MyD88 Inhibition Attenuates Cerebral Ischemia-reperfusion Injury by Regulating the Inflammatory Response and Reducing Blood-brain Barrier Damage.

Myeloid differentiation primary response gene 88 (MyD88), a downstream molecule directly linked to Toll-like receptor (TLRs) and IL1 receptor, has been implicated in ischemia-reperfusion injury across various organs. However, its role in cerebral ischemia-reperfusion injury (CIRI) remains unclear. Five transient middle cerebral artery occlusion (tMCAO) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. We screened these datasets for differentially expressed genes (DEGs) using the GSE35338 and GSE58720 datasets and performed weighted gene co-expression network analysis (WGCNA) using the GSE30655, GSE28731, and GSE32529 datasets to identify the core module related to tMCAO. A protein-protein interaction (PPI) network was constructed using the intersecting DEGs and genes in the core module. Finally, we identified Myd88 was the core gene. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) validated that TNFα, IL17, and MyD88 signaling pathways were significantly enriched in tMCAO. Subsequently, we investigated the mechanistic role of MyD88 in the tMCAO model using male C57BL/6 mice. MyD88 expression increased significantly 24 h after reperfusion. After intraperitoneal administration of TJ-M2010-5, a MyD88-specific inhibitor, during reperfusion, the infarction volumes in the mice were ameliorated. TJ-M2010-5 inhibits the activation of microglia and astrocytes. Moreover, it attenuates the upregulation of inflammatory cytokines TNFα, IL17, and MMP9 while preserving the expression level of ZO1 after tMCAO, thereby safeguarding against blood-brain barrier (BBB) disruption. Finally, our findings suggest that MyD88 regulates the IRAK4/IRF5 signaling pathway associated with microglial activation. MyD88 participates in CIRI by regulating the inflammatory response and BBB damage following tMCAO.

NRF2 activation ameliorates blood-brain barrier injury after cerebral ischemic stroke by regulating ferroptosis and inflammation.

Arterial occlusion-induced ischemic stroke (IS) is a highly frequent stroke subtype. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that modulates antioxidant genes. Its role in IS is still unelucidated. The current study focused on constructing a transient middle cerebral artery occlusion (tMCAO) model for investigating the NRF2-related mechanism underlying cerebral ischemia/reperfusion (I/R) injury. Each male C57BL/6 mouse was injected with/with no specific NRF2 activator post-tMCAO. Changes in blood-brain barrier (BBB)-associated molecule levels were analyzed using western-blotting, PCR, immunohistochemistry, and immunofluorescence analysis. NRF2 levels within cerebral I/R model decreased at 24-h post-ischemia. NRF2 activation improved brain edema, infarct volume, and neurological deficits after MCAO/R. Similarly, sulforaphane (SFN) prevented the down-regulated tight junction proteins occludin and zonula occludens 1 (ZO-1) and reduced the up-regulated aquaporin 4 (AQP4) and matrix metalloproteinase 9 (MMP9) after tMCAO. Collectively, NRF2 exerted a critical effect on preserving BBB integrity modulating ferroptosis and inflammation. Because NRF2 is related to BBB injury regulation following cerebral I/R, this provides a potential therapeutic target and throws light on the underlying mechanism for clinically treating IS.

Modulation of Dendritic Cell Function via Nanoparticle-Induced Cytosolic Calcium Changes.

Calcium nanoparticles have been investigated for applications, such as drug and gene delivery. Additionally, Ca2+ serves as a crucial second messenger in the activation of immune cells. However, few studies have systematically studied the effects of calcium nanoparticles on the calcium levels and functions within immune cells. In this study, we explore the potential of calcium nanoparticles as a vehicle to deliver calcium into the cytosol of dendritic cells (DCs) and influence their functions. We synthesized calcium hydroxide nanoparticles, coated them with a layer of silica to prevent rapid degradation, and further conjugated them with anti-CD205 antibodies to achieve targeted delivery to DCs. Our results indicate that these nanoparticles can efficiently enter DCs and release calcium ions in a controlled manner. This elevation in cytosolic calcium activates both the NFAT and NF-κB pathways, in turn promoting the expression of costimulatory molecules, antigen-presenting molecules, and pro-inflammatory cytokines. In mouse tumor models, the calcium nanoparticles enhanced the antitumor immune response and augmented the efficacy of both radiotherapy and chemotherapy without introducing additional toxicity. Our study introduces a safe nanoparticle immunomodulator with potential widespread applications in cancer therapy.

Study on Infection Control and Management of Acute and Critically Ill Patients in Tuberculosis Clinic.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a persistent infectious disease with significant global impact. Amidst the challenges presented by tuberculosis, optimizing infection control and management for acute and critically ill patients remains imperative. This study addresses this need by investigating the efficacy of standardized risk management in enhancing care outcomes. Objective: The study aims to investigate the impact of standardized risk management on infection control and the management of acute and critically ill patients in a tuberculosis clinic. Design: A randomized controlled experiment was employed for this study. Setting: The research took place at Qingdao Haici Medical Group. Participants: A total of 96 patients with acute and severe tuberculosis treated in the outpatient department from January 2020 to December 2022 were randomly assigned to the control group (CG) and the observation group (OG), with 48 cases in each group. Interventions: Patients in the CG received conventional management, while those in the OG underwent standardized risk management. Primary Outcome Measures: (1) incidence of infection events; (2) quality of management; (3) outpatient health indicators; and (4) patient satisfaction. Results: The OG exhibited a lower incidence of infection events compared to the CG (P < .05). Quality management scores were higher in the OG (P < .05). The OG demonstrated a higher qualified rate in air quality, disinfectant standards, hand hygiene, and mechanical use compared to the CG (P < .05). Patient satisfaction was higher in the OG (χ2=7.21, P < .05). Conclusions: The application of standardized risk management in infection control and management of acute and critically ill patients in tuberculosis clinics significantly reduced the incidence of infection events and improved patient satisfaction with nursing. This approach is considered worthy of widespread implementation.

Bone marrow mesenchymal stem cell-derived exosomal miR-193b-5p reduces pyroptosis after ischemic stroke by targeting AIM2.

BACKGROUND: Ischemic stroke represents a major factor causing global morbidity and death. Bone marrow mesenchymal stem cell (BMSC)-derived exosomes (Exos) have important effects on treating ischemic stroke. Here, we investigated the therapeutic mechanism by which BMSC-derived exosomal miR-193b-5p affects ischemic stroke. METHODS: luciferase assay was performed to evaluate the regulatory relationship of miR-193b-5p with absent in melanoma 2 (AIM2). Additionally, an oxygen-glucose deprivation/reperfusion (OGD/R) model was constructed for the in vitro assay, while a middle cerebral artery occlusion (MCAO) model was developed for the in vivo assay. After exosome therapy, lactate dehydrogenase and MTT assays were conducted to detect cytotoxicity and cell viability, while PCR, ELISA, western blotting assay, and immunofluorescence staining were performed to detect changes in the levels of pyroptosis-related molecules. TTC staining and TUNEL assays were performed to assess cerebral ischemia/reperfusion (I/R) injury. RESULTS: In the luciferase assay, miR-193b-5p showed direct binding to the 3'-untranslated region of AIM2. In both in vivo and in vitro assays, the injected exosomes could access the sites of ischemic injury and could be internalized. In the in vitro assay, compared to normal BMSC-Exos, miR-193b-5p-overexpressing BMSC-Exos showed greater effects on increasing cell viability and attenuating cytotoxicity; AIM2, GSDMD-N, and cleaved caspase-1 levels; and IL-1ß/IL-18 generation. In the in vivo assay, compared to normal BMSC-Exos, miR-193b-5p-overexpressing BMSC-Exos showed greater effects on decreasing the levels of these pyroptosis-related molecules and infarct volume. CONCLUSION: BMSC-Exos attenuate the cerebral I/R injury in vivo and in vitro by inhibiting AIM2 pathway-mediated pyroptosis through miR-193b-5p delivery.

Single-cell analysis reveals novel clonally expanded monocytes associated with IL1ß-IL1R2 pair in acute inflammatory demyelinating polyneuropathy.

Guillain-Barré syndrome (GBS) is an autoimmune disorder wherein the composition and gene expression patterns of peripheral blood immune cells change significantly. It is triggered by antigens with similar epitopes to Schwann cells that stimulate a maladaptive immune response against peripheral nerves. However, an atlas for peripheral blood immune cells in patients with GBS has not yet been constructed. This is a monocentric, prospective study. We collected 5 acute inflammatory demyelinating polyneuropathy (AIDP) patients and 3 healthy controls hospitalized in the First Affiliated Hospital of Harbin Medical University from December 2020 to May 2021, 3 AIDP patients were in the peak stage and 2 were in the convalescent stage. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from these patients. Furthermore, we performed cell clustering, cell annotation, cell-cell communication, differentially expressed genes (DEGs) identification and pseudotime trajectory analysis. Our study identified a novel clonally expanded CD14+ CD163+ monocyte subtype in the peripheral blood of patients with AIDP, and it was enriched in cellular response to IL1 and chemokine signaling pathways. Furthermore, we observed increased IL1ß-IL1R2 cell-cell communication between CD14+ and CD16+ monocytes. In short, by analyzing the single-cell landscape of the PBMCs in patients with AIDP we hope to widen our understanding of the composition of peripheral immune cells in patients with GBS and provide a theoretical basis for future studies.

Naringin@Metal-Organic Framework as a Multifunctional Bioplatform.

Naringin, a natural product, can be used as a therapeutic agent due to its low systemic toxicity and negligible adverse effect. However, due to its hydrophobic nature and thereby low solubility, high-dose treatment is required when used for human therapy. Herein, we demonstrate the employment of a metal-organic framework (MOF) as a nontoxic loading carrier to encapsulate naringin, and the afforded nairngin@MOF composite can serve as a multifunctional bioplatform capable of treating Gram-positive bacteria and certain cancers by slowly and progressively releasing the encapsulated naringin as well as improving and modulating immune system functions through synergy between naringin and the MOF.

Radiodynamic therapy with CsI(na)@MgO nanoparticles and 5-aminolevulinic acid.

BACKGROUND: Radiodynamic therapy (RDT) holds the potential to overcome the shallow tissue penetration issue associated with conventional photodynamic therapy (PDT). To this end, complex and sometimes toxic scintillator-photosensitizer nanoconjugates are often used, posing barriers for large-scale manufacturing and regulatory approval. METHODS: Herein, we report a streamlined RDT strategy based on CsI(Na)@MgO nanoparticles and 5-aminolevulinic acid (5-ALA). 5-ALA is a clinically approved photosensitizer, converted to protoporphyrin IX (PpIX) in cancer cells' mitochondria. CsI(Na)@MgO nanoparticles produce strong ~ 410 nm X-ray luminescence, which matches the Soret band of PpIX. We hypothesize that the CsI(Na)@MgO-and-5-ALA combination can mediate RDT wherein mitochondria-targeted PDT synergizes with DNA-targeted irradiation for efficient cancer cell killing. Because scintillator nanoparticles and photosensitizer are administered separately, the approach forgoes issues such as self-quenching or uncontrolled release of photosensitizers. RESULTS: When tested in vitro with 4T1 cells, the CsI(Na)@MgO and 5-ALA combination elevated radiation-induced reactive oxygen species (ROS), enhancing damages to mitochondria, DNA, and lipids, eventually reducing cell proliferation and clonogenicity. When tested in vivo in 4T1 models, RDT with the CsI(Na)@MgO and 5-ALA combination significantly improved tumor suppression and animal survival relative to radiation therapy (RT) alone. After treatment, the scintillator nanoparticles, made of low-toxic alkali and halide elements, were efficiently excreted, causing no detectable harm to the hosts. CONCLUSIONS: Our studies show that separately administering CsI(Na)@MgO nanoparticles and 5-ALA represents a safe and streamlined RDT approach with potential in clinical translation.

S1PR3, as a Core Protein Related to Ischemic Stroke, is Involved in the Regulation of Blood-Brain Barrier Damage.

Background: Ischemic stroke is the most common stroke incident. Sphingosine-1-phosphate (S1P) receptor 3 (S1PR3) is a member of the downstream G protein-coupled receptor family of S1P. The effect of S1PR3 on ischemic stroke remains elusive. Methods: We downloaded two middle cerebral artery occlusion (MCAO) microarray datasets from the Gene Expression Omnibus (GEO) database and screened differentially expressed genes (DEGs). Then, we performed a weighted gene coexpression network analysis (WGCNA) and identified the core module genes related to ischemic stroke. We constructed a protein-protein interaction (PPI) network for the core genes in which DEGs and WGCNA intersected. Finally, we discovered that S1PR3 was involved as the main member of the red proteome. Then, we explored the mechanism of S1PR3 in the mouse tMCAO model. The S1PR3-specific inhibitor CAY10444 was injected into the abdominal cavity of mice after cerebral ischemia/reperfusion (I/R) injury, and changes in the expression of blood-brain barrier-related molecules were measured using PCR, western blotting, and immunofluorescence staining. Results: Both GEO datasets showed that S1PR3 was upregulated during cerebral I/R in mice. WGCNA revealed that the light yellow module had the strongest correlation with the occurrence of IS. We determined the overlap with DEGs, identified 146 core genes that are potentially related to IS, and constructed a PPI network. Finally, S1PR3 was found to be the main member of the red proteome. In the mouse cerebral I/R model, S1PR3 expression increased 24 h after ischemia. After the administration of CAY10444, brain edema and neurological deficits in mice were ameliorated. CAY10444 rescued the decreased expression of the tight junction (TJ) proteins zonula occludens 1 (ZO1) and occludin after ischemia induced by transient MCAO (tMCAO) and reduced the increase in aquaporin 4 (AQP4) levels after tMCAO, preserving the integrity of the BBB. Finally, we found that S1PR3 is involved in regulating the mitogen-activated protein kinase (MAPK) and (phosphatidylinositol-3 kinase/serine-threonine kinase) PI3K-Akt signaling pathways. Conclusion: S1PR3 participates in the regulation of blood-brain barrier damage after cerebral I/R. S1PR3 is expected to be an indicator and predictor of cerebral ischemia, and drugs targeting S1PR3 may also provide new ideas for clinical medications.

Combining Doxorubicin-Conjugated Polymeric Nanoparticles and 5-Aminolevulinic Acid for Enhancing Radiotherapy against Lung Cancer.

Radiation therapy (RT) concurrent with chemotherapy improves local lung cancer control but may cause systemic toxicity. There is an unmet clinical need of treatments that can selectively sensitize cancer cells to RT. Herein, we explored a radiosensitizing strategy that combines doxorubicin (DOX)-encapsulated polyaspartamide nanoparticles and 5-aminolevulinic acid (5-ALA). The DOX-polyaspartamide nanoparticles were coupled with NTSmut, a ligand specific to neurotensin receptor type 1 (NTSR1), for lung cancer targeting. DOX was coupled to the polymer backbone through a pH-sensitive hydrazone linker, which allows for controlled release of the drug in an acidic tumor micromovement. Meanwhile, 5-ALA accumulates in the cancer cell's mitochondria, forming protoporphyrin (PpIX) that amplifies RT-induced oxidative stress. When tested in vitro in H1299 cells, DOX-encapsulated nanoparticles in conjugation with 5-ALA enhanced cancer cell killing owing to the complementary radiosensitizing effects of DOX and 5-ALA. In vivo studies confirmed that the combination improved tumor suppression relative to RT alone without causing toxicity to normal tissues. Overall, our study suggests an effective and selective radiosensitizing approach.

Inhibiting Sphingosine 1-Phosphate Receptor Subtype 3 Attenuates Brain Damage During Ischemia-Reperfusion Injury by Regulating nNOS/NO and Oxidative Stress.

BACKGROUND: Ischemic stroke (IS) is a common disease endangering human life and health. Cerebral ischemia triggers a series of complex harmful events, including excitotoxicity, inflammation and cell death, as well as increased nitric oxide production through the activation of nitric oxide synthase (NOS). Oxidative stress plays a major role in cerebral ischemia and reperfusion. Sphingosine 1-phosphate receptor subtype 3 (S1PR3), a member of S1P's G protein-coupled receptors S1PR1-S1PR5, is involved in a variety of biological effects in the body, and its role in regulating oxidative stress during cerebral ischemia and reperfusion is still unclear. METHODS: Transient middle cerebral artery occlusion (tMCAO) mice were selected as the brain ischemia-reperfusion (I/R) injury model. Male C57/BL6 mice were treated with or without a selective S1PR3 inhibition after tMCAO, and changes in infarct volume, Nissl staining, hematoxylin-eosin (H&E) staining and NOS protein, nitric oxide (NO), superoxide dismutase (SOD), and malondialdehyde (MDA) content after tMCAO were observed. RESULTS: In the cerebral ischemia-reperfusion model, inhibition of S1PR3 improved the infarct volume and neuronal damage in mice after tMCAO. Similarly, inhibition of S1PR3 can reduce the expression of NO synthase subtype neuronal NOS (nNOS) and reduce the production of NO after cerebral ischemia. After cerebral ischemia and reperfusion, the oxidative stress response was enhanced, and after the administration of the S1PR3 inhibitor, the SOD content increased and the MDA content decreased, indicating that S1PR3 plays an important role in regulating oxidative stress response. CONCLUSION: Inhibiting S1PR3 attenuates brain damage during I/R injury by regulating nNOS/NO and oxidative stress, which provides a potential new therapeutic target and mechanism for the clinical treatment of IS.

Comprehensive analysis of cuproptosis-related genes in immune infiltration in ischemic stroke.

Background: Immune infiltration plays an important role in the course of ischemic stroke (IS) progression. Cuproptosis is a newly discovered form of programmed cell death. To date, no studies on the mechanisms by which cuproptosis-related genes regulate immune infiltration in IS have been reported. Methods: IS-related microarray datasets were retrieved from the Gene Expression Omnibus (GEO) database and standardized. Immune infiltration was extracted and quantified based on the processed gene expression matrix. The differences between the IS group and the normal group as well as the correlation between the infiltrating immune cells and their functions were analyzed. The cuproptosis-related DEGs most related to immunity were screened out, and the risk model was constructed. Finally, Gene Ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and drug target were performed using the Enrichr website database. miRNAs were predicted using FunRich software. Finally, cuproptosis-related differentially expressed genes (DEGs) in IS samples were typed, and Gene Set Variation Analysis (GSVA) was used to analyze the differences in biological functions among the different types. Results: Seven Cuproptosis-related DEGs were obtained by merging the GSE16561 and GSE37587 datasets. Correlation analysis of the immune cells showed that NLRP3, NFE2L2, ATP7A, LIPT1, GLS, and MTF1 were significantly correlated with immune cells. Subsequently, these six genes were included in the risk study, and the risk prediction model was constructed to calculate the total score to analyze the risk probability of the IS group. KEGG analysis showed that the genes were mainly enriched in the following two pathways: D-glutamine and D-glutamate metabolism; and lipids and atherosclerosis. Drug target prediction found that DMBA CTD 00007046 and Lithocholate TTD 00009000 were predicted to have potential therapeutic effects of candidate molecules. GSVA showed that the TGF-ß signaling pathway and autophagy regulation pathways were upregulated in the subgroup with high expression of cuproptosis-related DEGs. Conclusions: NLRP3, NFE2L2, ATP7A, LIPT1, GLS and MTF1 may serve as predictors of cuproptosis and play an important role in the pathogenesis of immune infiltration in IS.

Identification and Clinical Validation of Key Extracellular Proteins as the Potential Biomarkers in Relapsing-Remitting Multiple Sclerosis.

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by autoimmunity. No objective clinical indicators are available for the diagnosis and prognosis of MS. Extracellular proteins are most glycosylated and likely to enter into the body fluid to serve as potential biomarkers. Our work will contribute to the in-depth study of the functions of extracellular proteins and the discovery of disease biomarkers. Methods: MS expression profiling data of the human brain was downloaded from the Gene Expression Omnibus (GEO). Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases. GO and KEGG were used to analyze the function and pathway of EP-DEGs. STRING, Cytoscape, MCODE and Cytohubba were used to construct a protein-protein interaction (PPI) network and screen key EP-DEGs. Key EP-DEGs levels were detected in the CSF of MS patients. ROC curve and survival analysis were used to evaluate the diagnostic and prognostic ability of key EP-DEGs. Results: We screened 133 EP-DEGs from DEGs. EP-DEGs were enriched in the collagen-containing extracellular matrix, signaling receptor activator activity, immune-related pathways, and PI3K-Akt signaling pathway. The PPI network of EP-DEGs had 85 nodes and 185 edges. We identified 4 key extracellular proteins IL17A, IL2, CD44, IGF1, and 16 extracellular proteins that interacted with IL17A. We clinically verified that IL17A levels decreased, but Del-1 and resolvinD1 levels increased. The diagnostic accuracy of Del-1 (AUC: 0.947) was superior to that of IgG (AUC: 0.740) with a sensitivity of 82.4% and a specificity of 100%. High Del-1 levels were significantly associated with better relapse-free and progression-free survival. Conclusion: IL17A, IL2, CD44, and IGF1 may be key extracellular proteins in the pathogenesis of MS. IL17A, Del-1, and resolvinD1 may co-regulate the development of MS and Del-1 is a potential biomarker of MS. We used bioinformatics methods to explore the biomarkers of MS and validated the results in clinical samples. The study provides a theoretical and experimental basis for revealing the pathogenesis of MS and improving the diagnosis and prognosis of MS.

Image-guided selection of Gd@C-dots as sensitizers to improve radiotherapy of non-small cell lung cancer.

BACKGROUND: Recently, gadolinium-intercalated carbon dots (Gd@C-dots) have demonstrated potential advantages over traditional high-Z nanoparticles (HZNPs) as radiosensitizers due to their high stability, minimal metal leakage, and remarkable efficacy. RESULTS: In this work, two Gd@C-dots formulations were fabricated which bore carboxylic acid (CA-Gd@C-dots) or amino group (pPD-Gd@C-dots), respectively, on the carbon shell. While it is critical to develop innovative nanomateirals for cancer therapy, determining their tumor accumulation and retention is equally important. Therefore, in vivo positron emission tomography (PET) was performed, which found that 64Cu-labeled pPD-Gd@C-dots demonstrated significantly improved tumor retention (up to 48 h post injection) compared with CA-Gd@C-dots. Indeed, cell uptake of 64Cu-pPD-Gd@C-dots reached close to 60% of total dose compared with ~ 5% of 64Cu-CA-Gd@C-dots. pPD-Gd@C-dots was therefore further evaluated as a new radiosensitizer for non-small cell lung cancer treatment. While single dose radiation plus intratumorally injected pPD-Gd@C-dots did lead to improved tumor suppression, the inhibition effect was further improved with two doses of radiation. The persistent retention of pPD-Gd@C-dots in tumor region eliminates the need of reinjecting radiosensitizer for the second radiation. CONCLUSIONS: PET offers a simple and straightforward way to study nanoparticle retention in vivo, and the selected pPD-Gd@C-dots hold great potential as an effective radiosensitizer.

Cell-type-specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila.

The impact of the Drosophila experimental system on studies of modern biology cannot be understated. The ability to tag endogenously expressed proteins is essential to maximize the use of this model organism. Here, we describe a method for labeling endogenous proteins with self-complementing split fluorescent proteins (split FPs) in a cell-type-specific manner in Drosophila A short fragment of an FP coding sequence is inserted into a specific genomic locus while the remainder of the FP is expressed using an available GAL4 driver line. In consequence, complementation fluorescence allows examination of protein localization in particular cells. Besides, when inserting tandem repeats of the short FP fragment at the same genomic locus, we can substantially enhance the fluorescence signal. The enhanced signal is of great value in live-cell imaging at the subcellular level. We can also accomplish a multicolor labeling system with orthogonal split FPs. However, other orthogonal split FPs do not function for in vivo imaging besides split GFP. Through protein engineering and in vivo functional studies, we report a red split FP that we can use for duplexed visualization of endogenous proteins in intricate Drosophila tissues. Using the two orthogonal split FP systems, we have simultaneously imaged proteins that reside in distinct subsynaptic compartments. Our approach allows us to study the proximity between and localization of multiple proteins endogenously expressed in essentially any cell type in Drosophila.

Ultrasmall Gd@Cdots as a radiosensitizing agent for non-small cell lung cancer.

High-Z nanoparticles (HZNPs) afford high cross-section for high energy radiation and have attracted wide attention as a novel type of radiosensitizer. However, conventional HZNPs are often associated with issues such as heavy metal toxicity, suboptimal pharmacokinetics, and low cellular uptake. Herein, we explore gadolinium-intercalated carbon dots (Gd@Cdots) as a dose-modifying agent for radiotherapy. Gd@Cdots are synthesized through a hydrothermal reaction with an ultrasmall size (∼3 nm) and a high Gd content. Gd@Cdots can significantly increase hydroxyl radical production under X-ray irradiation; this is attributed to not only the photoelectric effects of Gd, but also the surface catalytic effects of carbon. Because carbon is biologically and chemically inert, Gd@Cdots show low Gd leakage and minimal toxicity. In vitro studies confirm that Gd@Cdots can efficiently enhance radiation-induced cellular damage, causing elevated double strand breaks, lipid peroxidation, and mitochondrial depolarization. When tested in mice bearing non-small cell lung cancer H1299 tumors, intravenously injected Gd@Cdots plus radiation leads to improved tumor suppression and animal survival relative to radiation alone while causing no detectable toxicity. Our studies suggest a great potential of Gd@Cdots as a safe and efficient radiosensitizer.

Multiplexed labeling of cellular proteins with split fluorescent protein tags.

Self-complementing split fluorescent proteins (split FP1-10/11) have become an important labeling tool in live-cell protein imaging. However, current split FP systems to label multiple proteins in single cells have a fundamental limitation in the number of proteins that can be simultaneously labeled. Here, we describe an approach to expand the number of orthogonal split FP systems with spectrally distinct colors. By combining rational design and cycles of directed evolution, we expand the spectral color palette of FP1-10/11. We also circularly permutate GFP and synthesize the ß-strand 7, 8, or 10 system. These split GFP pairs are not only capable of labeling proteins but are also orthogonal to the current FP1-10/11 pairs, offering multiplexed labeling of cellular proteins. Our multiplexing approach, using the new orthogonal split FP systems, demonstrates simultaneous imaging of four distinct proteins in single cells; the resulting images reveal nuclear localization of focal adhesion protein Zyxin.