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Role of light emitting diode (LED) wavelengths on increase of protein productivity and free amino acid profile of Spirulina sp. cultures
摘要： LEDs have specific wavelengths that can positively influence the production of microalga biomass and biomolecules of interest. Filling the gaps in the literature, this study evaluated the effect of different LED wavelengths and photoperiods on protein productivities and free amino acid (FAA) profile of Spirulina sp. LEB 18 cultures. The best protein productivity results were obtained in red and green LED cultures using integral and partial photoperiods, respectively. In these experiments, protein productivities increased 2 and 1.6 times, respectively, compared to the control culture using fluorescent light. Green LEDs in partial photoperiod provided also the highest concentrations of essential and non-essential FAA, about 1.8 and 2.3 times higher, respectively, than control cultures. LEDs showed to be a promising sustainable light source for increasing protein productivity and FAA concentration in Spirulina sp. LEB 18 cultures.
关键词： Photoperiod,Microalgae,Protein productivity,Cyanobacteria,Light source
更新于2025-09-23 15:21:01
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LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution
摘要： Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.
关键词： protein translocation,eukaryotic cells,optogenetics,type III secretion system,LITESEC-T3SS
更新于2025-09-23 15:21:01
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Quantifying the Oligomeric States of Membrane Proteins in Cells through Super-Resolution Localizations
摘要： Transitions between different oligomeric states of membrane proteins are essential for proper cellular functions. However, the quantification of their oligomeric states in cells is technically challenging. Here we developed a new method to quantify oligomeric state(s) of highly-expressed membrane proteins using the probability density function of molecule density (PDFMD) calculated from super-resolution localizations. We provided the theoretical model of PDFMD, discussed the effects of protein concentration, cell geometry and photophysics of fluorescent proteins on PDFMD, and provided experimental criteria for proper quantification of oligomeric states. This method was further validated using simulated single-molecule fluorescent movies, and applied to two membrane proteins, UhpT and SbmA in E. coli. The study shows that PDFMD is useful in quantifying oligomeric states of membrane proteins in cells that can help in understanding cellular tasks. Potential applications to proteins with higher oligomeric states under high concentration and limitation of our methodology were also discussed.
关键词： PALM,Photophysics,Protein concentration,Cell geometry,Single molecule
更新于2025-09-23 15:21:01
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Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells
摘要： Monitoring virus assembly at the nanoscale in host cells remains a major challenge. Human immunodeficiency virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.
关键词： HIV-1,Gag protein,virus assembly,CD4+ T cells,single-molecule localisation microscopy
更新于2025-09-23 15:21:01
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Protective effect of RIPK1-inhibitory compound in in vivo models for retinal degenerative disease
摘要： Receptor interacting protein kinase 1 (RIPK1) plays a key role in necroptosis, which is a type of programmed necrosis that is involved in ocular diseases, including glaucoma and dry age-related macular degeneration (AMD). We previously introduced RIPK1-inhibitory compound (RIC), which has biochemical characteristics and a mode of action that are distinct from those of the prototype RIPK1 inhibitor necrostatin-1. The intraperitoneal administration of RIC exerts a protective effect on retinal ganglion cells against a glaucomatous insult. In this study, we examined the protective effect of RIC on retinal pigment epithelium (RPE) against sodium iodate (SI) insult, which is associated with dry AMD pathogenesis. The eye drop administration of RIC that reached on the retina prevented RPE loss in SI-induced retinal degeneration. RIC consistently demonstrated retinal protection in the funduscopy and electroretinogram analyses in SI-injected rabbits and iodoacetic acid-treated mini-pigs. Moreover, the in vivo protective effects of RIC were superior to those of ACU-4429 and doxycycline, which are other medications investigated in clinical trials for the treatment of dry AMD, and RIC did not induce retinal toxicity following topical administration in rats. Collectively, RIC displayed excellent retinal penetration and prevented retinal degeneration in the pathogenesis of dry AMD with a high in vivo efficacy.
关键词： RPE protection,Receptor interacting protein kinase1 (RIPK1),Dry AMD,Topical application,Retinal degeneration,RIPK1-inhibitory compound (RIC)
更新于2025-09-23 15:21:01
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Side chain torsion dictates planarity and ionizability of Green fluorescent protein’s chromophore leading to spectral perturbations.
摘要： Spectral characteristics of Fluorescent Proteins (FP) are well studied and through protein engineering, several FP variants constituting entire visible spectrum have been created. One of the most common mechanisms attributed to spectral shifts in FP are excited state proton transfer (ESPT), hydroxyl moiety protonation and deprotonation, along with chromophore Cis-Trans isomerism. Most widely studied FPs are those derived from avGFP (Aequorea victoria GFP) and Dsred (Discosoma coral). Apart from the above mechanism, certain interacting residues are said to play a vital role in altering the proton transfer pathway shift. The torsional changes of wild avGFP, S65T avGFP, and Dsred has been studied to van der Waals packing in Dsred is more compact than that of avGFP, thus creating a low solvent occupiable environment and reduced solvent interactions having higher red spectral hypothesize the torsional landscape and altered residual interactions are prominent factors for the spectral shifts. Through our 200 ns molecular dynamics investigation we prospect that leading to numerous spectral variants. Similarly, the hydrogen-bonded networks solely cannot dictate the energy landscape of FPs. Non-bonded interactions also can create secondary harmonic shifts by dipole-dipole inductions. Side chain contacts tend to alter the topological and torsional geometry, thereby disturbing the chromophore’s planarity. Side chain torsional variations have almost been unaccounted for their distortions in FP’s. We comprehend the inter-residual contact distance and the geometrical descriptors.
关键词： Green fluorescent protein,spectral perturbations,Sidechain torsional variations,Residual accessible area,Chromophore planarity
更新于2025-09-23 15:21:01
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[IEEE 2018 43rd International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz2018) - Nagoya, Japan (2018.9.9-2018.9.14)] 2018 43rd International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz) - Detection of Human Tumor Markers with THz Metamaterials
摘要： THz bioanalytical techniques are already proven as a viable tool for the label-free detection of biomolecules. But still, significant improvement of THz sensing techniques is needed to reach application relevant sensitivity levels. We present the label-free THz sensing of human tumor markers using a highly sensitive metamaterial based THz biosensor. High affinity binding of the specific protein to DNA is utilized for localized functionalization to further enhance the detection sensitivity.
关键词： tumor markers,DNA-protein interaction,THz sensing,metamaterials,biosensor
更新于2025-09-23 15:21:01
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Metal Nanoclusters: Engineering Functional Metal Materials at the Atomic Level (Adv. Mater. 47/2018)
摘要： Customizing functional metal materials at an atom-by-atom basis is one of the most ambitious dreams of materials scientists. In article number 1802751, Jianping Xie and co-workers summarize recent progress in introducing such fine capability of structural modulation into functional metal nanoclusters, based on a protein-like hierarchical structure scheme.
关键词： structural modulation,metal nanoclusters,protein-like hierarchical structure
更新于2025-09-23 15:21:01
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Systems toxicology assessment revealed the impact of graphenea??based materials on cell cycle regulators
摘要： Understanding the cellular and molecular toxicity of graphene and its derivatives is essential for their biomedical applications. Herein, gene expression profile of graphene-exposed cells was retrieved from the GEO database. Differentially expressed genes and their functional roles were then investigated through the pathway, protein-protein interaction network, and module analysis. High degree (hub) and high betweenness centrality (bottleneck) nodes were subsequently identified. The functional analysis of central genes indicated that these graphene-gene interactions could be of great value for further investigation. Accordingly, we also followed the expression of five hub-bottleneck genes in graphene-treated murine peritoneal macrophages and human breast cancer cell line by real-time PCR. The five hub-bottleneck genes related to graphene cytotoxicity; CDK1, CCNB1, PLK1, TOP2A, and CCNA2 were identified through network analysis, which were highly correlated with regulation of cell cycle processes. The module analysis indicated the cell cycle pathway to be the predominant one. Gene expression evaluation showed down-regulation of these genes in the macrophages and cancer cells treated with graphene. These results provided some new intuitions concerning the graphene-cell interactions and unveiled targeting critical cell cycle regulators. The present study indicated some toxic effects of graphene-based materials through systems toxicology assessment. Integrating gene expression and protein-protein interaction network may help explaining biological responses of graphene and lead to beneficial impacts in nanomedicine.
关键词： Cell cycle,Graphene,Protein-protein interaction network,Gene expression,Systems toxicology
更新于2025-09-23 15:19:57
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Impact of external amino acids on fluorescent protein chromophore biosynthesis revealed by molecular dynamics and mutagenesis studies
摘要： The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the β-barrel structure of the FP. It has been shown earlier that chromophore biosynthesis of the red FP from Zoanthus sp. (zoan2RFP) proceeds via an immature green state. At the same time, the green state is the final stage of chromophore biosynthesis of green FP (zoanGFP), which is highly homologous to zoan2RFP. It was also shown that a single N66D substitution in the chromophore-forming sequence of zoanGFP might trigger the synthesis of the red chromophore. However, in this case, the synthesis of the red chromophore is incomplete and occurs only at elevated temperatures. Here, we tried to uncover additional structural determinants that govern the biosynthesis of the red chromophore. A comparison of zoanGFP and zoan2RFP revealed intrabarrel amino acid differences at five positions. Exhaustive substitutions of these five positions in zoanGFP-N66D gave rise to zoanGFPmut with the same intrabarrel amino acid composition as zoan2RFP. zoanGFPmut showed only partial green-to-red chromophore transformation at elevated temperatures. To elucidate the extra factors that can affect red chromophore biosynthesis, we performed comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. The simulations revealed several external amino acids that might influence the arrangement and flexibility of the chromophore-surrounding amino acid residues in these proteins. Mutagenesis experiments confirmed the crucial role of these residues in red chromophore biosynthesis. The obtained zoanGFPmut2 exhibited complete green-to-red transformation, suggesting that the mutated amino acids exposed on the surface of the β-barrel contribute to red chromophore biosynthesis.
关键词： autocatalysis,chromophore,protein engineering,fluorescent protein,molecular dynamics simulation,long-range interaction
更新于2025-09-23 15:19:57