Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.

XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity.

The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

X-ray focusing below 3 nm with aberration-corrected multilayer Laue lenses.

Multilayer Laue lenses are volume diffractive optical elements for hard X-rays with the potential to focus beams to sizes as small as 1 nm. This ability is limited by the precision of the manufacturing process, whereby systematic errors that arise during fabrication contribute to wavefront aberrations even after calibration of the deposition process based on wavefront metrology. Such aberrations can be compensated by using a phase plate. However, current high numerical aperture lenses for nanometer resolution exhibit errors that exceed those that can be corrected by a single phase plate. To address this, we accumulate a large wavefront correction by propagation through a linear array of 3D-printed phase correcting elements. With such a compound refractive corrector, we report on a point spread function with a full-width at half maximum area of 2.9 × 2.8 nm2 at a photon energy of 17.5 keV.

Early-stage dynamics of chloride ion-pumping rhodopsin revealed by a femtosecond X-ray laser.

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

Imaging via Correlation of X-Ray Fluorescence Photons.

We demonstrate that x-ray fluorescence emission, which cannot maintain a stationary interference pattern, can be used to obtain images of structures by recording photon-photon correlations in the manner of the stellar intensity interferometry of Hanbury Brown and Twiss. This is achieved utilizing femtosecond-duration pulses of a hard x-ray free-electron laser to generate the emission in exposures comparable to the coherence time of the fluorescence. Iterative phasing of the photon correlation map generated a model-free real-space image of the structure of the emitters. Since fluorescence can dominate coherent scattering, this may enable imaging uncrystallised macromolecules.

Robust ptychographic X-ray speckle tracking with multilayer Laue lenses.

In recent years, X-ray speckle tracking techniques have emerged as viable tools for wavefront metrology and sample imaging applications, and have been actively developed for use at synchrotron light sources. Speckle techniques can recover an image free of aberrations and can be used to measure wavefronts with a high angular sensitivity. Since they are compatible with low-coherence sources they can be also used with laboratory X-ray sources. A new implementation of the ptychographic X-ray speckle tracking method, suitable for the metrology of highly divergent wavefields, such as those created by multilayer Laue lenses, is presented here. This new program incorporates machine learning techniques such as Huber and non-parametric regression and enables robust and quick wavefield measurements and data evaluation even for low brilliance X-ray beams, and the imaging of low-contrast samples. To realize this, a software suite was written in Python 3, with a C back-end capable of concurrent calculations for high performance. It is accessible as a Python module and is available as source code under Version 3 or later of the GNU General Public License.

Non-cryogenic structure of a chloride pump provides crucial clues to temperature-dependent channel transport efficiency.

Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.

Time-resolved spectra from millivolt EELS data.

The millivolt energy resolution now obtainable in electron energy-loss spectra (EELS) on the latest monochromated scanning transmission electron microscope corresponds, via the uncertainty principle, to a time range of 414 fs (for 10 meV resolution), and a time resolution of 0.138 fs (for energy range of 30 eV). (Thus, the width of an EELS peak is inversely related to the lifetime of an excitation.) This compares favorably with the latest X-ray free electron lasers. The time evolution of a Drude-Lorentz oscillator may be obtained from an EELS using logarithmic deconvolution followed by Kramers-Kronig analysis to extract the frequency-dependent dielectric function, and a final Fourier transform from frequency to time domain. This time-dependent dielectric function was interpreted as the impulse response of electrons, phonons, or ions based on the Drude-Lorentz theory. The time evolution of electronic oscillators from ice and protein, extracted from low resolution experimental data, were compared. Using higher energy resolution data we have also extracted the time-resolved spectra from excitons in an alkali halide, BaF2. Despite the small scanning transmission electron microscope probe size, delocalization limits the spatial resolution to about 50 nm, which is, nevertheless, better than the millimeter resolution of infrared absorption spectroscopy or Raman spectroscopy.

Reconciling the Cooperative-Competitive Patterns among Tumor and Immune Cells for Triple-Negative Breast Cancer Treatment Using Multimodule Nanocomplexes.

Targeting the competitive-cooperative relationships among tumor cells and various immune cells can efficiently reverse the immune-dysfunction microenvironment to boost the immunotherapies for the triple-negative breast cancer treatment. Hence, a bacterial outer membrane vesicle-based nanocomplex is designed for specifically targeting malignant cells and immune cells to reconcile the relationships based on metabolic-immune crosstalk. By uniquely utilizing the property of charge-reversal polymers to realize function separation, the nanocomplexes could synergistically regulate tumor cells and immune cells. This approach could reshape the immunosuppressive competition-cooperation pattern into one that is immune-responsive, showcasing significant potential for inducing tumor remission in TNBC models.

EGC enhances tumor antigen presentation and CD8+ T cell-mediated antitumor immunity via targeting oncoprotein SND1.

The Staphylococcal nuclease and Tudor domain containing 1 (SND1) has been identified as an oncoprotein. Our previous study demonstrated that SND1 impedes the major histocompatibility complex class I (MHC-I) assembly by hijacking the nascent heavy chain of MHC-I to endoplasmic reticulum-associated degradation. Herein, we aimed to identify inhibitors to block SND1-MHC-I binding, to facilitate the MHC-I presentation and tumor immunotherapy. Our findings validated the importance of the K490-containing sites in SND1-MHC-I complex. Through structure-based virtual screening and docking analysis, (-)-Epigallocatechin (EGC) exhibited the highest docking score to prevent the binding of MHC-I to SND1 by altering the spatial conformation of SND1. Additionally, EGC treatment resulted in increased expression levels of membrane-presented MHC-I in tumor cells. The C57BL/6J murine orthotopic melanoma model validated that EGC increases infiltration and activity of CD8+ T cells in both the tumor and spleen. Furthermore, the combination of EGC with programmed death-1 (PD-1) antibody demonstrated a superior antitumor effect. In summary, we identified EGC as a novel inhibitor of SND1-MHC-I interaction, prompting MHC-I presentation to improve CD8+ T cell response within the tumor microenvironment. This discovery presents a promising immunotherapeutic candidate for tumors.

Novel 3D-printing bilayer GelMA-based hydrogel containing BP,ß-TCP and exosomes for cartilage-bone integrated repair.

The integrated repair of cartilage and bone involves the migration and differentiation of cells, which has always been a difficult problem to be solved. We utilize the natural biomaterial gelatin to construct gelatin methacryloyl (GelMA), a hydrogel scaffold with high cell affinity. GelMA is mixed with different components to print a bi-layer porous hydrogel scaffold with different modulus and composition in upper and lower layers through three-dimensional (3D) printing technology. The upper scaffold adds black phosphorus (BP) and human umbilical cord mesenchymal stem cells (hUMSCs) exosomes (exos) in GelMA, which has a relatively lower elastic modulus and is conducive to the differentiation of BMSCs into cartilage. In the lower scaffold, in addition to BP and hUMSCs exos,ß-tricalcium phosphate (ß-TCP), which has osteoconductive and osteoinductive effects, is added to GelMA. The addition ofß-TCP significantly enhances the elastic modulus of the hydrogel scaffold, which is conducive to the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).In vitroexperiments have confirmed that the bi-layer scaffolds can promote osteogenesis and chondrogenic differentiation respectively. And in the rabbit cartilage-bone injury model, MRI and micro-CT results show that the 3D printed bi-layer GelMA composite scaffold has a repair effect close to normal tissue.

Nanomaterials with dual immunomodulatory functions for synergistic therapy of breast cancer brain metastases.

A long-standing paucity of effective therapies results in the poor outcomes of triple-negative breast cancer brain metastases. Immunotherapy has made progress in the treatment of tumors, but limited by the non-immunogenicity of tumors and strong immunosuppressive environment, patients with TNBC brain metastases have not yet benefited from immunotherapy. Dual immunoregulatory strategies with enhanced immune activation and reversal of the immunosuppressive microenvironment provide new therapeutic options for patients. Here, we propose a cocktail-like therapeutic strategy of microenvironment regulation-chemotherapy-immune synergistic sensitization and construct reduction-sensitive immune microenvironment regulation nanomaterials (SIL@T). SIL@T modified with targeting peptide penetrates the BBB and is subsequently internalized into metastatic breast cancer cells, releasing silybin and oxaliplatin responsively in the cells. SIL@T preferentially accumulates at the metastatic site and can significantly prolong the survival period of model animals. Mechanistic studies have shown that SIL@T can effectively induce immunogenic cell death of metastatic cells, activate immune responses and increase infiltration of CD8+ T cells. Meanwhile, the activation of STAT3 in the metastatic foci is attenuated and the immunosuppressive microenvironment is reversed. This study demonstrates that SIL@T with dual immunomodulatory functions provides a promising immune synergistic therapy strategy for breast cancer brain metastases.

Gradient tumor microenvironment-promoted penetrating micelles for hypoxia relief and immunosuppression reversion in pancreatic cancer treatment.

The tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) is the main block for the penetration of chemotherapy. In the tumor microenvironment, a dense matrix composed of fibrin is formed on the exterior, while the interior is featured by high reduction, hypoxia and low pH. How to match the special microenvironment to on-demand drug release is the key to improve chemotherapeutic efficacy. Herein, a microenvironment-responsive micellar system is developed to deepen tumoral penetration. Briefly, the conjugation of a fibrin-targeting peptide to PEG-poly amino acid has been utilized to achieve accumulation of micelles in the tumor stroma. By modification of micelles with hypoxia-reducible nitroimidazole which becomes protonated under acidic conditions, their surface charge is more positive, facilitating deeper penetration into tumors. Paclitaxel was loaded onto the micelles via a disulfide bond to enable glutathione (GSH)-responsive release. Therefore, the immunosuppressive microenvironment is relived through the alleviation of hypoxia and depletion of GSH. Hopefully, this work could establish paradigms by designing sophisticated drug-delivery systems to tactfully employ and retroact the tamed tumoral microenvironment to improve the therapeutic efficacy based on understanding the multiple hallmarks and learning the mutual regulation. STATEMENT OF SIGNIFICANCE: Tumor microenvironment(TME) is an unique pathological feature of pancreatic cancer and an inherent barrier to chemotherapy. Numerous studies regard TME as the targets for drug delivery. In this work, we propose a hypoxia-responsive nanomicellar drug delivery system that aiming hypoxia TME of pancreatic cancer. The nanodrug delivery system could respond to the hypoxic microenvironment and enhance the penetration of the inner tumor at the same time preserving the outer tumor stroma, thus achieving targeted treatment of PDAC by preserving the integrity of the outer stroma. Simultaneously, the responsive group can reverse the degree of hypoxia in TME by disrupting the redox balance in the tumor region, thus achieving precise treatment of PDAC by matching the pathological characteristics of TME. We believe our article would provide new design ideas for the future treatments for pancreatic cancer.

Smart hypoxia-responsive transformable and charge-reversible nanoparticles for the deep penetration and tumor microenvironment modulation of pancreatic cancer.

The compact extracellular matrix (ECM) of pancreatic ductal adenocarcinoma (PDAC) is the major physical barrier that hinders the delivery of anti-tumor drugs, leading to strong inherent chemotherapy resistance as well as establishing an immunosuppressive tumor microenvironment (TME). However, forcibly destroying the stroma barrier would break the balance of delicate signal transduction and dependence between tumor cells and matrix components. Uncontrollable growth and metastasis would occur, making PDAC more difficult to control. Hence, we design and construct an aptamer-decorated hypoxia-responsive nanoparticle s(DGL)n@Apt co-loading gemcitabine monophosphate and STAT3 inhibitor HJC0152. This nanoparticle can reverse its surficial charge in the TME, and reduce the size triggered by hypoxia. The released ultra-small DGL particles loading gemcitabine monophosphate exhibit excellent deep-tumor penetration, chemotherapy drugs endocytosis promotion, and autophagy induction ability. Meanwhile, HJC0152 inhibits overactivated STAT3 in both tumor cells and tumor stroma, softens the stroma barrier, and reeducates the TME into an immune-activated state. This smart codelivery strategy provides an inspiring opportunity in PDAC treatment.

Microenvironment-tailored micelles restrain carcinoma-astrocyte crosstalk for brain metastasis.

Breast-to-brain metastatic cells can interact with the surrounding cells, including astrocytes and microglia, to generate a pro-tumorigenic niche. Breast-to-brain metastasis can be treated using a dual strategy of eliminating metastatic tumor cells and normalizing their localized microenvironment. The effective accumulation of drugs at the action site of metastasis is crucial to realizing the above strategy, especially when dealing with the blood-brain barrier (BBB)-penetrating and tumor-targeting tactics. Here, we establish an in-situ microenvironment-tailored micelle (T-M/siRNA) to co-deliver therapeutic siRNA and paclitaxel (PTX) into the breast-to-brain metastasis. Anchored with a D-type cyclic peptide, T-M/siRNA can penetrate the BBB and subsequently target the brain metastases. Upon internalization by metastatic tumor cells, T-M/siRNA can release PTX in the high-level glutathione (GSH), resulting in killing cancer cells. Meanwhile, the micellar structure is dissociated, resulting in lowering the charge density to release the loaded siRNA that can targeted downregulate the expression of protocadherin 7 (PCDH7). Treatment of model mice revealed that T-M/siRNA can inhibit the abnormal activation of astrocytes and immunosuppressive activation of microglia, resulting in significantly enhanced synergistic anti-tumor efficacy. This study indicates that the micelle system can serve as a hopeful strategy to treat breast-to-brain metastasis.

A Micro-Environment Regulator for Filling the Clinical Treatment Gap after a Glioblastoma Operation.

The rapid postoperative recurrence and short survival time of glioblastoma (GBM) patients necessitate immediate and effective postoperative treatment. Herein, an immediate and mild postoperative local treatment strategy is developed that regulates the postoperative microenvironment and delays GBM recurrence. Briefly, an injectable hydrogel system (imGEL) loaded with Zn(II)2 -AMD3100 (AMD-Zn) and CpG oligonucleotide nanoparticles (CpG NPs) is injected into the operation cavity, with long-term function to block the recruitment of microglia/ macrophages and activate cytotoxic T cells. The finding indicated that the imGEL can regulate the immune microenvironment, inhibit GBM recurrence, and gain valuable time for subsequent adjuvant clinical chemotherapy.

The Ratio of NT-proBNP to CysC1.53 Predicts Heart Failure in Patients With Chronic Kidney Disease.

Background: The N-terminal pro B type natriuretic peptide (NT-proBNP) is important for prognosis of heart failure in patients with chronic kidney disease (CKD). However, the NT-proBNP level is easily affected by renal insufficiency, which limits its clinical use. Methods: This study included 396 patients with CKD. Plasma levels of NT-proBNP and cystatin C (CysC) were measured during hospitalization. The echocardiographic parameters were also detected. Patients were divided into the heart failure group and control group according to the European Society of Cardiology Guideline on Chronic Heart Failure 2021. Multiple modeling analysis of the values of NT-proBNP and CysC, including NT-proBNP/Cyscn and NT-proBNP/nCysC was performed. The receiver operating characteristic (ROC) curve, combined with the cardiac function, was used to determine the formula with the best diagnostic efficiency. Then, the sensitivity and specificity of new predictors for cardiac insufficiency in CKD patients were calculated. Pearson correlation analysis was used to analyze the relationship between new predictors and the NT-proBNP level. The clinical data of CKD patients from another local hospital were used to validate the new predictors and the cut-off values. Results: An elevated NT-proBNP/CysC1.53 ratio was an independent risk factor for cardiac dysfunction in CKD and the best predictor derived from multiple modeling analysis. There was no correlation between the NT-proBNP/CysC1.53 ratio and the NT-proBNP level (r = 0.376, p = 6.909). The area under the ROC curve for the NT-proBNP/CysC1.53 ratio was 0.815 (95% confidence interval: 0.772-0.858), and for a cut-off point of 847.964, this ratio had a sensitivity of 78.24%, and a specificity of 69.44%. When applied to the data of CKD patients from another local hospital, the NT-proBNP to CysC1.53 ratio had a sensitivity of 70.27% and a specificity of 67.74%. Conclusion: The NT-proBNP to CysC1.53 ratio was superior to NT-proBNP alone for predicting cardiac dysfunction in patients with CKD.

Biomimetic Dendrimer-Peptide Conjugates for Early Multi-Target Therapy of Alzheimer's Disease by Inflammatory Microenvironment Modulation.

Current therapeutic strategies for Alzheimer's disease (AD) treatments mainly focus on ß-amyloid (Aß) targeting. However, such therapeutic strategies have limited clinical outcomes due to the chronic and irreversible impairment of the nervous system in the late stage of AD. Recently, inflammatory responses, manifested in oxidative stress and glial cell activation, have been reported as hallmarks in the early stages of AD. Based on the crosstalk between inflammatory response and brain cells, a reactive oxygen species (ROS)-responsive dendrimer-peptide conjugate (APBP) is devised to target the AD microenvironment and inhibit inflammatory responses at an early stage. With the modification of the targeting peptide, this nanoconjugate can efficiently deliver peptides to the infected regions and restore the antioxidant ability of neurons by activating the nuclear factor (erythroid-derived 2)-like 2 signaling pathway. Moreover, this multi-target strategy exhibits a synergistic function of ROS scavenging, promoting Aß phagocytosis, and normalizing the glial cell phenotype. As a result, the nanoconjugate can reduce ROS level, decrease Aß burden, alleviate glial cell activation, and eventually enhance cognitive functions in APPswe/PSEN1dE9 model mice. These results indicate that APBP can be a promising candidate for the multi-target treatment of AD.

Sequentially Triggered Bacterial Outer Membrane Vesicles for Macrophage Metabolism Modulation and Tumor Metastasis Suppression.

Metabolic interactions between different cell types in the tumor microenvironment (TME) often result in reprogramming of the metabolism to be totally different from their normal physiological processes in order to support tumor growth. Many studies have attempted to inhibit tumor growth and activate tumor immunity by regulating the metabolism of tumors and other cells in TME. However, metabolic inhibitors often suffer from the heterogeneity of tumors, since the favorable metabolic regulation of malignant cells and other cells in TME is often inconsistent with each other. Therefore, we reported the design of a pH-sensitive drug delivery system that targets different cells in TME successively. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria were applied to coload paclitaxel (PTX) and regulated in development and DNA damage response 1 (Redd1)-siRNA and regulate tumor metabolism microenvironment and suppress tumor growth. Our siRNA@M-/PTX-CA-OMVs could first release PTX triggered by the tumor pH (pH 6.8). Then the rest of it would be taken in by M2 macrophages to increase their level of glycolysis. Great potential was observed in TAM repolarization, tumor suppression, tumor immune activation, and TME remolding in the triple-negative breast cancer model. The application of the OMV provided an insight for establishing a codelivery platform for chemical drugs and genetic medicines.

Macrophage-Disguised Manganese Dioxide Nanoparticles for Neuroprotection by Reducing Oxidative Stress and Modulating Inflammatory Microenvironment in Acute Ischemic Stroke.

Reperfusion injury is still a major challenge that impedes neuronal survival in ischemic stroke. However, the current clinical treatments are remained on single pathological process, which are due to lack of comprehensive neuroprotective effects. Herein, a macrophage-disguised honeycomb manganese dioxide (MnO2 ) nanosphere loaded with fingolimod (FTY) is developed to salvage the ischemic penumbra. In particular, the biomimetic nanoparticles can accumulate actively in the damaged brain via macrophage-membrane protein-mediated recognition with cell adhesion molecules that are overexpressed on the damaged vascular endothelium. MnO2 nanosphere can consume excess hydrogen peroxide (H2 O2 ) and convert it into desiderated oxygen (O2 ), and can be decomposed in acidic lysosome for cargo release, so as to reduce oxidative stress and promote the transition of M1 microglia to M2 type, eventually reversing the proinflammatory microenvironment and reinforcing the survival of damaged neuron. This biomimetic nanomedicine raises new strategy for multitargeted combined treatment of ischemic stroke.