Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing.

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.

Recent Advances in Fast-Decaying Metal Halide Perovskites Scintillators.

Fast-decaying scintillators show subnanoseconds or nanoseconds lifetime and high time resolution, making them important in nuclear physics, medical diagnostics, scientific research, and other fields. Metal halide perovskites (MHPs) show great potential for scintillator applications owing to their easy synthesis procedure and attractive optical properties. However, MHPs scintillators still need further improvement in decay lifetime. To optimize the decay lifetime, great progress has been achieved recently. In this Perspective, we first summarize the structural characteristics of MHPs in various dimensions, which brings different exciton behaviors. Then, recent advances in designing fast-decaying MHPs according to different exciton behaviors have been concluded, focusing on the photophysical mechanisms to achieve fast-decaying lifetimes. These advancements in decay lifetimes could facilitate the MHPs scintillators in advanced applications, such as time-of-flight positron emission tomography (TOF-PET), photon-counting computed tomography (PCCT), etc. Finally, the challenges and future opportunities are discussed to provide a roadmap for designing novel fast-decaying MHPs scintillators.

Clinicopathological spectrum, management, and outcome of ectopic parathyroid carcinoma: Experience with 24 cases.

PURPOSE: Ectopic parathyroid carcinoma (EPC) is a rare clinical entity with multiple diagnostic pitfalls, making surgical cures challenging. We assessed the clinicopathological spectrum and outcome of EPCs. METHODS: In this retrospective cohort study, 24 EPCs were identified from 133 PC patients treated at a tertiary referral center. The relationship between clinicopathological findings and locations was analyzed. RESULTS: The locations of EPCs were predominantly intrathyroidal (62.5%), followed by 16.7% in the mediastinum, 8.3% in the retropharyngeal space, 8.3% in the carotid sheath, and 4.2% in the upper neck. Intrathyroidal EPC patients experienced higher serum calcium (p = 0.020), a higher rate of vascular invasion (p = 0.040), and a slightly higher incidence of non-R0 initial resection (p = 0.092) than those in other ectopic locations. Intrathyroidal EPC patients also suffered a trend of higher upper aerodigestive tract (UAT) invasion rate (p = 0.070) and higher risks of distant metastasis (p = 0.037) than the other PC patients. The 5-year disease-free survival rate after surgery was slightly compromised at 41.5% in intrathyroidal EPC patients compared with 77.8% among those in other ectopic locations (p = 0.143) and 59.7% among the other PC patients (log-rank = 3.194; p = 0.074), though without statistical significance. CONCLUSION: Intrathyroidal EPC might cause a more biochemically and invasively distinct PC picture compared with other PCs. Special caution should be exercised in the preoperative diagnosis and management of such cases.

DNA-based nanosized functional ruthenium(II) complex as potential phosphorescent probe to track tumor cells.

Identifying tumor cells can be challenging due to cancer's complex and heterogeneous nature. Here, an efficacious phosphorescent probe that can precisely highlight tumor cells has been created. By combining the ruthenium(II) complex with oligonucleotides, we have developed a nanosized functional ruthenium(II) complex (Ru@DNA) with dimensions ranging from 300 to 500 nm. Our research demonstrates that Ru@DNA can readily traverse biomembranes via ATP-dependent endocytosis without carriers. Notably, the nanosized ruthenium(II) complex exhibits rapid and selective accumulation within tumor cells, possibly attributed to the nanoparticles' enhanced permeation and retention (EPR) effect. Ru@DNA can also effectively discern and label the transplanted cancer cells in the zebrafish model. Moreover, Ru@DNA is efficiently absorbed into the intestine and further distributed in the pancreas. Our findings underscore the potential of Ru@DNA as a DNA-based nanodevice derived from a functional ruthenium(II) complex. This innovative nanodevice holds promise as an efficient phosphorescent probe for both in vitro and in vivo imaging of living tumor cells.

GRACE: Unveiling Gene Regulatory Networks With Causal Mechanistic Graph Neural Networks in Single-Cell RNA-Sequencing Data.

Reconstructing gene regulatory networks (GRNs) using single-cell RNA sequencing (scRNA-seq) data holds great promise for unraveling cellular fate development and heterogeneity. While numerous machine-learning methods have been proposed to infer GRNs from scRNA-seq gene expression data, many of them operate solely in a statistical or black box manner, limiting their capacity for making causal inferences between genes. In this study, we introduce GRN inference with Accuracy and Causal Explanation (GRACE), a novel graph-based causal autoencoder framework that combines a structural causal model (SCM) with graph neural networks (GNNs) to enable GRN inference and gene causal reasoning from scRNA-seq data. By explicitly modeling causal relationships between genes, GRACE facilitates the learning of regulatory context and gene embeddings. With the learned gene signals, our model successfully decoding the causal structures and alleviates the accurate determination of multiple attributes of gene regulation that is important to determine the regulatory levels. Through extensive evaluations on seven benchmarks, we demonstrate that GRACE outperforms 14 state-of-the-art GRN inference methods, with the incorporation of causal mechanisms significantly enhancing the accuracy of GRN and gene causality inference. Furthermore, the application to human peripheral blood mononuclear cell (PBMC) samples reveals cell type-specific regulators in monocyte phagocytosis and immune regulation, validated through network analysis and functional enrichment analysis.

Risk Factors of Distant Metastatic Parathyroid Carcinoma and Insights into Therapeutic Perspectives.

BACKGROUND: Distant metastatic parathyroid carcinoma (DM-PC) is a rare but often lethal entity with limited data about prognostic indicators. We sought to investigate the risk factors, patterns, and outcomes of DM-PC. METHODS: In this observational cohort study, 126 patients who underwent surgery for PC at a tertiary referral center from 2010 to 2023 were enrolled, among whom 38 had DMs. Univariate and multivariate Cox regression analyses were used to assess the effects of prognostic factors on DM. RESULTS: The cumulative incidence of DM was 14.1%, 33.8%, and 66.9% at 5, 10, and 20 years in the duration of disease course, respectively. DM-PC patients suffered a worse 5-year overall survival of 37.1% compared with 89.8% in the non-DM patients (p < 0.001). DM-PC patients also suffered more previous operations (p < 0.001), higher preoperative serum calcium (p<0.001) and parathyroid hormone (PTH) levels (p < 0.001), lower frequencies of R0 resection (p < 0.001), higher rates of pathological vascular invasion (p = 0.020), thyroid infiltration (p = 0.027), extraglandular extension (p = 0.001), upper aerodigestive tract (UAT) invasion (p < 0.001), and lymph node metastasis (p < 0.001). Multivariate Cox regression revealed that non-R0 resection (HR 6.144, 95% CI 2.881-13.106, p < 0.001), UAT invasion (HR 3.718, 95% CI 1.782-7.756, p < 0.001), and higher preoperative PTH levels (HR 1.001, 95% CI 1.000-1.001, p = 0.012) were independent risk factors of DM. CONCLUSIONS: Upper aerodigestive tract invasion and higher preoperative PTH levels might be risk factors for possible metastatic involvement of PC. R0 resection and closer surveillance should be considered in such cases to minimize the risk of DM and to optimize patient care.

Efficient and durable seawater electrolysis with a V2O3-protected catalyst.

The ocean, a vast hydrogen reservoir, holds potential for sustainable energy and water development. Developing high-performance electrocatalysts for hydrogen production under harsh seawater conditions is challenging. Here, we propose incorporating a protective V2O3 layer to modulate the microcatalytic environment and create in situ dual-active sites consisting of low-loaded Pt and Ni3N. This catalyst demonstrates an ultralow overpotential of 80 mV at 500 mA cm-2, a mass activity 30.86 times higher than Pt-C and maintains at least 500 hours in seawater. Moreover, the assembled anion exchange membrane water electrolyzers (AEMWE) demonstrate superior activity and durability even under demanding industrial conditions. In situ localized pH analysis elucidates the microcatalytic environmental regulation mechanism of the V2O3 layer. Its role as a Lewis acid layer enables the sequestration of excess OH- ions, mitigate Cl- corrosion, and alkaline earth salt precipitation. Our catalyst protection strategy by using V2O3 presents a promising and cost-effective approach for large-scale sustainable green hydrogen production.

Detection and identification of circulating tumor cells in parathyroid tumors and correlation analysis with clinicopathological features.

INTRODUCTION: The differential diagnosis of parathyroid carcinoma (PC)/parathyroid adenoma (PA) in parathyroid tumors is critical for their management and prognosis. Circulating tumor cells (CTCs) identification in the peripheral blood of parathyroid tumors remains unknown. In this study, we proposed to investigate the differences of CTCs in PC/PA and the relationship with clinicopathologic features to assess its relevance to PC and value in identifying PC/PA. METHODS AND MATERIALS: Peripheral blood was collected from 27 patients with PC and 37 patients with PA treated in our hospital, and the number of chromosome 8 aberrant CTCs was detected by negative magnetic bead sorting fluorescence in situ hybridization (NE-FISH). The differences of CTCs in PC/PA peripheral blood were compared and their diagnostic efficacy was evaluated, and the correlation between CTCs and clinicopathological features of PC was further explored. RESULTS: CTCs differed significantly in PC/PA (p = 0.0008) and were up-regulated in PC, with good diagnostic efficacy. CTCs combined with alkaline phosphatase (ALP) assay improved the diagnostic efficacy in identifying PC/PA (AUC = 0.7838, p = 0.0001). The number of CTCs was correlated with tumor dimensions, but not significantly correlated with clinical markers such as calcium and PTH and pathological features such as vascular invasion, lymph node metastasis and distant metastasis. CONCLUSION: As a non-invasive liquid biopsy method, CTCs test combined with ALP test can be used as an important reference basis for timely and accurate identification and treatment of PC. It is of great significance to improve the current situation of PC diagnosis, treatment and prognosis.

Versican controlled by Lmx1b regulates hyaluronate density and hydration for semicircular canal morphogenesis.

During inner ear semicircular canal morphogenesis in zebrafish, patterned canal-genesis zones express genes for extracellular matrix component synthesis. These include hyaluronan and the hyaluronan-binding chondroitin sulfate proteoglycan Versican, which are abundant in the matrices of many developing organs. Charged hyaluronate polymers play a key role in canal morphogenesis through osmotic swelling. However, the developmental factor(s) that control the synthesis of the matrix components and regulation of hyaluronate density and swelling are unknown. Here, we identify the transcription factor, Lmx1b, as a positive transcriptional regulator of hyaluronan, Versican, and chondroitin synthesis genes crucial for canal morphogenesis. We show that Versican regulates hyaluronan density through its protein core, whereas the charged chondroitin side chains contribute to the osmotic swelling of hyaluronate. Versican-tuned properties of hyaluronate matrices may be a broadly used mechanism in morphogenesis with important implications for understanding diseases where these matrices are impaired, and for hydrogel engineering for tissue regeneration.

Iron-Nickel synergistic catalysis growth of (Fe,Ni)9S8/Ni3S2@N,S codoped carbon bridged nanowires enhanced oxygen evolution reaction performance.

Improving the conductivity of the electrocatalyst itself is essential for enhancing its performance. In this work, N, S-rich 6-thioguanine (TG) is selected as the ligand to synthesize a Fe, Ni bimetallic porous coordination polymer (PCP), which is then derived to fabricate N,S codoped carbon (NSC)-coated (Fe,Ni)9S8/Ni3S2 bridged nanowires. The (Fe,Ni)9S8/Ni3S2@NSC bridged nanowires obtained through bimetallic synergistic catalysis and self-sulfurization processes not only introduced additional electrocatalytic active sites but also significantly enhance the overall conductivity of the catalyst due to the interconnected nanowire structure. The resulting (Fe,Ni)9S8/Ni3S2@NSC demonstrates remarkable oxygen evolution reaction (OER) performance, exhibiting an overpotential as low as 252 mV at a current density of 10 mA cm-2. This work proposes a novel strategy for enhancing the overall conductivity of catalysts by growing bridged nanowires, providing valuable insights and inspiration for the design and preparation of advanced transition metal sulfide electrocatalysts.

Lipid metabolism mediates the association between body mass index change and bone mineral density: The Taizhou imaging study.

BACKGROUND: Limited research explores the impact of body mass index (BMI) change on osteoporosis, regarding the role of lipid metabolism. We aimed to cross-sectionally investigate these relationships in 820 Chinese participants aged 55-65 from the Taizhou Imaging Study. METHODS: We used the baseline data collected between 2013 and 2018. T-score was calculated by standardizing bone mineral density and was used for osteoporosis and osteopenia diagnosis. Multinomial logistic regression was used to examine the effect of BMI change on bone health status. Multivariable linear regression was employed to identify the metabolites corrected with BMI change and T-score. Exploratory factor analysis (EFA) and mediation analysis were conducted to ascertain the involvement of the metabolites. RESULTS: BMI increase served as a protective factor against osteoporosis (OR = 0.79[0.71-0.88], P-value<0.001) and osteopenia (OR = 0.88[0.82-0.95], P-value<0.001). Eighteen serum metabolites were associated with both BMI change and T-score. Specifically, high-density lipoprotein (HDL) substructures demonstrated negative correlations (ß = -0.08 to -0.06 and - 0.12 to -0.08, respectively), while very low-density lipoprotein (VLDL) substructions showed positive correlations (ß = 0.09 to 0.10 and 0.10 to 0.11, respectively). The two lipid factors (HDL and VLDL) extracted by EFA acted as mediators between BMI change and T-score (Prop. Mediated = 8.16% and 10.51%, all P-value<0.01). CONCLUSION: BMI gain among Chinese aged 55-65 is beneficial for reducing the risk of osteoporosis. The metabolism of HDL and VLDL partially mediates the effect of BMI change on bone loss. Our research offers novel insights into the prevention of osteoporosis, approached from the perspective of weight management and lipid metabolomics.

Metal-organic complex coating for enhanced corrosion control and biocompatibility on biodegradable magnesium alloy for orthopaedic implants.

Magnesium alloy is currently regarded as the most favourable biodegradable metal; however, obstacles remain to be overcome in terms of managing its corrosion and ensuring its biocompatibility. In this study, a metal-organic complex comprising Ca ions incorporated in tannic acid (TA) was prepared and used to coat magnesium alloy by chemical conversion and dipping processes, followed by modification with stearic acid (SA). This metal-organic complex coating was demonstrated to be homogeneous and compact, and it significantly improved the electrochemical corrosion resistance and long-term degradation behaviour of the coated samples. Consequently, the well-controlled release of Mg and Ca ions, as well as the osteo-compatible TA and SA molecules, promoted the proliferation of osteoblast cells. This metal-organic complex coating offers a promising modifying strategy for magnesium-based orthopaedic implants.

Natural gambogic acid-tuned self-assembly of nanodrugs towards synergistic chemophototherapy against breast cancer.

Gambogic acid (GA) as a naturally derived chemotherapeutic agent is of increasing interest for antitumor therapy. However, current research mainly focuses on improving the pharmacological properties to overcome the shortcomings in clinical applications or as a synergistic anticancer agent in combination with chemotherapy and chemophototherapy. Yet, the material properties of GA (e.g., self-assembly) are often neglected. Herein, we validated the self-assembly function of GA and its huge potential as a single-component active carrier for synergistic delivery using pyropheophorbide-a (PPa) as a drug model. The results showed that self-assembled GA drives the formation of nano-GA/PPa mainly through noncovalent interactions such as π-π stacking, hydrophobic interactions, and hydrogen bonding. Additionally, although no significant differences in cytotoxicity were found between the individual in vitro chemotherapy and combined chemophototherapy, the as-prepared nano-GA/PPa exhibits remarkably improved water solubility and multiple favorable therapeutic features, leading to a prominent in vivo photochemotherapy efficiency of 89.3% inhibition rate with reduced hepatotoxicity of GA. This work highlights the potential of self-assembled GA as a drug delivery carrier for synergistic biomedical applications.

Vertical Cross-Alignments of 2D Semiconductors with Steered Internal Electric Field for Urea Electrooxidation via Balancing Intermediates Adsorption.

Single-component electrocatalysts generally lead to unbalanced adsorption of OH- and urea during urea oxidation reaction (UOR), thus obtaining low activity and selectivity especially when oxygen evolution reaction (OER) competes at high potentials (>1.5 V). Herein, a cross-alignment strategy of in situ vertically growing Ni(OH)2 nanosheets on 2D semiconductor g-C3N4 is reported to form a hetero-structured electrocatalyst. Various spectroscopy measurements including in situ experiments indicate the existence of enhanced internal electric field at the interfaces of vertical Ni(OH)2 and g-C3N4 nanosheets, favorable for balancing adsorption of reaction intermediates. This heterojunction electrocatalyst shows high-selectivity UOR compared to pure Ni(OH)2, even at high potentials (>1.5 V) and large current density. The computational results show the vertical heterojunction could steer the internal electric field to increase the adsorption of urea, thus efficiently avoiding poisoning of strongly adsorbed OH- on active sites. A membrane electrode assembly (MEA)-based electrolyzer with the heterojunction anode could operate at an industrial-level current density of 200 mA cm-2. This work paves an avenue for designing high-performance electrocatalysts by vertical cross-alignments of active components.

Induction of therapeutic immunity and cancer eradication through biofunctionalized liposome-like nanovesicles derived from irradiated-cancer cells.

Immunotherapy has revolutionized the treatment of cancer. However, its efficacy remains to be optimized. There are at least two major challenges in effectively eradicating cancer cells by immunotherapy. Firstly, cancer cells evade immune cell killing by down-regulating cell surface immune sensors. Secondly, immune cell dysfunction impairs their ability to execute anti-cancer functions. Radiotherapy, one of the cornerstones of cancer treatment, has the potential to enhance the immunogenicity of cancer cells and trigger an anti-tumor immune response. Inspired by this, we fabricate biofunctionalized liposome-like nanovesicles (BLNs) by exposing irradiated-cancer cells to ethanol, of which ethanol serves as a surfactant, inducing cancer cells pyroptosis-like cell death and facilitating nanovesicles shedding from cancer cell membrane. These BLNs are meticulously designed to disrupt both of the aforementioned mechanisms. On one hand, BLNs up-regulate the expression of calreticulin, an "eat me" signal on the surface of cancer cells, thus promoting macrophage phagocytosis of cancer cells. Additionally, BLNs are able to reprogram M2-like macrophages into an anti-cancer M1-like phenotype. Using a mouse model of malignant pleural effusion (MPE), an advanced-stage and immunotherapy-resistant cancer model, we demonstrate that BLNs significantly increase T cell infiltration and exhibit an ablative effect against MPE. When combined with PD-1 inhibitor (α-PD-1), we achieve a remarkable 63.6% cure rate (7 out of 11) among mice with MPE, while also inducing immunological memory effects. This work therefore introduces a unique strategy for overcoming immunotherapy resistance.

Irradiated tumour cell-derived microparticles upregulate MHC-I expression in cancer cells via DNA double-strand break repair pathway.

Radiotherapy (RT) is used for over 50 % of cancer patients and can promote adaptive immunity against tumour antigens. However, the underlying mechanisms remain unclear. Here, we discovered that RT induces the release of irradiated tumour cell-derived microparticles (RT-MPs), which significantly upregulate MHC-I expression on the membranes of non-irradiated cells, enhancing the recognition and killing of these cells by T cells. Mechanistically, RT-MPs induce DNA double-strand breaks (DSB) in tumour cells, activating the ATM/ATR/CHK1-mediated DNA repair signalling pathway, and upregulating MHC-I expression. Inhibition of ATM/ATR/CHK1 reversed RT-MP-induced upregulation of MHC-I. Furthermore, phosphorylation of STAT1/3 following the activation of ATM/ATR/CHK1 is indispensable for the DSB-dependent upregulation of MHC-I. Therefore, our findings reveal the role of RT-MP-induced DSBs and the subsequent DNA repair signalling pathway in MHC-I expression and provide mechanistic insights into the regulation of MHC-I expression after DSBs.

Uridine-Modified Ruthenium(II) Complex as Lysosomal LIMP-2 Targeting Photodynamic Therapy Photosensitizer for the Treatment of Triple-Negative Breast Cancer.

Lysosome-targeted photodynamic therapy, which enhances reactive oxygen species (ROS)-responsive tumor cell death, has emerged as a promising strategy for cancer treatment. Herein, a uridine (dU)-modified Ru(II) complex (RdU) was synthesized by click chemistry. It was found that RdU exhibits impressive photo-induced inhibition against the growth of triple-negative breast cancer (TNBC) cells in normoxic and hypoxic microenvironments through ROS production. It was further revealed that RdU induces ferroptosis of MDA-MB-231 cells under light irradiation (650 nm, 300 mW/cm2). Additional experiments showed that RdU binds to lysosomal integral membrane protein 2 (LIMP-2), which was confirmed by the fact that RdU selectively localizes in the lysosomes of MDA-MB-231 cells and significantly augments the levels of LIMP-2. Molecular docking simulations and an isothermal titration calorimetry assay also showed that RdU has a high affinity to LIMP-2. Finally, in vivo studies in tumor-bearing (MDA-MB-231 cells) nude mice showed that RdU exerts promising photodynamic therapeutic effects on TNBC tumors. In summary, the uridine-modified Ru(II) complex has been developed as a potential LIMP-2 targeting agent for TNBC treatment through enhancing ROS production and promoting ferroptosis.