The biochemical pathways of apoptotic, necroptotic, pyroptotic, and ferroptotic cell death.

Apoptosis, the first regulated form of cell death discovered in mammalian cells, is executed by caspase-3/7, which are dormant in living cells but become activated by upstream caspase-8 or caspase-9 in responding to extracellular cytokines or intracellular stress signals, respectively. The same cell death-inducing cytokines also cause necroptosis when caspase-8 is inhibited, resulting in the activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates pseudokinase MLKL to trigger its oligomerization and membrane-disrupting activity. Caspase-1/4/5/11, known as inflammatory caspases, instead induce pyroptosis by cleaving gasdermin D, whose caspase-cleaved N terminus forms pores on the plasma membrane. The membrane protein NINJ1 amplifies the extent of membrane rupture initiated by gasdermin D. Additionally, disturbance of peroxidation of polyunsaturated fatty acid tails of membrane phospholipids triggers ferroptosis, an iron-dependent and caspases-independent necrotic death. This review will discuss how these regulated cell death pathways act individually and interconnectively in particular cell types to carry out specific physiological and pathological functions.

Discovery of cytosine deaminases enables base-resolution methylome mapping using a single enzyme.

Deaminases have important uses in modification detection and genome editing. However, the range of applications is limited by the small number of characterized enzymes. To expand the toolkit of deaminases, we developed an in vitro approach that bypasses a major hurdle with their toxicity in cells. We assayed 175 putative cytosine deaminases on a variety of substrates and found a broad range of activity on double- and single-stranded DNA in various sequence contexts, including CpG-specific deaminases and enzymes without sequence preference. We also characterized enzyme selectivity across six DNA modifications and reported enzymes that do not deaminate modified cytosines. The detailed analysis of diverse deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed SEM-seq, a non-destructive single-enzyme methylation sequencing method using a modification-sensitive double-stranded DNA deaminase. The streamlined protocol enables accurate, base-resolution methylome mapping of scarce biological material, including cell-free DNA and 10 pg input DNA.

Membrane Damage during Ferroptosis Is Caused by Oxidation of Phospholipids Catalyzed by the Oxidoreductases POR and CYB5R1.

Ferroptosis is a form of necrotic cell death caused by iron-dependent peroxidation of polyunsaturated phospholipids on cell membranes and is actively suppressed by the cellular antioxidant systems. We report here that oxidoreductases, including NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), transfer electrons from NAD(P)H to oxygen to generate hydrogen peroxide, which subsequently reacts with iron to generate reactive hydroxyl radicals for the peroxidation of the polyunsaturated fatty acid (PUFA) chains of membrane phospholipids, thereby disrupting membrane integrity during ferroptosis. Genetic knockout of POR and CYB5R1 decreases cellular hydrogen peroxide generation, preventing lipid peroxidation and ferroptosis. Moreover, POR knockdown in mouse liver prevents ConA-induced liver damage. Ferroptosis, therefore, is a result of incidental electron transfer carried out by POR/CYB5R1 oxidoreductase and thus needs to be constitutively countered by the antioxidant systems.

Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine.

Multiomics require concerted recording of independent information, ideally from a single experiment. In this study, we introduce RIMS-seq2, a high-throughput technique to simultaneously sequence genomes and overlay methylation information while requiring only a small modification of the experimental protocol for high-throughput DNA sequencing to include a controlled deamination step. Importantly, the rate of deamination of 5-methylcytosine is negligible and thus does not interfere with standard DNA sequencing and data processing. Thus, RIMS-seq2 libraries from whole- or targeted-genome sequencing show the same germline variation calling accuracy and sensitivity compared with standard DNA-seq. Additionally, regional methylation levels provide an accurate map of the human methylome.

Pretraining a foundation model for generalizable fluorescence microscopy-based image restoration.

Fluorescence microscopy-based image restoration has received widespread attention in the life sciences and has led to significant progress, benefiting from deep learning technology. However, most current task-specific methods have limited generalizability to different fluorescence microscopy-based image restoration problems. Here, we seek to improve generalizability and explore the potential of applying a pretrained foundation model to fluorescence microscopy-based image restoration. We provide a universal fluorescence microscopy-based image restoration (UniFMIR) model to address different restoration problems, and show that UniFMIR offers higher image restoration precision, better generalization and increased versatility. Demonstrations on five tasks and 14 datasets covering a wide range of microscopy imaging modalities and biological samples demonstrate that the pretrained UniFMIR can effectively transfer knowledge to a specific situation via fine-tuning, uncover clear nanoscale biomolecular structures and facilitate high-quality imaging. This work has the potential to inspire and trigger new research highlights for fluorescence microscopy-based image restoration.

Laser direct overall water splitting for H2 and H2O2 production.

Hydrogen (H2) and hydrogen peroxide (H2O2) play crucial roles as energy carriers and raw materials for industrial production. However, the current techniques for H2 and H2O2 production rely on complex catalysts and involve multiple intermediate steps. In this study, we present a straightforward, environmentally friendly, and highly efficient laser-induced conversion method for overall water splitting to simultaneously generate H2 and H2O2 at ambient conditions without any catalysts. The laser direct overall water splitting approach achieves an impressive light-to-hydrogen energy conversion efficiency of 2.1%, with H2 production rates of 2.2 mmol/h and H2O2 production rates of 65 µM/h in a limited reaction area (1 mm2) within a short real reaction time (0.36 ms/h). Furthermore, we elucidate the underlying physics and chemistry behind the laser-induced water splitting to produce H2 and H2O2. The laser-induced cavitation bubbles create an optimal microenvironment for water-splitting reactions because of the transient high temperatures (104 K) surpassing the chemical barrier required. Additionally, their rapid cooling rate (1010 K/s) hinders reverse reactions and facilitates H2O2 retention. Finally, upon bubble collapse, H2 is released while H2O2 remains dissolved in the water. Moreover, a preliminary amplification experiment demonstrates the potential industrial applications of this laser chemistry. These findings highlight that laser-based production of H2 and H2O2 from water holds promise as a straightforward, environmentally friendly, and efficient approach on an industrial scale beyond conventional chemical catalysis.

The Role of Fc Receptors in the Innate Immune System of Flounders Purported to Be Homologs of FcγRII and FcγRIII.

FcγR is a significant opsonin receptor located on the surface of immune cells, playing a crucial role in Ab-dependent cell-mediated immunity. Our previous work revealed opposite expression trends of FcγRII and FcγRIII in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda. This observation suggests that FcγRII and FcγRIII might serve distinct functions in Ig-opsonized immune responses. In this study, we prepared rFcγRIII as well as its corresponding Abs to investigate the potential roles of FcγRII and FcγRIII in the Ab-dependent immune response of IgM+ B cells. Our findings indicate that, unlike FcγRII, FcγRIII does not participate in Ab-dependent cellular phagocytosis. Instead, it is involved in cytokine production and bacterial killing in mIgM+ B lymphocytes. Additionally, we identified platelet-derived ADAM17 as a key factor in regulating FcγRIII shedding and cytokine release in mIgM+ B lymphocytes. These results elucidate the functions of FcγRII and FcγRIII in the innate immunology of mIgM+ B lymphocytes and contribute to an improved understanding of the regulatory roles of FcγRs in the phagocytosis of teleost B lymphocytes.

Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq.

Determination of eukaryotic transcription start sites (TSSs) has been based on methods that require the cap structure at the 5' end of transcripts derived from Pol II RNA polymerase. Consequently, these methods do not reveal TSSs derived from the other RNA polymerases that also play critical roles in various cell functions. To address this limitation, we developed ReCappable-seq, which comprehensively identifies TSS for both Pol II and non-Pol II transcripts at single-nucleotide resolution. The method relies on specific enzymatic exchange of 5' m7G caps and 5' triphosphates with a selectable tag. When applied to human transcriptomes, ReCappable-seq identifies Pol II TSSs that are in agreement with orthogonal methods such as CAGE. Additionally, ReCappable-seq reveals a rich landscape of TSSs associated with Pol III transcripts that have not previously been amenable to study at genome-wide scale. Novel TSS from non-Pol II transcription can be located in the nuclear and mitochondrial genomes. ReCappable-seq interrogates the regulatory landscape of coding and noncoding RNA concurrently and enables the classification of epigenetic profiles associated with Pol II and non-Pol II TSS.

Methyl-SNP-seq reveals dual readouts of methylome and variome at molecule resolution while enabling target enrichment.

Covalent modifications of genomic DNA are crucial for most organisms to survive. Amplicon-based high-throughput sequencing technologies erase all DNA modifications to retain only sequence information for the four canonical nucleobases, necessitating specialized technologies for ascertaining epigenetic information. To also capture base modification information, we developed Methyl-SNP-seq, a technology that takes advantage of the complementarity of the double helix to extract the methylation and original sequence information from a single DNA molecule. More specifically, Methyl-SNP-seq uses bisulfite conversion of one of the strands to identify cytosine methylation while retaining the original four-bases sequence information on the other strand. As both strands are locked together to link the dual readouts on a single paired-end read, Methyl-SNP-seq allows detecting the methylation status of any DNA even without a reference genome. Because one of the strands retains the original four nucleotide composition, Methyl-SNP-seq can also be used in conjunction with standard sequence-specific probes for targeted enrichment and amplification. We show the usefulness of this technology in a broad spectrum of applications ranging from allele-specific methylation analysis in humans to identification of methyltransferase specificity in complex bacterial communities.

Cortical atrophy in early-stage patients with anti-NMDA receptor encephalitis: a machine-learning MRI study with various feature extraction.

Conventional brain magnetic resonance imaging (MRI) of anti-N-methyl-D-aspartate-receptor encephalitis (NMDARE) is non-specific, thus showing little differential diagnostic value, especially for MRI-negative patients. To characterize patterns of structural alterations and facilitate the diagnosis of MRI-negative NMDARE patients, we build two support vector machine models (NMDARE versus healthy controls [HC] model and NMDARE versus viral encephalitis [VE] model) based on radiomics features extracted from brain MRI. A total of 109 MRI-negative NMDARE patients in the acute phase, 108 HCs and 84 acute MRI-negative VE cases were included for training. Another 29 NMDARE patients, 28 HCs and 26 VE cases were included for validation. Eighty features discriminated NMDARE patients from HCs, with area under the receiver operating characteristic curve (AUC) of 0.963 in validation set. NMDARE patients presented with significantly lower thickness, area, and volume and higher mean curvature than HCs. Potential atrophy predominately presented in the frontal lobe (cumulative weight = 4.3725, contribution rate of 29.86%), and temporal lobe (cumulative weight = 2.573, contribution rate of 17.57%). The NMDARE versus VE model achieved certain diagnostic power, with AUC of 0.879 in validation set. Our research shows potential atrophy across the entire cerebral cortex in acute NMDARE patients, and MRI machine learning model has a potential to facilitate the diagnosis MRI-negative NMDARE.

Immunosuppressant therapy averts rejection of allogeneic FKBP1A-disrupted CAR-T cells.

Chimeric antigen receptor (CAR) T cells from allogeneic donors promise "off-the-shelf" availability by overcoming challenges associated with autologous cell manufacturing. However, recipient immunologic rejection of allogeneic CAR-T cells may decrease their in vivo lifespan and limit treatment efficacy. Here, we demonstrate that the immunosuppressants rapamycin and tacrolimus effectively mitigate allorejection of HLA-mismatched CAR-T cells in immunocompetent humanized mice, extending their in vivo persistence to that of syngeneic humanized mouse-derived CAR-T cells. In turn, genetic knockout (KO) of FKBP prolyl isomerase 1A (FKBP1A), which encodes a protein targeted by both drugs, was necessary to confer CD19-specific CAR-T cells (19CAR) robust functional resistance to these immunosuppressants. FKBP1AKO 19CAR-T cells maintained potent in vitro functional profiles and controlled in vivo tumor progression similarly to untreated 19CAR-T cells. Moreover, immunosuppressant treatment averted in vivo allorejection permitting FKBP1AKO 19CAR-T cell-driven B cell aplasia. Thus, we demonstrate that genome engineering enables immunosuppressant treatment to improve the therapeutic potential of universal donor-derived CAR-T cells.

Blockage of MLKL prevents myelin damage in experimental diabetic neuropathy.

SignificanceDiabetic neuropathy is a commonly occurring complication of diabetes that affects hundreds of millions of patients worldwide. Patients suffering from diabetic neuropathy experience abnormal sensations and have damage in their peripheral nerve axons as well as myelin, a tightly packed Schwann cell sheath that wraps around axons to provide insulation and increases electrical conductivity along the nerve fibers. The molecular events underlying myelin damage in diabetic neuropathy are largely unknown, and there is no efficacious treatment for the disease. The current study, using a diabetic mouse model and human patient nerve samples, uncovered a molecular mechanism underlying myelin sheath damage in diabetic neuropathy and provides a potential treatment strategy for the disease.

The highest-elevation frog provides insights into mechanisms and evolution of defenses against high UV radiation.

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri. We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri. Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri. We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri. Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

Efficient and Rapid Hydrogen Extraction from Ammonia-Water via Laser Under Ambient Conditions without Catalyst.

As a good carrier of hydrogen, ammonia-water has been employed to extract hydrogen in many ways. Here, we demonstrate a simple, green, ultrafast, and highly efficient method for hydrogen extraction from ammonia-water by laser bubbling in liquids (LBL) at room temperature and ambient pressure without catalyst. A maximum apparent yield of 33.7 mmol/h and a real yield of 93.6 mol/h were realized in a small operating space, which were far higher than the yields of most hydrogen evolution reactions from ammonia-water under ambient conditions. We also established that laser-induced cavitation bubbles generated a transient high temperature, which enabled a very suitable environment for hydrogen extraction from ammonia-water. The laser used here can serve as a demonstration of potentially solar-pumped catalyst-free hydrogen extraction and other chemical synthesis. We anticipate that the LBL technique will open unprecedented opportunities to produce chemicals.

Catalyst-Free Activation and Fixation of Nitrogen by Laser-Induced Conversion.

Ultrafast N2 fixation reactions are quite challenging. Currently used methods for N2 fixation are limited, and strong dinitrogen bonds usually need to be activated via extreme temperature or pressure or by the use of an energy-consuming process with sophisticated catalysts. Herein, we report a novel laser-based chemical method for N2 fixation under ambient conditions without catalysts, this method is called laser bubbling in liquids (LBL), and it directly activates N2 in water (H2O) and efficiently converts N2 into valuable NH3 (max: 4.2 mmol h-1) and NO3- (0.17 mmol h-1). Remarkably, the highest yields of NH3 and NO3- are 4 orders of magnitude greater than the best values for electrocatalysis reported to date. Notably, we further validate the experimental mechanism by using optical emission spectroscopy to detect the production of intermediate plasma and by employing isotope tracing. We also establish that an extremely high-temperature environment far from thermodynamic equilibrium inside a laser-induced bubble and the kinetic process of rapid quenching of bubbles is crucial for N2 activation and fixation to generate NH3 and NOx via LBL. Based on these results, it is shown that LBL is a simple, safe, efficient, green, and sustainable technology that enables the rapid conversion of the renewable feedstocks H2O and N2 to NH3 and NO3-, facilitating new prospects for chemical N2 fixation.

Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.

Mining the interpretable prognostic features from pathological image of intrahepatic cholangiocarcinoma using multi-modal deep learning.

BACKGROUND: The advances in deep learning-based pathological image analysis have invoked tremendous insights into cancer prognostication. Still, lack of interpretability remains a significant barrier to clinical application. METHODS: We established an integrative prognostic neural network for intrahepatic cholangiocarcinoma (iCCA), towards a comprehensive evaluation of both architectural and fine-grained information from whole-slide images. Then, leveraging on multi-modal data, we conducted extensive interrogative approaches to the models, to extract and visualize the morphological features that most correlated with clinical outcome and underlying molecular alterations. RESULTS: The models were developed and optimized on 373 iCCA patients from our center and demonstrated consistent accuracy and robustness on both internal (n = 213) and external (n = 168) cohorts. The occlusion sensitivity map revealed that the distribution of tertiary lymphoid structures, the geometric traits of the invasive margin, the relative composition of tumor parenchyma and stroma, the extent of necrosis, the presence of the disseminated foci, and the tumor-adjacent micro-vessels were the determining architectural features that impacted on prognosis. Quantifiable morphological vector extracted by CellProfiler demonstrated that tumor nuclei from high-risk patients exhibited significant larger size, more distorted shape, with less prominent nuclear envelope and textural contrast. The multi-omics data (n = 187) further revealed key molecular alterations left morphological imprints that could be attended by the network, including glycolysis, hypoxia, apical junction, mTORC1 signaling, and immune infiltration. CONCLUSIONS: We proposed an interpretable deep-learning framework to gain insights into the biological behavior of iCCA. Most of the significant morphological prognosticators perceived by the network are comprehensible to human minds.

Pd@CuInP2S6 Core-Shell Nanospheres with Exceptional Hydrogen Evolution Capability and Stability in Both Alkaline and Acidic Media under Large Current Density Exceeding 1000 mA cm-2.

By combining Pd with 2D layered crystal CuInP2S6 (CIPS) via laser irradiation in liquids, low-loading Pd@CIPS core-shell nanospheres are fabricated as an efficient and robust electrocatalysts for HER in both alkaline and acidic media under large current density (⩾1000 mA cm-2). Pd@CIPS core-shell nanosphere has two structural features, i) the out-shell is the nanocomposite of PdHx and PdInHx, and ii) there is a kind of dendritic structure on the surface of nanospheres, while the dendritic structure porvides good gas desorption pathway and cause the Pd@CIPS system to maintain higher HER activity and stability than that of commercial Pt/C under large current densities. Pd@CIPS exhibits very low overpotentials of -218 and -313 mV for the large current density of 1000 mA cm-2, and has a small Tafel slope of 29 and 63 mV dec-1 in 0.5 m H2SO4 and 1 m KOH condition, respectively. Meanwhile, Pd@CIPS has an excellent stability under -10 and -500 mA cm-2 current densities and 50 000 cycles cyclic voltammetry tests in 0.5 m H2SO4 and 1 m KOH, respectively, which being much superior to that of commercial Pt/C. Density functional theory (DFT) reveals that engineering electronic structure of PdHx and PdInHx nanostructure can strongly weaken the Pd─H bonding.

Observation of off-axis solitary waves propagating along a specific trajectory in photorefractive crystals.

We report the propagation dynamics of swallowtail beams (SBs) within a photorefractive crystal. In the nonlinear regime, the self-accelerating and secondary self-focusing features of the swallowtail beams are influenced, and a solitary wave is generated. The main lobe energy of the swallowtail beams is guided to a specific inclined trajectory, leading to a stable solitary wave, and we control the output position of the solitary wave by changing the launch angle. Our results are supported by the corresponding experiments. In addition, we demonstrate that a Gaussian beam can be effectively guided in swallowtail optical waveguide structures. Our research represents an interesting interaction between the swallowtail beams and nonlinear medium, which may find potential applications in photonic integrated devices and optical information transmission.

Full-space metasurfaces for independent manipulation of transmission and reflection.

In recent years, beam manipulation using metasurfaces has evolved from being limited to either a transmission or reflection space to encompassing a full space. However, existing methods still inevitably require complex systems and are unable to achieve continuous and arbitrary phase manipulation. Here, one type of a bilayer metasurface is proposed to simultaneously manipulate reflection and transmission phases continuously and independently, which also makes the optical system more compact without requiring any analyzers and enhances the degree of freedom for full-space beam manipulation. As a proof-of-concept demonstration, one device is designed to show different holograms in transmission and reflection spaces. Additionally, the Dammann grating designed in the reflection hologram increases the information capacity. The proposed method may pave the way toward achieving a variety of applications such as multi-channel beam manipulation and multifunctional optical devices.