Chromatographic purification of the plasmin-like enzyme from clamworm (Perinereis aibuhitensis Grub) and its fibrinolytic activity by metal ions.

In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.

Difluoromethylated Difunctionalization of Alkenes under Visible Light.

Difluoromethylated compounds usually act as bioisosteres for alcohol functional groups and show unique physicochemical and biological properties. The cyano-difluoromethylation of alkenes using 5-((difluoromethyl)sulfonyl)-1-phenyl-1H-tetrazole as a CF2H radical difluoromethyl precursor was developed to afford nitriles including a CF2H group. A low-cost, stable, easily handled 5-((difluoromethyl)sulfonyl)-1-methyl-1H-tetrazole (DFSMT) was synthesized and applied as the radical CF2H reagent. Using DFSMT as the radical CF2H precursor, the oxyl-difluoromethylation of alkenes was developed to obtain difluoromethylated ether products. All of the reactions showed good functional group tolerability. Initial mechanistic experiments indicated that the CF2H radical was involved as the key active intermediate.

α-Cyanostilbene-based Molecule with the Synergistical Mechanisms of AIE, ESIPT and TICT: A New Schiff Base Probe for Selective Detection of Fe3+ and Reversible Response to HCl/NH3 Vapor.

We designed and synthesized a new Schiff base probe, which incorporated the salicylaldehyde-analogue α-cyanostilbene and benzophenone hydrazone by the imine linkage. Its chemical structure was verified by FT-IR, MALDI-TOF-MS, HR-MS and 1H/13C NMR technologies. It could exhibit a red fluorescence based on the synergistical effects of aggregation-induce emission (AIE), excited-state intramolecular proton transfer (ESIPT) and twisted intramolecular charge-transfer (TICT) in the aggregation or solid states. Interestingly, the TLC-based test strip loaded with the target compound showed the reversible fluorescence response to amine/acid vapor and on-site visual fluorescence quenching response to Fe3+. In THF/water mixtures (fw = 90%, 10 µM, pH = 7.4), the detection limit (DL) and the binding constant (Ka) of the developed probe towards Fe3+ were evaluated as 5.50 × 10- 8 M and 1.69 × 105, respectively. The developed probe was successfully applied for the detection of Fe3+ with practical, reliable, and satisfying results.

Genome Mining Leads to Diverse Sesquiterpenes with Anti-inflammatory Activity from an Arctic-Derived Fungus.

With the advancement of bioinformatics, the integration of genome mining with efficient separation technology enables the discovery of a greater number of novel bioactive compounds. The deletion of the key gene responsible for triterpene cyclase biosynthesis in the polar strain Eutypella sp. D-1 instigated metabolic shunting, resulting in the activation of dormant genes and the subsequent production of detectable, new compounds. Fifteen sesquiterpenes were isolated from the mutant strain, with eight being new compounds. The structural elucidation of these compounds was obtained through a combination of HRESIMS, NMR spectroscopy, and ECD calculations, revealing six distinct skeleton types. Compound 7 possessed a unique skeleton of 5/10 macrocyclic ether structure. Based on the gene functions and newly acquired secondary metabolites, the metabolic shunting pathway in the mutant strain was inferred. Compounds 6, 8, 11, 14, and 15 exhibited anti-inflammatory effects without cytotoxicity through the release of nitric oxide from lipopolysaccharide-stimulated RAW264.7 cells. Notably, acorane-type sesquiterpene 8 inhibited nitric oxide production and modulated the MAPK and NLRP3/caspase-1 signaling pathways. Compound 8 also alleviated the CuSO4-induced systemic neurological inflammation symptoms in a transgenic fluorescent zebrafish model.

Genetic loss of Nrf1 and Nrf2 leads to distinct metabolism reprogramming of HepG2 cells by opposing regulation of the PI3K-AKT-mTOR signalling pathway.

As a vital hallmarker of cancer, the metabolic reprogramming has been shown to play a pivotal role in tumour occurrence, metastasis and drug resistance. Amongst a vast variety of signalling molecules and metabolic enzymes involved in the regulation of cancer metabolism, two key transcription factors Nrf1 and Nrf2 are required for redox signal transduction and metabolic homeostasis. However, the regulatory effects of Nrf1 and Nrf2 (both encoded by Nfe2l1 and Nfe2l2, respectively) on the metabolic reprogramming of hepatocellular carcinoma cells have been not well understood to date. Here, we found that the genetic deletion of Nrf1 and Nrf2 from HepG2 cells resulted in distinct metabolic reprogramming. Loss of Nrf1α led to enhanced glycolysis, reduced mitochondrial oxygen consumption, enhanced gluconeogenesis and activation of the pentose phosphate pathway in the hepatocellular carcinoma cells. By striking contrast, loss of Nrf2 attenuated the glycolysis and gluconeogenesis pathways, but with not any significant effects on the pentose phosphate pathway. Moreover, knockout of Nrf1α also caused fat deposition and increased amino acid synthesis and transport, especially serine synthesis, whilst Nrf2 deficiency did not cause fat deposition, but attenuated amino acid synthesis and transport. Further experiments revealed that such distinctive metabolic programming of between Nrf1α-/- and Nrf2-/- resulted from substantial activation of the PI3K-AKT-mTOR signalling pathway upon the loss of Nrf1, leading to increased expression of critical genes for the glucose uptake, glycolysis, the pentose phosphate pathway, and the de novo lipid synthesis, whereas deficiency of Nrf2 resulted in the opposite phenomenon by inhibiting the PI3K-AKT-mTOR pathway. Altogether, these provide a novel insight into the cancer metabolic reprogramming and guide the exploration of a new strategy for targeted cancer therapy.

Purification and Properties of a Plasmin-like Marine Protease from Clamworm (Perinereis aibuhitensis).

Marine organisms are a rich source of enzymes that exhibit excellent biological activity and a wide range of applications. However, there has been limited research on the proteases found in marine mudflat organisms. Based on this background, the marine fibrinolytic enzyme FELP, which was isolated and purified from clamworm (Perinereis aibuhitensis), has exhibited excellent fibrinolytic activity. We demonstrated the FELP with a purification of 10.61-fold by precipitation with ammonium sulfate, ion-exchange chromatography, and gel-filtration chromatography. SDS-PAGE, fibrin plate method, and LC-MS/MS indicated that the molecular weight of FELP is 28.9 kDa and identified FELP as a fibrinolytic enzyme-like protease. FELP displayed the maximum fibrinolytic activity at pH 9 (407 ± 16 mm2) and 50 °C (724 ± 27 mm2) and had excellent stability at pH 7-11 (50%) or 30-60 °C (60%), respectively. The three-dimensional structure of some amino acid residues of FELP was predicted with the SWISS-MODEL. The fibrinolytic and fibrinogenolytic assays showed that the enzyme possessed direct fibrinolytic activity and indirect fibrinolysis via the activation of plasminogen; it could preferentially degrade Aα-chains of fibrinogen, followed by Bß- and γ-chains. Overall, the fibrinolytic enzyme was successfully purified from Perinereis aibuhitensis, a marine Annelida (phylum), with favorable stability that has strong fibrinolysis activity in vitro. Therefore, FELP appears to be a potent fibrinolytic enzyme with an application that deserves further investigation.

A multi-stimuli-responsive fluorescence material based on 1,8-naphthalimide.

A pair of 1,8-naphthalimides (NPIs) were designed and successfully synthesized through embellishing amino-containing NPI with 4-diethylaminosalicyladehyde and 4-diethylaminobenzaldehyde, respectively. Their structures were fully confirmed by 1H/13C NMR, HR-MS and FT-IR spectroscopic studies. Their photophysical properties were systematically investigated in different solvents of varied polarity, in THF/water mixtures with varying water fractions (fw), and in THF solvent with varying concentrations of NPIs. It inferred that the distinct differences in emission between two NPIs during self-assembled process could be ascribed that the hydroxyl-containing NPI allowed the excited-state intramolecular proton transfer process between -OH and CH=N units in the aggregation state. Interestingly, the solid of 4-diethylaminosalicyladehyde-functionalized NPI exhibited multi-stimuli-responsive fluorescence changes involving mechanofluorochromism and HCl/NH3 vapor stimulus-induced conversion. However, no remarkable change was observed in the photoluminescence (PL) spectra for the solid of 4-diethylaminobenzaldehyde-functionalized NPI under the stimuli of mechanical force and organic solvent.

Methyl α-D-galactopyranosyl-(1→3)-ß-D-galactopyranoside and methyl ß-D-galactopyranosyl-(1→3)-ß-D-galactopyranoside: Glycosidic linkage conformation determined from MA'AT analysis.

MA'AT analysis has been applied to two biologically-important O-glycosidic linkages in two disaccharides, α-D-Galp-(1→3)-ß-D-GalpOMe (3) and ß-D-Galp-(1→3)-ß-D-GalpOMe (4). Using density functional theory (DFT) to obtain parameterized equations relating a group of trans-O-glycosidic NMR spin-couplings to either phi (ϕ') or psi (ψ'), and experimental 3JCOCH, 2JCOC, and 3JCOCC spin-couplings measured in aqueous solution in 13C-labeled isotopomers, probability distributions of ϕ' and ψ' in each linkage were determined and compared to those determined by aqueous 1-µs molecular dynamics (MD) simulation. Good agreement was found between the MA'AT and single-state MD conformational models of these linkages for the most part, with modest (approximately <15°) differences in the mean values of ϕ' and ψ', although the envelope of allowed angles (encoded in circular standard deviations or CSDs) is consistently larger for ϕ' determined from MA'AT analysis than from MD for both linkages. The MA'AT model of the α-Galp-(1→3)-ß-Galp linkage agrees well with those determined previously using conventional NMR methods (3JCOCH values and/or 1H-1H NOEs), but some discrepancy was observed for the ß-Galp-(1→3)-ß-Galp linkage, which may arise from errors in the conventions used to describe the linkage torsion angles. Statistical analyses of X-ray crystal structures show ranges of ϕ' and ψ' for both linkages that include the mean angles determined from MA'AT analyses, although both angles adopt a wide range of values in the crystalline state, with ϕ' in ß-Galp-(1→3)-ß-Galp linkages showing greater-than-expected conformational variability.

Evaluation of XQ528 tartrate on embryo-fetus developmental toxicity in SD rats and genotoxicity.

The effects of XQ528 tartrate on the embryonic and fetal development of fertile Sprague-Dawley (SD) rats, along with their embryos and littermates, were evaluated using an embryo-fetus developmental toxicity assay. fertile SD rats exhibited no significant general toxic effects when administered doses of 0.25, 1.25, and 5.0 mg/kg intranasally from days 6 to 15 of gestation. The genotoxicity of the compound was evaluated through an amalgam of tests that included the Ames test, the Chinese hamster ovary (CHO) cell chromosome aberration test, and the micronucleus test in ICR mice. The results from the Ames test indicated non-mutagenicity at concentrations of 5000, 500, 50.0, 5.0, and 0.5 µg/dish across strains TA97, TA98, TA100, TA102, and TA1535. Additionally, the chromosomal aberration rates in CHO cells were not significantly altered at concentrations of 50.5, 101.0, and 202.0 µg/mL. No micronuclei induction was observed in ICR mice at dosage levels of 11.25, 22.50, and 45.00 mg/kg post intranasal administration. In conclusion, the no observed adverse effect level (NOAEL) for developmental toxicity of XQ528 tartrate in fertile SD rats, embryos, and littermates under the test conditions in this study was established at 5.0 mg/kg/day. Under these test conditions, XQ528 tartrate did not exhibit any significant genotoxic or carcinogenic potential.

Abnormal Interherispheric Functional Connectivity in Acute Pericoronitis: A Resting-State MRI Study.

OBJECTIVE: Acute pericoronitis (AP) is a prevalent cause of odontogenic toothache which can significantly impact brain function. Previous research has predominantly concentrated on localized brain activity. However, the synergistic changes between brain hemispheres induced by toothache and resulting abnormal functional connectivity across the brain have not been comprehensively studied. METHODS: A total of 34 patients with AP and 34 healthy individuals, matched for age, sex, and education were recruited for this study. All participants underwent resting-state functional magnetic resonance imaging (rs-MRI) scans. The voxel mirror homotopic connectivity (VMHC) method was used to identify intergroup differences. Brain regions exhibiting statistically significant differences were selected as regions of interest for further functional connectivity analysis. The partial correlation method was utilized to assess the correlation between abnormal VMHC values in different regions and clinical parameters, with age and sex included as covariates. RESULTS: Patients with AP exhibited reduced VMHC values in the thalamus and elevated VMHC values in the inferior frontal gyrus compared with healthy controls. Subsequent functional connectivity analyses revealed extensive changes in functional networks, predominantly affecting the default, frontoparietal, cerebellar, and pain networks. CONCLUSION: Changes in functional patterns across these brain networks offer novel insights into the neurophysiological mechanisms underlying pain information processing.