Controlling organoid symmetry breaking uncovers an excitable system underlying human axial elongation.

The human embryo breaks symmetry to form the anterior-posterior axis of the body. As the embryo elongates along this axis, progenitors in the tail bud give rise to tissues that generate spinal cord, skeleton, and musculature. This raises the question of how the embryo achieves axial elongation and patterning. While ethics necessitate in vitro studies, the variability of organoid systems has hindered mechanistic insights. Here, we developed a bioengineering and machine learning framework that optimizes organoid symmetry breaking by tuning their spatial coupling. This framework enabled reproducible generation of axially elongating organoids, each possessing a tail bud and neural tube. We discovered that an excitable system composed of WNT/FGF signaling drives elongation by inducing a neuromesodermal progenitor-like signaling center. We discovered that instabilities in the excitable system are suppressed by secreted WNT inhibitors. Absence of these inhibitors led to ectopic tail buds and branches. Our results identify mechanisms governing stable human axial elongation.

Multimodal charting of molecular and functional cell states via in situ electro-sequencing.

Paired mapping of single-cell gene expression and electrophysiology is essential to understand gene-to-function relationships in electrogenic tissues. Here, we developed in situ electro-sequencing (electro-seq) that combines flexible bioelectronics with in situ RNA sequencing to stably map millisecond-timescale electrical activity and profile single-cell gene expression from the same cells across intact biological networks, including cardiac and neural patches. When applied to human-induced pluripotent stem-cell-derived cardiomyocyte patches, in situ electro-seq enabled multimodal in situ analysis of cardiomyocyte electrophysiology and gene expression at the cellular level, jointly defining cell states and developmental trajectories. Using machine-learning-based cross-modal analysis, in situ electro-seq identified gene-to-electrophysiology relationships throughout cardiomyocyte development and accurately reconstructed the evolution of gene expression profiles based on long-term stable electrical measurements. In situ electro-seq could be applicable to create spatiotemporal multimodal maps in electrogenic tissues, potentiating the discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Spatiotemporally resolved transcriptomics reveals the subcellular RNA kinetic landscape.

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Inhibition of human peptide deformylase by actinonin sensitizes glioblastoma cells to temozolomide chemotherapy.

Glioblastoma multiforme (GBM) is a common intracranial primary tumor of the central nervous system with high malignancy, poor prognosis, and short survival. Studies have shown that mitochondrial energy metabolism plays an important role in GBM chemotherapy resistance, suggesting that interrupting mitochondrial oxidative phosphorylation (OXPHOS) may improve GBM treatment. Human peptide deformylase (HsPDF) is a mitochondrial deformylase that removes the formylated methionine from the N-terminus of proteins encoded by mitochondrial DNA (mtDNA), thereby contributing to correct protein folding and participating in the assembly of the electron respiratory chain complex. In this study, we found that the expression of mtDNA-encoded proteins was significantly downregulated after treatment of GBM cells U87MG and LN229 with the HsPDF inhibitor, actinonin. In combination with temozolomide, a preferred chemotherapeutic medicine for GBM, the OXPHOS level decreased, mitochondrial protein homeostasis was unbalanced, mitochondrial fission increased, and the integrated stress response was activated to promote mitochondrial apoptosis. These findings suggest that HsPDF inhibition is an important strategy for overcoming chemoresistance of GBM cells.

Interfering with mitochondrial dynamics sensitizes glioblastoma multiforme to temozolomide chemotherapy.

Glioblastoma multiforme (GBM) is a primary tumour of the central nervous system (CNS) that exhibits the highest degree of malignancy. Radiotherapy and chemotherapy are essential to prolong the survival time of patients. However, clinical work has demonstrated that sensitivity of GBM to chemotherapy decreases with time. The phenomenon of multi-drug resistance (MDR) reminds us that there may exist some fundamental mechanisms in the process of chemo-resistance. We tried to explore the mechanism of GBM chemo-resistance from the perspective of energy metabolism. First, we found that the oxidative phosphorylation (OXPHOS) level of SHG44 and U87 cells increased under TMZ treatment. In further studies, it was found that the expression of PINK1 and mitophagy flux downstream was downregulated in GBM cells, which were secondary to the upregulation of TP53 in tumour cells under TMZ treatment. At the same time, we examined the mitochondrial morphology in tumour cells and found that the size of mitochondria in tumour cells increased under the treatment of TMZ, which originated from the regulation of AMPK on the subcellular localization of Drp1 under the condition of unbalanced energy supply and demand in tumour cells. The accumulation of mitochondrial mass and the optimization of mitochondrial quality accounted for the increased oxidative phosphorylation, and interruption of the mitochondrial fusion process downregulated the efficiency of oxidative phosphorylation and sensitized GBM cells to TMZ, which was also confirmed in the in vivo experiment. What is more, interfering with this process is an innovative strategy to overcome the chemo-resistance of GBM cells.

Adaptive Changes Allow Targeting of Ferroptosis for Glioma Treatment.

Ferroptosis is a type of regulated cell death that plays an essential role in various brain diseases, including cranial trauma, neuronal diseases, and brain tumors. It has been reported that cancer cells rely on their robust antioxidant capacity to escape ferroptosis. Therefore, ferroptosis exploitation could be an effective strategy to prevent tumor proliferation and invasion. Glioma is a common malignant craniocerebral tumor exhibiting complicated drug resistance and survival mechanisms, resulting in a high mortality rate and short survival time. Recent studies have determined that metabolic alterations in glioma offer exploitable therapeutic targets. These metabolic alterations allow targeted therapy to achieve some initial efficacy but have failed to inhibit glioma growth, invasion, and drug resistance effectively. It has been proposed that the reason for the high malignancy and drug resistance observed with glioma is that these tumors can effectively evade ferroptosis. Ferroptosis-inducing drugs were found to exert a positive effect by targeting this particular characteristic of glioma cells. Moreover, gliomas develop enhanced drug resistance through anti-ferroptosis mechanisms. In this study, we provided an overview of the mechanisms by which glioma aggressiveness and drug resistance are mediated by the evasion of ferroptosis. This information might provide new targets for glioma therapy as well as new insights and ideas for future research.

Coniferyl aldehyde alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via JAK2-STAT1 pathway in acute pneumonia.

Pneumonia is a kind of inflammatory disease characterized by pathogen infection of lower respiratory track. Lipopolysaccharide (LPS) is the main bioactive component of Gram-negative bacteria responsible for inflammatory response. Recently, coniferyl aldehyde (CA) has been reported to play a crucial role because of its anti-inflammatory activity. However, the effect and mechanisms of CA in ameliorating symptoms of acute pneumonia remain unknown. Evaluating and identifying the value and exploring the mechanisms of CA on LPS-mediated WI-38 apoptosis and inflammation were the aims of this study. Here, CCK-8 cell viability assay was applied on WI-38 after treatment with or without LPS at different doses of CA to verify that CA can increase LPS-induced cell viability. Then, quantitative polymerase chain reaction (qPCR) and enzyme-linked-immunosorbent serologic assays (ELISA) suggested that LPS treatment dramatically decreased the expression level of IL-10 (anti-inflammatory factor) while strikingly increasing the expression levels of IL-1ß, IL-6, and TNF-α (tumor necrosis factor-α; proinflammatory factor) whereas CA treatment attenuates LPS-induced inflammation of WI-38. Further, flow cytometry and Western blot assay verified that LPS treatment dramatically promoted apoptosis of WI-38 cells, while administration of CA notably inhibited apoptosis of WI-38 cells. Moreover, the Western blot assay hinted that CA could inactivate LPS-induced JAK2-STAT1 signaling pathway. These findings indicated that CA could alleviate LPS-mediated WI-38 apoptosis and inflammation injury through JAK2-STAT1 pathway in acute pneumonia.

ACTN4 and the pathways associated with cell motility and adhesion contribute to the process of lung cancer metastasis to the brain.

BACKGROUND: The aim of this study was to identify critical gene pathways that are associated with lung cancer metastasis to the brain. METHODS: The RNA-Seq approach was used to establish the expression profiles of a primary lung cancer, adjacent benign tissue, and metastatic brain tumor from a single patient. The expression profiles of these three types of tissues were compared to define differentially expressed genes, followed by serial-cluster analysis, gene ontology analysis, pathway analysis, and knowledge-driven network analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the expression of essential candidate genes in tissues from ten additional patients. RESULTS: Differential gene expression among these three types of tissues was classified into multiple clusters according to the patterns of their alterations. Further bioinformatic analysis of these expression profile data showed that the network of the signal transduction pathways related to actin cytoskeleton reorganization, cell migration, and adhesion was associated with lung cancer metastasis to the brain. The expression of ACTN4 (actinin, alpha 4), a cytoskeleton protein gene essential for cytoskeleton organization and cell motility, was significantly elevated in the metastatic brain tumor but not in the primary lung cancer tissue. CONCLUSIONS: The signaling pathways involved in the regulation of cytoskeleton reorganization, cell motility, and focal adhesion play a role in the process of lung cancer metastasis to the brain. The contribution of ACTN4 to the process of lung cancer metastasis to the brain could be mainly through regulation of actin cytoskeleton reorganization, cell motility, and focal adhesion.

Tuning transition metals layered-electroplated on bimetallic MxCu1-x crystallites (M = Fe, Co, Ni, and Zn) to boost ammonia yield in electrocatalytic reduction of nitrate wastewaters.

Nitrate-containing wastewaters have been recognized as an important source for recovering valuable ammonia. This work targets integrating a series of transition metals (M = Fe, Co, Ni, and Zn) onto Cu crystallites through a layered-plating method. The strategy to promote the nitrate reduction reaction (NO3-RR) involves tuning M surfaces in specific ratios for the hydrogenation of nitrogenous species on MxCu1-x electrodes. Electrochemical analysis and operando Raman spectra identified that a solid-state Cu2O-to-Cu0 transition acted as the primary mediator, while its high corrosion resistance protected the M metals or metal oxides from inactivation in nitrate-to-ammonia pathways. Among bimetals, FeCu was the best combination, with the order of performance in constant potential electrolysis, Fe0.36Cu0.64 > Ni0.73Cu0.27 > Co0.34Cu0.66 > Zn0.64Cu0.36. The collaboration of Cu and M in deoxygenating nitrate and subsequently hydrogenating NOx at respective overpotentials is key to enhancing ammonia yield. Nitrate removal (96 %), NH3 selectivity (93 %), and Faradaic efficiency (92 %) were optimized on Fe0.36Cu0.64 electrode at -0.6 V (vs. RHE). A steady yield as high as 14,080 µg h-1 mg-1 was achieved at 30 mA cm-2 using a real water sample (NO3- ∼ 500 mg-N L-1, pH 4) as the input stream, continuously operated for 96 h.

Triggering of endoplasmic reticulum stress via ATF4-SPHK1 signaling promotes glioblastoma invasion and chemoresistance.

Despite advances in therapies, glioblastoma (GBM) recurrence is almost inevitable due to the aggressive growth behavior of GBM cells and drug resistance. Temozolomide (TMZ) is the preferred drug for GBM chemotherapy, however, development of TMZ resistance is over 50% cases in GBM patients. To investigate the mechanism of TMZ resistance and invasive characteristics of GBM, analysis of combined RNA-seq and ChIP-seq was performed in GBM cells in response to TMZ treatment. We found that the PERK/eIF2α/ATF4 signaling was significantly upregulated in the GBM cells with TMZ treatment, while blockage of ATF4 effectively inhibited cell migration and invasion. SPHK1 expression was transcriptionally upregulated by ATF4 in GBM cells in response to TMZ treatment. Blockage of ATF4-SPHK1 signaling attenuated the cellular and molecular events in terms of invasive characteristics and TMZ resistance. In conclusion, GBM cells acquired chemoresistance in response to TMZ treatment via constant ER stress. ATF4 transcriptionally upregulated SPHK1 expression to promote GBM cell aggression and TMZ resistance. The ATF4-SPHK1 signaling in the regulation of the transcription factors of EMT-related genes could be the underlying mechanism contributing to the invasion ability of GBM cells and TMZ resistance. ATF4-SPHK1-targeted therapy could be a potential strategy against TMZ resistance in GBM patients.

Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.

Characterizing the transcriptional and translational gene expression patterns at the single-cell level within their three-dimensional (3D) tissue context is essential for revealing how genes shape tissue structure and function in health and disease. However, most existing spatial profiling techniques are limited to 5-20 µm thin tissue sections. Here, we developed Deep-STARmap and Deep-RIBOmap, which enable 3D in situ quantification of thousands of gene transcripts and their corresponding translation activities, respectively, within 200-µm thick tissue blocks. This is achieved through scalable probe synthesis, hydrogel embedding with efficient probe anchoring, and robust cDNA crosslinking. We first utilized Deep-STARmap in combination with multicolor fluorescent protein imaging for simultaneous molecular cell typing and 3D neuron morphology tracing in the mouse brain. We also demonstrate that 3D spatial profiling facilitates comprehensive and quantitative analysis of tumor-immune interactions in human skin cancer.

Brain implantation of tissue-level-soft bioelectronics via embryonic development.

The design of bioelectronics capable of stably tracking brain-wide, single-cell, and millisecond-resolved neural activities in the developing brain is critical to the study of neuroscience and neurodevelopmental disorders. During development, the three-dimensional (3D) structure of the vertebrate brain arises from a 2D neural plate 1,2 . These large morphological changes previously posed a challenge for implantable bioelectronics to track neural activity throughout brain development 3-9 . Here, we present a tissue-level-soft, sub-micrometer-thick, stretchable mesh microelectrode array capable of integrating into the embryonic neural plate of vertebrates by leveraging the 2D-to-3D reconfiguration process of the tissue itself. Driven by the expansion and folding processes of organogenesis, the stretchable mesh electrode array deforms, stretches, and distributes throughout the entire brain, fully integrating into the 3D tissue structure. Immunostaining, gene expression analysis, and behavioral testing show no discernible impact on brain development or function. The embedded electrode array enables long-term, stable, brain-wide, single-unit-single-spike-resolved electrical mapping throughout brain development, illustrating how neural electrical activities and population dynamics emerge and evolve during brain development.

Explainable multi-task learning for multi-modality biological data analysis.

Current biotechnologies can simultaneously measure multiple high-dimensional modalities (e.g., RNA, DNA accessibility, and protein) from the same cells. A combination of different analytical tasks (e.g., multi-modal integration and cross-modal analysis) is required to comprehensively understand such data, inferring how gene regulation drives biological diversity and functions. However, current analytical methods are designed to perform a single task, only providing a partial picture of the multi-modal data. Here, we present UnitedNet, an explainable multi-task deep neural network capable of integrating different tasks to analyze single-cell multi-modality data. Applied to various multi-modality datasets (e.g., Patch-seq, multiome ATAC + gene expression, and spatial transcriptomics), UnitedNet demonstrates similar or better accuracy in multi-modal integration and cross-modal prediction compared with state-of-the-art methods. Moreover, by dissecting the trained UnitedNet with the explainable machine learning algorithm, we can directly quantify the relationship between gene expression and other modalities with cell-type specificity. UnitedNet is a comprehensive end-to-end framework that could be broadly applicable to single-cell multi-modality biology. This framework has the potential to facilitate the discovery of cell-type-specific regulation kinetics across transcriptomics and other modalities.

Soft bioelectronics for cardiac interfaces.

Bioelectronics for interrogation and intervention of cardiac systems is important for the study of cardiac health and disease. Interfacing cardiac systems by using conventional rigid bioelectronics is limited by the structural and mechanical disparities between rigid electronics and soft tissues as well as their limited performance. Recently, advances in soft electronics have led to the development of high-performance soft bioelectronics, which is flexible and stretchable, capable of interfacing with cardiac systems in ways not possible with conventional rigid bioelectronics. In this review, we first review the latest developments in building flexible and stretchable bioelectronics for the epicardial interface with the heart. Next, we introduce how stretchable bioelectronics can be integrated with cardiac catheters for a minimally invasive in vivo heart interface. Then, we highlight the recent progress in the design of soft bioelectronics as a new class of biomaterials for integration with different in vitro cardiac models. In particular, we highlight how these devices unlock opportunities to interrogate the cardiac activities in the cardiac patch and cardiac organoid models. Finally, we discuss future directions and opportunities using soft bioelectronics for the study of cardiac systems.

The MAMs Structure and Its Role in Cell Death.

The maintenance of cellular homeostasis involves the participation of multiple organelles. These organelles are associated in space and time, and either cooperate or antagonize each other with regards to cell function. Crosstalk between organelles has become a significant topic in research over recent decades. We believe that signal transduction between organelles, especially the endoplasmic reticulum (ER) and mitochondria, is a factor that can influence the cell fate. As the cellular center for protein folding and modification, the endoplasmic reticulum can influence a range of physiological processes by regulating the quantity and quality of proteins. Mitochondria, as the cellular "energy factory," are also involved in cell death processes. Some researchers regard the ER as the sensor of cellular stress and the mitochondria as an important actuator of the stress response. The scientific community now believe that bidirectional communication between the ER and the mitochondria can influence cell death. Recent studies revealed that the death signals can shuttle between the two organelles. Mitochondria-associated membranes (MAMs) play a vital role in the complex crosstalk between the ER and mitochondria. MAMs are known to play an important role in lipid synthesis, the regulation of Ca2+ homeostasis, the coordination of ER-mitochondrial function, and the transduction of death signals between the ER and the mitochondria. Clarifying the structure and function of MAMs will provide new concepts for studying the pathological mechanisms associated with neurodegenerative diseases, aging, and cancers. Here, we review the recent studies of the structure and function of MAMs and its roles involved in cell death, especially in apoptosis.

ClusterMap for multi-scale clustering analysis of spatial gene expression.

Quantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we introduce an unsupervised and annotation-free framework, termed ClusterMap, which incorporates the physical location and gene identity of RNAs, formulates the task as a point pattern analysis problem, and identifies biologically meaningful structures by density peak clustering (DPC). Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and performs consistently on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell niche, and tissue organization principles from images with high-dimensional transcriptomic profiles.

Clinical Characteristics of Hospitalized Neonates With Hypofibrinogenemia: A Retrospective Cohort Study.

Background: Neonatal hypofibrinogenemia is often asymptomatic but can manifest as hemorrhage. Objective: This study was conducted to characterize clinical characteristics of neonates with hypofibrinogenemia and identify factors associated with hemorrhage. Methods: This was a retrospective study of neonates with plasma fibrinogen level (FIB) ≤1.0 g/L who were hospitalized at the Neonatology Department, People's Hospital, Chongqing, China, from January 2012 to December 2017. Based on severity, patients were grouped into severe, moderate, and mild hypofibrinogenemia (FIB < 0.5 g/L, 0.5 g/L ≤ FIB < 0.7 g/L, and 0.7 g/L ≤ FIB ≤ 1.0 g/L, respectively). Clinical characteristics associated with hemorrhage were analyzed. Results: Among 330 neonates, 52.7% showed mild hypofibrinogenemia, 25.5% had moderate hypofibrinogenemia, and 21.8% had severe hypofibrinogenemia. Severe hypofibrinogenemia was not associated with gestational age, but the mild form was frequent in neonates with low/normal birthweight (P = 0.018). Approximately 80.6% of neonates presented hypofibrinogenemia as variable combinations of thrombocytopenia or coagulopathies. Hemorrhage occurred in 38.8% of the cases, 60.9% of which were mild. Hemorrhage manifested as puncture site bleeding (47.7%) or spontaneous skin/mucous membrane bleeding (34.2%). The degree of hypofibrinogenemia was not associated with the severity or occurrence of hemorrhage. Among patients with hypofibrinogenemia and bleeding, 53.4% of the cases with coagulopathies showed mild hemorrhage, 85.7% of the cases with thrombocytopenia had moderate bleeding, while 53.8% of the cases with coagulopathy and thrombocytopenia showed severe hemorrhage. Conclusion: Neonatal hypofibrinogenemia is often comorbid and occurs with thrombocytopenia and/or coagulopathies. Although hemorrhage is not associated with the degree of hypofibrinogenemia, it may be severe when hypofibrinogenemia co-occurs with coagulopathies and/or thrombocytopenia.

Inhibition of pyruvate dehydrogenase kinase­1 by dicoumarol enhances the sensitivity of hepatocellular carcinoma cells to oxaliplatin via metabolic reprogramming.

The Warburg effect is a unique metabolic feature of the majority of tumor cells and is closely related to chemotherapeutic resistance. Pyruvate dehydrogenase kinase 1 (PDK1) is considered a 'switch' that controls the fate of pyruvate in glucose metabolism. However, to date, to the best of our knowledge, there are only a few studies to available which had studied the reduction of chemotherapeutic resistance via the metabolic reprogramming of tumor cells with PDK1 as a target. In the present study, it was found dicoumarol (DIC) reduced the phosphorylation of pyruvate dehydrogenase (PDH) by inhibiting the activity of PDK1, which converted the metabolism of human hepatocellular carcinoma (HCC) cells to oxidative phosphorylation, leading to an increase in mitochondrial reactive oxygen species ROS (mtROS) and a decrease in mitochondrial membrane potential (MMP), thereby increasing the apoptosis induced by oxaliplatin (OXA). Furthermore, the present study elucidated that the targeting of PDK1 may be a potential strategy for targeting metabolism in the chemotherapy of HCC. In addition, DIC as an 'old drug' exhibits novel efficacy, bringing new hope for antitumor therapy.