Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes.

The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.

Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase.

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

A Red-Emission Fluorescent Probe with Large Stokes Shift for Detection of Viscosity in Living Cells and Tumor-Bearing Mice.

Abnormal viscosity is closely related to the occurrence of many diseases, such as cancer. Therefore, real-time detection of changes in viscosity in living cells is of great importance. Fluorescent molecular rotors play a critical role in detecting changes in cellular viscosity. Developing red emission viscosity probes with large Stokes shifts and high sensitivity and specificity remains an urgent and important topic. Herein, a novel viscosity-sensitive fluorescent probe (TCF-VIS1) with a large stokes shift and red emission was prepared based on the 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) skeleton. Due to intramolecular rotation, the probe itself does not fluorescence at low viscosity. With the increase in viscosity, the rotation of TCF-VIS1 is limited, and its fluorescence is obviously enhanced. The probe has the advantages of simple preparation, large Stokes shift, good sensitivity and selectivity, and low cytotoxicity, which make it successfully used for viscosity detection in living cells. Moreover, TCF-VIS1 showed its potential for cancer diagnosis at the cell level and in tumor-bearing mice by detecting viscosity. Therefore, the probe is expected to enrich strategies for the detection of viscosity in biological systems and offer a potential tool for cancer diagnosis.

Acid-Triggered Degradation of Three-In-One Ag2S Quantum Dots for In Situ Ratiometric NIR-II Fluorescence Imaging-Guided Ion/Gas Combination Therapy.

Smart theranostic nanoprobes with the integration of multiple therapeutic modalities are preferred for precise diagnosis and efficient therapy of tumors. However, it remains a big challenge to arrange the imaging and two or more kinds of therapeutic agents without weakening the intended performances. In addition, most existing fluorescence (FL) imaging agents suffer from low spatiotemporal resolution due to the short emission wavelength (<900 nm). Here, novel three-in-one Ag2S quantum dot (QD)-based smart theranostic nanoprobes were proposed for in situ ratiometric NIR-II FL imaging-guided ion/gas combination therapy of tumors. Under the acidic tumor microenvironment, three-in-one Ag2S QDs underwent destructive degradation, generating toxic Ag+ and H2S. Meanwhile, their FL emission at 1270 nm was weakened. Upon introduction of a downconversion nanoparticle (DCNP) as the delivery carrier and NIR-II FL reference signal unit, the formed Ag2S QD-based theranostic nanoprobes could achieve precise diagnosis of tumors through ratiometric NIR-II FL signals. Also, the generated Ag+ and H2S enabled specific ion/gas combination therapy toward tumors. By combining the imaging and therapeutic functions, three-in-one Ag2S QDs may open a simple yet reliable avenue to design theranostic nanoprobes.

Plant-derived exosome-like nanovesicles: A novel nanotool for disease therapy.

Exosomes are extracellular vesicles comprising bilayer phospholipid membranes and are secreted by eukaryotic cells. They are released via cellular exocytosis, contain DNA, RNA, proteins, and other substances, and participate in various cellular communications between tissues and organs. Since the discovery of exosomes in 1983, animal-derived exosomes have become a research focus for small-molecule drug delivery in biology, medicine, and other fields owing to their good biocompatibility and homing effects. Recent studies have found that plant-derived exosome-like nanovesicles (PELNVs) exhibit certain biological effects, such as anti-inflammatory and anti-tumor abilities, and have minimal toxic side effects. Because they are rich in active lipid molecules with certain pharmacological effects, PELNVs could be novel carriers for drug delivery. In this review, the biological formation and effects, isolation, and extraction of PELNVs, as well as characteristics of transporting drugs as carriers are summarized to provide new ideas and methods for future research on plant-derived exosome-like nanovesicles.

Lycium barbarum polysaccharide remodels colon inflammatory microenvironment and improves gut health.

Aim: Disturbed intestinal microbiota has been implicated in the inflammatory microenvironment of the colon, which usually results in ulcerative colitis (UC). Given the limitations of these drugs, it is important to explore alternative means of protecting the gut health from UC. This study aimed to investigate the potential of polysaccharides as beneficial nutrients in the regulation of the gut microbiota, which determines the inflammatory microenvironment of the colon. Materials and methods: Mice were treated with dextran sulfate sodium (DSS) to evaluate the effects and mechanisms of Lycium barbarum polysaccharide (LBP) in remodeling the inflammatory microenvironment and improving gut health. Body weight and disease activity indices were monitored daily. Hematoxylin and eosin staining was used to analyze colon dynamics. The levels of inflammatory indicators and expression of MUC-2, claudin-1, ZO-1, and G-protein-coupled receptor 5 (TGR5) were determined using assay kits and immunohistochemistry, respectively. 16S rRNA high-throughput sequencing of the intestinal microbiota and liquid chromatography-tandem mass spectrometry for related bile acids were used. Results: LBP significantly improved the colonic tissue structure by upregulating MUC-2, claudin-1, and ZO-1 protein expression. The bacterial genus Dubosiella was dominant in healthy mice, but significantly decreased in mice treated with DSS. LBP rehabilitated Dubosiella in the sick guts of DSS mice to a level close to that of healthy mice. The levels of other beneficial bacterial genera Akkermansia and Bifidobacterium were also increased, whereas those of the harmful bacterial genera Turicibacter, Clostridium_sensu_stricto_1, Escherichia-Shigella, and Faecalibaculum decreased. The activity of beneficial bacteria promoted the bile acids lithocholic and deoxycholic acids in mice with UC, which improved the gut barrier function through the upregulation of TGR5. Conclusion: The inflammatory microenvironment in the gut is determined by the balance of the gut microbiota. LBP showed great potential as a beneficial nutrient for rehabilitating Dubosiella which is dominant in the gut of healthy mice. Nutrient-related LBP may play an important role in gut health management.

Photochemical three-component assembly of tri-substituted oxazoles through a carbenic phosphorus-nitrile hybrid ylide formation/trapping cascade.

Construction of complex molecular skeletons with ubiquitous chemical feedstocks in a single transformation is highly appealing in organic synthesis. We report a novel visible-light-induced three-component reaction for the construction of complex 2,4,5-trisubstituted oxazoles, which are valuable in medicinal chemistry, from simple and readily available iodonium-phosphonium hybrid ylides, carboxylic acids, and nitriles. This reaction features a carbenic phosphorus-nitrile hybrid ylide formation/trapping cascade, in which a photo-generated α-phosphonium carbene acts as a sequence trigger. This catalyst- and additive-free transformation exhibits high efficiency and broad substrate scope for synthesizing diverse oxazoles.

Manganese-based nanomaterials in diagnostics and chemodynamic therapy of cancers: new development.

In the realm of cancer treatment, traditional modalities like radiotherapy and chemotherapy have achieved certain advancements but continue to grapple with challenges including harm to healthy tissues, resistance to treatment, and adverse drug reactions. The swift progress in nanotechnology recently has opened avenues for investigating innovative approaches to cancer therapy. Especially, chemodynamic therapy (CDT) utilizing metal nanomaterials stands out as an effective cancer treatment choice owing to its minimal side effects and independence from external energy sources. Transition metals like manganese are capable of exerting anti-tumor effects through a Fenton-like mechanism, with their distinctive magnetic properties playing a crucial role as contrast agents in tumor diagnosis and treatment. Against this backdrop, this review emphasizes the recent five-year advancements in the application of manganese (Mn) metal ions within nanomaterials, particularly highlighting their unique capabilities in catalyzing CDT and enhancing MRI imaging. Initially, we delineate the biomedical properties of manganese, followed by an integrated discussion on the utilization of manganese-based nanomaterials in CDT alongside multimodal therapies, and delve into the application and future outlook of manganese-based nanomaterial-mediated MRI imaging techniques in cancer therapy. By this means, the objective is to furnish novel viewpoints and possibilities for the research and development in future cancer therapies.

Primary bone solitary B-cell lymphoblastic lymphoma aberrant expression Fli-1 and ERG: A case report.

ABSTRACT: Primary non-Hodgkin bone lymphoma is a rare disease, and within this category, B-cell lymphoblastic lymphoma (B-LBL) is an even rare clinical entity that has only been reported in small case series or individual case reports. B-LBL can mimic Ewing's sarcoma both clinically and histologically, leading to misdiagnosis. We present a case of primary B-LBL of the bone in a 7-year-old girl. The tumor cells are uniform and small with a diffuse and infiltrative growth pattern similar to EWS. Immunohistochemical results are positive for CD99, Fli-1, ERG, TDT, PAX5, and CD79α, but negative for leukocyte common antigen, CD3, CD20, and NKX2.2. No other lesions are found on positron emission tomography/computed tomography imaging. Finally, primary solitary bone B-LBL of the right tibia was diagnosed.

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Dexmedetomidine inhibited arrhythmia susceptibility to adrenergic stress in RyR2R2474S mice through regulating the coupling of membrane potential and intracellular calcium.

BACKGROUND: Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear. METHODS: Ryanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2R2474S/+ mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX. RESULTS: DEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2R2474S/+ mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD80) and calcium transient duration (CaTD80) in both RyR2R2474S/+ and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2R2474S/+ hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency. CONCLUSION: DEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca2+. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.

Targeted gene delivery systems for T-cell engineering.

T lymphocytes are indispensable for the host systems of defense against pathogens, tumors, and environmental threats. The therapeutic potential of harnessing the cytotoxic properties of T lymphocytes for antigen-specific cell elimination is both evident and efficacious. Genetically engineered T-cells, such as those employed in CAR-T and TCR-T cell therapies, have demonstrated significant clinical benefits in treating cancer and autoimmune disorders. However, the current landscape of T-cell genetic engineering is dominated by strategies that necessitate in vitro T-cell isolation and modification, which introduce complexity and prolong the development timeline of T-cell based immunotherapies. This review explores the complexities of gene delivery systems designed for T cells, covering both viral and nonviral vectors. Viral vectors are known for their high transduction efficiency, yet they face significant limitations, such as potential immunogenicity and the complexities involved in large-scale production. Nonviral vectors, conversely, offer a safer profile and the potential for scalable manufacturing, yet they often struggle with lower transduction efficiency. The pursuit of gene delivery systems that can achieve targeted gene transfer to T cell without the need for isolation represents a significant advancement in the field. This review assesses the design principles and current research progress of such systems, highlighting the potential for in vivo gene modification therapies that could revolutionize T-cell based treatments. By providing a comprehensive analysis of these systems, we aim to contribute valuable insights into the future development of T-cell immunotherapy.

Strategies in product engineering of mesenchymal stem cell-derived exosomes: unveiling the mechanisms underpinning the promotive effects of mesenchymal stem cell-derived exosomes.

Articular cartilage injuries present a significant global challenge, particularly in the aging population. These injuries not only restrict movement due to primary damage but also exacerbate elderly degenerative lesions, leading to secondary cartilage injury and osteoarthritis. Addressing osteoarthritis and cartilage damage involves overcoming several technical challenges in biological treatment. The use of induced mesenchymal stem cells (iMSCs) with functional gene modifications emerges as a solution, providing a more stable and controllable source of Mesenchymal Stem Cells (MSCs) with reduced heterogeneity. Furthermore, In addition, this review encompasses strategies aimed at enhancing exosome efficacy, comprising the cultivation of MSCs in three-dimensional matrices, augmentation of functional constituents within MSC-derived exosomes, and modification of their surface characteristics. Finally, we delve into the mechanisms through which MSC-exosomes, sourced from diverse tissues, thwart osteoarthritis (OA) progression and facilitate cartilage repair. This review lays a foundational framework for engineering iMSC-exosomes treatment of patients suffering from osteoarthritis and articular cartilage injuries, highlighting cutting-edge research and potential therapeutic pathways.

Innexin hemichannel activation by Microplitis bicoloratus ecSOD monopolymer reduces ROS.

The extracellular superoxide dismutases (ecSODs) secreted by Microplitis bicoloratus reduce the reactive oxygen species (ROS) stimulated by the Microplitis bicoloratus bracovirus. Here, we demonstrate that the bacterial transferase hexapeptide (hexapep) motif and bacterial-immunoglobulin-like (BIg-like) domain of ecSODs bind to the cell membrane and transiently open hemichannels, facilitating ROS reductions. RNAi-mediated ecSOD silencing in vivo elevated ROS in host hemocytes, impairing parasitoid larva development. In vitro, the ecSOD-monopolymer needed to be membrane bound to open hemichannels. Furthermore, the hexapep motif in the beta-sandwich of ecSOD49 and ecSOD58, and BIg-like domain in the signal peptides of ecSOD67 were required for cell membrane binding. Hexapep motif and BIg-like domain deletions induced ecSODs loss of adhesion and ROS reduction failure. The hexapep motif and BIg-like domain mediated ecSOD binding via upregulating innexins and stabilizing the opened hemichannels. Our findings reveal a mechanism through which ecSOD reduces ROS, which may aid in developing anti-redox therapy.

Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing.

BACKGROUND: Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application. AIM: To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs. METHODS: The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification. RESULTS: In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation. CONCLUSION: Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.

Tenovin 3 induces apoptosis and ferroptosis in EGFR 19del non small cell lung cancer cells.

Epidermal growth factor receptor (EGFR) exon 19 deletion is a major driver for the drug resistance of non-small cell lung cancer (NSCLC). Identification small inhibitor capable of selectively inhibiting EGFR-19del NSCLC is a desirable strategy to overcome drug resistance in NSCLC. This study aims to screen an inhibitor for EGFR exon 19 deletion cells and explore its underlying mechanism. High through-put screen was conducted to identify an inhibitor for EGFR-19del NSCLC cells. And tenovin-3 was identified as a selective inhibitor of PC9 cells, an EGFR-19del NSCLC cells. Tenovin-3 showed particular inhibition effect on PC9 cells proliferation through inducing apoptosis and ferroptosis. Mechanistically, tenovin-3 might induce the apoptosis and ferroptosis of PC9 cells through mitochondrial pathway, as indicated by the change of VDAC1 and cytochrome c (cyt c). And bioinformatics analyses showed that the expression levels of SLC7A11 and CPX4 were correlated with NSCLC patient's survival. Our findings provide evidences for tenovin-3 to be developed into a novel candidate agent for NSCLC with EGFR exon 19 deletion. Our study also suggests that inducing ferroptosis may be a therapeutic strategy for NSCLC with EGFR exon 19 deletion.

Narrow linewidth laser based on a sidewall grating active distributed Bragg reflector.

We demonstrated a narrow linewidth semiconductor laser based on a deep-etched sidewall grating active distributed Bragg reflector (SG-ADBR). The coupling coefficients and reflectance were numerically simulated for deep-etched fifth-order SG-ADBR, and a reflectance of 0.86 with a bandwidth of 1.04 nm was obtained by the finite element method for a 500-period SG-ADBR. Then the fifth-order SG-ADBR lasers were fabricated using projection i-line lithography processes. Single-mode lasing at 1537.9 nm was obtained with a high side-mode suppression ratio (SMSR) of 65 dB, and a continuous tuning range of 10.3 nm was verified with SMSRs greater than 53 dB. Furthermore, the frequency noise power spectral density was characterized, from which a Lorentzian linewidth of 288 kHz was obtained.

In vivo safety evaluation method for nanomaterials for cancer therapy.

Nanomaterials are extensively used in the diagnosis and treatment of cancer and other diseases because of their distinctive physicochemical properties, including the small size and ease of modification. The approval of numerous nanomaterials for clinical treatment has led to a significant increase in human exposure to these materials. When nanomaterials enter organisms, they interact with DNA, cells, tissues, and organs, potentially causing various adverse effects, such as genotoxicity, reproductive toxicity, immunotoxicity, and damage to tissues and organs. Therefore, it is crucial to elucidate the side effects and toxicity mechanisms of nanomaterials thoroughly before their clinical applications. Although methods for in vitro safety evaluation of nanomaterials are well established, systematic methods for in vivo safety evaluation are still lacking. This review focuses on the in vivo safety evaluation of nanomaterials and explores their potential effects. In addition, the experimental methods for assessing such effects in various disciplines, including toxicology, pharmacology, physiopathology, immunology, and bioinformatics are also discussed.