Maximizing the Accuracy of Equilibrium Dissociation Constants for Affinity Complexes: From Theory to Practical Recommendations.

The equilibrium dissociation constant (Kd) is a major characteristic of affinity complexes and one of the most frequently determined physicochemical parameters. Despite its significance, the values of Kd obtained for the same complex under similar conditions often exhibit considerable discrepancies and sometimes vary by orders of magnitude. These inconsistencies highlight the susceptibility of Kd determination to large systematic errors, even when random errors are small. It is imperative to both minimize and quantitatively assess the systematic errors inherent in Kd determination. Traditionally, Kd values are determined through nonlinear regression of binding isotherms. This analysis utilizes three variables: concentrations of two reactants and a fraction R of unbound limiting reactant. The systematic errors in Kd arise directly from systematic errors in these variables. Therefore, to maximize the accuracy of Kd, this study thoroughly analyzes the sources of systematic errors within the three variables, including (i) non-additive signals to calculate R, (ii) mis-calibrated experimental instruments, (iii) inaccurate calibration parameters, (iv) insufficient incubation time, (v) unsaturated binding isotherm, (vi) impurities in the reactants, and (vii) solute adsorption onto surfaces. Through this analysis, we illustrate how each source contributes to inaccuracies in the determination of Kd and propose strategies to minimize these contributions. Additionally, we introduce a method for quantitatively assessing the confidence intervals of systematic errors in concentrations, a crucial step toward quantitatively evaluating the accuracy of Kd. While presenting original findings, this paper also reiterates the fundamentals of Kd determination, hence guiding researchers across all proficiency levels. By shedding light on the sources of systematic errors and offering strategies for their mitigation, our work will help researchers enhance the accuracy of Kd determination, thereby making binding studies more reliable and the conclusions drawn from such studies more robust.

Mesoporous MOFs with ROS scavenging capacity for the alleviation of inflammation through inhibiting stimulator of interferon genes to promote diabetic wound healing.

Excessive production of reactive oxygen species (ROS) and inflammation are the key problems that impede diabetic wound healing. In particular, dressings with ROS scavenging capacity play a crucial role in the process of chronic wound healing. Herein, Zr-based large-pore mesoporous metal-organic frameworks (mesoMOFs) were successfully developed for the construction of spatially organized cascade bioreactors. Natural superoxide dismutase (SOD) and an artificial enzyme were spatially organized in these hierarchical mesoMOFs, forming a cascade antioxidant defense system, and presenting efficient intracellular and extracellular ROS scavenging performance. In vivo experiments demonstrated that the SOD@HMUiO-MnTCPP nanoparticles (S@M@H NPs) significantly accelerated diabetic wound healing. Transcriptomic and western blot results further indicated that the nanocomposite could inhibit fibroblast senescence and ferroptosis as well as the stimulator of interferon genes (STING) signaling pathway activation in macrophages mediated by mitochondrial oxidative stress through ROS elimination. Thus, the biomimetic multi-enzyme cascade catalytic system with spatial ordering demonstrated a high potential for diabetic wound healing, where senescence, ferroptosis, and STING signaling pathways may be potential targets.

Structure and distinct supramolecular organization of a PSII-ACPII dimer from a cryptophyte alga Chroomonas placoidea.

Cryptophyte algae are an evolutionarily distinct and ecologically important group of photosynthetic unicellular eukaryotes. Photosystem II (PSII) of cryptophyte algae associates with alloxanthin chlorophyll a/c-binding proteins (ACPs) to act as the peripheral light-harvesting system, whose supramolecular organization is unknown. Here, we purify the PSII-ACPII supercomplex from a cryptophyte alga Chroomonas placoidea (C. placoidea), and analyze its structure at a resolution of 2.47 Å using cryo-electron microscopy. This structure reveals a dimeric organization of PSII-ACPII containing two PSII core monomers flanked by six symmetrically arranged ACPII subunits. The PSII core is conserved whereas the organization of ACPII subunits exhibits a distinct pattern, different from those observed so far in PSII of other algae and higher plants. Furthermore, we find a Chl a-binding antenna subunit, CCPII-S, which mediates interaction of ACPII with the PSII core. These results provide a structural basis for the assembly of antennas within the supercomplex and possible excitation energy transfer pathways in cryptophyte algal PSII, shedding light on the diversity of supramolecular organization of photosynthetic machinery.

Experimental Study on Liquid Metal Embrittlement of Al-Zn-Mg Aluminum Alloy (7075): From Macromechanical Property Experiment to Microscopic Characterization.

This study is a multiscale experimental investigation into the embrittlement of Al-Zn-Mg aluminum alloy (7075-T6) caused by liquid metal gallium. The results of the experiment demonstrate that the tensile strength of the 7075-T6 aluminum alloy significantly weakens with an increase in the embrittlement temperature and a prolonged embrittlement time, whereas it improves with an increase in the strain rate. On the basis of the analysis of the experimental data, the sensitivity of the embrittlement of 7075-T6 aluminum alloy by liquid gallium to the loading strain rate is significantly higher compared to other environmental factors. In addition, this study also includes several experiments for microscopic observation, such as Scanning Electron Microscope (SEM) observation, Energy-Dispersive Spectrometer (EDS) spectroscopy, and Electron Back Scatter Diffraction (EBSD) analysis. The experimental observations confirmed the following: (1) gallium is enriched in the intergranular space of aluminum; (2) the fracture mode of 7075-T6 aluminum alloy changes from ductile to brittle fracture; and (3) the infiltration of liquid gallium into aluminum alloys and its enrichment in the intergranular space result in the formation of new dislocation nucleation sites, in addition to the original dislocations cutting and entanglement. This reduces the material's ability to undergo plastic deformation, intensifies stress concentration at the dislocation nucleation point, and, ultimately, leads to the evolution of dislocations into cracks.

Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.

TERT mediates the U-shape of glucocorticoids effects in modulation of hippocampal neural stem cells and associated brain function.

BACKGROUND: Glucocorticoids (GCs) are steroidal hormones produced by the adrenal cortex. A physiological-level GCs have a crucial function in maintaining many cognitive processes, like cognition, memory, and mood, however, both insufficient and excessive GCs impair these functions. Although this phenomenon could be explained by the U-shape of GC effects, the underlying mechanisms are still not clear. Therefore, understanding the underlying mechanisms of GCs may provide insight into the treatments for cognitive and mood-related disorders. METHODS: Consecutive administration of corticosterone (CORT, 10 mg/kg, i.g.) proceeded for 28 days to mimic excessive GCs condition. Adrenalectomy (ADX) surgery was performed to ablate endogenous GCs in mice. Microinjection of 1 µL of Ad-mTERT-GFP virus into mouse hippocampus dentate gyrus (DG) and behavioral alterations in mice were observed 4 weeks later. RESULTS: Different concentrations of GCs were shown to affect the cell growth and development of neural stem cells (NSCs) in a U-shaped manner. The physiological level of GCs (0.01 µM) promoted NSC proliferation in vitro, while the stress level of GCs (10 µM) inhibited it. The glucocorticoid synthesis blocker metyrapone (100 mg/kg, i.p.) and ADX surgery both decreased the quantity and morphological development of doublecortin (DCX)-positive immature cells in the DG. The physiological level of GCs activated mineralocorticoid receptor and then promoted the production of telomerase reverse transcriptase (TERT); in contrast, the stress level of GCs activated glucocorticoid receptor and then reduced the expression of TERT. Overexpression of TERT by AD-mTERT-GFP reversed both chronic stresses- and ADX-induced deficiency of TERT and the proliferation and development of NSCs, chronic stresses-associated depressive symptoms, and ADX-associated learning and memory impairment. CONCLUSION: The bidirectional regulation of TERT by different GCs concentrations is a key mechanism mediating the U-shape of GC effects in modulation of hippocampal NSCs and associated brain function. Replenishment of TERT could be a common treatment strategy for GC dysfunction-associated diseases.

Structures and organizations of PSI-AcpPCI supercomplexes from red tidal and coral symbiotic photosynthetic dinoflagellates.

Marine photosynthetic dinoflagellates are a group of successful phytoplankton that can form red tides in the ocean and also symbiosis with corals. These features are closely related to the photosynthetic properties of dinoflagellates. We report here three structures of photosystem I (PSI)-chlorophylls (Chls) a/c-peridinin protein complex (PSI-AcpPCI) from two species of dinoflagellates by single-particle cryoelectron microscopy. The crucial PsaA/B subunits of a red tidal dinoflagellate Amphidinium carterae are remarkably smaller and hence losing over 20 pigment-binding sites, whereas its PsaD/F/I/J/L/M/R subunits are larger and coordinate some additional pigment sites compared to other eukaryotic photosynthetic organisms, which may compensate for the smaller PsaA/B subunits. Similar modifications are observed in a coral symbiotic dinoflagellate Symbiodinium species, where two additional core proteins and fewer AcpPCIs are identified in the PSI-AcpPCI supercomplex. The antenna proteins AcpPCIs in dinoflagellates developed some loops and pigment sites as a result to accommodate the changed PSI core, therefore the structures of PSI-AcpPCI supercomplex of dinoflagellates reveal an unusual protein assembly pattern. A huge pigment network comprising Chls a and c and various carotenoids is revealed from the structural analysis, which provides the basis for our deeper understanding of the energy transfer and dissipation within the PSI-AcpPCI supercomplex, as well as the evolution of photosynthetic organisms.

Charaoteristics and clinical significance of immune microenvironment in nonsmall cell lung cancer tissues / 中国肿瘤生物治疗杂志

@# Objective: To investigate the characteristics and clinical significance of the immunomicroenvironment typing based on the expression of programmed death-ligand 1 (PD-L1) and the infiltration of CD8+ T cells in the stroma in patients with non-small cell lung cancer (NSCLC). Methods: Paraffin tissue specimens and relevant clinicopathological data of 74 NSCLC patients admitted to our hospital from January 2016 to July 2018 were collected.All patients received EGFR gene test, and none received radiotherapy, chemotherapy or targeted therapy. Immunohistochemistry was used to detect the expression of PD-L1 in tissues and the infiltration of CD8+T cells in interstitium, and the relationship between PD-L1, CD8+T cells, and the immune microenvironment typing based on both, and the pathological parameters and the survival of patients was analyzed. Results: PD-L1 expression in the primary tumor of NSCLC patients showed statistical differences in gender, pathological type, smoking history, EFGR gene mutation status ( P <0.05). The infiltration of CD8+ T lymphocytes in tumor microenvironment showed statistically significant differences in different TNM stage and lymph node metastasis ( P <0.05), PD-L1 expression was significantly correlated with EGFR mutation ( P =0.000), while CD8+T lymphocyte infiltration was not correlated with EGFR mutation ( P =0.605). The immunomicroenvironment of EGFR wild-type patients was mainly (CD8+ PD-L1+) (type I), and the mutants were mainly (CD8-PD-L1-) (type II) and (CD8+PD-L1-) (type IV). The distribution of immune microenvironmental typing in each group with different EGFR mutation, smoking history and pathological differentiation degree was significantly different ( P <0.05) and significantly correlated with EGFR mutation ( P <0.05). Follow-up showed that the patients with disease free survival, recurrence and metastasis and death were the most in type I, type II and type I, respectively. Conclusions: In this study, the distribution of tumor immunomicroenvironmental typing in NSCLC patients was mainly the highest in type I and the lowest in type Ⅲ, which was related to EGFR mutation, smoking history and pathological differentiation. Patients with EGFR mutations were mainly of type Ⅱand type Ⅳ, and were associated with low expression of PD-L1. ··