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Methods in Enzymology 2024Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl...
Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.
Topics: Penicillium; Alkyl and Aryl Transferases; Cryoelectron Microscopy; Diterpenes; Fungal Proteins; Dimethylallyltranstransferase
PubMed: 38942500
DOI: 10.1016/bs.mie.2023.11.002 -
The Journal of Neuroscience : the... Jun 2024During navigation, the neocortex must actively integrate learned spatial context with current sensory experience to guide behaviours. However, the relative encoding of...
During navigation, the neocortex must actively integrate learned spatial context with current sensory experience to guide behaviours. However, the relative encoding of spatial and sensorimotor information among cortical cells, and whether hippocampal feedback continues to modify these properties in familiar environments, remains poorly understood. Thus, two-photon microscopy of male and female Thy1-GCaMP6s mice was used to longitudinally image neurons spanning superficial retrosplenial cortex and layers II-Va of primary and secondary motor cortices before and after bilateral dorsal hippocampal lesions. During behaviour on a familiar cued treadmill, the locations of two added obstacles were interchanged to decouple place-tuning from cue-tuning among the position correlated cells with fields at those locations. The subpopulations of place- and cue-tuned cells each formed interareal gradients such that higher-level cortical regions exhibited higher fractions of place cells, whereas lower-level regions exhibited higher fractions of cue cells. Position correlated cells in motor cortex also formed translaminar gradients; cells closer to the cortical surface were more likely to exhibit fields and were more sparsely and precisely tuned than deeper cells. After dorsal hippocampal lesions, a neural representation of the learned environment persisted but retrosplenial cortex exhibited significantly increased cue-tuning and, in motor cortices, both position correlated cell recruitment and population activity at the unstable obstacle locations became more homogeneously elevated across laminae. Altogether, these results support that the hippocampus continues to modulate cortical responses in familiar environments, and the relative impact of top-down feedback obeys hierarchical interareal and interlaminar gradients opposite to the flow of bottom-up sensory inputs. During learning, the hippocampus imparts spatial context to memory representations throughout the superficial neocortex. However, the post-learning role of the hippocampus has not been well defined. The results of this study suggest that, during navigation of a familiar environment, the hippocampus continues to link unreliable sensory attributes to a stable contextual framework, effectively updating the learned model of the environment. The results are also consistent with top-down suppression of sensory-evoked activity during behaviour, which varied in strength according to hierarchical proximity to the hippocampus. This effect was abolished by bilateral lesions of the dorsal hippocampus, supporting that the hippocampus plays an ongoing role in propagating context-dependent predictions throughout the cortical hierarchy, a core hypothesis of the predictive coding theoretical framework.
PubMed: 38942472
DOI: 10.1523/JNEUROSCI.1619-23.2024 -
International Journal of Biological... Jun 2024Cellulose modified hydrogels can be produced directly from raw biopolymers in novel cellulose solvents such as NaOH/urea aqueous solution. The effect of cellulose...
Cellulose modified hydrogels can be produced directly from raw biopolymers in novel cellulose solvents such as NaOH/urea aqueous solution. The effect of cellulose characteristics on the synthesis of a cellulose-graft-(net-poly(acrylamide-co-acrylic acid)) and its performance as water absorbent/methylene blue dye removal material is analyzed. Three cellulose samples, one analytical grade and two obtained from teak wood sawdust with different pretreatments (one alkaline and the other, a novel one known as (gas phase) acid pretreatment) were compared. The starting raw celluloses were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and viscosity in cupri ethylenediamine hydroxide (CED) solution, whereas the chemically modified materials were characterized by SEM, FTIR, and TGA. The pretreatment used influences composition, crystallinity index and degree of polymerization (DP) of the cellulose obtained. The modified material produced with cellulose from alkaline pretreatment showed the highest swelling ratio in water absorption tests at room temperature (12,714 %); in contrast, the one with cellulose from acid pretreatment showed the lowest swelling ratio (7470 %). However, this difference is not so significative in dye removal tests, where absorption capacity is 139 and 140 mg/g, respectively. The results indicate that cellulose composition, particularly structures with significant hemicellulose and lignin remaining content, has a major effect on the performance of modified materials for water absorption, and degree of polymerization has a major effect on adsorption capacity of methylene blue.
PubMed: 38942409
DOI: 10.1016/j.ijbiomac.2024.133482 -
The Science of the Total Environment Jun 2024Thawing permafrost forms "thaw ponds" that accumulate and transport organic carbon (OC), redox-active iron (Fe), and other elements. Although Fe has been shown to act as...
Thawing permafrost forms "thaw ponds" that accumulate and transport organic carbon (OC), redox-active iron (Fe), and other elements. Although Fe has been shown to act as a control on the microbial degradation of OC in permafrost soils, the role of iron in carbon cycling in thaw ponds remains poorly understood. Here, we investigated Fe-OC interactions in thaw ponds in partially and fully thawed soils ("bog" and "fen" thaw ponds, respectively) in a permafrost peatland complex in Abisko, Sweden, using size separation (large particulate fraction (LPF), small particulate fraction (SPF), and dissolved fraction (DF)), acid extractions, scanning electron microscopy (SEM), Fe K-edge X-ray absorption spectroscopy (XAS), and Fourier Transform Infrared (FTIR) spectroscopy. The bulk total Fe (total suspended Fe) in the bogs ranged from 1 to 35 mg/L (mean = 13 mg/L) whereas the fens exhibited higher total Fe (1.5 to 212 mg/L, mean = 30 mg/L). The concentration of bulk total OC (TOC) in the bog thaw ponds ranged from 50 to 352 mg/L (mean = 170 mg/L), higher than the TOC concentration in the fen thaw ponds (8.5 to 268 mg/L, mean = 17 mg/L). The concentration of 1 M HCl-extractable Fe in the bog ponds was slightly lower than that in the fens (93 ± 1.2 and 137 ± 3.5 mg/L Fe, respectively) with Fe predominantly (>75 %) in the DF in both thaw stages. Fe K-edge XAS analysis showed that while Fe(II) was the predominant species in LPF, Fe(III) was more abundant in the DF, indicating that the stage of thawing and particle size may control Fe redox state. Furthermore, Fe(II) and Fe(III) were partially complexed with natural organic matter (NOM, 8 to 80 %) in both thaw ponds. Results of our work suggest that Fe and OC released during permafrost thaw into thaw ponds (re-)associate, potentially protecting OC from microbial decomposition while also stabilizing the redox state of Fe.
PubMed: 38942322
DOI: 10.1016/j.scitotenv.2024.174321 -
Virus Research Jun 2024In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured...
In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture.
PubMed: 38942295
DOI: 10.1016/j.virusres.2024.199428 -
Journal of Proteomics Jun 2024Tuberculosis (TB) is an infectious disease that remains one of the major global public health concerns. Early detection of Active Pulmonary TB is therefore of utmost... (Review)
Review
Tuberculosis (TB) is an infectious disease that remains one of the major global public health concerns. Early detection of Active Pulmonary TB is therefore of utmost importance for controlling lethality and disease spreading. Currently available TB diagnostics can be broadly categorized into microscopy, culture-based, and molecular approaches, all of which come with compromised sensitivity, limited efficacy, and high expenses. Hence, rapid, sensitive, and affordable diagnostic methods for TB is the current prerequisite for disease management. This review summarizes the proteomics investigations for host-specific biomarkers from serum, sputum, saliva, and urine samples of TB patients, along with patients having comorbidity. Thorough data mining from available literature led us to conclude that the host-specific proteins involved in immunity and defense, metabolic regulation, cellular adhesion, and motility, inflammatory responses, and tissue remodelling have shown significant deregulation upon Mycobacterium tuberculosis (Mtb) infection. Notably, the immunoregulatory protein orosomucoid (ORM) was up-regulated in active TB compared to non-TB individuals, as observed in multiple studies from diverse sample types. Mannose receptor C type 2 (MRC2) was identified as an upregulated, treatment response biomarker in two independent serum proteomics investigations. Thorough mechanistic investigation on these candidate proteins would be fascinating to dig into potential drug targets and customized therapeutics for TB patients, along with their diagnostic potentials.
PubMed: 38942234
DOI: 10.1016/j.jprot.2024.105245 -
The Journal of Investigative Dermatology Jun 2024Early detection of melanoma is a major determinant in disease outcome and drives the number of (over)excised naevi in clinical practice. This study aimed to evaluate...
Early detection of melanoma is a major determinant in disease outcome and drives the number of (over)excised naevi in clinical practice. This study aimed to evaluate demographic features and melanoma risk of clinically suspicious, mainly flat naevus subtypes. Based on the methodology of ex vivo dermoscopy and derm dotting, the 12 most prevalent naevus subtypes were identified in a collection of over 7000 naevi excised for medical reason. Dermoscopical, histopathological and clinical features of these subtypes were described. In addition, the association with melanoma history, histopathological atypia and melanoma occurrence within naevi was compared. Nearly half of the naevi removed for medical reasons were of the hypermelanotic subtype with no or mild histopathological atypia and low melanoma association, suggesting overtreatment in daily practice. Contrarily, the subtypes atypical lentiginous naevus and orange pulverocytic flat naevus were associated with higher proportions of (severe) atypia and melanoma (history). We believe these subtypes may reflect different tumoural and/or (germline) genetic entities with different melanoma risk. The data from this study may direct further prospective research on specific naevus subtypes in order to obtain better insights in associated clinical/genetic factors and melanoma risk.
PubMed: 38942231
DOI: 10.1016/j.jid.2024.03.046 -
Journal of Ethnopharmacology Jun 2024The traditional medicinal formulation, Qifu-yin (QFY), has been widely prescribed for Alzheimer's disease (AD) treatment in China, yet the comprehensive mechanisms...
ETHNOPHARMACOLOGICAL RELEVANCE
The traditional medicinal formulation, Qifu-yin (QFY), has been widely prescribed for Alzheimer's disease (AD) treatment in China, yet the comprehensive mechanisms through which QFY mitigates AD pathology remain to be fully delineated.
AIM OF THE STUDY
This study aimed to explore the therapeutic implications of QFY on the synaptic injury and oxidative stress in the hippocampus of APPswe/PS1dE9 (APP/PS1) mice, with a concerted effort to elucidate the molecular mechanisms related to synaptic preservation and memory improvement.
MATERIALS AND METHODS
The components of QFY were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The neuroprotective effects of QFY was evaluated using six-month-old male APP/PS1 mice. Subsequent to a 15 days of QFY regimen, spatial memory was assessed utilizing the Morris water maze (MWM) test. Amyloid-beta (Aβ) aggregation was detected via immunostaining, while the quantification of Aβ and Aβ was achieved through enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was used to investigate the synaptic structure and mitochondrial morphology. Golgi staining was applied to examine dendritic spine density. Reactive oxygen species (ROS), 3-nitrotyrosine (3-NT) and 4-hydroxy-nonenal (4-HNE) assays were employed to assess oxidative stress. The expression profiles of Aβ metabolism-associated enzymes and the Keap1/Nrf2/ARE signaling pathway were determined by Western blot.
RESULTS
A total of 20 principal compounds in QFY were identified. QFY mitigated memory deficits of APP/PS1 mice, including reducing escape latency and search distance and increasing the time and distance spent in the target quadrant. In addition, QFY increased platform crossings of APP/PS1 mice in the probe trial of MWM tests. TEM analysis showed that QFY increased synapse number in the CA1 region of APP/PS1 mice. Further studies indicated that QFY elevated the expression levels of Post synaptic density protein 95 (PSD95) and synaptophysin, and mitigated the loss of dendritic spine density in the hippocampus of APP/PS1 mice. QFY has been shown to ameliorated the structural abnormalities of mitochondria, including mitochondrial dissolution and degradation, up-regulate ATP synthesis and membrane potential in the hippocampus of APP/PS1 mice. Moreover, QFY activated the Keap1/Nrf2/ARE signaling pathway in the hippocampus of APP/PS1 mice, which might contribute to the neuroprotective effects of QFY.
CONCLUSION
QFY activates the Keap1/Nrf2/ARE signaling, and protects against synaptic and mitochondrial dysfunction in APP/PS1 mice, proposing a potential alternative therapeutic strategy for AD management.
PubMed: 38942156
DOI: 10.1016/j.jep.2024.118497 -
Journal of the American Chemical Society Jun 2024Identifying the active phase with the highest activity, which is long-believed to be a steady state of the catalyst, is the basis of rational design of heterogeneous...
Identifying the active phase with the highest activity, which is long-believed to be a steady state of the catalyst, is the basis of rational design of heterogeneous catalysis. In this work, we performed detailed investigations, successfully capturing the instantaneous structure-activity change in oscillating Pd nanocatalysts during methane oxidation, which reveals an unprecedented oscillatory active state. Combining quantitative environmental transmission electron microscopy and highly sensitive online mass spectrometry, we identified two distinct phases for the reaction: one where the Pd nanoparticles refill with oxygen, and the other, a period of abrupt pumping of oxygen and boosted methane oxidation within about 1 s. It is the rapid reduction process that shows the highest activity for total oxidation of methane, not a PdO or Pd steady state under the conditions applied here (methane:oxygen = 5:1). This observation challenges the traditional understanding of the active phase and requires a completely different strategy for catalyst optimization.
PubMed: 38942067
DOI: 10.1021/jacs.4c02830 -
European Journal of Dentistry Jun 2024This study evaluated surface roughness, biaxial flexural strength, and phase transformation of 5Y-PSZ after grinding and polishing with different protocols.
Effect of Grinding and Polishing Protocols on Surface Roughness, Flexural Strength, and Phase Transformation of High-Translucent 5 mol% Yttria-Partially Stabilized Zirconia.
OBJECTIVES
This study evaluated surface roughness, biaxial flexural strength, and phase transformation of 5Y-PSZ after grinding and polishing with different protocols.
MATERIAL AND METHODS
Two commercial 5Y-PSZ, Lava Esthetic (L) and Cercon xt (C), were used and divided into 3 groups: LC and CC represented unpolished control groups; LE and CE were polished with protocol I (EVE DIASYNT® PLUS HP following with EVE DIACERA RA); and LJ and CJ were polished with protocol II (Superfine diamond bur following with Jota ZIR Gloss polishing kit). Surface roughness was evaluated after polishing step-by-step with a contact-type profilometer. After high-gross polishing, the specimens were subjected to biaxial flexural strength test, crystallographic microstructure analysis using an X-ray diffractometer (XRD), and surface micro-topography using scanning electron microscopy (SEM).
STATISTICAL ANALYSIS
Surface roughness differences after each step and biaxial flexural strength between groups were evaluated with one-way ANOVA, followed by Bonferroni post-hoc analysis. Changes in surface roughness across four different time points within groups were assessed using one-way repeated measures ANOVA, followed by Bonferroni post-hoc analysis.
RESULTS
After high-gross polishing, both polishing protocols showed significantly lower surface roughness than the grinding group ( < 0.05). The LE and CE groups exhibited the highest surface roughness values, which were significant differences from the LJ and CJ groups ( < 0.05). The LE group showed significantly lower biaxial flexural strength compared to the LC group ( < 0.05). However, there was no statistically significant difference in the CE and CJ groups compared to the control group ( > 0.05). Furthermore, all polishing protocols did not change the phase transformation of zirconia.
CONCLUSION
Polishing protocol II provided a smoother surface than the protocol I after high-gross polishing, while the biaxial flexural strength of materials remained unaffected.
PubMed: 38942054
DOI: 10.1055/s-0044-1787001