Multiple-scattering simulator-trained neural network for intensity diffraction tomography.

Recovering 3D phase features of complex biological samples traditionally sacrifices computational efficiency and processing time for physical model accuracy and reconstruction quality. Here, we overcome this challenge using an approximant-guided deep learning framework in a high-speed intensity diffraction tomography system. Applying a physics model simulator-based learning strategy trained entirely on natural image datasets, we show our network can robustly reconstruct complex 3D biological samples. To achieve highly efficient training and prediction, we implement a lightweight 2D network structure that utilizes a multi-channel input for encoding the axial information. We demonstrate this framework on experimental measurements of weakly scattering epithelial buccal cells and strongly scattering C. elegans worms. We benchmark the network's performance against a state-of-the-art multiple-scattering model-based iterative reconstruction algorithm. We highlight the network's robustness by reconstructing dynamic samples from a living worm video. We further emphasize the network's generalization capabilities by recovering algae samples imaged from different experimental setups. To assess the prediction quality, we develop a quantitative evaluation metric to show that our predictions are consistent with both multiple-scattering physics and experimental measurements.

Fourier ptychographic topography.

Topography measurement is essential for surface characterization, semiconductor metrology, and inspection applications. To date, performing high-throughput and accurate topography remains challenging due to the trade-off between field-of-view (FOV) and spatial resolution. Here we demonstrate a novel topography technique based on the reflection-mode Fourier ptychographic microscopy, termed Fourier ptychograhpic topography (FPT). We show that FPT provides both a wide FOV and high resolution, and achieves nanoscale height reconstruction accuracy. Our FPT prototype is based on a custom-built computational microscope consisting of programmable brightfield and darkfield LED arrays. The topography reconstruction is performed by a sequential Gauss-Newton-based Fourier ptychographic phase retrieval algorithm augmented with total variation regularization. We achieve a synthetic numerical aperture (NA) of 0.84 and a diffraction-limited resolution of 750 nm, increasing the native objective NA (0.28) by 3×, across a 1.2 × 1.2 mm2 FOV. We experimentally demonstrate the FPT on a variety of reflective samples with different patterned structures. The reconstructed resolution is validated on both amplitude and phase resolution test features. The accuracy of the reconstructed surface profile is benchmarked against high-resolution optical profilometry measurements. In addition, we show that the FPT provides robust surface profile reconstructions even on complex patterns with fine features that cannot be reliably measured by the standard optical profilometer. The spatial and temporal noise of our FPT system is characterized to be 0.529 nm and 0.027 nm, respectively.

Phase separation of YAP mediated by coiled-coil domain promotes hepatoblastoma proliferation via activation of transcription.

AIM AND BACKGROUND: Yes-associated protein (YAP), a key transcriptional co-activator associated with cell fate and tumor progression, has been reported to be a powerful driver of hepatoblastoma (HB). In this study, we investigated the mechanism underlying oncogenic role of YAP in HB. METHODS: The expression of YAP in HB tissues was measured through WB and qRT-PCR. The IHC and IF were performed to determine the distribution of YAP. The phase separation of YAP was proved by living cell imaging and FRAP experiment. The effect of YAP phase separation in HB cells in vitro an in vivo were tested using CCK8, flow cytometry, and xenograft tumors. RESULTS: YAP was overexpressed and activated in HB. Nuclear YAP formed an active transcriptional site via LLPS to recruit the crucial transcription factor TEAD4. Thus, YAP phase separation facilitated transcription of oncogenic genes and subsequently mediated chemoresistance of HB. Mechanistically, the phase separation ability of YAP depends on the coiled-coil domain, which is a typical phase separation domain. The electrostatic interactions and hydrophobic interactions within YAP are also vital to YAP phase separation. More importantly, YAP inhibitor verteporfin is potential treatment for HB and combination with cisplatin enhanced therapeutic efficacy. CONCLUSIONS: Highly expressed and active YAP exerts an oncogenic effect in HB via phase separation and provides new insights for the treatment of HB.

IL-27 improves adoptive CD8+ T cells' antitumor activity via enhancing cell survival and memory T cell differentiation.

IL-27 is an anti-inflammatory cytokine that triggers enhanced antitumor immunity, particularly cytotoxic T lymphocyte responses. In the present study, we sought to develop IL-27 into a therapeutic adjutant for adoptive T cell therapy using our well-established models. We have found that IL-27 directly improved the survival status and cytotoxicity of adoptive OT-1 CD8+ T cells in vitro and in vivo. Meanwhile, IL-27 treatment programs memory T cell differentiation in CD8+ T cells, characterized by upregulation of genes associated with T cell memory differentiation (T-bet, Eomes, Blimp1, and Ly6C). Additionally, we engineered the adoptive OT-1 CD8+ T cells to deliver IL-27. In mice, the established tumors treated with OT-1 CD8+ T-IL-27 were completely rejected, which demonstrated that IL-27 delivered via tumor antigen-specific T cells enhances adoptive T cells' cancer immunity. To our knowledge, this is the first application of CD8+ T cells as a vehicle to deliver IL-27 to treat tumors. Thus, this study demonstrates IL-27 is a feasible approach for enhancing CD8+ T cells' antitumor immunity and can be used as a therapeutic adjutant for T cell adoptive transfer to treat cancer.

High-fidelity intensity diffraction tomography with a non-paraxial multiple-scattering model.

We propose a novel intensity diffraction tomography (IDT) reconstruction algorithm based on the split-step non-paraxial (SSNP) model for recovering the 3D refractive index (RI) distribution of multiple-scattering biological samples. High-quality IDT reconstruction requires high-angle illumination to encode both low- and high- spatial frequency information of the 3D biological sample. We show that our SSNP model can more accurately compute multiple scattering from high-angle illumination compared to paraxial approximation-based multiple-scattering models. We apply this SSNP model to both sequential and multiplexed IDT techniques. We develop a unified reconstruction algorithm for both IDT modalities that is highly computationally efficient and is implemented by a modular automatic differentiation framework. We demonstrate the capability of our reconstruction algorithm on both weakly scattering buccal epithelial cells and strongly scattering live C. elegans worms and live C. elegans embryos.

Large-scale holographic particle 3D imaging with the beam propagation model.

We develop a novel algorithm for large-scale holographic reconstruction of 3D particle fields. Our method is based on a multiple-scattering beam propagation method (BPM) combined with sparse regularization that enables recovering dense 3D particles of high refractive index contrast from a single hologram. We show that the BPM-computed hologram generates intensity statistics closely matching with the experimental measurements and provides up to 9× higher accuracy than the single-scattering model. To solve the inverse problem, we devise a computationally efficient algorithm, which reduces the computation time by two orders of magnitude as compared to the state-of-the-art multiple-scattering based technique. We demonstrate the superior reconstruction accuracy in both simulations and experiments under different scattering strengths. We show that the BPM reconstruction significantly outperforms the single-scattering method in particular for deep imaging depths and high particle densities.

Ginsenoside Rg1 impairs homologous recombination repair by targeting CtBP-interacting protein and sensitizes hepatoblastoma cells to DNA damage.

The ginsenoside Rg1, the primary pharmacologically active ingredient of the traditional Chinese herb ginseng, is widely used in the clinical treatment of diseases of the immune and nervous systems. Recent studies have shown that it also has an antitumor effect. In this study, we explored the effects of Rg1 on hepatoblastoma (HB) and its underlying mechanisms. We demonstrated that Rg1 significantly inhibited HB cell growth both in vivo and in vitro. Mechanistic studies revealed that Rg1 impaired homologous recombination and triggered double-strand breaks in HB cells by directly targeting CtBP-interacting protein (CtIP), a key double-strand break repair factor, which is highly expressed in HB tissues. Moreover, we also demonstrated that Rg1 sensitized HB cells to DNA-damaging agents both in vitro and in vivo. In conclusion, our data not only demonstrate the potential clinical application of Rg1 as a novel chemotherapeutic candidate but also offer a mechanism-based therapeutic option by which DNA-damaging agents can be used in combination with Rg1 to target HB.

Sirtuin 6 plays an oncogenic role and induces cell autophagy in esophageal cancer cells.

Sirtuin 6, a member of sirtuin family, is generally regarded as a tumor suppressor as it participates in suppressing hypoxia-inducible factor 1α and MYC transcription activity by deacetylating H3K9 (histone H3 lysine 9) and H3K56 (histone H3 lysine) at promoters of target genes, leading to the aerobic glycolysis inhibition and cell growth suppression. However, its expression has recently been reported to be highly elevated in a series of tumors, including prostate cancer, breast cancer, and non-small cell lung cancer, indicating that sirtuin 6 plays dual roles in tumorigenicity in a cell/tumor type-specific manner. To our knowledge, the biological roles of sirtuin 6 in esophageal cancer cells have still been underestimated. In the study, data from quantitative reverse transcriptase polymerase chain reaction-based assays and immunohistochemical assays revealed that sirtuin 6 was remarkably overexpressed in esophageal squamous tumor tissues. Moreover, its upregulation was closely related with clinical features, such as gender, pathology, tumor-node-metastasis, and cell differentiation. Subsequently, the biological tests showed that it promoted cell proliferation and induced the expression of Bcl2, a key anti-apoptotic factor, in esophageal carcinoma cells. Moreover, using the ratio of LC3II/I, a widely recognized autophagy biomarker, we showed that it apparently induced cell autophagy, which was further confirmed by the autophagy flux assays. In addition, results from western blotting assays and immunoprecipitation assays displayed that sirtuin 6 specifically interacted with ULK1 and positively regulated its activity by inhibiting its upstream factor mammalian target of rapamycin activity. In summary, our studies shed insights into the crucial functions of sirtuin 6 in esophageal carcinoma cells and provide evidence supporting sirtuin 6-based personalized therapies in esophageal carcinoma cell patients.

Causal Relationship between Sex Hormone-Binding Globulin and Risk of Neuroblastoma: A Bidirectional Two-Sample Mendelian Randomization Study.

BACKGROUND: The causal relationship between sex hormone-binding globulin (SHBG) and neuroblastoma remains unknown. This study aimed to explore the causality between SHBG and the risk of neuroblastoma using bidirectional two-sample Mendelian randomization (MR) study. METHODS: Instrumental variables associated with SHBG were obtained from the genome-wide association study (GWAS) of European containing 214,989 females and 185,221 males from the UK Biobank. Summary-level data for neuroblastoma were derived from the IEU OpenGWAS project with 1,627 patients and 3,254 controls. The inverse-variance weighted (IVW) method served as the primary analytic tool. RESULTS: The IVW method revealed a significant positive causal relationship between male SHBG and the risk of neuroblastoma [OR, 2.169; 95% confidence interval (CI), 1.207-3.897; P = 0.010]. Conversely, female SHBG showed no significant causal link with neuroblastoma (IVW OR, 1.004; 95% CI, 0.542-1.860; P = 0.990). No significant reverse causality was detected. Sensitivity analyses validated these findings. CONCLUSIONS: Elevated SHBG levels in males, but not in females, can causally increase the risk of neuroblastoma. This gender-specific effect indicates a potential differential role of SHBG in the etiology of neuroblastoma. Further research is needed to elucidate the underlying mechanisms of this gender disparity. Monitoring SHBG levels, especially in males, could be pivotal in neuroblastoma risk assessment and management. IMPACT: This study highlights a novel gender-specific aspect in the risk of neuroblastoma, emphasizing the potential role of male SHBG levels in neuroblastoma incidence, and sets the stage for targeted preventative strategies and further investigation into gender-based biological mechanisms.

Mid-infrared Chemical Imaging of Intracellular Tau Fibrils using Fluorescence-guided Computational Photothermal Microscopy.

Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases. However, it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment. To address this challenge, we developed a computational chemical microscope integrating 3D mid-infrared photothermal imaging with fluorescence imaging, termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography (FBS-IDT). Based on a low-cost and simple optical design, FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific mid-IR fingerprint spectroscopic analysis of tau fibrils, an important type of amyloid protein aggregates, in their intracellular environment. Label-free volumetric chemical imaging of human cells with/without seeded tau fibrils is demonstrated to show the potential correlation between lipid accumulation and tau aggregate formation. Depth-resolved mid-infrared fingerprint spectroscopy is performed to reveal the protein secondary structure of the intracellular tau fibrils. 3D visualization of the \b{eta}-sheet for tau fibril structure is achieved.

Mid-infrared chemical imaging of intracellular tau fibrils using fluorescence-guided computational photothermal microscopy.

Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases. However, it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment. To address this challenge, we developed a computational chemical microscope integrating 3D mid-infrared photothermal imaging with fluorescence imaging, termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography (FBS-IDT). Based on a low-cost and simple optical design, FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific mid-IR fingerprint spectroscopic analysis of tau fibrils, an important type of amyloid protein aggregates, in their intracellular environment. Label-free volumetric chemical imaging of human cells with/without seeded tau fibrils is demonstrated to show the potential correlation between lipid accumulation and tau aggregate formation. Depth-resolved mid-infrared fingerprint spectroscopy is performed to reveal the protein secondary structure of the intracellular tau fibrils. 3D visualization of the ß-sheet for tau fibril structure is achieved.

Anti-ferroptotic PRKAA2 serves as a potential diagnostic and prognostic marker for hepatoblastoma.

Background: The incidence rate of hepatoblastoma (HB), which is the most prevalent malignant tumour among children, rises each year. According to recent studies, a number of neoplastic disorders and ferroptosis are intimately connected. This study aims to identify key ferroptosis-related genes in HB and explore new directions for the diagnosis and treatment of HB. Methods: Differentially expressed ferroptosis-related genes were identified using the Gene Expression Omnibus datasets. The functional annotation of candidate genes was evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Machine learning and receiver operating characteristic (ROC) curves revealed protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2), tribbles homolog 2 (TRIB2), and liver-type glutaminase (GLS2) as potential diagnostic genes of HB. By using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry, relative expression of PRKAA2 was examined. The effect of PRKAA2 on proliferation, apoptosis, and ferroptosis of HB cells was verified in vitro and in vivo. Fisher's exact test was used to evaluate the clinical significance of PRKAA2 in HB. Results: The prognostic indicators had a substantial correlation with PRKAA2 expression, which rose dramatically in HB tissues. PRKAA2 promotes proliferation and inhibits ferroptosis in HB cells. PRKAA2 plays a role in ferroptosis by regulating hypoxia-inducible factor 1α (HIF-1α) and transferrin receptor 1 (TFR1). Conclusions: PRKAA2 functions as a tumor-promoting factor in HB by promoting cell proliferation and prohibiting ferroptosis. Ferroptosis-related genes PRKAA2 is a potential diagnostic and prognostic marker for HB as well as a novel therapeutic target in the future.

Alternative Splicing of lncRNAs From SNHG Family Alters snoRNA Expression and Induces Chemoresistance in Hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is a common pediatric malignant liver tumor that is characterized by a low level of genetic mutations. Alternative splicing (AS) has been shown to be closely associated with cancer progression, especially in tumors with a low mutational burden. However, the role of AS in HB remains unknown. METHODS: Transcriptome sequencing was performed on 5 pairs of HB tissues and matched non-tumor tissues to delineate the AS landscape in HB. AS events were validated in 92 samples from 46 patients. RNA pull-down and RNA immunoprecipitation assays were carried out to identify splicing factors that regulate the AS of small nucleolar RNA host genes (SNHG). Patient-derived organoids (PDOs) were established to investigate the role of the splicing factor polyadenylate-binding nuclear protein 1 (PABPN1). RESULTS: This study uncovered aberrant alternative splicing in HB, including lncRNAs from SNHG family that undergo intron retention in HB. Further investigations revealed that PABPN1, a significantly upregulated RNA binding protein, interacts with splicing machinery in HB, inducing the intron retention of these SNHG RNAs and the downregulation of intronic small nucleolar RNAs (snoRNAs). Functionally, PABPN1 acts as an oncofetal splicing regulator in HB by promoting cell proliferation and DNA damage repair via inducing the intron retention of SNHG19. Knock-down of PABPN1 increases the cisplatin sensitivity of HB PDOs. CONCLUSIONS: Our findings revealed the role of intron retention in regulating snoRNA expression in hepatoblastoma, explained detailed regulatory mechanism between PABPN1 and the intron retention of SNHG RNAs, and provided insight into the development of new HB treatment options.

SNORA14A inhibits hepatoblastoma cell proliferation by regulating SDHB-mediated succinate metabolism.

Hepatoblastoma (HB) is the most common paediatric liver malignancy. Dysregulation of small nucleolar RNAs (snoRNAs) is a critical inducer of tumour initiation and progression. However, the association between snoRNAs and HB remains unknown. Here, we conducted snoRNA expression profiling in HB by snoRNA sequencing and identified a decreased level of SNORA14A, a box H/ACA snoRNA, in HB tissues. Low expression of SNORA14A was correlated with PRETEXT stage and metastasis in patients. Functionally, overexpression of SNORA14A suppressed HB cell proliferation and triggered cell apoptosis and G2/M phase arrest. Mechanistically, SNORA14A overexpression promoted the processing and maturation of the 18 S ribosomal RNA (rRNA) precursor to increase succinate dehydrogenase subunit B (SDHB) protein levels. In accordance with SNORA14A downregulation, SDHB protein expression was significantly reduced in HB tissues and cells, accompanied by abnormal accumulation of succinate. Overexpression of SDHB showed antiproliferative and proapoptotic effects and the capacity to induce G2/M phase arrest, while succinate dose-dependently stimulated HB cell growth. Furthermore, the inhibition of SNORA14A in HB malignant phenotypes was mediated by SDHB upregulation-induced reduction of cellular succinate levels. Therefore, the SNORA14A/18 S rRNA/SDHB axis suppresses HB progression by preventing cellular accumulation of the oncometabolite succinate and provides promising prognostic biomarkers and novel therapeutic targets for HB.

O-GlcNAcylated LARP1 positively regulated by circCLNS1A facilitates hepatoblastoma progression through DKK4/ß-catenin signalling.

BACKGROUND: Accumulating studies have shown that La-related protein 1 (LARP1) is involved in the occurrence and development of various tumours. However, the expression pattern and biological role of LARP1 in hepatoblastoma (HB) remain unclear so far. METHODS: LARP1 expression level in HB and adjacent normal liver tissues was analysed by qRT-PCR, Western blotting and immunohistochemistry assays. The prognostic significance of LARP1 was evaluated by Kaplan-Meier method and multivariate Cox regression analysis. In vitro and in vivo functional assays were implemented to clarify the biological effects of LARP1 on HB cells. Mechanistically, the regulatory roles of O-GlcNAcylation and circCLNS1A in LARP1 expression were investigated by co-immunoprecipitation (co-IP), immunofluorescence, RNA immunoprecipitation (RIP), RNA pull-down and protein stability assays. Moreover, RNA-sequencing, co-IP, RIP, mRNA stability and poly(A)-tail length assays were performed to investigate the association between LARP1 and DKK4. The expression and diagnostic significance of plasma DKK4 protein in multi-centre cohorts were evaluated by ELISA and ROC curves. RESULTS: LARP1 mRNA and protein levels were remarkably elevated in HB tissues and associated with worse prognosis of HB patients. LARP1 knockdown abolished cell proliferation, triggered cell apoptosis in vitro as well as prohibited tumour growth in vivo, whereas LARP1 overexpression incited HB progression. Mechanistically, O-GlcNAcylation of LARP1 Ser672 by O-GlcNAc transferase strengthened its binding to circCLNS1A and then protected LARP1 from TRIM-25-mediated ubiquitination and proteolysis. LARP1 upregulation subsequently led to DKK4 mRNA stabilisation by competitively interacting with PABPC1 to prevent DKK4 mRNA from B-cell translocation gene 2-dependent deadenylation and degradation, thus facilitating ß-catenin protein expression and nuclear import. CONCLUSION: This study indicates that upregulated protein level of O-GlcNAcylated LARP1 mediated by circCLNS1A promotes the tumorigenesis and progression of HB through LARP1/DKK4/ß-catenin axis. Hence, LARP1 and DKK4 are promising therapeutical target and diagnostic/prognostic plasma biomarker for HB.

SNORA56-mediated pseudouridylation of 28 S rRNA inhibits ferroptosis and promotes colorectal cancer proliferation by enhancing GCLC translation.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis. METHODS: The dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe2+/Fe3+, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis. RESULTS: Here, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation. CONCLUSIONS: Therefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC.

SNORD11B-mediated 2'-O-methylation of primary let-7a in colorectal carcinogenesis.

Evidence indicates that small nucleolar RNAs (snoRNAs) participate in tumorigenesis and development and could be promising biomarkers for colorectal cancer (CRC). Here, we examine the profile of snoRNAs in CRC and find that expression of SNORD11B is increased in CRC tumor tissues and cell lines, with a significant positive correlation between SNORD11B expression and that of its host gene NOP58. SNORD11B promotes CRC cell proliferation and invasion and inhibits apoptosis. Mechanistically, SNORD11B promotes the processing and maturation of 18 S ribosomal RNA (rRNA) by mediating 2'-O-methylated (Nm) modification on the G509 site of 18 S rRNA. Intriguingly, SNORD11B mediates Nm modification on the G225 site of MIRLET7A1HG (pri-let-7a) with a canonical motif, resulting in degradation of pri-let-7a, inhibition of DGCR8 binding, reduction in mature tumor suppressor gene let-7a-5p expression, and upregulation of downstream oncogene translation. SNORD11B performs comparably to CEA and CA199 in diagnosing CRC. High expression of SNORD11B is significantly correlated with a more advanced TNM stage and lymph node metastasis, which indicates poor prognosis.

Bond-selective intensity diffraction tomography.

Recovering molecular information remains a grand challenge in the widely used holographic and computational imaging technologies. To address this challenge, we developed a computational mid-infrared photothermal microscope, termed Bond-selective Intensity Diffraction Tomography (BS-IDT). Based on a low-cost brightfield microscope with an add-on pulsed light source, BS-IDT recovers both infrared spectra and bond-selective 3D refractive index maps from intensity-only measurements. High-fidelity infrared fingerprint spectra extraction is validated. Volumetric chemical imaging of biological cells is demonstrated at a speed of ~20 s per volume, with a lateral and axial resolution of ~350 nm and ~1.1 µm, respectively. BS-IDT's application potential is investigated by chemically quantifying lipids stored in cancer cells and volumetric chemical imaging on Caenorhabditis elegans with a large field of view (~100 µm x 100 µm).

O-GlcNAcylation enhances sensitivity to RSL3-induced ferroptosis via the YAP/TFRC pathway in liver cancer.

Ferroptosis is a form of regulated cell death characterized by iron-dependent accumulation of lipid hydroperoxides to lethal levels. YAP has been reported to play a pivotal role in controlling ferroptotic death, and the expression of YAP is enhanced and stabilized by O-GlcNAcylation. However, whether O-GlcNAcylation can increase the sensitivity of hepatocellular carcinoma (HCC) cells to ferroptosis remains unknown. In the present study, we found that O-GlcNAcylation increased the sensitivity of HCC cells to ferroptosis via YAP. Moreover, YAP increased the iron concentration in HCC cells through transcriptional elevation of TFRC via its O-GlcNAcylation. With YAP knockdown or YAP-T241 mutation, the increased sensitivity to ferroptosis induced by O-GlcNAcylation was abolished. In addition, the xenograft assay confirmed that O-GlcNAcylation increased ferroptosis sensitivity via TFRC in vivo. In summary, we are the first to find that O-GlcNAcylation can increase ferroptosis sensitivity in HCC cells via YAP/TFRC. Our work will provide a new basis for clinical therapeutic strategies for HCC patients.

Anatomical Modeling of Brain Vasculature in Two-Photon Microscopy by Generalizable Deep Learning.

Objective and Impact Statement. Segmentation of blood vessels from two-photon microscopy (2PM) angiograms of brains has important applications in hemodynamic analysis and disease diagnosis. Here, we develop a generalizable deep learning technique for accurate 2PM vascular segmentation of sizable regions in mouse brains acquired from multiple 2PM setups. The technique is computationally efficient, thus ideal for large-scale neurovascular analysis. Introduction. Vascular segmentation from 2PM angiograms is an important first step in hemodynamic modeling of brain vasculature. Existing segmentation methods based on deep learning either lack the ability to generalize to data from different imaging systems or are computationally infeasible for large-scale angiograms. In this work, we overcome both these limitations by a method that is generalizable to various imaging systems and is able to segment large-scale angiograms. Methods. We employ a computationally efficient deep learning framework with a loss function that incorporates a balanced binary-cross-entropy loss and total variation regularization on the network's output. Its effectiveness is demonstrated on experimentally acquired in vivo angiograms from mouse brains of dimensions up to 808×808×702 µm. Results. To demonstrate the superior generalizability of our framework, we train on data from only one 2PM microscope and demonstrate high-quality segmentation on data from a different microscope without any network tuning. Overall, our method demonstrates 10× faster computation in terms of voxels-segmented-per-second and 3× larger depth compared to the state-of-the-art. Conclusion. Our work provides a generalizable and computationally efficient anatomical modeling framework for brain vasculature, which consists of deep learning-based vascular segmentation followed by graphing. It paves the way for future modeling and analysis of hemodynamic response at much greater scales that were inaccessible before.