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Current Opinion in Structural Biology Jun 2024Specimen preparation is a critical but challenging step in high-resolution cryogenic electron microscopy (cryo-EM) structural analysis of macromolecules. In the past... (Review)
Review
Specimen preparation is a critical but challenging step in high-resolution cryogenic electron microscopy (cryo-EM) structural analysis of macromolecules. In the past decade, graphene has gained much recognition as the supporting substrate to optimize cryo-EM specimen preparation. It improves macromolecule embedding in ice, reduces beam-induced motion, while imposing negligible background noise. Various types of graphene-coated cryo-EM grids were implemented to improve the robustness and efficiency of specimen preparation. Graphene functionalization by different means has been proved specifically useful in addressing challenges related to the air-water interface (AWI), such as preferential orientation and sample denaturation. Graphene sandwich specimen preparation sets a new direction to explore in cryo-EM analysis of biological specimens. In this review, we discuss the current challenges and future prospects of graphene application in cryo-EM analysis of macromolecules.
Topics: Cryoelectron Microscopy; Graphite; Macromolecular Substances; Specimen Handling
PubMed: 38688075
DOI: 10.1016/j.sbi.2024.102823 -
Frontiers in Immunology 2024Skin injuries represent a prevalent form of physical trauma, necessitating effective therapeutic strategies to expedite the wound healing process. Hesperidin, a...
INTRODUCTION
Skin injuries represent a prevalent form of physical trauma, necessitating effective therapeutic strategies to expedite the wound healing process. Hesperidin, a bioflavonoid naturally occurring in citrus fruits, exhibits a range of pharmacological attributes, including antimicrobial, antioxidant, anti-inflammatory, anticoagulant, and analgesic properties. The main objective of the study was to formulate a hydrogel with the intention of addressing skin conditions, particularly wound healing.
METHODS
This research introduces a methodology for the fabrication of a membrane composed of a Polyvinyl alcohol - Sodium Alginate (PVA/A) blend, along with the inclusion of an anti-inflammatory agent, Hesperidin (H), which exhibits promising wound healing capabilities. A uniform layer of a homogeneous solution comprising PVA/A was cast. The process of crosslinking and the enhancement of hydrogel characteristics were achieved through the application of gamma irradiation at a dosage of 30 kGy. The membrane was immersed in a Hesperidin (H) solution, facilitating the permeation and absorption of the drug. The resultant system is designed to deliver H in a controlled and sustained manner, which is crucial for promoting efficient wound healing. The obtained PVA/AH hydrogel was evaluated for cytotoxicity, antioxidant and free radical scavenging activities, anti-inflammatory and membrane stability effect. In addition, its action on oxidative stress, and inflammatory markers was evaluated on BJ-1 human normal skin cell line.
RESULTS AND DISCUSSION
We determined the effect of radical scavenging activity PVA/A (49 %) and PVA/AH (87%), the inhibition of Human red blood cell membrane hemolysis by PVA/AH (81.97 and 84.34 %), hypotonicity (83.68 and 76.48 %) and protein denaturation (83.17 and 85.8 %) as compared to 250 μg/ml diclofenac (Dic.) and aspirin (Asp.), respectively. Furthermore, gene expression analysis revealed an increased expression of genes associated with anti-oxidant and anti-inflammatory properties and downregulated TNFα, NFκB, iNOS, and COX2 by 67, 52, 58 and 60%, respectively, by PVA/AH hydrogel compared to LPS-stimulated BJ-1 cells. The advantages associated with Hesperidin can be ascribed to its antioxidant and anti-inflammatory attributes. The incorporation of Hesperidin into hydrogels offers promise for the development of a novel, secure, and efficient strategy for wound healing. This innovative approach holds potential as a solution for wound healing, capitalizing on the collaborative qualities of PVA/AH and gamma irradiation, which can be combined to establish a drug delivery platform for Hesperidin.
Topics: Hesperidin; Polyvinyl Alcohol; Humans; Alginates; NF-kappa B; Tumor Necrosis Factor-alpha; Hydrogels; Signal Transduction; Anti-Inflammatory Agents; Wound Healing; Cyclooxygenase 2; Nitric Oxide Synthase Type II; Antioxidants; Inflammation
PubMed: 38686374
DOI: 10.3389/fimmu.2024.1347420 -
Foods (Basel, Switzerland) Apr 2024is a species of octopus that is favored by consumers due to its rich nutrient profile. To investigate the influence of different thawing methods on the quality of...
is a species of octopus that is favored by consumers due to its rich nutrient profile. To investigate the influence of different thawing methods on the quality of octopus meat, we employed four distinct thawing methods: air thawing (AT), hydrostatic thawing (HT), flowing water thawing (FWT), and microwave thawing (MT). We then explored the differences in texture, color, water retention, pH, total volatile basic nitrogen (TVB-N), total sulfhydryl content, Ca-ATPase activity, and myofibrillar protein, among other quality indicators in response to these methods, and used a low-field nuclear magnetic resonance analyzer to assess the water migration that occurred during the thawing process. The results revealed that AT had the longest thawing time, leading to oxidation-induced protein denaturation, myofibrillar protein damage, and a significant decrease in water retention. Additionally, when this method was utilized, the content of TVB-N was significantly higher than in the other three groups. HT, to a certain extent, isolated the oxygen in the meat and thus alleviated protein oxidation, allowing higher levels of Ca-ATPase activity, sulfhydryl content, and springiness to be maintained. However, HT had a longer duration: 2.95 times that of FWT, resulting in a 9.84% higher cooking loss and a 28.21% higher TVB-N content compared to FWT. MT had the shortest thawing time, yielding the lowest content of TVB-N. However, uneven heating and in some cases overcooking occurred, severely damaging the protein structure, with a concurrent increase in thawing loss, W value, hardness, and shear force. Meanwhile, FWT improved the , and values of octopus meat, enhancing its color and water retention. The myofibrillar protein (MP) concentration was also the highest after FWT, with clearer subunit bands in SDS-PAGE electrophoresis, indicating that less degradation occurred and allowing greater springiness, increased Ca-ATPase activity, and a higher sulfhydryl content to be maintained. This suggests that FWT has an inhibitory effect on oxidation, alleviating protein oxidation degradation and preserving the quality of the meat. In conclusion, FWT outperformed the other three thawing methods, effectively minimizing adverse changes during thawing and successfully maintaining the quality of octopus meat.
PubMed: 38672906
DOI: 10.3390/foods13081234 -
Foods (Basel, Switzerland) Apr 2024Fresh pork tenderloin was stored at -3 °C under different static magnetic fields (SMF) of 0, 4, and 10 mT (control, MF-4, and MF-10) to investigate their...
Fresh pork tenderloin was stored at -3 °C under different static magnetic fields (SMF) of 0, 4, and 10 mT (control, MF-4, and MF-10) to investigate their physicochemical properties changes during storage of 8 days. The initial equilibrium temperature of the samples stored with 4 mT MF was found to be -2.3 °C, which was slightly lower (0.3 °C) than that the control value. The super-chilling phenomenon on the pork was then observed, as the samples stored under the magnetic field did not freeze throughout storage period, but the control experienced a sudden change in temperature after 138 h and then froze. The preservation effect of MF-4 on meat quality was the best in all treatment groups. MF-4 achieved a higher water-retention rate, with drip and cook losses of 6.5% and 29.0% lower than the control, respectively. Meanwhile, the MF-4 effectively delayed the color change in the meat during the storage and the texture hardening after cooking, and effectively controlled the growth of the total volatile saline nitrogen content on the samples. In addition, MF-4 delayed the reduction in myofibrillar protein solubility, sulfhydryl content, and emulsification capacity, indicating that this field inhibited the denaturation of myofibrillar protein. This study can be considered as an application reference of magnetic fields during meat storage at a super-chilled temperature.
PubMed: 38672878
DOI: 10.3390/foods13081205 -
Biomedicines Apr 2024A series of novel 1,5-diaryl pyrazole derivatives targeting the COX enzyme were designed by combined ligand and structure-based approach. The designed molecules were...
A series of novel 1,5-diaryl pyrazole derivatives targeting the COX enzyme were designed by combined ligand and structure-based approach. The designed molecules were then further subjected to ADMET and molecular docking studies. Out of 34 designed compounds, the top-10 molecules from the computation studies were synthesized, characterized, and evaluated for COX-2 inhibition and anti-cancer activity. Initially, the target compounds were screened for the protein denaturation assay. The results of the top-five molecules T2, T3, T5, T6, and T9 were further subjected to in vitro COX-2 enzymatic assay and anti-cancer activity. As far as COX-2 inhibitory activity is considered, two compounds, T3 and T5, exhibited the half maximum inhibitory concentration (IC) at 0.781 µM and 0.781 µM respectively. Further, the two compounds T3 and T5, when evaluated for COX-1 inhibition, exhibited excellent inhibitory activity with T3 IC of 4.655μM and T5 with IC of 5.596 μM. The compound T5 showed more significant human COX-2 inhibition, with a selectivity index of 7.16, when compared with T3, which had a selectivity index of 5.96. Further, in vitro anti-cancer activity was screened against two cancer cell lines in which compounds T2 and T3 were active against A549 cell lines and T6 was active against the HepG2 cell line. Stronger binding energy was found by comparing MM-PBSA simulations with molecular docking, which suggests that compounds T3 and T5 have a better possibility of being effective compounds, in which T5 showed higher binding affinity. The results suggest that these compounds have the potential to develop effective COX-2 inhibitors as anti-cancer agents.
PubMed: 38672144
DOI: 10.3390/biomedicines12040788 -
Bioengineering (Basel, Switzerland) Mar 2024Femtosecond (fs) laser technology is currently being considered in innovative fields such as osteotomy and treatment of hard tissue thanks to the achievable high...
Femtosecond (fs) laser technology is currently being considered in innovative fields such as osteotomy and treatment of hard tissue thanks to the achievable high resolution and ability to prevent tissue damage. In a previous study, suitable process parameters were obtained to achieve competitive ablation rates on pork femur processing. Nevertheless, a better control of thermal accumulation in the tissue during laser ablation could further improve the postoperative regeneration of the treated bone compared with conventional procedures and push forward the exploitation of such technology. This study presents methods for real time analyses of bone tissue temperature and composition during fs laser ablation and highlights the importance of implementing an efficient cooling method of bone tissue in order to achieve optimized results. Results show that it is possible to achieve a larger process window for bone tissue ablation where bone tissue temperature remains within the protein denaturation temperature in water-based processing environment. This is a key outcome towards a clinical exploitation of the presented technology, where higher process throughputs are necessary. The effects of process parameters and environments on bone tissue were confirmed by LIBS technique, which proved to be an efficient method by which to record real-time variation of bone tissue composition during laser irradiation.
PubMed: 38671731
DOI: 10.3390/bioengineering11040309 -
Antibiotics (Basel, Switzerland) Apr 2024has widespread use in folk medicine on the Balkan peninsula. In order to scientifically justify this use, the composition and biological activity of aqueous, ethanol,...
has widespread use in folk medicine on the Balkan peninsula. In order to scientifically justify this use, the composition and biological activity of aqueous, ethanol, and acetone extract were investigated in this study. Moreover, acetone and ethanol extracts were obtained from the plant material previously exhausted by water extraction. A total of 27 compounds were detected in extracts by UHPLC-DAD-MS/MS analysis, with all of them present in acetone and ethanol extracts. Consequentially, the acetone and ethanol extracts showed higher contents of total phenols of 23% and 18%, respectively, compared to the water extract. The results indicated high biological potential in the investigated extracts. Among all extracts, the aqueous extract showed slightly higher antimicrobial potential, especially against Gram-positive strains, probably due to the release of components soluble in water from the dry unexhausted plant material. On the other hand, the acetone and ethanol extracts had significantly higher antioxidative (by 20%), anti-inflammatory activity (up to 3 and 4 times higher, respectively), and α-glucosidase inhibitory potential (3 times higher) than the aqueous extract. The results of this investigation reveal the great potential of the use of in various branches of food, cosmetics, and the pharmaceutical industry. An important part of this research is a confirmation that, once exhausted by water extraction, for example by hydrodistillation, plant material can be reused for obtaining valuable products with a wide range of biological activities.
PubMed: 38667034
DOI: 10.3390/antibiotics13040358 -
Chemical Science Apr 2024The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass...
The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals -phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) was developed to enhance detection sensitivity. RAPID with OPA successfully identified structural changes induced by the allosteric drug TEPP-46 on its target protein PKM2 and was applied to profile the conformation change of the proteome occurring in cells during thermal denaturation. The application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting enables the identification of ligand-binding sites and detection of protein structural changes occurring in cells.
PubMed: 38665522
DOI: 10.1039/d4sc00032c -
Nature Methods Jun 2024Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation...
Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air-water or graphene-water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues.
Topics: Cryoelectron Microscopy; Specimen Handling; Spectrometry, Mass, Electrospray Ionization; Proteins; Humans; Macromolecular Substances
PubMed: 38664529
DOI: 10.1038/s41592-024-02247-0 -
Nature Communications Apr 2024The development of unconventional long-wavelength fluorescent polymer hydrogels without using polycyclic aromatic hydrocarbons or extended π-conjugation is a...
The development of unconventional long-wavelength fluorescent polymer hydrogels without using polycyclic aromatic hydrocarbons or extended π-conjugation is a fundamental challenge in luminescent materials owing to a lack of understanding regarding the spatial interactions induced inherent clustering-triggered emission under water-rich conditions. Inspired by the color change of protein astaxanthin as a result of heat-induced denaturation, we propose a thermodynamically driven strategy to develop red fluorescence (~610 nm) by boiling multiple hydrogen-bonded poly(N-acryloylsemicarbazide) hydrogels in a water bath. We reveal that thermodynamically driven conformational changes of polymer chains from isolated hydrogen bonding donor-acceptor structures to through-space interaction structures induce intrinsic fluorescence shifts from blue to red during clustering-triggered emission. The proposed multiple hydrogen-bonding supramolecular hydrogel shows good fluorescence stability, mechanical robustness, and 3D printability for customizable shaping. We provide a viable method to prepare nonconventional long-wavelength fluorescent hydrogels towards soft fluorescent devices without initially introducing any fluorescent components.
PubMed: 38664408
DOI: 10.1038/s41467-024-47880-7