Cullin 4B-RING E3 ligase negatively regulates the immunosuppressive capacity of mesenchymal stem cells by suppressing iNOS.

Mesenchymal stem cells (MSCs) are multipotent stem cells that can exert immunomodulatory capacity upon stimulation with pro-inflammatory cytokines. Our previous work has identified Cullin 4B (CUL4B), a scaffold protein in the CUL4B-RING E3 ligase (CRL4B) complex, as a key regulator in the differentiation of MSCs. Here, we demonstrate the critical role of CUL4B in regulating the immunosuppressive function of MSCs. When stimulated with pro-inflammatory cytokines, MSCs lacking CUL4B display enhanced immunosuppressive capacity, which is mediated by the elevated inducible nitric oxide synthase (iNOS). TGF-ß signaling can suppress iNOS by inhibiting its transcription as well as promoting its protein degradation. We show that the CRL4B complex cooperates with PRC2 complex and HDACs to repress transcription of Dlx1 and Pmepa1, two inhibitors of TGF-ß signaling, leading to decreased expression and accelerated degradation of iNOS. Our study unveils the CRL4B complex as a potential therapeutic target in promoting the immunosuppressive capacity of MSCs.

Structural Characterization of the Metalized Radical Cations of Adenosine ([Ade+Li-H]•+ and [Ade+Na-H]•+) by Infrared Multiphoton Dissociation Spectroscopy and Theoretical Studies.

Nucleoside radicals are key intermediates in the process of DNA damage, and alkali metal ions are a common group of ions in living organisms. However, so far, there has been a significant lack of research on the structural effects of alkali metal ions on nucleoside free radicals. In this study, we report a new method for generating metalized nucleoside radical cations in the gas phase. The radical cations [Ade+M-H]•+ (M = Li, Na) are generated by the 280 nm ultraviolet photodissociation (UVPD) of the precursor ions of lithiated and sodiated ions of 2-iodoadenine in a Fourier transform ion cyclotron resonance (FT ICR) cell. Further infrared multiphoton dissociation (IRMPD) spectra of both radical cations were recorded in the region of 2750-3750 cm-1. By combining these results with theoretical calculations, the most stable isomers of both radicals can be identified, which share the common characteristics of triple coordination patterns of the metal ions. For both radical species, the lowest-energy isomers undergo hydrogen transfer. Although the sugar ring in the most stable isomer of [Ade+Li-H]•+ is in a (South, syn) conformation similar to that of [Ado+Na]+, [Ade+Na-H]•+ is distinguished by the unexpected opening of the sugar ring. Their theoretical spectra are in good agreement with experimental spectra. However, due to the flexibility of the structures and the complexity of their potential energy surfaces, the hydrogen transfer pathways still need to be further studied. Considering that the free radicals formed directly after C-I cleavage have some similar spectral characteristics, the existence of these corresponding isomers cannot be ruled out. The findings imply that the structures of nucleoside radicals may be significantly influenced by the attached alkali metal ions. More detailed experiments and theoretical calculations are still crucial.

Optimal cut-off values of haematoma volume for predicting haematoma expansion at different intracerebral haemorrhage locations.

BACKGROUND: Haematoma expansion (HE) is a frequent manifestation of acute intracerebral haemorrhage (ICH) and is associated with early disease progression and poor functional status. Approximately 30 % of patients with ICH experience substantial HE within the first few hours of onset. OBJECTIVES: This study aimed to investigate the relationship between HE and initial volume at different locations in patients with ICH. METHODS: We investigated consecutive patients with ICH admitted to the emergency room at Xiangyang No. 1 People's Hospital between January 2018 and June 2022. Haematoma volume was calculated using a three-dimensional slicer platform. Prediction models were assessed using a logistic regression model. The Youden index was used to assess the haematoma volume cut-off values for predicting HE. RESULTS: This study included 306 patients: 161 had basal ganglia ICH, 41 lobar ICH, and 104 thalamic ICH. The area under the ROC curve (AUC) for the thalamic ICH score in predicting intraventricular haemorrhage (IVH) expansion ≥ 1 mL or delayed IVH expansion was 0.786, and the best cut-off value was 7.05 mL (specificity, 85.3 %; sensitivity, 62.8 %; and accuracy, 76.0 %). The AUC for the thalamic ICH and lobar ICH scores in predicting haematoma or IVH expansion were 0.756 and 0.653, respectively; the best cut-offs were 7.05 mL for the thalamus (specificity, 84.8 %; sensitivity, 60.0 %; and accuracy, 74.0 %) and 31.89 mL in the lobar area (specificity, 81.8 %; sensitivity, 52.3 %; and accuracy, 68.3 %). CONCLUSIONS: Initial ICH volume predicted haematoma or IVH expansion at different locations. Moreover, it can assist clinicians in determining whether patients are suitable for future surgical interventions or other procedures.

New IMB16-4 Hot-Melt Extrusion Preparation Improved Oral Bioavailability and Enhanced Anti-Cholestatic Effect on Rats.

Background: Cholestasis is challenging to treat due to lacked effective drugs. N-(3,4,5-trichlorophenyl)-2 (3-nitrobenzenesulfonamido) benzamide, abbreviated as IMB16-4, which may be effective for the treatment of cholestasis. However, its poor solubility and bioavailability seriously obstruct the research programs. Methods: A hot-melt extrusion (HME) preparation was first applied to increase the bioavailability of IMB16-4, the oral bioavailability, anti-cholestatic effect and vitro cytotoxicity of IMB16-4 and IMB16-4-HME were evaluated. Meanwhile, the molecular docking and qRT-PCR were used to validate the mechanism behind. Results: The oral bioavailability of IMB16-4-HME improved 65-fold compared with that of pure IMB16-4. Pharmacodynamics results demonstrated that IMB16-4-HME prominently decreased the serum levels of total bile acid (TBA) and alkaline phosphatase (ALP), but elevated the level of total bilirubin (TBIL) and direct bilirubin (DBIL). Histopathology revealed that IMB16-4-HME at lower dose exhibited stronger anti-cholestatic effect compared with pure IMB16-4. In addition, molecular docking demonstrated that IMB16-4 has great affinity with PPARα, and qRT-PCR results revealed that IMB16-4-HME significantly elevated the mRNA expression level of PPARα, but decreased the mRNA level of CYP7A1. Cytotoxicity assays demonstrated that the hepatotoxicity of IMB16-4-HME was absolutely attributed to IMB16-4, and the excipients of IMB16-4-HME may increase the drug load within HepG2 cells. Conclusion: The HME preparation significantly increased the oral bioavailability and anti-cholestatic effect of pure IMB16-4, but caused liver injury at high dose, which require a dose balance between the curative effect and safety in the future research.

Identifying a selective oligopeptide clamp in the gas phase.

Oligopeptide foldamers are promising as minimalist functional analogues to proteins. Herein, we report a versatile and cost-effective experimental scheme in the gas phase that can facilely identify selective oligopeptides and unambiguously resolve the corresponding folding conformations. Based on this methodology, a stereoselective oligopeptide clamp targeting ß2-blockers is successfully identified.

A novel method for photon unfolding spectroscopy of protein ions in the gas phase.

In this study, a new experimental method for photon unfolding spectroscopy of protein ions based on a Fourier transform ion cyclotron resonance (FT ICR) mass spectrometer was developed. The method of short-time Fourier transform has been applied here to obtain decay curves of target ions trapped in the cell of the FT ICR mass spectrometer. Based on the decay constants, the collision cross sections (CCSs) of target ions were calculated using the energetic hard-sphere model. By combining a tunable laser to the FT ICR mass spectrometer, the changes of CCSs of the target ions were recorded as a function of the wavelengths; thus, the photon isomerization spectrum was obtained. As one example, the photon isomerization spectrum of [Cyt c + 13H]13+ was recorded as the decay constants relative to the applied wavelengths of the laser in the 410-480 nm range. The spectrum shows a maximum at 426 nm, where an unfolded structure induced by a 4 s irradiation can be deduced. The strong peak at 426 nm was also observed for another ion of [Cyt c + 15H]15+, although some difference at 410 nm between the two spectra was found at the same time. This novel method can be expanded to ultraviolet or infrared region, making the experimental study of wavelength-dependent photon-induced structural variation of a variety of organic or biological molecules possible.

Geometrical Structures and Dissociation Channels of CuP2n + (n = 2-11): Studied by Mass Spectrometry and Theoretical Calculations.

Transition metal phosphorus cluster cations CuP2n + (2 ≤ n ≤ 11) were studied by laser ablation mass spectrometry and collision-induced dissociation (CID). The magic-numbered cluster ion of CuP8 + was identified experimentally, and cluster ions of CuP14 + and CuP18 + were also found to be generated with high abundance. CID results show that the dissociation channels of CuP2n + (n = 4 and 6-10) are all characterized by the loss of the P4 unit. Theoretical calculations combining global minima searching with the basin-hopping method and density functional theory (DFT) optimizations were performed for these clusters. Among them, the magic-numbered cluster CuP8 + was characterized by a D2d symmetry, with the Cu atom bridging two P4 units. The most stable isomer of CuP14 + was found to be characterized by a C2v symmetry. Calculations also reflect that the dissociation channels of the loss of the P4 unit are more energetically favorable than those of the loss of the P2 unit for CuP2n + (n = 4 and 6-10), which are in good consistent with the experimental results.

Differentiation of disaccharide isomers via a combination of IR and UV photodissociation mass spectrometry.

RATIONALE: The challenge of glycan identification due to their structural complexity and diversity has profited enormously from recent developments in mass spectrometry (MS)-related methods. For photodissociation MS, infrared (IR) and ultraviolet (UV) lasers can generate complementary fragment ions, so an effective combination of the two methods may provide rich and valuable fragmentation patterns for glycan analysis. METHODS: A 7.0 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a double-beam laser system was applied for the experiments. 3,5-Diiodo-L-tyrosine was selected as the assistant molecule to form complex ions with ten isomeric disaccharides through electrospray ionization. The complex ions were further isolated and irradiated by IR and UV lasers separately or continuously in the FTICR cell. RESULTS: By combining the two complementary fragment spectra generated from the IR and UV lasers, a clear identification of all the ten isomers was achieved using their binary codes based on their fragmentation patterns. The double-beam method simplifies the experiment by introducing the two lasers sequentially in one experiment, providing richer fragmentation patterns and making the full discrimination easier. CONCLUSIONS: This study demonstrates the capabilities of the combination of IR and UV photodissociation MS in the identification of diverse glycan isomers. The double-beam photodissociation method described here distinguished compositional, configurational and connectivity disaccharide isomers successfully. Compared with the data accumulation method based on separate IR and UV experiments, this method is simpler, faster, more flexible and also characterized by richer fragmentation patterns.

Versatile Double-Beam Confocal Laser System Combined with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for Photodissociation Mass Spectrometry and Spectroscopy.

Both infrared multiphoton dissociation (IRMPD) and ultraviolet photodissociation (UVPD) play important roles in tandem mass spectrometry and the action spectroscopy of organic and biological molecules. A flexible combination of the two methods may provide researchers with more versatile and powerful ion activation/dissociation choices for structural characterization and spectroscopic studies. Here, we report the integration of two tunable lasers with a Fourier transform ion cyclotron resonance mass spectrometer in a confocal mode, which offers multiple capabilities for photon activation/dissociation experiments. The two overlapped beams can be introduced into the cell individually, sequentially, or simultaneously, providing highly flexible and diverse activation schemes. The setup can also measure the UVPD or IRMPD action spectra of fragment ions generated by previous photon dissociation processes. In addition, the multistage tandem-in-time mass spectrometry performance up to MS4, including three different activation methods in a single cell, has also been demonstrated.

Application of Infrared Multiple Photon Dissociation (IRMPD) Spectroscopy in Chiral Analysis.

In recent years, methods based on photodissociation in the gas phase have become powerful means in the field of chiral analysis. Among them, infrared multiple photon dissociation (IRMPD) spectroscopy is a very attractive one, since it can provide valuable spectral and structural information of chiral complexes in addition to chiral discrimination. Experimentally, the method can be fulfilled by the isolation of target diastereomeric ions in an ion trap followed by the irradiation of a tunable IR laser. Chiral analysis is performed by comparing the difference existing in the spectra of enantiomers. Combined with theoretical calculations, their structures can be further understood on the molecular scale. By now, lots of chiral molecules, including amino acids and peptides, have been studied with the method combined with theoretical calculations. This review summarizes the relative experimental results obtained, and discusses the limitation and prospects of the method.