Native Structure Research Articles

Hydrogels in Cutaneous Wound Healing: Insights into Characterization, Properties, Formulation and Therapeutic Potential.

Hydrogels are polymeric materials that possess a set of characteristics meeting various requirements of an ideal wound dressing, making them promising for wound care. These features include, among others, the ability to absorb and retain large amounts of water and the capacity to closely mimic native structures, such as the extracellular matrix, facilitating various cellular processes like proliferation and differentiation. The polymers used in hydrogel formulations exhibit a broad spectrum of properties, allowing them to be classified into two main categories: natural polymers like collagen and chitosan, and synthetic polymers such as polyurethane and polyethylene glycol. This review offers a comprehensive overview and critical analysis of the key polymers that can constitute hydrogels, beginning with a brief contextualization of the polymers. It delves into their function, origin, and chemical structure, highlighting key sources of extraction and obtaining. Additionally, this review encompasses the main intrinsic properties of these polymers and their roles in the wound healing process, accompanied, whenever available, by explanations of the underlying mechanisms of action. It also addresses limitations and describes some studies on the effectiveness of isolated polymers in promoting skin regeneration and wound healing. Subsequently, we briefly discuss some application strategies of hydrogels derived from their intrinsic potential to promote the wound healing process. This can be achieved due to their role in the stimulation of angiogenesis, for example, or through the incorporation of substances like growth factors or drugs, such as antimicrobials, imparting new properties to the hydrogels. In addition to substance incorporation, the potential of hydrogels is also related to their ability to serve as a three-dimensional matrix for cell culture, whether it involves loading cells into the hydrogel or recruiting cells to the wound site, where they proliferate on the scaffold to form new tissue. The latter strategy presupposes the incorporation of biosensors into the hydrogel for real-time monitoring of wound conditions, such as temperature and pH. Future prospects are then ultimately addressed. As far as we are aware, this manuscript represents the first comprehensive approach that brings together and critically analyzes fundamental aspects of both natural and synthetic polymers constituting hydrogels in the context of cutaneous wound healing. It will serve as a foundational point for future studies, aiming to contribute to the development of an effective and environmentally friendly dressing for wounds.

Structural determinants of odorant-binding proteins affecting their ability to form amyloid fibrils

The formation of amyloid fibrils is associated with many severe pathologies as well as the execution of essential physiological functions by proteins. Despite the diversity, all amyloids share a similar morphology and consist of stacked β-strands, suggesting high amyloidogenicity of native proteins enriched with β-structure. Such proteins include those with a β-barrel-like structure with β-strands arranged into a cylindrical β-sheet. However, the mechanisms responsible for destabilization of the native state and triggering fibrillogenesis have not thoroughly explored yet. Here we analyze the structural determinants of fibrillogenesis in proteins with β-barrel structures on the example of odorant-binding protein (OBP), whose amyloidogenicity was recently demonstrated in vitro. We reveal a crucial role in the fibrillogenesis of OBPs for the “open” conformation of the molecule. This conformation is achieved by disrupting the interaction between the β-barrel and the C-terminus of protein monomers or dimers, which exposes “sticky” amyloidogenic sites for interaction. The data suggest that the “open” conformation of OBPs can be induced by destabilizing the native β-barrel structure through the disruption of: 1) intramolecular disulfide cross-linking and non-covalent contacts between the C-terminal fragment and β-barrel in the protein's monomeric form, or 2) intermolecular contacts involved in domain swapping in the protein's dimeric form.

Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A.

Protein aggregation is related to numerous pathological conditions like Alzheimer's and Parkinson's disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen β-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native β-barrel conformation to β-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the β-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.

Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection.

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.

Biomaterial engineering strategies for modeling the Bruch's membrane in age-related macular degeneration.

Age-related macular degeneration, a multifactorial inflammatory degenerative retinal disease, ranks as the leading cause of blindness in the elderly. Strikingly, there is a scarcity of curative therapies, especially for the atrophic advanced form of age-related macular degeneration, likely due to the lack of models able to fully recapitulate the native structure of the outer blood retinal barrier, the prime target tissue of age-related macular degeneration. Standard in vitro systems rely on 2D monocultures unable to adequately reproduce the structure and function of the outer blood retinal barrier, integrated by the dynamic interaction of the retinal pigment epithelium, the Bruch's membrane, and the underlying choriocapillaris. The Bruch's membrane provides structural and mechanical support and regulates the molecular trafficking in the outer blood retinal barrier, and therefore adequate Bruch's membrane-mimics are key for the development of physiologically relevant models of the outer blood retinal barrier. In the last years, advances in the field of biomaterial engineering have provided novel approaches to mimic the Bruch's membrane from a variety of materials. This review provides a discussion of the integrated properties and function of outer blood retinal barrier components in healthy and age-related macular degeneration status to understand the requirements to adequately fabricate Bruch's membrane biomimetic systems. Then, we discuss novel materials and techniques to fabricate Bruch's membrane-like scaffolds for age-related macular degeneration in vitro modeling, discussing their advantages and challenges with a special focus on the potential of Bruch's membrane-like mimics based on decellularized tissue.

Extraction of canola protein via natural deep eutectic solvents compared to alkaline treatments: Isolate characteristics and protein structural and functional properties

The capacity of four formulations of natural deep eutectic solvents (NDESs) for extraction of canola protein was compared to alkaline extractions at pH 12 and pH 9. NDESs were prepared with a 1:2:1 ratio of choline chloride/d-sorbitol, glycerol, d-glucose or urea/water. The urea formulation showed the lowest viscosity and density and highest polarity; and extracted a bright-yellow isolate with extraction efficiency of 52.89% and protein content of 84.03%. The rest of NDESs extracted light-green isolates with efficiencies of approximately 44%. Furthermore, All NDESs offered lower extraction efficiencies than pH 12 (68.34%, dark-green isolate) but higher than pH 9 (39.14%, yellow isolate). Molecular analysis via SE-HPLC and SDS-PAGE showed the presence of cruciferins and napins in all isolates, but higher napin contents for NDES isolates with cruciferin-to-napin ratios of around 60:40. NDESs also altered the secondary and tertiary structure of the protein to a lesser extent. The functionality analyses at pH range of 3–7 revealed better solubility for pH 9 (72.09%–39.35%) and glycerol formulation (68.61%–39.05%) isolates; and better foaming properties for all NDES isolates, possibly due to their higher napin contents. NDES isolates also showed better emulsifying properties at pH 5 and 7, while alkaline isolates produced more stable emulsions at pH 3, possibly due to the better solubility of their cruciferins at pH 3. Overall, NDES isolates offered higher extraction efficiencies and a close functionality compared to pH 9 isolate, better color and functionality than pH 12 isolate, and better preservation of native protein structure compared to both alkaline treatments.

Deciphering the interaction of bacteria inoculants with the recipient endophytic community in grapevine micropropagated plants.

Engineering the plant microbiome with beneficial endophytic bacteria can improve the growth, health, and productivity of the holobiont. Here, we administered two beneficial bacterial strains, Kosakonia VR04 sp. and Rhizobium GR12 sp., to micropropagated grapevine cuttings obtained via somatic embryogenesis. While both strains colonized the plant endosphere, only Rhizobium GR12 sp. increased root biomass under nutritional-deficit conditions, as supported by the plant growth promotion traits detected in its genome. Phylogenetic and co-occurrence analyses revealed that the plant native bacterial community, originally dominated by Streptococcaceae and Micrococcaceae, dramatically changed depending on the inoculation treatments, as invading strains differently affected the relative abundance and the interactions of pre-existing taxa. After 30 days of plantlets' growth, Pantoea became a predominant taxon, and considering untreated plantlets as references, Rhizobium sp. GR12 showed a minor impact on the endophytic bacterial community. On the other hand, Kosakonia sp. VR04 caused a major change in community composition, suggesting an opportunistic colonization pattern. Overall, the results corroborate the importance of preserving the native endophytic community structure and functions during plant microbiome engineering.IMPORTANCEA better comprehension of bacterial colonization processes and outcomes could benefit the use of plant probiotics in the field. In this study, we applied two different beneficial bacteria to grapevine micropropagated plantlets and described how the inoculation of these strains impacts endophytic microbiota assembly. We showed that under nutritional deficit conditions, the response of the receiving endophytic bacterial communities to the invasion of the beneficial strains related to the manifestation of plant growth promotion effects by the inoculated invading strains. Rhizobium sp. GR12 was able to preserve the native microbiome structure despite its effective colonization, highlighting the importance of the plant-endophyte associations for the holobiont performance. Moreover, our approach showed that the use of micropropagated plantlets could be a valuable strategy to study the interplay among the plant, its native microbiota, and the invader on a wider portfolio of species besides model plants, facilitating the application of new knowledge in agriculture.

Correlation between in vitro starch digestibility and starch structure/physicochemical properties in rice

Natural resistant starch (RS) in rice provides human health benefits, and its concentration in rice is influenced by the structure and physicochemical properties of starch. The native starch structures and physicochemical properties of three rice varieties, QR, BR58, and BR50, and their relationships to in vitro digestibility were studied. The starch granules in all three varieties were irregular or polyhedral in shape. There were a few oval granules and a few pinhole structures in QR, no oval granules but a higher number of pinholes in BR58, and no oval granules and pinholes in BR50. QR is a low-amylose (13.8 %), low-RS (0.2 %) variety. BR58 is a low-amylose (15.3 %), high-RS (6.5 %) variety. BR50 is a high-amylose (26.7 %), high-RS (8.3 %) variety. All three starches exhibited typical A-type diffraction patterns. Starch molecular weight, chain length distribution, starch branching degree, pasting capabilities, and thermal properties differed considerably between the rice starches. The RS contents of the rice starch varieties were positively correlated with AAC, Mw/Mn, Mz/Mn, peak 3, B, PTime, and Tp and negatively correlated with Mn, peak 2, DB, PV, and BD, according to Pearson's correlation analysis. These findings may be helpful for the breeding and development of high-RS rice varieties.

Simulations of Functional Motions of Super Large Biomolecules with a Mixed-Resolution Model.

Many large protein machines function through an interplay between large-scale movements and intricate conformational changes. Understanding functional motions of these proteins through simulations becomes challenging for both all-atom and coarse-grained (CG) modeling techniques because neither approach alone can readily capture the full details of these motions. In this study, we develop a multiscale model by employing the popular MARTINI CG model to represent a heterogeneous environment and structurally stable proteins and using the united-atom (UA) model PACE to describe proteins undergoing subtle conformational changes. PACE was previously developed to be compatible with the MARTINI solvent and membrane. Here, we couple the protein descriptions of the two models by directly mixing UA and CG interaction parameters to greatly simplify parameter determination. Through extensive validations with diverse protein systems in solution or membrane, we demonstrate that only additional parameter rescaling is needed to enable the resulting model to recover the stability of native structures of proteins under mixed representation. Moreover, we identify the optimal scaling factors that can be applied to various protein systems, rendering the model potentially transferable. To further demonstrate its applicability for realistic systems, we apply the model to a mechanosensitive ion channel Piezo1 that has peripheral arms for sensing membrane tension and a central pore for ion conductance. The model can reproduce the coupling between Piezo1's large-scale arm movement and subtle pore opening in response to membrane stress while consuming much less computational costs than all-atom models. Therefore, our model shows promise for studying functional motions of large protein machines.

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar.

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.

Kinetic evidence for multiple aggregation pathways in antibody light chain variable domains.

Aggregation of antibody light chain proteins is associated with the progressive disease light chain amyloidosis. Patient-derived amyloid fibrils are formed from light chain variable domain residues in non-native conformations, highlighting a requirement that light chains unfold from their native structures in order to aggregate. However, mechanistic studies of amyloid formation have primarily focused on the self-assembly of natively unstructured peptides, and the role of native state unfolding is less well understood. Using a well-studied light chain variable domain protein known as WIL, which readily aggregates in vitro under conditions where the native state predominates, we asked how the protein concentration and addition of pre-formed fibril "seeds" alter the kinetics of aggregation. Monitoring aggregation with thioflavin T fluorescence revealed a distinctly non-linear dependence on concentration, with a maximum aggregation rate observed at 8 μM protein. This behavior is consistent with formation of alternate aggregate structures in the early phases of amyloid formation. Addition of N- or C-terminal peptide tags, which did not greatly affect the folding or stability of the protein, altered the concentration dependence of aggregation. Aggregation rates increased in the presence of pre-formed seeds, but this effect did not eliminate the delay before aggregation and became saturated when the proportion of seeds added was greater than 1 in 1600. The complexity of aggregation observed in vitro highlights how multiple species may contribute to amyloid pathology in patients.

A Tale of Two Chains: Geometries of a Chain Model and Protein Native State Structures.

Linear chain molecules play a central role in polymer physics with innumerable industrial applications. They are also ubiquitous constituents of living cells. Here, we highlight the similarities and differences between two distinct ways of viewing a linear chain. We do this, on the one hand, through the lens of simulations for a standard polymer chain of tethered spheres at low and high temperatures and, on the other hand, through published experimental data on an important class of biopolymers, proteins. We present detailed analyses of their local and non-local structures as well as the maps of their closest contacts. We seek to reconcile the startlingly different behaviors of the two types of chains based on symmetry considerations.

Identification of allergens from Aspergillus fumigatus-Potential association with lung damage in asthma.

Component-resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2-dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained. We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti-human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract. Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus-sensitized people with asthma, 23 non-fungal-sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose-bisphosphate aldolase class II (FBA2) (33%), NAD-dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens. In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.

HOPping up with the homeostasis engine in plant development and stress

Abstract Proteins need to acquire their native structure in order to become fully functional. In specific cases, active conformation is obtained spontaneously; nevertheless, many proteins need the assistance of chaperones and co-chaperones to be properly folded. These proteins help to maintain protein homeostasis under control conditions and under different stresses. HOP (HSP70-HSP90 Organizing Protein) is a highly conserved family of co-chaperones that assist HSP70 and HSP90 in the folding of specific proteins. In the last few years, exciting findings in mammals and yeast have revealed novel functions of HOP and re-defined the role of HOP in protein folding. In this article, we give an overview of the most important aspects of HOP regulation and function in other eukaryotes and analyze if these aspects are conserved in plants. In addition, we put special emphasis on the HOP clients described in plants and on the role of HOP in plant development and stress response.

Structural basis of ribosomal 30S subunit degradation by RNase R.

Protein synthesis is a major energy-consuming process of the cell that requires the controlled production1-3 and turnover4,5 of ribosomes. Although the past few years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3' to 5' exonuclease ribonuclease R (RNase R). The structures reveal that RNase R binds at first to the 30S platform to facilitate the degradation of the functionally important anti-Shine-Dalgarno sequence and the decoding-site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S subunit, and this is overcome by a major structural rearrangement of the 30S head, involving the loss of ribosomal proteins. RNase R parallels this movement and relocates to the decoding site by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also indicate that the enzyme alone is sufficient for complete degradation of 30S subunits. Collectively, our results provide a mechanistic basis for the degradation of 30S mediated by RNase R, and reveal that RNase R targets orphaned 30S subunits using a dynamic mechanism involving an anchored switching of binding sites.

The Effect of Concavity Restoration on Glenohumeral Stability in a Glenoid Bone Loss Model: Comparing the Distal Tibial Allograft Reconstruction and the Latarjet Procedure

Objectives: Glenoid bone loss occurs in more than 80% of patients with recurrent glenohumeral instability. The Latarjet procedure has become the gold standard for restoration of bone loss despite a high (15-30%) complication rate.1–3 Distal tibial allograft (DTA) reconstruction has recently gained interest due to its ability to restore width and concavity to the glenoid while providing a cartilage surface.4 The purpose of this study is to evaluate the restoration of glenohumeral anterior stability and glenoid concavity after DTA reconstruction when compared to the Latarjet procedure in a glenoid bone loss model. Methods: Five human cadaveric specimens (mean age: 62.2, range 57-69; 80% male) underwent preoperative computed tomography (CT) scans to assess native glenoid concavity as determined by the glenoid depth and bony shoulder stability ratio (BSSR). Stability testing was performed using a cadaveric shoulder simulator that allows 6 DoF at the glenohumeral joint. The specimens were placed with the scapula at 30° upward rotation and humerus at 60° glenohumeral abduction with a constant compressive force created by loading the supraspinatus, infraspinatus, teres minor, and subscapularis muscles (2lbs, 3lbs, 1lb and 4lbs respectively). A Kuka robot applied a 40N anterior force through the pectoralis tendon with the humerus in neutral as well as 60 degrees of external rotation. A motion capture system recorded humeral head translations for each condition. To create the Latarjet and DTA conditions, the coracoid was first harvested and the thickness was measured. A distal tibial allograft was then contoured to an identical size. A glenoid defect was created to match the thickness of the graft since both Latarjet and DTA aimed for a 100% glenoid restoration.. The following conditions were tested: 1. intact, 2. Bankart lesion, 3. bone loss model with DTA reconstruction, 4. Latarjet procedure without the conjoint tendon loaded, and 5. Latarjet with conjoint tendon loading (‘sling effect’). All specimens underwent post-testing CT scan to measure the BSSR of the DTA and Latarjet reconstructions. A repeated measures ANOVA was performed to determine if there was a significant difference in maximum anterior translation or BSSR between the distal tibial allograft reconstruction and Latarjet procedure (with or without the sling effect). Results: The mean preoperative glenoid depth measured 2.61±1.03mm with a BSSR of 0.40±0.16. However, postoperative measurements showed that BSSR for the DTA was greater than the Latarjet (0.46±0.11 vs 0.35±0.5). The maximum anterior translation was significantly lower after the DTA reconstruction than the Latarjet procedure without the sling effect (5.5mm vs 9.6mm, p = 0.044). However, there was no difference in anterior translation between the DTA reconstruction and Latarjet procedure with the sling effect applied (5.5mm vs 4.7mm, p = 0.36). The DTA reconstructed the glenoid with a significantly greater concavity than the Latarjet procedure (BSSR: 0.46 vs 0.35, p = 0.036). Conclusions: When addressing bone loss in glenohumeral instability, additional factors besides the restoration of glenoid width contribute to stability. The concave reconstruction produced by the distal tibial allograft decreases anterior translation compared to the flat reconstruction produced by the Latarjet, but there is no difference in translation between the DTA and Latarjet procedure when the sling effect is applied. The DTA reconstruction provides an alternative to the Latarjet procedure with equivalent biomechanical properties and less morbidity or change to the native shoulder structure.

3dDNAscoreA: A scoring function for evaluation of DNA 3D structures

DNA molecules are vital macromolecules that play a fundamental role in many cellular processes and have broad applications in medicine. For example, DNA aptamers have been rapidly developed for diagnosis, biosensors, and clinical therapy. Recently, we proposed a computational method of predicting DNA 3D structures, called 3dDNA. However, it lacks a scoring function to evaluate the predicted DNA 3D structures, and so they are not ranked for users. Here, we report a scoring function, 3dDNAscoreA, for evaluation of DNA 3D structures based on a deep learning model ARES for RNA 3D structure evaluation but using a new strategy for training. 3dDNAscoreA is benchmarked on two test sets to show its ability to rank DNA 3D structures and select the native and near-native structures.

Making Use of Averaging Methods in MODELLER for Protein Structure Prediction.

Recent advances in protein structure prediction, driven by AlphaFold 2 and machine learning, demonstrate proficiency in static structures but encounter challenges in capturing essential dynamic features crucial for understanding biological function. In this context, homology-based modeling emerges as a cost-effective and computationally efficient alternative. The MODELLER (version 10.5, accessed on 30 November 2023) algorithm can be harnessed for this purpose since it computes intermediate models during simulated annealing, enabling the exploration of attainable configurational states and energies while minimizing its objective function. There have been a few attempts to date to improve the models generated by its algorithm, and in particular, there is no literature regarding the implementation of an averaging procedure involving the intermediate models in the MODELLER algorithm. In this study, we examined MODELLER's output using 225 target-template pairs, extracting the best representatives of intermediate models. Applying an averaging procedure to the selected intermediate structures based on statistical potentials, we aimed to determine: (1) whether averaging improves the quality of structural models during the building phase; (2) if ranking by statistical potentials reliably selects the best models, leading to improved final model quality; (3) whether using a single template versus multiple templates affects the averaging approach; (4) whether the "ensemble" nature of the MODELLER building phase can be harnessed to capture low-energy conformations in holo structures modeling. Our findings indicate that while improvements typically fall short of a few decimal points in the model evaluation metric, a notable fraction of configurations exhibit slightly higher similarity to the native structure than MODELLER's proposed final model. The averaging-building procedure proves particularly beneficial in (1) regions of low sequence identity between the target and template(s), the most challenging aspect of homology modeling; (2) holo protein conformations generation, an area in which MODELLER and related tools usually fall short of the expected performance.

Native collagen sheet mask improves skin health and appearance: A comprehensive clinical evaluation.

Collagen, a critical structural protein found abundantly in animal skin and bones, has become increasingly recognized for its potential therapeutic role in skincare. Despite growing interest, the scientific evidence for the efficacy of collagen sheet masks remains limited. The principal objective of our study was to provide insights into the multifaceted role of collagen in skin health, with a specific focus on its application in collagen sheet masks. The effects of a collagen sheet mask consisting of >92% native bovine collagen were investigated. The soluble protein components of the collagen matrix were analyzed and the influence of soluble collagen components on fibroblast regulation was examined. Scanning Electron Microscope (SEM) analysis was performed for structural analysis and effect on irritated skin. Five different clinical studies were conducted, including a comparison of the diversity of the skin microbiome, the tolerance and local irritating reactions in atopic dermatitis, an evaluation of skin redness after UV radiation, wrinkle reduction, and hydration and skin roughness of the collagen mask in comparison to a pre-soaked cellulose sheet mask. The collagen mask contains soluble protein components, including small collagen peptides. The mask showed potential for promoting fibroblast activity. SEM analysis showed a native collagen structure similar to human dermis. The mask maintained the skin microbiome diversity and decreased skin pH levels. It demonstrated good tolerability on both intact and lesional skin and had a significant effect in reducing erythema caused by UV radiation compared to other skincare products. It showed significant improvements in skin hydration and the volume of eye wrinkles and was more effective than pre-soaked cellulose sheet masks. Collagen sheet masks have the potential to positively impact skin health and appearance by increasing hydration, reducing erythema, minimizing wrinkles, and maintaining a healthy skin microbiome and skin barrier.

Epoxide-Mediated Trans-Thioglycosylation and Application to the Synthesis of Oligosaccharides Related to the Capsular Polysaccharides of C. jejuni HS:4.

The enzyme-resistant thioglycosides are highly valuable immunogens because of their enhanced metabolic stability. We report the first synthesis of a family of thiooligosaccharides related to the capsular polysaccharides (CPS) of Campylobacter jejuni HS:4 for potential use in conjugate vaccines. The native CPS structures of the pathogen consist of a challenging repeating disaccharide formed with β(1→4)-linked 6-deoxy-β-D-ido-heptopyranoside and N-acetyl-D-glucosamine; the rare 6-deoxy-ido-heptopyranosyl backbone and β-anomeric configuration of the former monosaccharide makes the synthesis of this family of antigens very challenging. So far, no synthesis of the thioanalogs of the CPS antigens have been reported. The unprecedented synthesis presented in this work is built on an elegant approach by using β-glycosylthiolate as a glycosyl donor to open the 2,3-epoxide functionality of pre-designed 6-deoxy-β-D-talo-heptopyranosides. Our results illustrated that this key trans-thioglycosylation can be designed in a modular and regio and stereo-selective manner. Built on the success of this novel approach, we succeeded the synthesis of a family of thiooligosaccharides including a thiohexasaccharide which is considered to be the desired antigen length and complexity for immunizations. We also report the first direct conversion of base-stable but acid-labile 2-trimethylsilylethyl glycosides to glycosyl-1-thioacetates in a one-pot manner.