The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes.

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

Comparative and functional analysis unveils the contribution of photoperiod to DNA methylation, sRNA accumulation, and gene expression variations in short-day and long-day grasses.

Photoperiod employs complicated networks to regulate various developmental processes in plants, including flowering transition. However, the specific mechanisms by which photoperiod affects epigenetic modifications and gene expression variations in plants remain elusive. In this study, we conducted a comprehensive analysis of DNA methylation, small RNA (sRNA) accumulation, and gene expressions under different daylengths in facultative long-day (LD) grass Brachypodium distachyon and short-day (SD) grass rice. Our results showed that while overall DNA methylation levels were minimally affected by different photoperiods, CHH methylation levels were repressed under their favorable light conditions, particularly in rice. We identified numerous differentially methylated regions (DMRs) that were influenced by photoperiod in both plant species. Apart from differential sRNA clusters, we observed alterations in the expression of key components of the RNA-directed DNA methylation pathway, DNA methyltransferases, and demethylases, which may contribute to the identified photoperiod-influenced CHH DMRs. Furthermore, we identified many differentially expressed genes in response to different daylengths, some of which were associated with the DMRs. Notably, we discovered a photoperiod-responsive gene MYB11 in the transcriptome of B. distachyon, and further demonstrated its role as a flowering inhibitor by repressing FT1 transcription. Together, our comparative and functional analysis sheds light on the effects of daylength on DNA methylation, sRNA accumulation, and gene expression variations in LD and SD plants, thereby facilitating better designing breeding programs aimed at developing high-yield crops that can adapt to local growing seasons.

Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d.

The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1,319-fold increase in the activation of the ASCL1 gene. The activity can be further increased by tethering DNA-binding protein Sso7d to miniCRa and generating SminiCRa, which reached a 5,628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression of ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi used to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In summary, miniCRa, SminiCRa, miniCRi, and SminiCRi are robust miniature transcriptional modulators with high specificity that expand the toolbox for biomedical research and therapeutic applications.

OKseqHMM: a genome-wide replication fork directionality analysis toolkit.

During each cell division, tens of thousands of DNA replication origins are co-ordinately activated to ensure the complete duplication of the human genome. However, replication fork progression can be challenged by many factors, including co-directional and head-on transcription-replication conflicts (TRC). Head-on TRCs are more dangerous for genome integrity. To study the direction of replication fork movement and TRCs, we developed a bioinformatics toolkit called OKseqHMM (https://github.com/CL-CHEN-Lab/OK-Seq, https://doi.org/10.5281/zenodo.7428883). Then, we used OKseqHMM to analyse a large number of datasets obtained by Okazaki fragment sequencing to directly measure the genome-wide replication fork directionality (RFD) and to accurately predict replication initiation and termination at a fine resolution in organisms including yeast, mouse and human. We also successfully applied our analysis to other genome-wide sequencing techniques that also contain RFD information (e.g. eSPAN, TrAEL-seq). Our toolkit can be used to predict replication initiation and fork progression direction genome-wide in a wide range of cell models and growth conditions. Comparing the replication and transcription directions allows identifying loci at risk of TRCs, particularly head-on TRCs, and investigating their role in genome instability by checking DNA damage data, which is of prime importance for human health.

Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation, glucolipid metabolism, and inflammation. Thiazolidinediones are PPARγ full agonists with potent insulin-sensitizing effects, whereas their oral usage is restricted because of unwanted side effects, including obesity and cardiovascular risks. Here, via virtual screening, microscale thermophoresis analysis, and molecular confirmation, we demonstrate that diosmin, a natural compound of wide and long-term clinical use, is a selective PPARγ modulator that binds to PPARγ and blocks PPARγ phosphorylation with weak transcriptional activity. Local diosmin administration in subcutaneous fat (inguinal white adipose tissue [iWAT]) improved insulin sensitivity and attenuated obesity via enhancing browning of white fat and energy expenditure. Besides, diosmin ameliorated inflammation in WAT and liver and reduced hepatic steatosis. Of note, we determined that iWAT local administration of diosmin did not exhibit obvious side effects. Taken together, the present study demonstrated that iWAT local delivery of diosmin protected mice from diet-induced insulin resistance, obesity, and fatty liver by blocking PPARγ phosphorylation, without apparent side effects, making it a potential therapeutic agent for the treatment of metabolic diseases.

Activated factor X delivered by adeno-associated virus significantly inhibited bleeding and alleviated hemophilic synovitis in hemophilic mice.

In hemophilia, deficiency of factor VIII or IX prevents the activation of the common coagulation pathway, and inhibits the conversion of FX to activated FXa, which is required for thrombin generation. We hypothesized that the direct expressed FXa has the potential to activate the common pathway and restore coagulation in hemophilia patients. In this study, the cassettes that expressed FXa, FXaop and FXa-FVII were packaged into an engineered AAV capsid, AAV843, and were delivered into hemophilia A and B mice by intravenous injection. AAV-FXaop could be stably expressed in vivo and showed the best immediate and prolonged hemostatic effects, similar to those of commercial drugs (Xyntha and Benefix). AAV-FXaop also significantly inhibited bleeding in hemophilia A mice with inhibitors. In addition, FXa expression in joints significantly alleviated the occurrence of hemophilic synovitis. AAV-delivered FXa may be a novel target for treating hemophilic and hemophilic synovitis.

Sildenafil increases AAV9 transduction after a systemic administration and enhances AAV9-dystrophin therapeutic effect in mdx mice.

Adeno-associated virus (AAV) vectors have been successfully used to deliver genes for treating rare diseases. However, the systemic administration of high AAV vector doses triggers several adverse effects, including immune response, the asymptomatic elevation of liver transaminase levels, and complement activation. Thus, improving AAV transduction and reducing AAV dosage for treatment is necessary. Recently, we found that a phosphodiesterase-5 inhibitor significantly promoted AAV9 transduction in vitro by regulating the caveolae and macropinocytosis pathways. When AAV9-Gaussian luciferase (AAV9-Gluc) and AAV9-green fluorescent protein (AAV9-GFP) were injected intravenously into mice pre-treated with sildenafil, the expressions of Gluc in the plasma and GFP in muscle tissues significantly increased (P < 0.05). Sildenafil also improved Evans blue permeation in tissues. Additionally, we found that sildenafil promoted Treg proliferation, inhibited B-cell activation, and decreased anti-AAV9 IgG levels (P < 0.05). Furthermore, sildenafil significantly promoted Duchenne muscular dystrophy gene therapy efficacy using AAV9 in mdx mice; it increased micro-dystrophin gene expression, forelimb grip strength, and time spent on the rotarod test, decreased serum creatine kinase levels, and ameliorated histopathology by improving muscle cell morphology and reducing fibrosis (P < 0.05). These results show that sildenafil significantly improved AAV transduction, suppressed the levels of anti-AAV9 IgG, and enhanced the efficacy of gene therapy.

Automated individual cortical parcellation via consensus graph representation learning.

Cortical parcellation plays a pivotal role in elucidating the brain organization. Despite the growing efforts to develop parcellation algorithms using functional magnetic resonance imaging, achieving a balance between intra-individual specificity and inter-individual consistency proves challenging, making the generation of high-quality, subject-consistent cortical parcellations particularly elusive. To solve this problem, our paper proposes a fully automated individual cortical parcellation method based on consensus graph representation learning. The method integrates spectral embedding with low-rank tensor learning into a unified optimization model, which uses group-common connectivity patterns captured by low-rank tensor learning to optimize subjects' functional networks. This not only ensures consistency in brain representations across different subjects but also enhances the quality of each subject's representation matrix by eliminating spurious connections. More importantly, it achieves an adaptive balance between intra-individual specificity and inter-individual consistency during this process. Experiments conducted on a test-retest dataset from the Human Connectome Project (HCP) demonstrate that our method outperforms existing methods in terms of reproducibility, functional homogeneity, and alignment with task activation. Extensive network-based comparisons on the HCP S900 dataset reveal that the functional network derived from our cortical parcellation method exhibits greater capabilities in gender identification and behavior prediction than other approaches.

Auxochrome Dimethyl-Dihydroacridine Improves Fluorophores for Prolonged Live-Cell Super-Resolution Imaging.

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.

Blocking donor liver Pannexin 1 channels facilitates mitochondria protection during liver transplantation（3966）.

Static cold storage (SCS) is the standard technique for organ preservation during transplantation, resulting in cold ischemic injury. Hypoxia can induce Panx1 channels open, leading to release of ATP. However, it is unknown whether Panx1 play a role in SCS. Our research demonstrates that livers from Panx1-/- mice exhibited reduced ATP release, resulting in protected hepatocytes during preservation. The donor liver damage is decreased during SCS with blocking Panx1. Transmission electron microscopy revealed a decreased mitochondria-associated ER membranes (MAMs) and an improved mitochondria morphology. Mechanistically, Panx1 blockade upregulated the PI3K-AKT pathway and increased Bcl2 level to combat apoptosis during liver preservation. The data indicate that blocking Panx1 during preservation of the donor liver can effectively improve mitochondrial function, reducing cellular stress damage. Then cold ischemia and reperfusion-related injuries are obviously decreased in liver transplantation.

Prior-guided individualized thalamic parcellation based on local diffusion characteristics.

Comprising numerous subnuclei, the thalamus intricately interconnects the cortex and subcortex, orchestrating various facets of brain functions. Extracting personalized parcellation patterns for these subnuclei is crucial, as different thalamic nuclei play varying roles in cognition and serve as therapeutic targets for neuromodulation. However, accurately delineating the thalamic nuclei boundary at the individual level is challenging due to intersubject variability. In this study, we proposed a prior-guided parcellation (PG-par) method to achieve robust individualized thalamic parcellation based on a central-boundary prior. We first constructed probabilistic atlas of thalamic nuclei using high-quality diffusion MRI datasets based on the local diffusion characteristics. Subsequently, high-probability voxels in the probabilistic atlas were utilized as prior guidance to train unique multiple classification models for each subject based on a multilayer perceptron. Finally, we employed the trained model to predict the parcellation labels for thalamic voxels and construct individualized thalamic parcellation. Through a test-retest assessment, the proposed prior-guided individualized thalamic parcellation exhibited excellent reproducibility and the capacity to detect individual variability. Compared with group atlas registration and individual clustering parcellation, the proposed PG-par demonstrated superior parcellation performance under different scanning protocols and clinic settings. Furthermore, the prior-guided individualized parcellation exhibited better correspondence with the histological staining atlas. The proposed prior-guided individualized thalamic parcellation method contributes to the personalized modeling of brain parcellation.

The role of the cytochrome P450 superfamily in the skin.

In mammals, the skin acts as a barrier to prevent harmful environmental stimuli from entering the circulation. CYP450s are involved in drug biotransformation, exogenous and endogenous substrate metabolism, and maintaining the normal physiological function of the skin, as well as facilitating homeostasis of the internal environment. The expression pattern of CYP450s in the skin is tissue-specific and thus differs from the liver and other organs. The development of skin topical medications, and knowledge of the toxicity and side effects of these medications require a detailed understanding of the expression and function of skin-specific CYP450s. Thus, we summarized the expression of CYP450s in the skin, their function in endogenous metabolic physiology, aberrant CYP450 expression in skin diseases and the influence of environmental variables and medications. This information will serve as a crucial foundation for future studies on the skin, as well as for the design and development of new drugs for skin diseases including topical medications.

Advances in Drug Delivery Systems for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a common and catastrophic hematological neoplasm with high mortality rates. Conventional therapies, including chemotherapy, hematopoietic stem cell transplantation (HSCT), immune therapy, and targeted agents, have unsatisfactory outcomes for AML patients due to drug toxicity, off-target effects, drug resistance, drug side effects, and AML relapse and refractoriness. These intrinsic limitations of current treatments have promoted the development and application of nanomedicine for more effective and safer leukemia therapy. In this review, the classification of nanoparticles applied in AML therapy, including liposomes, polymersomes, micelles, dendrimers, and inorganic nanoparticles, is reviewed. In addition, various strategies for enhancing therapeutic targetability in nanomedicine, including the use of conjugating ligands, biomimetic-nanotechnology, and bone marrow targeting, which indicates the potential to reverse drug resistance, are discussed. The application of nanomedicine for assisting immunotherapy is also involved. Finally, the advantages and possible challenges of nanomedicine for the transition from the preclinical phase to the clinical phase are discussed.

Aflibercept Loaded Eye-Drop Hydrogel Mediated with Cell-Penetrating Peptide for Corneal Neovascularization Treatment.

Corneal neovascularization (CoNV) is a major cause of visual impairment worldwide. Currently, available treatment options have limited efficacy and are associated with adverse effects due to biological barriers and clearance mechanisms. To address this challenge, a novel topical delivery system is developed-Gel 2_1&Eylea-an aflibercept-loaded eye-drop hydrogel mediated with cell-penetrating peptide 1. Gel 2_1&Eylea demonstrates superior membrane permeability, increased stability, and prolonged drug retention time on the ocular surface, and thus may improve drug efficacy. In a rabbit CoNV model, Gel 2_1&Eylea significantly reduces the density of neovascularization with no adverse effects on normal corneoscleral limbal vessels, demonstrating high efficacy and biocompatibility. This work identifies a promising treatment for CoNV which has the potential to benefit other ocular neovascular diseases.

A major facilitator superfamily transporter MdtH in Escherichia coli is involved in anthocyanin biosynthesis and secretion.

Anthocyanin cyanidin 3-O-glucoside (C3G) is a natural pigment widely used in food and nutraceutical industries. Its microbial synthesis in Escherichia coli is a promising and efficient way toward large-scale production. The current production titer is low partly due to the accumulation of C3G inside the producing microbes; thus, it is important to explore native transporters responsible for anthocyanin secretion. Currently, there has been only one native E. coli transporter identified with C3G-transporting capability, and its overexpression has a very limited effect on the promotion of extracellular C3G production. In this study, we report the identification and verification of an efficient intrinsic C3G efflux transporter MdtH in E. coli through transcriptomic analysis and genetic/biochemical studies. MdtH could bind C3G with high affinity, and its overexpression increased the extracellular C3G biosynthesis in E. coli by 110%. Our study provides a new regulation target for microbial biosynthesis of C3G and other anthocyanins. IMPORTANCE: Cyanidin 3-O-glucoside (C3G) is a natural colorant with health-promoting activities and is, hence, widely used in food, cosmetic, and nutraceutical industries. Its market supply is currently dependent on extraction from plants. As an alternative, C3G can be produced by the microbe Escherichia coli in a green and sustainable way. However, a large portion of this compound is retained inside the cell of E. coli, thus complicating the purification process and limiting the high-level production. We have identified and verified an efficient native transporter named MdtH in E. coli that can export C3G to the cultivation medium. Overexpression of MdtH could improve extracellular C3G production by 110% without modifications of the metabolic pathway genes or enzymes. This study reveals a new regulation target for C3G production in bacteria and provides guidance to the microbial biosynthesis of related compounds.

Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.

Reproductive factors and risk of lung cancer among 300,000 Chinese female never-smokers: evidence from the China Kadoorie Biobank study.

BACKGROUND: Lung cancer is the leading cause of cancer mortality among Chinese females despite the low smoking prevalence among this population. This study assessed the roles of reproductive factors in lung cancer development among Chinese female never-smokers. METHODS: The prospective China Kadoorie Biobank (CKB) recruited over 0.5 million Chinese adults (0.3 million females) from 10 geographical areas in China in 2004-2008 when information on socio-demographic/lifestyle/environmental factors, physical measurements, medical history, and reproductive history collected through interviewer-administered questionnaires. Cox proportional hazard regression was used to estimate adjusted hazard ratios (HRs) of lung cancer by reproductive factors. Subgroup analyses by menopausal status, birth year, and geographical region were performed. RESULTS: During a median follow-up of 11 years, 2,284 incident lung cancers occurred among 282,558 female never-smokers. Ever oral contraceptive use was associated with a higher risk of lung cancer (HR = 1.16, 95% CI: 1.02-1.33) with a significant increasing trend associated with longer duration of use (p-trend = 0.03). Longer average breastfeeding duration per child was associated with a decreased risk (0.86, 0.78-0.95) for > 12 months compared with those who breastfed for 7-12 months. No statistically significant association was detected between other reproductive factors and lung cancer risk. CONCLUSION: Oral contraceptive use was associated with an increased risk of lung cancer in Chinese female never-smokers. Further studies are needed to assess lung cancer risk related to different types of oral contraceptives in similar populations.

Progress on Copper-Based Anode Materials for Sodium-Ion Batteries.

Fossil fuels have clearly failed to meet people's growing energy needs due to their limited reserves, potential pollution of the environment, and high costs. The development of cleaner, renewable energy sources as well as secondary batteries for energy storage is imminent, in a modern society where energy demand is soaring. Sodium-ion batteries (SIBs) have become the focus of large-scale energy storage systems as a promising alternative to lithium-ion batteries. The development of SIBs relies on the construction of high performance electrode materials. The design of low cost and high performance anode materials is a key link in this regard. Copper-based anodes are characterized by high theoretical capacity, abundant reserves, low cost and environmental friendliness. A variety of copper-based anode materials, which include cobalt oxides, sulfides, selenides and phosphides, have been synthesized and evaluated in the scientific literature for sodium storage. In detail, the preparation methods, response mechanisms, strengths and weaknesses, the relationship between morphology structure and electrochemical performance are discussed, as well as highlighting strategies to improve the electrochemical performance of copper-based anode materials. Finally, we offer our perspective on the challenges and potential for the development of copper-based anodes as a means of developing practical and high performing SIBs.

Engineering of bacteria towards programmed autolysis: why, how, and when?

Programmed autolytic bacteria, also termed controlled self-disruptive or self-destructive bacteria, are bacterial systems that express certain lytic genes and undergo cell lysis at a predetermined time point to release the intracellular contents or to commit suicide. Such systems have wide applications in high-throughput screening of protein libraries, synthesis and recovery of bio-products, population control of heterogeneous cultures or synthetic co-cultures, drug delivery, and food fermentation. Recently, great achievements have been reported regarding on-demand control of cell autolysis for different purposes, highlighting the potential of autolytic strains in biomanufacturing and biomedicine. In this review article, we first introduce the various applications of such bacteria, followed by a summarization of the approaches used in the establishment of autolytic bacterial systems, including cell autolysis mediated by cell wall hydrolases with or without facilitating proteins and by membrane-disturbing proteins. Next, we describe in detail the methodologies adopted to control and initiate cell lysis, including induction by chemical inducers, stimulation by physical signals, auto-induction by metabolic status or nutrient limitation, and constitutive expression of the lytic genes. This article is ended with discussions on the remaining problems and possible future directions. This review provides comprehensive information on autolytic bacteria and insightful guidance to the development of highly efficient, robust, and smart autolytic bacterial platforms.