-
Biochimie Jan 2024Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion...
Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion bodies (IB). Unlike mouse TSPO, BcTSPO purified in SDS detergent from IB is well structured and can bind various ligands such as high-affinity PK 11195, protoporphyrin IX (PPIX) and δ-aminolevulinic acid (ALA). For each of the three ligands, H-N HSQC titration NMR experiments suggest that different amino acids of BcTSPO binding cavity are involved in the interaction. PPIX, an intermediate of heme biosynthesis, binds to the cavity of BcTSPO and its fluorescence can be significantly reduced in the presence of light and oxygen. The light irradiation leads to two products that have been isolated and characterized as photoporphyrins. They result from the addition of singlet oxygen to the two vinyl groups hence leading to the formation of hydroxyaldehydes. The involvement of water molecules, recently observed along with the binding of heme in Rhodobacter sphaeroides (RsTSPO) is highly probable. Altogether, these results raise the question of the role of TSPO in heme biosynthesis regulation as a possible scavenger of reactive intermediates.
PubMed: 38280504
DOI: 10.1016/j.biochi.2024.01.011 -
Microbial Biotechnology Apr 2024Microorganisms known as psychrophiles/psychrotrophs, which survive in cold climates, constitute majority of the biosphere on Earth. Their capability to produce... (Review)
Review
Microorganisms known as psychrophiles/psychrotrophs, which survive in cold climates, constitute majority of the biosphere on Earth. Their capability to produce cold-active enzymes along with other distinguishing characteristics allows them to survive in the cold environments. Due to the relative ease of large-scale production compared to enzymes from plants and animals, commercial uses of microbial enzyme are alluring. The ocean depths, polar, and alpine regions, which make up over 85% of the planet, are inhabited to cold ecosystems. Microbes living in these regions are important for their metabolic contribution to the ecosphere as well as for their enzymes, which may have potential industrial applications. Cold-adapted microorganisms are a possible source of cold-active enzymes that have high catalytic efficacy at low and moderate temperatures at which homologous mesophilic enzymes are not active. Cold-active enzymes can be used in a variety of biotechnological processes, including food processing, additives in the detergent and food industries, textile industry, waste-water treatment, biopulping, environmental bioremediation in cold climates, biotransformation, and molecular biology applications with great potential for energy savings. Genetically manipulated strains that are suitable for producing a particular cold-active enzyme would be crucial in a variety of industrial and biotechnological applications. The potential advantage of cold-adapted enzymes will probably lead to a greater annual market than for thermo-stable enzymes in the near future. This review includes latest updates on various microbial source of cold-active enzymes and their biotechnological applications.
Topics: Cold Temperature; Biotechnology; Bacteria; Enzymes; Enzyme Stability
PubMed: 38656876
DOI: 10.1111/1751-7915.14467 -
Tau-RNA complexes inhibit microtubule polymerization and drive disease-relevant conformation change.Brain : a Journal of Neurology Aug 2023Alzheimer's disease and related disorders feature neurofibrillary tangles and other neuropathological lesions composed of detergent-insoluble tau protein. In recent...
Alzheimer's disease and related disorders feature neurofibrillary tangles and other neuropathological lesions composed of detergent-insoluble tau protein. In recent structural biology studies of tau proteinopathy, aggregated tau forms a distinct set of conformational variants specific to the different types of tauopathy disorders. However, the constituents driving the formation of distinct pathological tau conformations on pathway to tau-mediated neurodegeneration remain unknown. Previous work demonstrated RNA can serve as a driver of tau aggregation, and RNA associates with tau containing lesions, but tools for evaluating tau/RNA interactions remain limited. Here, we employed molecular interaction studies to measure the impact of tau/RNA binding on tau microtubule binding and aggregation. To investigate the importance of tau/RNA complexes (TRCs) in neurodegenerative disease, we raised a monoclonal antibody (TRC35) against aggregated tau/RNA complexes. We showed that native tau binds RNA with high affinity but low specificity, and tau binding to RNA competes with tau-mediated microtubule assembly functions. Tau/RNA interaction in vitro promotes the formation of higher molecular weight tau/RNA complexes, which represent an oligomeric tau species. Coexpression of tau and poly(A)45 RNA transgenes in Caenorhabditis elegans exacerbates tau-related phenotypes including neuronal dysfunction and pathological tau accumulation. TRC35 exhibits specificity for Alzheimer's disease-derived detergent-insoluble tau relative to soluble recombinant tau. Immunostaining with TRC35 labels a wide variety of pathological tau lesions in animal models of tauopathy, which are reduced in mice lacking the RNA binding protein MSUT2. TRC-positive lesions are evident in many human tauopathies including Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease. We also identified ocular pharyngeal muscular dystrophy as a novel tauopathy disorder, where loss of function in the poly(A) RNA binding protein (PABPN1) causes accumulation of pathological tau in tissue from post-mortem human brain. Tau/RNA binding drives tau conformational change and aggregation inhibiting tau-mediated microtubule assembly. Our findings implicate cellular tau/RNA interactions as modulators of both normal tau function and pathological tau toxicity in tauopathy disorders and suggest feasibility for novel therapeutic approaches targeting TRCs.
Topics: Humans; Mice; Animals; tau Proteins; Alzheimer Disease; RNA; Neurodegenerative Diseases; Detergents; Polymerization; Tauopathies; Brain; RNA, Messenger; Caenorhabditis elegans; Microtubules; Poly(A)-Binding Protein I
PubMed: 36732296
DOI: 10.1093/brain/awad032 -
Langmuir : the ACS Journal of Surfaces... Aug 2023Cultural heritage is a crucial resource to increase our society's resilience. However, degradation processes, enhanced by environmental and anthropic risks, inevitably... (Review)
Review
Cultural heritage is a crucial resource to increase our society's resilience. However, degradation processes, enhanced by environmental and anthropic risks, inevitably affect works of art, hindering their accessibility and socioeconomic value. In response, interfacial and colloidal chemistry has proposed valuable solutions over the past decades, overcoming the limitations of traditional restoration materials and granting cost- and time-effective remedial conservation of the endangered artifacts. Ranging from inorganic nanoparticles to hybrid composites and soft condensed matter (gels, microemulsions), a wide palette of colloidal systems has been made available to conservators worldwide, targeting the consolidation, cleaning, and protection of works of art. The effectiveness and versatility of the proposed solutions allow the safe and effective treatment of masterpieces belonging to different cultural and artistic productions, spanning from classic ages to the Renaissance and modern/contemporary art. Despite these advancements, the formulation of materials for the preservation of cultural heritage is still an open, exciting field, where recent requirements include coping with the imperatives of the Green Deal to foster the production of sustainable, low-toxicity, and environmentally friendly systems. This review gives a critical overview starting from pioneering works up to the latest advancements in colloidal systems for art conservation, a challenging topic where effective solutions can be transversal to multiple sectors even beyond cultural heritage preservation, from the pharmaceutical and food industry, to cosmetics, tissue engineering, and detergency.
PubMed: 37487238
DOI: 10.1021/acs.langmuir.3c01324 -
International Journal of Pharmaceutics:... Dec 2023To ensure the stability of biologicals over their entire shelf-life, non-ionic surface-active compounds (surfactants) are added to protect biologics from denaturation... (Review)
Review
To ensure the stability of biologicals over their entire shelf-life, non-ionic surface-active compounds (surfactants) are added to protect biologics from denaturation and particle formation. In this context, polysorbate 20 and 80 are the most used detergents. Despite their benefits of low toxicity and high biocompatibility, specific factors are influencing the intrinsic stability of polysorbates, leading to degradation, loss in efficacy, or even particle formation. Polysorbate degradation can be categorized into chemical or enzymatic hydrolysis and oxidation. Under pharmaceutical relevant conditions, hydrolysis is commonly originated from host cell proteins, whereas oxidative degradation may be caused by multiple factors such as light, presence of residual metal traces, peroxides, or temperature, which can be introduced upon manufacturing or could be already present in the raw materials. In this review, we provide an overview of the current knowledge on polysorbates with a focus on oxidative degradation. Subsequently, degradation products and key characteristics of oxidative-mediated polysorbate degradation in respect of different types and grades are summarized, followed by an extensive comparison between polysorbate 20 and 80. A better understanding of the radical-induced oxidative PS degradation pathway could support specific mitigation strategies. Finally, buffer conditions, various stressors, as well as appropriate mitigation strategies, reagents, and alternative stabilizers are discussed. Prior manufacturing, careful consideration and a meticulous risk-benefit analysis are highly recommended in terms of polysorbate qualities, buffers, storage conditions, as well as mitigation strategies.
PubMed: 37680877
DOI: 10.1016/j.ijpx.2023.100202 -
Life (Basel, Switzerland) Nov 2023Alfalfa ( L.), one of the most extensively grown forage crops, is sensitive to saline soils. We measured the breeding efficiency for increased salt tolerance in alfalfa...
Alfalfa ( L.), one of the most extensively grown forage crops, is sensitive to saline soils. We measured the breeding efficiency for increased salt tolerance in alfalfa by comparing lines selected from BC79S, CS, and SII populations with their unselected parental means for forage mass and associated changes in stem length, leaf-to-stem ratio (LSR), number of nodes per stem, crude protein (CP) content, and neutral detergent fiber (NDF) content. The overall forage mass in the non-salt-stressed test (9562 kg ha) was greater ( < 0.001) than under salt stress (5783 kg ha), with a 40% production advantage. In the non-salt-stressed test, the BC79S and CS lines averaged at a 4% lower production than their parents, while SII lines had on average a 9% greater production. Conversely, in the salt-stressed test, all lines showed a 20% overall greater seasonal production than their parents. Some selected lines produced more forage mass in both the non-stressed and salt-stressed tests than their parents. The stem length, LSR, node number, CP content, and NDF content of the selected lines varied with respect to non-stressed vs. stressed, but they tended not to differ greatly from their respective parental means under either non- or salt-stressed conditions. The selection protocol provided a universal increase in forage mass under salt-stressed field conditions of the selected lines. Furthermore, we identified lines with forage mass values greater than their parental means under non- and salt-stressed field conditions.
PubMed: 38004328
DOI: 10.3390/life13112188 -
BioRxiv : the Preprint Server For... Oct 2023Colloidal aggregation is one of the largest contributors to false-positives in early drug discovery and chemical biology. Much work has focused on its impact on...
Colloidal aggregation is one of the largest contributors to false-positives in early drug discovery and chemical biology. Much work has focused on its impact on pure-protein screens; here we consider aggregations role in cell-based infectivity assays in Covid-19 drug repurposing. We began by investigating the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal-particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate antiviral efficacy of the drugs in cells we used spike pseudotyped lentivirus and pre-saturation of the colloids with BSA. The antiviral potency of the aggregators was diminished by at least 10-fold and often entirely eliminated in the presence of BSA, suggesting antiviral activity can be attributed to the non-specific nature of the colloids. In confocal microscopy, the aggregates induced fluorescent puncta of labeled spike protein, consistent with sequestration of the protein on the colloidal particles. Addition of either non-ionic detergent or of BSA disrupted these puncta. These observations suggest that colloidal aggregation is common among cell-based anti-viral drug repurposing, and perhaps cell-based assays more broadly, and offers rapid counter-screens to detect and eliminate these artifacts, allowing the community invest resources in compounds with true potential as a Covid-19 therapeutic.
PubMed: 37961552
DOI: 10.1101/2023.10.27.564435 -
Bio-protocol Nov 2023The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the...
The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the membrane and interacting with membrane-embedded proteins to alternate their activity and stability. Like lipids, membrane proteins are amphipathic and are stabilized by the hydrophobic forces of the lipid bilayer. Membrane protein-lipid interactions are difficult to investigate since membrane proteins need to be reconstituted in a lipid-mimicking environment. A common and well-established approach is the detergent-based solubilization of the membrane proteins in detergent micelles. Nowadays, nanodiscs and liposomes are used to mimic the lipid bilayer and enable the work with membrane proteins in a more natural environment. However, these protocols need optimization and are labor intensive. The present protocol describes straightforward instructions on how the preparation of lipids is performed and how the lipid detergent mixture is integrated with the membrane protein MARCH5. The lipidation protocol was performed prior to an activity assay specific to membrane-bound E3 ubiquitin ligases and a stability assay that could be used for any membrane protein of choice.
PubMed: 38026763
DOI: 10.21769/BioProtoc.4887 -
Polymers May 2024Natural rubber (NR) is utilized in more than 40,000 products, and the demand for NR is projected to reach $68.5 billion by 2026. The primary commercial source of NR is... (Review)
Review
Natural rubber (NR) is utilized in more than 40,000 products, and the demand for NR is projected to reach $68.5 billion by 2026. The primary commercial source of NR is the latex of . NR is produced by the sequential cis-condensation of isopentenyl diphosphate (IPP) through a complex known as the rubber transferase (RTase) complex. This complex is associated with rubber particles, specialized organelles for NR synthesis. Despite numerous attempts to isolate, characterize, and study the RTase complex, definitive results have not yet been achieved. This review proposes an innovative approach to overcome this longstanding challenge. The suggested method involves isolating the RTase complex without using detergents, instead utilizing the native membrane lipids, referred to as "natural nanodiscs", and subsequently reconstituting the complex on liposomes. Additionally, we recommend the adaptation of large nanodiscs for the incorporation and reconstitution of the RTase complex, whether it is in vitro transcribed or present within the natural nanodiscs. These techniques show promise as a viable solution to the current obstacles. Based on our experimental experience and insights from published literature, we believe these refined methodologies can significantly enhance our understanding of the RTase complex and its role in in vitro NR synthesis.
PubMed: 38891415
DOI: 10.3390/polym16111468 -
ACS Omega Aug 2023In this study, we explored the potential of hydrodynamic cavitation (HC) for use in dissolution of liquid and powder detergents. For this, microfluidic and polyether...
In this study, we explored the potential of hydrodynamic cavitation (HC) for use in dissolution of liquid and powder detergents. For this, microfluidic and polyether ether ketone (PEEK) tube HC reactors with different configurations were employed, and the results from the reactors were compared with a magnetic stirrer, as well as a tergotometer. According to our results PEEK tube HC reactors present the best performance for dissolution of liquid and powder detergents. In the case of liquid detergent, for the same level of initial concentration and comparable final dissolution, the PEEK tube consumed 16.7 and 70% of the energy and time of a tergotometer and 16.7 and 14.8% of that of a magnetic stirrer, respectively. In the case of powder detergent, the PEEK tube used 12% less power than a tergotometer and 81.2% less power than a magnetic stirrer. Additionally, the time required to dissolve the detergent was reduced significantly from 1200 s in the tergotometer and 1800 s in the magnetic stirrer to just 50 s in the PEEK tube. These results suggest that HC could significantly improve the dissolution rate of liquid and powder detergents and energy consumption in washing machines.
PubMed: 37599931
DOI: 10.1021/acsomega.3c03517