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Methods in Molecular Biology (Clifton,... 2024With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk cells. However, such bulk measurement obscures...
With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk cells. However, such bulk measurement obscures cell-to-cell heterogeneity, precluding proteome profiling of single cells and small numbers of cells of interest. To address this issue, in the recent 5 years, there has been a surge of small sample preparation methods developed for robust and effective collection and processing of single cells and small numbers of cells for in-depth MS-based proteome profiling. Based on their broad accessibility, they can be categorized into two types: methods based on specific devices and those based on standard PCR tubes or multi-well plates. In this chapter, we describe the detailed protocol of our recently developed, easily adoptable, Surfactant-assisted One-Pot (SOP) sample preparation coupled with MS method termed SOP-MS for label-free single-cell and nanoscale proteomics. SOP-MS capitalizes on the combination of an MS-compatible surfactant, n-dodecyl-β-D-maltoside (DDM), and standard low-bind PCR tube or multi-well plate for "all-in-one" one-pot sample preparation without sample transfer. With its robust and convenient features, SOP-MS can be readily implemented in any MS laboratory for single-cell and nanoscale proteomics. With further improvements in MS detection sensitivity and sample throughput, we believe that SOP-MS could open an avenue for single-cell proteomics with broad applicability in biological and biomedical research.
Topics: Proteomics; Surface-Active Agents; Single-Cell Analysis; Humans; Mass Spectrometry; Proteome; Nanotechnology; Glucosides
PubMed: 38907149
DOI: 10.1007/978-1-0716-3934-4_8 -
Journal of Proteomics Jun 2024Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in...
Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SIGNIFICANCE: Trypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as "surra" in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.
PubMed: 38906247
DOI: 10.1016/j.jprot.2024.105231 -
Biomaterials Science Jun 2024Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein,...
Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or β-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.
PubMed: 38904161
DOI: 10.1039/d4bm00250d -
International Journal of Environmental... Jun 2024Sodium dodecylbenzene sulfonate (SDBS), a predominant component in detergents, requires an evaluation of its toxicological potential due to its hazardous environmental...
Sodium dodecylbenzene sulfonate (SDBS), a predominant component in detergents, requires an evaluation of its toxicological potential due to its hazardous environmental levels. Therefore, we evaluated the toxicological effects of SDBS on the liver and kidney of male . Therefore, we evaluated the toxicological effects of SDBS on the liver and kidney of male D. rerio. The fish were divided into three groups: 0.0 (control), 0.25, and 0.5 mg/L of SDBS with exposure for up to 96 hours. After exposure, histopathological, histochemical (hepatic glycogen content), and biochemical analyses (SOD and CAT enzyme analysis) were performed on both organs. The results showed significant histopathological effects, such as circulatory disturbances and progressive and regressive alterations, leading to an altered histopathological alteration index. SOD and CAT enzymes exhibited prominent changes. Thus, it became clear that the surfactant SDBS can cause serious hepatic and renal problems in fish, even with short-term exposure, necessitating more stringent control and regulation in the disposal of this surfactant.
PubMed: 38902975
DOI: 10.1080/09603123.2024.2369221 -
Insect Molecular Biology Jun 2024Bombyx mori cecropin A (Bmcecropin A) has antibacterial, antiviral, anti-filamentous fungal and tumour cell inhibition activities and is considered a potential...
Bombyx mori cecropin A (Bmcecropin A) has antibacterial, antiviral, anti-filamentous fungal and tumour cell inhibition activities and is considered a potential succedaneum for antibiotics. We clarified the antibacterial mechanism and structure-activity relationships and then directed the structure-activity optimization of Bmcecropin A. Firstly, we found Bmcecropin A shows a strong binding force and permeability to cell membranes like a detergent; Bmcecropin A could competitively bind to the cell membrane with the cell membrane-specific dye DiI, then damaged the membrane for the access of DiI into the cytoplasm and leading to the leakage of electrolyte and proteins. Secondly, we found Bmcopropin A could also bind to and degrade DNA; furthermore, DNA library polymerase chain reaction (PCR) results indicated that Bmcecropin A inhibited DNA replication by non-specific binding. In addition, we have identified C-terminus amidation and serine-lysine- glycine (SLG) amino acids of Bmcecropin A played critical roles in the membrane damage and DNA degradation. Based on the above results, we designed a mutant of Bmcecropin A (E to H, D to K, K to A), which showed higher antibacterial activity, thermostability and pH stability than ampicillin but no haemolytic activity. Finally, we speculated that Bmcecropin A damaged the cell membrane through a carpet model and drew the schematic diagram of its antibacterial mechanism, based on the antibacterial mechanism and the three-dimensional configuration. These findings yield insights into the mechanism of antimicrobial peptide-pathogen interaction and beneficial for the development of new antibiotics.
PubMed: 38898565
DOI: 10.1111/imb.12934 -
Biotechnology For Biofuels and... Jun 2024Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and...
Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.
PubMed: 38898475
DOI: 10.1186/s13068-024-02523-3 -
Applied Biochemistry and Biotechnology Jun 2024Detergents are used as a part of our daily life routine. Though they are widely used but their active ingredients which are highly toxic and persist in the environment...
Detergents are used as a part of our daily life routine. Though they are widely used but their active ingredients which are highly toxic and persist in the environment for long are an important cause of environmental pollution. In our current work, we have studied the harmful effects of a combination of some commonly used detergents which find their way into the water bodies especially the pond ecosystem through everyday activities like washing clothes, utensils, and bathing in water. This water is the home to many flora and fauna especially the fishes like Cirrhinus mrigala. In our work, we have analysed the levels of the hepatic enzymes Alanine Transaminase and Aspartate Aminotransferase as well as the histology of gill and liver tissues. We have also analysed the presence of micronucleus in the fish blood. It was observed that the presence of detergents has increased the enzyme level as well as resulted in destruction of gill and liver tissue morphology. Detergents also increased the presence of micronucleus in fish blood. These results are indicators of deterioration of fish health due to detergent pollution.
PubMed: 38896369
DOI: 10.1007/s12010-024-04983-7 -
Current Protocols Jun 2024U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes....
U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.
Topics: Escherichia coli; Humans; Ribonucleoprotein, U1 Small Nuclear; Phosphorylation; Recombinant Proteins; Gene Expression; Protein Domains
PubMed: 38896106
DOI: 10.1002/cpz1.1059 -
BioRxiv : the Preprint Server For... Jun 2024RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fu sed in s arcoma (FUS) is...
RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fu sed in s arcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic FTLD. Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.
PubMed: 38895337
DOI: 10.1101/2024.06.03.593639 -
ACS Medicinal Chemistry Letters Jun 2024The SARS-COV-2 virus is a deadly agent of inflammatory respiratory disease. Since 2020, studies have focused on developing new therapies based on galactose-rich IgA...
The SARS-COV-2 virus is a deadly agent of inflammatory respiratory disease. Since 2020, studies have focused on developing new therapies based on galactose-rich IgA antibodies. Clinical surveys have also revealed that galactose-deficient IgA1 polymerizes in serum, producing IgA nephropathy, which is a common cause of kidney failure in young adults. Here we show that IgA1-IgA2 dimers are efficiently and economically purified in solution via conjugated nonionic surfactant micellar aggregates. Quantitative capture at pH 7 and extraction at pH 6.5 can avoid antibody exposure to acidic, potentially denaturing conditions. Brij-O20 aggregates lead to the highest process yields (88-91%) and purity (94%). Recovered IgA dimers preserve their native secondary structure and do not self-associate. Increasing the reaction volume has little impact on yield or purity. By introducing an efficient, inexpensive IgA purification protocol, we assist pharmaceutical firms and research laboratories in developing new IgA-based therapies as well as in increasing our understanding of IgA1 polymerization.
PubMed: 38894919
DOI: 10.1021/acsmedchemlett.4c00128