Search

Aim: Neurodegenerative diseases are characterized by early increased beta-amyloid (Aβ) and decreased cerebrovascular reactivity. We investigated Aβ in gingiva, serum or hippocampus and neurovascular reactivity in basilar artery (BA) of periodontitis rats, to test the impact of Aβ on BA vasoreactivity ex vivo., Materials and Methods: Periodontitis was induced in 32 rats using silk-ligation. Rats were sacrificed at weeks 0, 1, 2 and 4. Gingival TNF-α, IL-1β and Aβ were quantified via immunoblotting. Alveolar bone destruction was examined by micro-computed tomography. Serum and hippocampus Aβ values were measured by enzyme-linked immunosorbent assay and fluorescence staining, respectively. Vasoreactivity was measured by myography on isolated BA., Results: From Week 1, gingival TNF-α and IL-1β and bone destruction increased. Gingiva, serum and hippocampus Aβ values increased from Week 2. Nicotine-induced BA relaxation declined from Week 2, while acetylcholine-induced relaxation decreased by Week 4. Bone loss correlated with Aβ and nicotine-induced relaxation. Correlations were observed between Aβs in tissues, between two induced BA relaxations and between Aβ expressions and the induced relaxations. Ex vivo, Aβ reduced nicotine- and isoproterenol-induced relaxations but not electrical stimulated relaxation., Conclusions: Periodontitis may increase Aβ expressions and reduce BA neurovascular reactivity, with Aβ contributing to this abnormal neurovascular coupling., (© 2025 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T 3 , and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future., (© 2024 Wiley Periodicals LLC.)

Secondary carbonate precipitation plays a crucial role in determining the CO 2 outgassing from carbonate-saturated rivers and influences the short-term terrestrial carbon cycle. The fractionation of stable isotopes has been extensively employed to quantify post-weathering reactions in rivers and assess the quantities of metals removed by secondary carbonates. However, a major challenge is that water isotopic compositions usually reflect both lithological mixing and biogeochemical fractionation, which complicates distinguishing between these signals. In this study, we applied triple Sr isotopes ( 87 Sr/ 86 Sr and δ 88/86 Sr) to determine the "unfractionated" δ 88/86 Sr of water. This method allowed us to precisely quantify the precipitation-derived fractionation. Our results show that a median of 48 % of Sr was incorporated into carbonates in southwestern (SW) Taiwan, corresponding to the removal of 69 % of the originally weathered Ca. This process is closely related to a significant increase in HCO 3 - concentration in the water. In SW Taiwan, high river discharge during wet periods typically enhances carbonate dissolution, leading to increased carbonate precipitation along with CO 2 outgassing. Thus, hydrology and river discharge constraints significantly influence CaCO 3 precipitation and CO 2 outgassing processes and their associated CO 2 budget. Using a machine learning model, we estimated that the precipitation-derived CO 2 outgassing from rivers is +7.9 × 10 8  mol per year. This flux is nearly twice the long-term (~10 kyr) emission flux of +4.2 ± 1.4 × 10 8  mol per year in this region. This indicates that precipitation-driven CO 2 emissions from rivers constitute a significant carbon source within the terrestrial carbon cycle of orogenic regions., Competing Interests: Declaration of competing interest Hou-Chun Liu reports financial support was provided by National Science and Technology Council. Chen-Feng You reports financial support was provided by National Science and Technology Council. Kuo-Fang Huang reports financial support was provided by National Science and Technology Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine's moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole's electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin's B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo .

Chemical weathering is a pivotal geochemical process that shapes the carbon cycling and climates in the critical zone. Among its critical drivers, river discharge holds a particular significance, especially in the orogenic landscapes. Here, we examined the impact of discharge on mineral weathering in southwestern (SW) Taiwan by analyzing river water chemistry across a wide discharge range. Current observations indicated that carbonate contributes significantly to total weathering (50-80 %), with sulfuric acid accounting for one-half to two-thirds of carbonate weathering. A statistically strong correlation between river discharge and sulfuric acid-mediated carbonate weathering was highlighted, while the silicate weathering remained constant. This relationship suggests an increased influx of fresh minerals, such as pyrite, into the weathering regime as water flow increases. Our model identifies a critical discharge threshold of 4.6 m 3  s -1 , determining whether mineral weathering acts as a net source or sink of CO 2 . Consequently, mineral weathering in SW Taiwan acts as a net CO 2 sink during dry periods but turns into a net source during wet periods. Through analyzing a decade of daily discharge data, we found mineral weathering in SW Taiwan is a net CO 2 source, with a 2.6-fold increase in annual mean discharge causing a 3.8-fold increase in net CO 2 flux. This pattern is likely to be applicable to other similar minerals containing mountain-building regions, highlighting the significant role of hydrology in determining weathering sources and their potential impact on the carbon cycle balance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hou-Chun Liu reports financial support was provided by National Science and Technology Council. Chen-Feng You reports financial support was provided by National Science and Technology Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Hard-to-heal diabetic foot ulcers (DFUs) are associated with higher mortality rates and an increased medical burden for patients. ON101, a new topical cream, exhibited better healing efficacy than the control dressing in a Phase III trial. In this post hoc analysis, we further identify whether ON101 can improve the healing of ulcers with hard-to-heal risk factors in this cohort of DFU patients. Approach: To compare the efficacy of ON101 with absorbent dressing among various hard-to-heal wounds in patients with DFU, a post hoc analysis of a randomized Phase III trial that included 276 DFU patients was performed by subgrouping those patients based on ulcer depth, location, size, duration, and patients' glycated hemoglobin (HbA1c) levels and body mass index (BMI). Results: In the full analysis set, the proportion of patients achieving healing was 61.7% in the ON101 group and 37.0% in the comparator ( p = 0.0001). In subgroup analysis according to risk factors, ON101 demonstrated superior healing capacity on Wagner grade 2 ulcers ( p < 0.0001); plantar ulcers ( p = 0.0016), ulcer size ≥5 cm 2 ( p = 0.0122), ulcer duration ≥3 months ( p = 0.0043); for patients with HbA1c ≥9% ( p = 0.0285); and patients with BMI ≥25 ( p = 0.0005). Innovation: ON101, a novel therapeutic drug, can modulate the functions of macrophages and demonstrate superior healing rates to conventional absorbent dressing in patients with hard-to-heal DFUs. Conclusions: The results of this post hoc study suggest that ON101 is a better therapeutic option than conventional dressing used in treatment for DFU patients with higher HbA1c, BMI, or ulcers with complex conditions such as longer duration, deeper wounds, larger size, and plantar location.

Background: Trace metals such as iron, nickel, copper, zinc, and cadmium (Fe, Ni, Cu, Zn, and Cd) are essential micronutrients (and sometimes toxins) for phytoplankton, and the analysis of trace-metal stable isotopes in seawater is a valuable tool for exploring the biogeochemical cycling of these elements in the ocean. However, the complex and often time-consuming chromatography process required to purify these elements from seawater has limited the number of trace-metal isotope samples which can be easily processed in biogeochemical studies. To facilitate the trace-metal stable isotope analysis, here, we describe a new rapid procedure that utilizes automated chromatography for extracting and purifying Ni and Cu from seawater for isotope analysis using a prepFAST-MC™ system (Elemental Scientific Inc.)., Results: We have tested the matrix removal effectiveness, recoveries, and procedural blanks of the new purification procedure with satisfactory results. A nearly complete recovery of Ni and a quantitative recovery of Cu are achieved. The total procedural blanks are 0.33 ± 0.24 ng for Ni and 0.42 ± 0.18 ng for Cu, which is negligible for natural seawater samples. The new procedure cleanly separates Ni and Cu from key seawater matrix elements that may cause interferences during mass spectrometry analysis. When the new procedure was used to purify seawater samples for Ni and Cu stable isotope analysis by multi-collector ICP-MS, we achieved an overall uncertainty of 0.07 ‰ for δ 60 Ni and 0.09 ‰ for δ 65 Cu (2 SD). The new purification procedure was also tested using natural seawater samples from the South Pacific, for comparison of δ 60 Ni and δ 65 Cu achieved in the same samples purified by traditional hand columns. Both methods produced similar results, and the results from both methods are consistent with analyses of δ 60 Ni and δ 65 Cu from other ocean locations as reported by other laboratories., Significance: This study presents a new rapid procedure for seawater stable-metal isotope analysis by automating the chromatography step. We anticipate that the automated chromatography described here will facilitate the rapid and accurate analysis of seawater δ 60 Ni and δ 65 Cu in future studies, and may be adapted in the future to automate chromatographic purification of Fe, Zn, and Cd isotopes from seawater., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Jude Sakowski reports a relationship with Elemental Scientific, Inc. that includes: employment., (Copyright © 2024 Elsevier B.V. All rights reserved.)

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli , which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5 , Pax3/7 , and Cyclin D1 but strongly repressed that of MyoD , suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD . In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD . Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.

The use of pharmacological chaperones (PCs) to stabilize specific enzymes and impart a therapeutic benefit is an emerging strategy in drug discovery. However, designing molecules that can bind optimally to their targets at physiological pH remains a major challenge. Our previous study found that dibasic polyhydroxylated pyrrolidine 5 exhibited superior pH-selective inhibitory activity and chaperoning activity for human α-galactosidase A (α-Gal A) compared with its monobasic parent molecule, 4 . To further investigate the role of different C-2 moieties on the pH-selectivity and protecting effects of these compounds, we designed and synthesized a library of monobasic and dibasic iminosugars, screened them for α-Gal A-stabilizing activity using thermal shift and heat-induced denaturation assays, and characterized the mechanistic basis for this stabilization using X-ray crystallography and binding assays. We noted that the dibasic iminosugars 5 and 20 protect α-Gal A from denaturation and inactivation at lower concentrations than monobasic or other N -substituted derivatives; a finding attributed to the nitrogen on the C-2 methylene of 5 and 20 , which forms the bifurcated salt bridges (BSBs) with two carboxyl residues, E203 and D231. Additionally, the formation of BSBs at pH 7.0 and the electrostatic repulsion between the vicinal ammonium cations of dibasic iminosugars at pH 4.5 are responsible for their pH-selective binding to α-Gal A. Moreover, compounds 5 and 20 demonstrated promising results in improving enzyme replacement therapy and exhibited significant chaperoning effects in Fabry cells. These findings suggest amino-iminosugars 5 and 20 as useful models to demonstrate how an additional exocyclic amino group can improve their pH-selectivity and protecting effects, providing new insights for the design of pH-selective PCs., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 7 1 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Retinal ischemia followed by reperfusion (IR) is a common cause of many ocular disorders, such as age-related macular degeneration (AMD), which leads to blindness in the elderly population, and proper therapies remain unavailable. Retinal pigment epithelial (RPE) cell death is a hallmark of AMD. Hyperbaric oxygen (HBO) therapy can improve IR tissue survival by inducing ischemic preconditioning responses. We conducted an in vitro study to examine the effects of HBO preconditioning on oxygen-glucose deprivation (OGD)-induced IR-injured RPE cells. RPE cells were treated with HBO (100% O 2 at 3 atmospheres absolute for 90 min) once a day for three consecutive days before retinal IR onset. Compared with normal cells, the IR-injured RPE cells had lower cell viability, lower peroxisome proliferator activator receptor-alpha (PPAR-α) expression, more severe oxidation status, higher blood-retinal barrier disruption and more elevated apoptosis and autophagy rates. HBO preconditioning increased PPAR-α expression, improved cell viability, decreased oxidative stress, blood-retinal barrier disruption and cellular apoptosis and autophagy. A specific PPAR-α antagonist, GW6471, antagonized all the protective effects of HBO preconditioning in IR-injured RPE cells. Combining these observations, HBO therapy can reverse OGD-induced RPE cell injury by activating PPAR-α signalling., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

A high-peak-power sub-500-fs mode-locked optically pumped semiconductor laser is innovatively developed with only three components of a semiconductor gain chip, a semiconductor saturable absorber, and a focusing lens. The developed laser near the threshold pump power of 3.9 W can be operated with stable fundamental mode locking. The laser output can be naturally turned into the stable harmonic mode locking (HML) with the order gradually changing from 2nd to 8th by increasing the pump power from 4.0 W to 5.0 W. Due to the onset of the high-order transverse modes, the order of HML is fixed at 8th for a pump power greater than 5.0 W. For the HML with order less than 8th, the overall peak power and pulse width in the HML are approximately 0.36 kW and 550 fs, respectively. In the operation of 8th-order HML, the minimum pulse width and maximum peak power can reach 480 fs and 0.95 kW, respectively.

Mitochondria (MITO) and peroxisomes (PEXO) are the major organelles involved in the oxidative metabolism of cells, but detailed examination of their dynamics and functional adaptations during skeletal muscle (SKM) development (myogenesis) is still lacking. In this study, we found that during myogenesis, MITO DNA, ROS level, and redox ratio increased in myotubes, but the membrane potential (Δψm) and ATP content reduced, implying that the MITO efficiency might reduce during myogenesis. The PEXO number and density both increased during myogenesis, which probably resulted from the accumulation and increased biogenesis of PEXO. The expression of PEXO biogenesis factors was induced during myogenesis in vitro and in utero, and their promoters were also activated by MyoD. Knockdown of the biogenesis factors Pex3 repressed not only the PEXO density and functions but also the levels of MITO genes and functions, suggesting a close coupling between PEXO biogenesis and MITO functions. Surprisingly, Pex3 knockdown by the CRISPRi system repressed myogenic differentiation, indicating critical involvement of PEXO biogenesis in myogenesis. Taken together, these observations suggest that the dynamics and functions of both MITO and PEXO are coupled with each other and with the metabolic changes that occur during myogenesis, and these metabolic couplings are critical to myogenesis.

Patagonian aquatic environments have been invaded since the end of the last century by different species of salmonids. Knowing the natal origin and homing/straying rate of the salmonids in colonised environments is essential to understanding the dispersal mechanisms and developing management plans. In the last two decades, Chinook salmon Oncorhynchus tshawytscha showed the greatest natural dispersal capacity in Patagonia. The main goal of this study was to evaluate the environmental strontium isotope ratio ( 87 Sr/ 86 Sr) as a potential natural tag to infer the natal origin and ontogenetic habitat use of salmonids in Patagonia, specifically Chinook salmon. 87 Sr/ 86 Sr ratio was determined in water samples from 26 sites distributed in 14 Atlantic and Pacific basins in low and high water seasons. Environmental 87 Sr/ 86 Sr showed greater spatial than temporal variation, revealing great potential as a tool to infer the natal origin and life history of several migratory fish species in Patagonia. Otolith core-to-edge 87 Sr/ 86 Sr profiles were also analysed in 108 Chinook salmon from six basins. A cluster analysis based on the Unweighted Pair Group method (UPGMA) and Euclidean distances without prior classification grouped the sampled rivers into five main groups with significantly different (p < 0.05) isotopic ratios, sometimes integrated basins with different slopes (Atlantic or Pacific). The cluster analysis based on the natal 87 Sr/ 86 Sr period in otolith (∼natal origin) showed clear segregation between the Atlantic and Pacific samples. A mismatch between water and otolith natal 87 Sr/ 86 Sr ratio was detected in some Atlantic basins (e.g. De las Vueltas River in Santa Cruz Basin) and Pacific (e.g. Liquiñe Basin) and, which could be explained either by straying behaviours or by large geochemical variability between tributaries, within river systems. Our results showed that 87 Sr/ 86 Sr is a useful natural tag to trace the life history of migratory fishes in Patagonia, especially for invasive species such as Chinook salmon., Competing Interests: Declaration of competing interest Authors have no competing interests to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Based on the birefringent effect of the gain medium, a diode-pumped Nd-doped vanadate laser with nearly hemispherical cavity is exploited to emulate the quantum Green functions of two-dimensional commensurate harmonic oscillators. By matching the theoretical calculations to the far-field patterns of lasing modes, the resonant transverse frequencies can be accurately determined up to extremely high orders. The Shannon entropy is further employed to calculate the spatial entanglement of the quantum Green function as a function the transverse frequency. From the resonant transverse frequencies, all lasing modes are confirmed to be in excellent agreement with the maximum entropy states. This discovery implies that the formation of lasing modes is relevant to the coupling interaction between the pump source and the laser cavity.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD •- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD •- . Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In a Josephson junction (JJ) at zero bias, Cooper pairs are transported between two superconducting contacts via the Andreev bound states (ABSs) formed in the Josephson channel. Extending JJs to multiple superconducting contacts, the ABSs in the Josephson channel can coherently hybridize Cooper pairs among different superconducting electrodes. Biasing three-terminal JJs with antisymmetric voltages, for example, results in a direct current (DC) of Cooper quartet (CQ), which involves a four-fermion entanglement. Here, we report half a flux periodicity in the interference of CQ formed in graphene based multi-terminal (MT) JJs with a magnetic flux loop. We observe that the quartet differential conductance associated with supercurrent exhibits magneto-oscillations associated with a charge of 4e, thereby presenting evidence for interference between different CQ processes. The CQ critical current shows non-monotonic bias dependent behavior, which can be modeled by transitions between Floquet-ABSs. Our experimental observation for voltage-tunable non-equilibrium CQ-ABS in flux-loop-JJs significantly extends our understanding of MT-JJs, enabling future design of topologically unique ABS spectrum., (© 2022. The Author(s).)

Bacterial polyynes are highly active natural products with a broad spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. By integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), in the biosynthesis gene cluster of antifungal polyynes (massilin A 1, massilin B 2, collimonin C 3, and collimonin D 4) of Massilia sp. YMA4. Crystallographic analysis indicated that bacterial polyynes serve as covalent inhibitors of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited the cell viability of Candida albicans by targeting ERG10, the homolog of MasL. Thus, this study demonstrated that acetyl-CoA acetyltransferase is a potential target for developing antifungal agents., (© 2022. The Author(s).)

Most known cellulase-associated carbohydrate-binding modules (CBMs) are attached to the N- or C-terminus of the enzyme or are expressed separately and assembled into multi-enzyme complexes (for example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR-220, a GH5-family endo-β-1,4-glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A-like catalytic domain with a (β/α) 8 TIM-barrel fold and an inserted CBM29-like noncatalytic domain with a β-jelly-roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full-length and CBM-deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full-length and CBM-deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single-chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross-domain groove. Subsequent site-specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase., (open access.)

Background: K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge., Methods: We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes., Results: We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1-3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose., Conclusions: Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism., (© 2022. The Author(s).)

The resonant modes generated from the modern Chladni experiment are systematically confirmed to intimately correspond to the maximum entropy states obtained from the inhomogeneous Helmholtz equation for the square and equilateral triangle plates. To investigate the origin of maximum entropy states, the inhomogeneous Helmholtz equation is modified to consider the point interaction coming from the driving oscillator. The coupling strength associated with the point interaction is characterized by a dimensionless factor α . The δ potential of the point interaction is numerically modelled by a truncated basis with an upper index N . The asymptotic behavior for the upper index N is thoroughly explored to verify that the coupling strength of α = 1.0 can make the theoretical resonant modes agree excellently with the maximum entropy states as N→∞. It is further authenticated that nearly the same resonant modes can be obtained by using a larger coupling strength α when a smaller upper index N is exploited in the calculation.