Functional stability of water wire-carbonyl interactions in an ion channel.

Water wires are critical for the functioning of many membrane proteins, as in channels that conduct water, protons, and other ions. Here, in liquid crystalline lipid bilayers under symmetric environmental conditions, the selective hydrogen bonding interactions between eight waters comprising a water wire and a subset of 26 carbonyl oxygens lining the antiparallel dimeric gramicidin A channel are characterized by 17O NMR spectroscopy at 35.2 T (or 1,500 MHz for 1H) and computational studies. While backbone 15N spectra clearly indicate structural symmetry between the two subunits, single site 17O labels of the pore-lining carbonyls report two resonances, implying a break in dimer symmetry caused by the selective interactions with the water wire. The 17O shifts document selective water hydrogen bonding with carbonyl oxygens that are stable on the millisecond timescale. Such interactions are supported by density functional theory calculations on snapshots taken from molecular dynamics simulations. Water hydrogen bonding in the pore is restricted to just three simultaneous interactions, unlike bulk water environs. The stability of the water wire orientation and its electric dipole leads to opposite charge-dipole interactions for K+ ions bound at the two ends of the pore, thereby providing a simple explanation for an ∼20-fold difference in K+ affinity between two binding sites that are ∼24 Šapart. The 17O NMR spectroscopy reported here represents a breakthrough in high field NMR technology that will have applications throughout molecular biophysics, because of the acute sensitivity of the 17O nucleus to its chemical environment.

Anti-Cancer Effect of Chlorophyllin-Assisted Photodynamic Therapy to Induce Apoptosis through Oxidative Stress on Human Cervical Cancer.

Photodynamic therapy is an alternative approach to treating tumors that utilizes photochemical reactions between a photosensitizer and laser irradiation for the generation of reactive oxygen species. Currently, natural photosensitive compounds are being promised to replace synthetic photosensitizers used in photodynamic therapy because of their low toxicity, lesser side effects, and high solubility in water. Therefore, the present study investigated the anti-cancer efficacy of chlorophyllin-assisted photodynamic therapy on human cervical cancer by inducing apoptotic response through oxidative stress. The chlorophyllin-assisted photodynamic therapy significantly induced cytotoxicity, and the optimal conditions were determined based on the results, including laser irradiation time, laser power density, and chlorophyllin concentration. In addition, reactive oxygen species generation and Annexin V expression level were detected on the photodynamic reaction-treated HeLa cells under the optimized conditions to evaluate apoptosis using a fluorescence microscope. In the Western blotting analysis, the photodynamic therapy group showed the increased protein expression level of the cleaved caspase 8, caspase 9, Bax, and cytochrome C, and the suppressed protein expression level of Bcl-2, pro-caspase 8, and pro-caspase 9. Moreover, the proposed photodynamic therapy downregulated the phosphorylation of AKT1 in the HeLa cells. Therefore, our results suggest that the chlorophyllin-assisted photodynamic therapy has potential as an antitumor therapy for cervical cancer.

Dieckol induces cell cycle arrest by down-regulating CDK2/cyclin E in response to p21/p53 activation in human tracheal fibroblasts.

The phlorotannin derivative dieckol isolated from Ecklonia cava has been shown to exhibit anti-inflammatory, anti-bacterial, anti-oxidative anti-adipogenic and anti-stenosis activity. However, the role of dieckol in cyclin-dependent kinase 2 (CDK2)/cyclin E signalling, which regulates fibrosis development, has not yet been determined. In this study, we report that dieckol-suppressed cell proliferation through the cell cycle arrest of Hs680.Tr human tracheal fibroblasts. Following consecutive purification, dieckol was identified as a potent bioactive compound. The results showed that dieckol had significant anti-proliferative activity against Hs680.Tr human tracheal fibroblastsWestern blotting analysis also found that dieckol dose-dependently induced the cell cycle arrest of Hs680.Tr fibroblasts in the G0/G1 phase, accompanied by the downregulation of CDK2 and cyclin E and the upregulation of p21 and p53. As attested by molecular docking study, the dieckol interacted with the core interface residues in transforming growth factor-ß receptor with high affinity. These findings suggest that dieckol from E. cava inhibits the cell proliferation of Hs680.Tr, potentially through p21- and p53-mediated G0/G1 cell cycle arrest.

A 3D-Printed Polycaprolactone/Marine Collagen Scaffold Reinforced with Carbonated Hydroxyapatite from Fish Bones for Bone Regeneration.

In bone tissue regeneration, extracellular matrix (ECM) and bioceramics are important factors, because of their osteogenic potential and cell-matrix interactions. Surface modifications with hydrophilic material including proteins show significant potential in tissue engineering applications, because scaffolds are generally fabricated using synthetic polymers and bioceramics. In the present study, carbonated hydroxyapatite (CHA) and marine atelocollagen (MC) were extracted from the bones and skins, respectively, of Paralichthys olivaceus. The extracted CHA was characterized using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis, while MC was characterized using FTIR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The scaffolds consisting of polycaprolactone (PCL), and different compositions of CHA (2.5%, 5%, and 10%) were fabricated using a three-axis plotting system and coated with 2% MC. Then, the MC3T3-E1 cells were seeded on the scaffolds to evaluate the osteogenic differentiation in vitro, and in vivo calvarial implantation of the scaffolds was performed to study bone tissue regeneration. The results of mineralization confirmed that the MC/PCL, 2.5% CHA/MC/PCL, 5% CHA/MC/PCL, and 10% CHA/MC/PCL scaffolds increased osteogenic differentiation by 302%, 858%, 970%, and 1044%, respectively, compared with pure PCL scaffolds. Consequently, these results suggest that CHA and MC obtained from byproducts of P. olivaceus are superior alternatives for land animal-derived substances.

Marine Biological Macromolecules and Chemically Modified Macromolecules; Potential Anticoagulants.

Coagulation is a potential defense mechanism that involves activating a series of zymogens to convert soluble fibrinogen to insoluble fibrin clots to prevent bleeding and hemorrhagic complications. To prevent the extra formation and diffusion of clots, the counterbalance inhibitory mechanism is activated at levels of the coagulation pathway. Contrariwise, this system can evade normal control due to either inherited or acquired defects or aging which leads to unusual clots formation. The abnormal formations and deposition of excess fibrin trigger serious arterial and cardiovascular diseases. Although heparin and heparin-based anticoagulants are a widely prescribed class of anticoagulants, the clinical use of heparin has limitations due to the unpredictable anticoagulation, risk of bleeding, and other complications. Hence, significant interest has been established over the years to investigate alternative therapeutic anticoagulants from natural sources, especially from marine sources with good safety and potency due to their unique chemical structure and biological activity. This review summarizes the coagulation cascade and potential macromolecular anticoagulants derived from marine flora and fauna.

Gating Mechanism of the Voltage-Gated Proton Channel Studied by Molecular Dynamics Simulations.

The voltage-gated proton channel Hv1 has important roles in proton extrusion, pH homeostasis, sperm motility, and cancer progression. The Hv1 channel has also been found to be highly expressed in cell lines and tissue samples from patients with breast cancer. A high-resolution closed-state structure has been reported for the mouse Hv1 chimera channel (mHv1cc), solved by X-ray crystallography, but the open-state structure of Hv1 has not been solved. Since Hv1 is a promising drug target, various groups have proposed open conformations by molecular modeling and simulation studies. However, the gating mechanism and the open-state conformation under the membrane potential are still debate. Here, we present a molecular dynamics study considering membrane potential and pH conditions. The closed-state structure of mHv1cc was used to run molecular dynamics (MD) simulations with respect to electric field and pH conditions in order to investigate the mechanism of proton transfer. We observed a continuous hydrogen bond chain of water molecules called a water-wire to be formed through the channel pore in the channel opening, triggered by downward displacement of the S2 helix and upward movement of the S4 helix relative to other helices. Due to the movement of the S2 and S4 helices, the internal salt bridge network was rearranged, and the hydrophobic gating layers were destroyed. In line with previous experimental and simulation observations, our simulation results led us to propose a new gating mechanism for the Hv1 proton channel, and may provide valuable information for novel drug discovery.

Erratum: Phan, T.T.V., et al. Spontaneous Hinge-Bending Motions of Angiotensin I Converting Enzyme: Role in Activation and Inhibition. Molecules 2020, 25, 1288.

The authors wish to make the following corrections to the paper [...].

Spontaneous Hinge-Bending Motions of Angiotensin I Converting Enzyme: Role in Activation and Inhibition.

The inhibition of human angiotensin I converting enzyme (ACE) has been regarded as a promising approach for the treatment of hypertension. Despite research attempts over many years, our understanding the mechanisms of activation and inhibition of ACE is still far from complete. Here, we present results of all atom molecular dynamics simulations of ACE with and without ligands. Two types of inhibitors, competitive and mixed non-competitive, were used to model the ligand bound forms. In the absence of a ligand the simulation showed spontaneous large hinge-bending motions of multiple conversions between the closed and open states of ACE, while the ligand bound forms were stable in the closed state. Our simulation results imply that the equilibrium between pre-existing backbone conformations shifts in the presence of a ligand. The hinge-bending motion of ACE is considered as an essential to the enzyme function. A mechanistic model of activation and the inhibition may provide valuable information for novel inhibitors of ACE.

Both Ligands and Macromolecular Crowders Preferentially Bind to Closed Conformations of Maltose Binding Protein.

In cellular environments, proteins not only interact with their specific partners but also encounter a high concentration of bystander macromolecules, or crowders. Nonspecific interactions with macromolecular crowders modulate the activities of proteins, but our knowledge about the rules of nonspecific interactions is still very limited. In previous work, we presented experimental evidence that macromolecular crowders acted competitively in inhibiting the binding of maltose binding protein (MBP) with its ligand maltose. Competition between a ligand and an inhibitor may result from binding to either the same site or different conformations of the protein. Maltose binds to the cleft between two lobes of MBP, and in a series of mutants, the affinities increased with an increase in the extent of lobe closure. Here we investigated whether macromolecular crowders also have a conformational or site preference when binding to MBP. The affinities of a polymer crowder, Ficoll70, measured by monitoring tryptophan fluorescence were 3-6-fold higher for closure mutants than for wild-type MBP. Competition between the ligand and crowder, as indicated by fitting of titration data and directly by nuclear magnetic resonance spectroscopy, and their similar preferences for closed MBP conformations further suggest the scenario in which the crowder, like maltose, preferentially binds to the interlobe cleft of MBP. Similar observations were made for bovine serum albumin as a protein crowder. Conformational and site preferences in MBP-crowder binding allude to the paradigm that nonspecific interactions can possess hallmarks of molecular recognition, which may be essential for intracellular organizations including colocalization of proteins and liquid-liquid phase separation.

Fish bone peptide promotes osteogenic differentiation of MC3T3-E1 pre-osteoblasts through upregulation of MAPKs and Smad pathways activated BMP-2 receptor.

Fish bone, a by-product of fishery processing, is composed of protein, calcium, and other minerals. The objective of this study was to investigate the effects of a bioactive peptide isolated from the bone of the marine fish, Johnius belengerii, on the osteoblastic differentiation of MC3T3-E1 pre-osteoblasts. Post consecutive purification by liquid chromatography, a potent osteogenic peptide, composed of 3 amino acids, Lys-Ser-Ala (KSA, MW: 304.17 Da), was identified. The purified peptide promoted cell proliferation, alkaline phosphatase activity, mineral deposition, and expression levels of phenotypic markers of osteoblastic differentiation in MC3T3-E1 pre-osteoblast. The purified peptide induced phosphorylation of mitogen-activated protein kinases, including p38 mitogen-activated protein kinase, extracellular regulated kinase, and c-Jun N-terminal kinase as well as Smads. As attested by molecular modelling study, the purified peptide interacted with the core interface residues in bone morphogenetic protein receptors with high affinity. Thus, the purified peptide could serve as a potential pharmacological substance for controlling bone metabolism.

Target-based drug discovery through inversion of quantitative structure-drug-property relationships and molecular simulation: CA IX-sulphonamide complexes.

In this work, a target-based drug screening method is proposed exploiting the synergy effect of ligand-based and structure-based computer-assisted drug design. The new method provides great flexibility in drug design and drug candidates with considerably lower risk in an efficient manner. As a model system, 45 sulphonamides (33 training, 12 testing ligands) in complex with carbonic anhydrase IX were used for development of quantitative structure-activity-lipophilicity (property)-relationships (QSPRs). For each ligand, nearly 5,000 molecular descriptors were calculated, while lipophilicity (logkw) and inhibitory activity (logKi) were used as drug properties. Genetic algorithm-partial least squares (GA-PLS) provided a QSPR model with high prediction capability employing only seven molecular descriptors. As a proof-of-concept, optimal drug structure was obtained by inverting the model with respect to reference drug properties. 3509 ligands were ranked accordingly. Top 10 ligands were further validated through molecular docking. Large-scale MD simulations were performed to test the stability of structures of selected ligands obtained through docking complemented with biophysical experiments.

Silver-Lactoferrin Nanocomplexes as a Potent Antimicrobial Agent.

The process of silver immobilization onto and/or into bovine lactoferrin (LTF), the physicochemical properties of bovine lactoferrin and obtained silver-lactoferrin complexes, as well as antibacterial activity of silver-lactoferrin complexes were investigated in this work. Kinetic study of the silver immobilization into lactoferrin was carried out using batch sorption techniques. Spectrometric (MALDI-TOF/TOF-MS, ICP-MS), spectroscopic (FTIR, SERS), electron microscopic (TEM) and electrophoretic (I-DE) techniques, as well as zeta potential measurements, were applied for characterization of LTF and binding nature of silver in Ag-LTF complexes. On the basis of the results of the kinetics study, it was established that the silver binding to LTF is a heterogeneous process involving two main stages: (i) internal diffusion and sorption onto external surface of lactoferrin globules; and (ii) internal diffusion and binding into lactoferrin globule structure. Spectroscopic techniques combined with TEM analysis confirmed the binding process. Molecular dynamics (MD) analysis was carried out in order to simulate the mechanism of the binding process, and locate potential binding sites, as well as complement the experimental findings. Quantum mechanics (QM) simulations were performed utilizing density functional theory (DFT) in order to support the reduction mechanism of silver ions to elemental silver. Antimicrobial activity of synthesized lactoferrin complexes against selected clinical bacteria was confirmed using flow cytometry and antibiograms.

Influence of solubilizing environments on membrane protein structures.

Membrane protein structures are stabilized by weak interactions and are influenced by additional interactions with the solubilizing environment. Structures of influenza virus A M2 protein, a proven drug target, have been determined in three different environments, thus providing a unique opportunity to assess environmental influences. Structures determined in detergents and detergent micelles can have notable differences from those determined in lipid bilayers. These differences make it imperative to validate membrane protein structures.

Leveraging Deep Learning for Fine-Grained Categorization of Parkinson's Disease Progression Levels through Analysis of Vocal Acoustic Patterns.

Speech impairments often emerge as one of the primary indicators of Parkinson's disease (PD), albeit not readily apparent in its early stages. While previous studies focused predominantly on binary PD detection, this research explored the use of deep learning models to automatically classify sustained vowel recordings into healthy controls, mild PD, or severe PD based on motor symptom severity scores. Popular convolutional neural network (CNN) architectures, VGG and ResNet, as well as vision transformers, Swin, were fine-tuned on log mel spectrogram image representations of the segmented voice data. Furthermore, the research investigated the effects of audio segment lengths and specific vowel sounds on the performance of these models. The findings indicated that implementing longer segments yielded better performance. The models showed strong capability in distinguishing PD from healthy subjects, achieving over 95% precision. However, reliably discriminating between mild and severe PD cases remained challenging. The VGG16 achieved the best overall classification performance with 91.8% accuracy and the largest area under the ROC curve. Furthermore, focusing analysis on the vowel /u/ could further improve accuracy to 96%. Applying visualization techniques like Grad-CAM also highlighted how CNN models focused on localized spectrogram regions while transformers attended to more widespread patterns. Overall, this work showed the potential of deep learning for non-invasive screening and monitoring of PD progression from voice recordings, but larger multi-class labeled datasets are needed to further improve severity classification.

Differences in gene organization between type I and type II crustins in the morotoge shrimp, Pandalopsis japonica.

Crustins are cysteine-rich cationic antimicrobial peptides (AMPs) found in decapod crustaceans. Six novel crustin genes (Paj-CrusIc, Id, Ie, If, IIb and IIc) were identified in the morotoge shrimp, Pandalopsis japonica. Deduced amino acid sequences of isolated Paj-Crus genes ranged from 99 to 178 amino acid residues (10.6-17.8 kDa). Sequence analysis of nine isolated Paj-Crus genes and 100 different crustins from various decapod crustaceans revealed that a splice site and KXXXCP motif within the WAP domain may be the main criteria for classifying type I and II crustins, suggesting that the two types of crustin genes may have been generated by different processes. We also identified three intron-less crustin I genes (Paj-Crus Id, Ie and If) for the first time, which may have been generated by gene duplication. The tissue distribution profiles showed that Paj-CrusI genes were expressed predominantly in the gill and epidermis, whereas Paj-CrusII genes were expressed ubiquitously, suggesting that the two types of crustins may play different roles in various tissues or under different physiological conditions. Differing from previous results, hemocyte-specific crustin was not isolated from Pandalopsis japonica. This study showed that both types of crustin genes (types I and II) exist in decapod crustaceans and their primary structure and expression profiles differ from each other, suggesting that they may play different biological roles. This will help to extend our knowledge of the crustacean innate immune response, which will provide important basic information of shrimp immunity against various pathogens.

Marine-derived biopolymers as potential bioplastics, an eco-friendly alternative.

The manufacturing and consumption of plastic products have steadily increased over the past decades due to rising global demand, resulting not only in the depletion of petroleum resources but also increased environmental pollution due to the non-biodegradable nature of conventional plastics. Moreover, despite being introduced into the market as an alternative to conventional petroleum-based plastics, biobased plastics are mainly manufactured from agricultural crop-based sources, which has negative impacts on the environment and the livelihoods of people. Marine-derived bioplastics are becoming a promising and cost-effective solution to the rising demand for plastic products. The physicochemical, biological, and degradation properties of marine-derived bioplastics have made them promising substances for many applications. However, more research is required for their large-scale implementation. Therefore, this review summarizes the raw materials of marine-derived bioplastics such as algae, animals, and microorganisms, as well as their extraction processes and properties. These insights could thus accelerate the production of marine-derived bioplastics as a novel alternative to prevailing bioplastics by taking advantage of marine biomass.

Mitochondrial DNA Corroborates the Genetic Variability of Clarias Catfishes (Siluriformes, Clariidae) from Cameroon.

The airbreathing walking catfish (Clariidae: Clarias) comprises 32 species that are endemic to African freshwater systems. The species-level identification of this group is challenging due to their complex taxonomy and polymorphism. Prior to this study, the biological and ecological studies were restricted to a single species, Clarias gariepinus, resulting in a biased view of their genetic diversity in African waters. Here, we generated the 63-mitochondrial Cytochrome c oxidase subunit 1 (COI) gene sequences of Clarias camerunensis and Clarias gariepinus from the Nyong River in Cameroon. Both C. camerunensis and C. gariepinus species maintained adequate intra-species (2.7% and 2.31%) and inter-species (6.9% to 16.8% and 11.4% to 15.1%) genetic distances with other Clarias congeners distributed in African and Asian/Southeast Asian drainages. The mtCOI sequences revealed 13 and 20 unique haplotypes of C. camerunensis and C. gariepinus, respectively. The TCS networks revealed distinct haplotypes of C. camerunensis and shared haplotypes of C. gariepinus in African waters. The multiple species delimitation approaches (ABGD and PTP) revealed a total of 20 and 22 molecular operational taxonomic units (MOTUs), respectively. Among the two Clarias species examined, we found more than one MOTU in C. camerunensis, which is consistent with population structure and tree topology results. The phylogeny generated through Bayesian Inference analysis clearly separated C. camerunensis and C. gariepinus from other Clarias species with high posterior probability supports. The present study elucidates the occurrence of possible cryptic diversity and allopatric speciation of C. camerunensis in African drainages. Further, the present study confirms the reduced genetic diversity of C. gariepinus across its native and introduced range, which might have been induced by unscientific aquaculture practices. The study recommends a similar approach to the same and related species from different river basins to illuminate the true diversity of Clarias species in Africa and other countries.

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation.

The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solid-state NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak approximately 10 degrees in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, approximately 0 degrees and 40 degrees . Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior.

Automated Structural Analysis and Quantitative Characterization of Scar Tissue Using Machine Learning.

An analysis of scar tissue is necessary to understand the pathological tissue conditions during or after the wound healing process. Hematoxylin and eosin (HE) staining has conventionally been applied to understand the morphology of scar tissue. However, the scar lesions cannot be analyzed from a whole slide image. The current study aimed to develop a method for the rapid and automatic characterization of scar lesions in HE-stained scar tissues using a supervised and unsupervised learning algorithm. The supervised learning used a Mask region-based convolutional neural network (RCNN) to train a pattern from a data representation using MMDetection tools. The K-means algorithm characterized the HE-stained tissue and extracted the main features, such as the collagen density and directional variance of the collagen. The Mask RCNN model effectively predicted scar images using various backbone networks (e.g., ResNet50, ResNet101, ResNeSt50, and ResNeSt101) with high accuracy. The K-means clustering method successfully characterized the HE-stained tissue by separating the main features in terms of the collagen fiber and dermal mature components, namely, the glands, hair follicles, and nuclei. A quantitative analysis of the scar tissue in terms of the collagen density and directional variance of the collagen confirmed 50% differences between the normal and scar tissues. The proposed methods were utilized to characterize the pathological features of scar tissue for an objective histological analysis. The trained model is time-efficient when used for detection in place of a manual analysis. Machine learning-assisted analysis is expected to aid in understanding scar conditions, and to help establish an optimal treatment plan.

Enhanced wound-healing capability with inherent antimicrobial activities of usnic acid incorporated poly(ε-caprolactone)/decellularized extracellular matrix nanofibrous scaffold.

An extracellular matrix-mimicking, biodegradable tissue-engineered skin substitute with improved antibacterial, antibiofilm, and wound healing capabilities is essential in skin tissue regeneration applications. The purpose of this study was to develop a novel biodegradable composite nanofibrous poly(ε-caprolactone) (PCL)/decellularized extracellular matrix (dECM) scaffolds loaded with usnic acid (UA); (PEU), where UA is employed as an antibacterial agent as well as a wound-healing accelerator. The architecture and fiber structure of the scaffolds were examined using scanning electron microscopy, and the results revealed that the average diameters decreased as the dECM content increased. The chemical composition, changes in the crystalline structure, homogeneity, and thermal stability of the nanofiber scaffolds with different material compositions were determined using Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis, respectively. The composite nanofibrous scaffolds exhibited strong antibacterial activity against various bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Cutibactrium acnes, and fungal pathogens (such as Candida albicans). Additionally, the composite nanofibrous scaffolds exhibited biofilm inhibition properties against Klebsiella pneumoniae and Pseudomonas aeruginosa. An evaluation of the appearance of in vivo full-thickness excisional wounds treated with the composite nanofiber scaffolds, as well as a histological analysis of the wounds 21 days after surgery, revealed that treatment with nanofibrous PEU scaffolds enhanced wound healing. This study reveals that the proposed composite nanofibrous PEU scaffold has substantial potential for treating infectious full-thickness wounds.