A plant-derived natural photosynthetic system for improving cell anabolism.

Insufficient intracellular anabolism is a crucial factor involved in many pathological processes in the body1,2. The anabolism of intracellular substances requires the consumption of sufficient intracellular energy and the production of reducing equivalents. ATP acts as an 'energy currency' for biological processes in cells3,4, and the reduced form of NADPH is a key electron donor that provides reducing power for anabolism5. Under pathological conditions, it is difficult to correct impaired anabolism and to increase insufficient levels of ATP and NADPH to optimum concentrations1,4,6-8. Here we develop an independent and controllable nanosized plant-derived photosynthetic system based on nanothylakoid units (NTUs). To enable cross-species applications, we use a specific mature cell membrane (the chondrocyte membrane (CM)) for camouflage encapsulation. As proof of concept, we demonstrate that these CM-NTUs enter chondrocytes through membrane fusion, avoid lysosome degradation and achieve rapid penetration. Moreover, the CM-NTUs increase intracellular ATP and NADPH levels in situ following exposure to light and improve anabolism in degenerated chondrocytes. They can also systemically correct energy imbalance and restore cellular metabolism to improve cartilage homeostasis and protect against pathological progression of osteoarthritis. Our therapeutic strategy for degenerative diseases is based on a natural photosynthetic system that can controllably enhance cell anabolism by independently providing key energy and metabolic carriers. This study also provides an enhanced understanding of the preparation and application of bioorganisms and composite biomaterials for the treatment of disease.

A novel inhalable nanobody targeting IL-4Rα for the treatment of asthma.

BACKGROUND: Inhalable biologics represent a promising approach to improve the efficacy and safety of asthma treatment. Although several mAbs targeting IL-4 receptor α chain (IL-4Rα) have been approved or are undergoing clinical trials, the development of inhalable mAbs targeting IL-4Rα presents significant challenges. OBJECTIVE: Capitalizing on the distinctive advantages of nanobodies (Nbs) in maintaining efficacy during storage and administration, we sought to develop a novel inhalable IL-4Rα Nb for effectively treating asthma. METHODS: Three IL-4Rα immunized Nb libraries were used to generate specific and functional IL-4Rα Nbs. LQ036, a bivalent Nb comprising 2 HuNb103 units, was constructed with a high affinity and specificity for human IL-4Rα. The efficacy, pharmacokinetics, and safety of inhaled LQ036 were evaluated in B-hIL4/hIL4RA humanized mice. RESULTS: LQ036 inhibited secreted embryonic alkaline phosphatase reporter activity, inhibited TF-1 cell proliferation, and suppressed phosphorylated signal transducer and activator of transduction 6 in T cells from patients with asthma. Crystal structure analysis revealed a binding region similar to dupilumab but with higher affinity, leading to better efficacy in blocking the signaling pathway. HuNb103 competed with IL-4 and IL-13 for IL-4Rα binding. Additionally, LQ036 significantly inhibited ovalbumin-specific IgE levels in serum, CCL17 levels in bronchoalveolar lavage fluid, bronchial mucous cell hyperplasia, and airway goblet cell hyperplasia in B-hIL4/hIL4RA humanized mice. Inhaled LQ036 exhibited favorable pharmacokinetics, safety, and tissue distribution, with higher concentrations observed in the lungs and bronchi. CONCLUSIONS: These findings from preclinical studies establish the safety and efficacy of inhaled LQ036, underscoring its potential as a pioneering inhalable biologic therapy for asthma.

Structural Insights into the Binding of Red Fluorescent Protein mCherry-Specific Nanobodies.

Red fluorescent proteins (RFPs) have broad applications in life science research, and the manipulation of RFPs using nanobodies can expand their potential uses. However, the structural information available for nanobodies that bind with RFPs is still insufficient. In this study, we cloned, expressed, purified, and crystallized complexes formed by mCherry with LaM1, LaM3, and LaM8. Then, we analyzed the biochemical properties of the complexes using mass spectrometry (MS), fluorescence-detected size exclusion chromatography (FSEC), isothermal titration calorimetry (ITC), and bio-layer interferometry (BLI) technology. We determined the crystal structure of mCherry-LaM1, mCherry-LaM3, and mCherry-LaM8, with resolutions of 2.05 Å, 3.29 Å, and 1.31 Å, respectively. In this study, we systematically compared various parameters of several LaM series nanobodies, including LaM1, LaM3, and LaM8, with previously reported data on LaM2, LaM4, and LaM6, specifically examining their structural information. After designing multivalent tandem LaM1-LaM8 and LaM8-LaM4 nanobodies based on structural information, we characterized their properties, revealing their higher affinity and specificity to mCherry. Our research provides novel structural insights that could aid in understanding nanobodies targeting a specific target protein. This could provide a starting point for developing enhanced mCherry manipulation tools.

Structural insights into the binding of nanobody Rh57 to active RhoA-GTP.

RhoA protein is a small GTPase that acts as a molecular switch. When bound to guanosine triphosphate (GTP), RhoA can activate several key signal pathways. Recently, nanobody Rh57 specific binding with GTP bound active RhoA was discovered and developed as a BRET biosensor without cytotoxicity. To further clarify the nanobody Rh57's mechanism of action, we co-expressed, purified, and crystallized the RhoA-Rh57 nanobody complex and solved the structure by X-ray diffraction with a resolution of 2.76 Å. The structure showed that the interaction is mainly through hydrogen bonds, salt bridges, aromatic-aromatic interactions, and hydrophobic interactions. The involved regions include CDR3 and non-hypervariable loop of Rh57, and the SWI switch loops of RhoA, respectively. The different SWI conformation of inactivated RhoA-GDP prevented the Rh57's binding. The possible explanation of Rh57 as a non-cytotoxic BRET intracellular tracer is that Rh57's binding did not overlap with downstream PRK1 and thus did not interfere with the downstream signaling pathway. Our research provides an in-depth understanding of how nanobodies recognize activated RhoA-GTP while not binding inactivated RhoA-GDP. This structural information may also provide critical information for further optimization of relevant nanobodies.

Structural insights into two distinct nanobodies recognizing the same epitope of green fluorescent protein.

Green fluorescent protein (GFP) and its derivatives are widely used in biomedical research, and the manipulation of GFP-tagged proteins by GFP-specific binders is highly desired. However, structural information on how these binders bind with GFP is still lacking. In this study, we determined the crystal structure of the nanobody Nb2 complexed with superfolder GFP (sfGFP) at a resolution of 2.2 Å. Interestingly, although the complementarity-determining regions (CDRs) of Nb2 and LaG16 sequences were only 29.7% identical, they both bound to the same epitope of GFP and existed in the same orientation. Structural analysis indicated that they achieved similar binding characteristics through different mechanisms. We further verified the kinetics and thermodynamics of binding by biolayer interferometry (BLI) and isothermal titration calorimetry (ITC). Nb2 showed a slightly higher binding affinity for sfGFP than LaG16. The stability of GFP-specific nanobodies was verified by nano differential scanning fluorimetry (nanoDSF). Nb2 exhibited the highest melting temperature (Tm); thus, Nb2 is a promising GFP nanobody candidate for use in applications requiring harsh testing conditions. We also compared the binding sites of available GFP nanobodies and showed that some of them can simultaneously bind with GFP and assemble into multifunctional complexes to manipulate GFP-tagged target proteins. Our results provide atomic-scale binding information for Nb2-sfGFP, which is important for the further development of GFP-nanobody based fusion protein manipulation techniques.

Epigallocatechin-3-gallate inhibits the growth and increases the apoptosis of human thyroid carcinoma cells through suppression of EGFR/RAS/RAF/MEK/ERK signaling pathway.

BACKGROUND: Thyroid cancer is the most common type of endocrine malignancy and the incidence rate is rapidly increasing worldwide. Epigallocatechin-3-gallate (EGCG) could suppress cancer growth and induce apoptosis in many types of cancer cells. However, the mechanism of action of EGCG on the growth of human thyroid carcinoma cells has not been fully illuminated. METHODS: Cell proliferation and viability were detected by EdU and MTS assays. Cell cycle distribution was measured by flow cytometry. Migration and invasion were evaluated by scratch and transwell assays. Apoptotic levels were detected by TUNEL staining and western blotting. The protein levels of EGFR/RAS/RAF/MEK/ERK signaling pathway were detected by western blotting. The in vivo results were determined by tumor xenografts in nude mice. The in vivo proliferation, tumor microvessel density, and apoptosis were detected by immunohistochemistry. RESULTS: EGCG inhibited the proliferation, viability, and cell cycle progression in human thyroid carcinoma cells. EGCG decreased the migration and invasion, but increased the apoptosis of human thyroid carcinoma cells. EGCG reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), H-RAS, p-RAF, p-MEK1/2, and p-extracellular signal-regulated protein kinase 1/2 (ERK1/2) in human thyroid carcinoma cells. EGCG inhibited the growth of human thyroid carcinoma xenografts by inducing apoptosis and down-regulating angiogenesis. CONCLUSIONS: EGCG could reduce the growth and increase the apoptosis of human thyroid carcinoma cells through suppressing the EGFR/RAS/RAF/MEK/ERK signaling pathway. EGCG can be developed as an effective therapeutic agent for the treatment of thyroid cancer.

Exogenous hydrogen sulfide mitigates LPS + ATP-induced inflammation by inhibiting NLRP3 inflammasome activation and promoting autophagy in L02 cells.

The aim of this study is to investigate whether exogenous hydrogen sulfide (H2S) could mitigate lipopolysaccharide (LPS) + Adenosine Triphosphate (ATP)-induced inflammation by inhibiting nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome activation and promoting autophagy in L02 cells. We stimulated L02 cells with different concentrations of LPS, then the cell viability, cell apoptosis, and the protein level of NLRP3 inflammasome were detected by MTT and western blot to determine the appropriate LPS concentration used in this study. The cells were divided into 4 group: the cells in control group were cultured with RPMI-1640 for 23.5 h; the cells in LPS + ATP group were cultured with RPMI-1640 for 0.5 h, then were stimulated with 100 ng/ml LPS for 18 h followed by stimulation with 5 mM ATP for 5 h; the cells in Sodium hydrosulfide (NaHS) + LPS + ATP group were pretreated with NaHS for 0.5 h before exposure to LPS for 18 h and ATP for 5 h; the cells in NaHS group were treated with NaHS for 0.5 h, then were cultured with RPMI-1640 for 23 h. Subsequently, the cells in each group were collected, the protein levels of NLRP3, pro-caspase-1, cleaved caspase-1, P62, toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), LC3, Beclin-1, and interleukin (IL)-1 beta (ß) were detected by western blot and enzyme-linked immunosorbent assay. Our results showed that exogenous H2S reduced the protein levels of NLRP3, cleaved caspase-1, TLR4, NF-κB, P62, and IL-1ß induced by LPS + ATP and increased the ratio of LC3-II/I and the protein levels of Beclin 1 suppressed by LPS + ATP. This study demonstrated that H2S might suppress LPS + ATP-induced inflammation by inhibiting NLRP3 inflammasome and promoting autophagy. In conclusion, H2S might have potential applications in the treatment of aseptic hepatitis.

Structural insights into the binding of nanobodies to the Staphylococcal enterotoxin B.

Staphylococcal Enterotoxin Type B (SEB), produced by Staphylococcus aureus bacteria, is notorious for inducing severe food poisoning and toxic shock syndrome. While nanobody-based treatments hold promises for combating SEB-induced diseases, the lack of structural information between SEB and nanobodies has hindered the development of nanobody-based therapeutics. Here, we present crystal structures of SEB-Nb3, SEB-Nb6, SEB-Nb8, SEB-Nb11, and SEB-Nb20 at resolutions ranging from 1.59 Å to 2.33 Å. Crystallographic analysis revealed that Nb3, Nb8, Nb11, and Nb20 bind to SEB at the T-cell receptor (TCR) interface, while Nb6 binds at the major histocompatibility complex (MHC) interface, suggesting their potential to inhibit SEB function by disrupting interactions with TCR or MHC molecules. Molecular biological analyses confirmed the thermodynamic and kinetic parameters of Nb3, Nb5, Nb6, Nb8, Nb11, Nb15, Nb18, and Nb20 to SEB. The competitive inhibition was further confirmed by cell-based experiments demonstrating nanobody neutralization. These findings elucidate the structural basis for developing specific nanobodies to neutralize SEB threats, providing crucial insights into the underlying mechanisms and offering significant assistance for further optimization towards future therapeutic strategies.

Biological functions and biomedical applications of extracellular vesicles derived from blood cells.

There is a growing interest in using extracellular vesicles (EVs) for therapeutic applications. EVs are composed of cytoplasmic proteins and nucleic acids and an external lipid bilayer containing transmembrane proteins on their surfaces. EVs can alter the state of the target cells by interacting with the receptor ligand of the target cell or by being internalised by the target cell. Blood cells are the primary source of EVs, and 1 µL of plasma contains approximately 1.5 × 107 EVs. Owing to their easy acquisition and the avoidance of cell amplification in vitro, using blood cells as a source of therapeutic EVs has promising clinical application prospects. This review summarises the characteristics and biological functions of EVs derived from different blood cell types (platelets, erythrocytes, and leukocytes) and analyses the prospects and challenges of using them for clinical therapeutic applications. In summary, blood cell-derived EVs can regulate different cell types such as immune cells (macrophages, T cells, and dendritic cells), stem cells, and somatic cells, and play a role in intercellular communication, immune regulation, and cell proliferation. Overall, blood cell-derived EVs have the potential for use in vascular diseases, inflammatory diseases, degenerative diseases, and injuries. To promote the clinical translation of blood cell-derived EVs, researchers need to perform further studies on EVs in terms of scalable and reproducible isolation technology, quality control, safety, stability and storage, regulatory issues, cost-effectiveness, and long-term efficacy.

Epigallocatechin-3-Gallate Alleviates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via Inhibition of Apoptosis and Promotion of Autophagy through the ROS/MAPK Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) represents one of the most common chronic liver diseases in the world. It has been reported that epigallocatechin-3-gallate (EGCG) plays important biological and pharmacological roles in mammalian cells. Nevertheless, the mechanism underlying the beneficial effect of EGCG on the progression of NAFLD has not been fully elucidated. In the present study, the mechanisms of action of EGCG on the growth, apoptosis, and autophagy were examined using oleic acid- (OA-) treated liver cells and the high-fat diet- (HFD-) induced NAFLD mouse model. Administration of EGCG promoted the growth of OA-treated liver cells. EGCG could reduce mitochondrial-dependent apoptosis and increase autophagy possibly via the reactive oxygen species- (ROS-) mediated mitogen-activated protein kinase (MAPK) pathway in OA-treated liver cells. In line with in vitro findings, our in vivo study verified that treatment with EGCG attenuated HFD-induced NAFLD through reduction of apoptosis and promotion of autophagy. EGCG can alleviate HFD-induced NAFLD possibly by decreasing apoptosis and increasing autophagy via the ROS/MAPK pathway. EGCG may be a promising agent for the treatment of NAFLD.

Structural insights into the binding of nanobodies LaM2 and LaM4 to the red fluorescent protein mCherry.

Red fluorescent proteins (RFPs) are powerful tools used in molecular biology research. Although RFP can be easily monitored in vivo, manipulation of RFP by suitable nanobodies binding to different epitopes of RFP is still desired. Thus, it is crucial to obtain structural information on how the different nanobodies interact with RFP. Here, we determined the crystal structures of the LaM2-mCherry and LaM4-mCherry complexes at 1.4 and 1.9 Å resolution. Our results showed that LaM2 binds to the side of the mCherry ß-barrel, while LaM4 binds to the bottom of the ß-barrel. The distinct binding sites of LaM2 and LaM4 were further verified by isothermal titration calorimetry, fluorescence-based size exclusion chromatography, and dynamic light scattering assays. Mutation of the residues at the LaM2 or LaM4 binding interface to mCherry significantly decreased the binding affinity of the nanobody to mCherry. Our results also showed that LaM2 and LaM4 can bind to mCherry simultaneously, which is crucial for recruiting multiple operation elements to the RFP. The binding of LaM2 or LaM4 did not significantly change the chromophore environment of mCherry, which is important for fluorescence quantification assays, while several GFP nanobodies significantly altered the fluorescence. Our results provide atomic resolution interaction information on the binding of nanobodies LaM2 and LaM4 with mCherry, which is important for developing detection and manipulation methods for RFP-based biotechnology.

Hydrogen Sulfide Attenuates High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Inhibiting Apoptosis and Promoting Autophagy via Reactive Oxygen Species/Phosphatidylinositol 3-Kinase/AKT/Mammalian Target of Rapamycin Signaling Pathway.

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide. Hydrogen sulfide (H2S) is involved in a wide range of physiological and pathological processes. Nevertheless, the mechanism of action of H2S in NAFLD development has not been fully clarified. Here, the reduced level of H2S was observed in liver cells treated with oleic acid (OA). Administration of H2S increased the proliferation of OA-treated cells. The results showed that H2S decreased apoptosis and promoted autophagy through reactive oxygen species (ROS)-mediated phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) cascade in OA-treated cells. In addition, administration of H2S relieved high-fat diet (HFD)-induced NAFLD via inhibition of apoptosis and promotion of autophagy. These findings suggest that H2S could ameliorate HFD-induced NAFLD by regulating apoptosis and autophagy through ROS/PI3K/AKT/mTOR signaling pathway. Novel H2S-releasing donors may have therapeutic potential for the treatment of NAFLD.

Exogenous hydrogen sulfide mitigates NLRP3 inflammasome-mediated inflammation through promoting autophagy via the AMPK-mTOR pathway.

The aim of this study was to investigate whether exogenous hydrogen sulfide (H2S) could mitigate NLRP3 inflammasome-mediated inflammation through promoting autophagy via the AMPK-mTOR pathway in L02 cells. L02 cells were stimulated with different concentrations of oleic acid (OA), then cell viability and the protein expression of NLRP3 and pro-caspase-1 were detected by MTT and western blot, respectively, to determine appropriate OA concentration in this study. The cells were divided into four groups: the cells in the control group were cultured with RPMI-1640 for 24.5 h; the cells in the OA group were cultured with RPMI-1640 for 0.5 h, then were stimulated with 1.2 mmol/l OA for 24 h; the cells in the NaHS+OA group were pretreated with sodium hydrogen sulfide (NaHS, a donor of H2S) for 0.5 h before exposure to OA for 24 h; and the cells in the NaHS group were treated with NaHS 0.5 h, then were cultured with RPMI-1640 for 24 h. Subsequently, the cells in every group were collected and the protein expression of NLRP3, procaspase-1, cleaved caspase-1, P62, LC3, Beclin1, T-AMPK, P-AMPK, T-mTOR, P-mTOR and the level of IL-1ß were detected by western blot and ElISA, respectively. Exogenous H2S reduced the level of NLRP3, caspase-1, P62, IL-1ß and the ratio of P-mTOR/T-mTOR induced by OA and increased the ratio of LC3 II/I and the protein expression of Beclin1 suppressed by OA. This study demonstrates for the first time that H2S might suppress NLRP3 inflammasome-mediated inflammation induced by OA through promoting autophagy via the AMPK-mTOR pathway. It provides a theoretical basis for the further study of the anti-inflammatory mechanism of H2S.

Structural insights into the mechanism of single domain VHH antibody binding to cortisol.

To date, few structural models of VHH antibody binding to low molecular weight haptens have been reported. Here, we report the crystal structure of cortisol binding to its VHH antibody NbCor at pH 3.5 and 10.5. Cortisol binds to NbCor mainly by burying itself under the tunnel formed by the complementarity determining region 1 (CDR1) of NbCor. The affinity of NbCor binding to cortisol and similar compounds was also verified by a microscale thermophoresis assay. Combining our findings with several previously reported structures of hapten-VHH antibody complexes, we propose that VHH antibodies exhibit a special mechanism of binding small haptens by encapsulating them in a tunnel formed by CDR1. Our findings provide useful structural information for the further development and optimization of hapten-specific VHH antibodies.

Exogenous Hydrogen Sulfide Regulates the Growth of Human Thyroid Carcinoma Cells.

Hydrogen sulfide (H2S) is involved in the development and progression of many types of cancer. However, the effect and mechanism of H2S on the growth of human thyroid carcinoma cells remain unknown. In the present study, we found that the proliferation, viability, migration, and invasion of human thyroid carcinoma cells were enhanced by 25-50 µM NaHS (an H2S donor) and inhibited by 200 µM NaHS. However, H2S showed no obvious effects on the proliferation, viability, and migration of human normal thyroid cells. Administration of 50 µM NaHS increased the expression levels of CBS, SQR, and TST, while 200 µM NaHS showed reverse effects in human thyroid carcinoma cells. After treatment with 25-50 µM NaHS, the ROS levels were decreased and the protein levels of p-PI3K, p-AKT, p-mTOR, H-RAS, p-RAF, p-MEK1/2, and p-ERK1/2 were increased, whereas 200 µM NaHS exerted opposite effects in human thyroid carcinoma cells. Furthermore, 1.4-2.8 mg/kg/day NaHS promoted the tumor growth and blood vessel formation in human thyroid carcinoma xenograft tumors, while 11.2 mg/kg/day NaHS inhibited the tumor growth and angiogenesis. In conclusion, our results demonstrate that exogenous H2S regulates the growth of human thyroid carcinoma cells through ROS/PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways. Novel H2S-releasing donors/drugs can be designed and applied for the treatment of thyroid cancer.

Blocking CXCR3 with AMG487 ameliorates the blood-retinal barrier disruption in diabetic mice through anti-oxidative.

Oxidative stress and blood-retinal barrier (BRB) damage induced by hyperglycemia are the principal processes involved in the early stages of diabetic retinopathy (DR). CXC chemokine receptor 3 (CXCR3)-mediated inflammatory infiltration exists in many disease models. The main objective of the present study was to determine whether AMG487, a CXCR3 antagonist, can ameliorate BRB disruption and reactive oxygen species generation in the DR model. The retinal endothelial cell and ganglion cell ultrastructures were observed using a transmission electron microscope. The pericyte marker PDGFR-ß, tight junction occludin, and leaking albumin were evaluated. The oxidative stress level, CCAAT-enhancer-binding protein homologous protein (CHOP), and p-p38 expression were also investigated in vivo and in vitro. The results indicated that AMG487 application might alleviate PDGFR-ß and occludin loss, and decreased the residual content of retinal albumin in the streptozocin-induced DR mouse model via the inhibition of oxidative and endoplasmic reticulum stress, in which p38 activation was also involved. Thus, CXCR3 inhibition might be a target to prevent the early stage of DR injury.