted onto conductive MALDI slides and sublimed with an analyte specific matrix, recrystallised and analyzed in a Bruker UltrafleXtreme MALDI TOF/TOF. Results/conclusion: Here we present a general guide that serves as the first comprehensive, explanatory index for curation and verification of both sample preparation and data generation during the MALDI-MSI process. Lay abstract The field of mass spectrometry tissue imaging is a complex field that is designed to provide a map of the molecules on the surface of tissue sections. It often requires a significant investment of time and resources before useful data can be generated; therefore, this paper provides a visual troubleshooting guide that will act as a reference point for a range of sample preparation mistakes and explanations for unusual or suboptimal data. Elimination of the lag phase associated with the development of new techniques will help to expedite the growth and application of this technology
A comprehensive guide for performing sample preparation and top-down protein analysis
© 2017 by the authors. Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform0s abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism0s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer
Quantitative Proteomic Profiling of Small Molecule Treated Mesenchymal Stem Cells Using Chemical Probes.
The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation
What is Normalization? The Strategies Employed in Top-Down and Bottom-Up Proteome Analysis Workflows.
The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process
OXSR1 inhibits inflammasome activation by limiting potassium efflux during mycobacterial infection.
Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1β, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections
Association analysis of PRNP gene region with chronic wasting disease in Rocky Mountain elk
<p>Abstract</p> <p>Background</p> <p>Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids including white-tailed (<it>Odocoileus virginianus</it>) and mule deer (<it>Odocoileus hemionus</it>), Rocky Mountain elk (<it>Cervus elaphus nelsoni</it>), and moose (<it>Alces alces</it>). A leucine variant at position 132 (132L) in prion protein of Rocky Mountain elk confers a long incubation time with CWD, but not complete resistance. However, variants in regulatory regions outside the open reading frame of <it>PRNP </it>have been associated with varying degrees of susceptibility to prion disease in other species, and some variants have been observed in similar regions of Rocky Mountain elk <it>PRNP</it>. Thus, additional genetic variants might provide increased protection, either alone or in combination with 132L.</p> <p>Findings</p> <p>This study provided genomic sequence of all exons for <it>PRNP </it>of Rocky Mountain elk. Many functional sites in and around the <it>PRNP </it>gene region were sequenced, and this report approximately doubled (to 75) the number of known variants in this region. A haplotype-tagging approach was used to reduce the number of genetic variants required to survey this variation in the <it>PRNP </it>gene region of 559 Rocky Mountain elk. Eight haplotypes were observed with frequencies over 1.0%, and one haplotype was present at 71.2% frequency, reflecting limited genetic diversity in the <it>PRNP </it>gene region.</p> <p>Conclusions</p> <p>The presence of 132L cut odds of CWD by more than half (Odds Ratio = 0.43; P = 0.0031), which was similar to a previous report. However after accounting for 132L, no association with CWD was found for any additional variants in the <it>PRNP </it>region (P > 0.05).</p
Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.
Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic
The Caenorhabditis elegans Mucin-Like Protein OSM-8 Negatively Regulates Osmosensitive Physiology Via the Transmembrane Protein PTR-23
The molecular mechanisms of animal cell osmoregulation are poorly understood. Genetic studies of osmoregulation in yeast have identified mucin-like proteins as critical regulators of osmosensitive signaling and gene expression. Whether mucins play similar roles in higher organisms is not known. Here, we show that mutations in the Caenorhabditis elegans mucin-like gene osm-8 specifically disrupt osmoregulatory physiological processes. In osm-8 mutants, normal physiological responses to hypertonic stress, such as the accumulation of organic osmolytes and activation of osmoresponsive gene expression, are constitutively activated. As a result, osm-8 mutants exhibit resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. To identify genes required for Osm-8 phenotypes, we performed a genome-wide RNAi osm-8 suppressor screen. After screening ∼18,000 gene knockdowns, we identified 27 suppressors that specifically affect the constitutive osmosensitive gene expression and Osr phenotypes of osm-8 mutants. We found that one suppressor, the transmembrane protein PTR-23, is co-expressed with osm-8 in the hypodermis and strongly suppresses several Osm-8 phenotypes, including the transcriptional activation of many osmosensitive mRNAs, constitutive glycerol accumulation, and osmotic stress resistance. Our studies are the first to show that an extracellular mucin-like protein plays an important role in animal osmoregulation in a manner that requires the activity of a novel transmembrane protein. Given that mucins and transmembrane proteins play similar roles in yeast osmoregulation, our findings suggest a possible evolutionarily conserved role for the mucin-plasma membrane interface in eukaryotic osmoregulation
Factors Associated With the Quality of Life of Nursing Home Residents During the COVID-19 Pandemic: A Cross-Sectional Study
Objectives
Quality of life (QoL) of nursing home (NH) residents is critical, yet understudied, particularly during the COVID-19 pandemic. Our objective was to examine whether COVID-19 outbreaks, lack of access to geriatric professionals, and care aide burnout were associated with NH residents' QoL.
Design
Cross-sectional study (July to December 2021).
Setting and Participants
We purposefully selected 9 NHs in Alberta, Canada, based on their COVID-19 exposure (no or minor/short outbreaks vs repeated or extensive outbreaks). We included data for 689 residents from 18 care units.
Methods
We used the DEMQOL-CH to assess resident QoL through video-based care aide interviews. Independent variables included a COVID-19 outbreak in the NH in the past 2 weeks (health authority records), care unit-levels of care aide burnout (9-item short-form Maslach Burnout Inventory), and resident access to geriatric professionals (validated facility survey). We ran mixed-effects regression models, adjusted for facility and care unit (validated surveys), and resident covariates (Resident Assessment Instrument–Minimum Data Set 2.0).
Results
Recent COVID-19 outbreaks (β = 0.189; 95% CI: 0.058–0.320), higher proportions of emotionally exhausted care aides on a care unit (β = 0.681; 95% CI: 0.246–1.115), and lack of access to geriatric professionals (β = 0.216; 95% CI: 0.003–0.428) were significantly associated with poorer resident QoL.
Conclusions and Implications
Policies aimed at reducing infection outbreaks, better supporting staff, and increasing access to specialist providers may help to mitigate how COVID-19 has negatively affected NH resident QoL
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