A programmable targeted protein-degradation platform for versatile applications in mammalian cells and mice.

Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.

CRISPR-Cas9 knockin mice for genome editing and cancer modeling.

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

CELLULOSE SYNTHASE-LIKE C proteins modulate cell wall establishment during ethylene-mediated root growth inhibition in rice.

The cell wall shapes plant cell morphogenesis and affects the plasticity of organ growth. However, the way in which cell wall establishment is regulated by ethylene remains largely elusive. Here, by analyzing cell wall patterns, cell wall composition and gene expression in rice (Oryza sativa, L.) roots, we found that ethylene induces cell wall thickening and the expression of cell wall synthesis-related genes, including CELLULOSE SYNTHASE-LIKE C1, 2, 7, 9, 10 (OsCSLC1, 2, 7, 9, 10) and CELLULOSE SYNTHASE A3, 4, 7, 9 (OsCESA3, 4, 7, 9). Overexpression and mutant analyses revealed that OsCSLC2 and its homologs function in ethylene-mediated induction of xyloglucan biosynthesis mainly in the cell wall of root epidermal cells. Moreover, OsCESA-catalyzed cellulose deposition in the cell wall was enhanced by ethylene. OsCSLC-mediated xyloglucan biosynthesis likely plays an important role in restricting cell wall extension and cell elongation during the ethylene response in rice roots. Genetically, OsCSLC2 acts downstream of ETHYLENE-INSENSITIVE3-LIKE1 (OsEIL1)-mediated ethylene signaling, and OsCSLC1, 2, 7, 9 are directly activated by OsEIL1. Furthermore, the auxin signaling pathway is synergistically involved in these regulatory processes. These findings link plant hormone signaling with cell wall establishment, broadening our understanding of root growth plasticity in rice and other crops.

Evolutionary and biomedical insights from a marmoset diploid genome assembly.

The accurate and complete assembly of both haplotype sequences of a diploid organism is essential to understanding the role of variation in genome functions, phenotypes and diseases1. Here, using a trio-binning approach, we present a high-quality, diploid reference genome, with both haplotypes assembled independently at the chromosome level, for the common marmoset (Callithrix jacchus), an primate model system that is widely used in biomedical research2,3. The full spectrum of heterozygosity between the two haplotypes involves 1.36% of the genome-much higher than the 0.13% indicated by the standard estimation based on single-nucleotide heterozygosity alone. The de novo mutation rate is 0.43 × 10-8 per site per generation, and the paternal inherited genome acquired twice as many mutations as the maternal. Our diploid assembly enabled us to discover a recent expansion of the sex-differentiation region and unique evolutionary changes in the marmoset Y chromosome. In addition, we identified many genes with signatures of positive selection that might have contributed to the evolution of Callithrix biological features. Brain-related genes were highly conserved between marmosets and humans, although several genes experienced lineage-specific copy number variations or diversifying selection, with implications for the use of marmosets as a model system.

Platypus and echidna genomes reveal mammalian biology and evolution.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

Heterozygous deletion of the autism-associated gene CHD8 impairs synaptic function through widespread changes in gene expression and chromatin compaction.

Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.

Accurate integration of multiple heterogeneous single-cell RNA-seq data sets by learning contrastive biological variation.

For most biological and medical applications of single-cell transcriptomics, an integrative study of multiple heterogeneous single-cell RNA sequencing (scRNA-seq) data sets is crucial. However, present approaches are unable to integrate diverse data sets from various biological conditions effectively because of the confounding effects of biological and technical differences. We introduce single-cell integration (scInt), an integration method based on accurate, robust cell-cell similarity construction and unified contrastive biological variation learning from multiple scRNA-seq data sets. scInt provides a flexible and effective approach to transfer knowledge from the already integrated reference to the query. We show that scInt outperforms 10 other cutting-edge approaches using both simulated and real data sets, particularly in the case of complex experimental designs. Application of scInt to mouse developing tracheal epithelial data shows its ability to integrate development trajectories from different developmental stages. Furthermore, scInt successfully identifies functionally distinct condition-specific cell subpopulations in single-cell heterogeneous samples from a variety of biological conditions.

Pax4-Ghrelin mediates the conversion of pancreatic ε-cells to ß-cells after extreme ß-cell loss in zebrafish.

Pancreatic ε-cells producing ghrelin are one type of endocrine cell found in islets, which have been shown to influence other intra-islet cells, especially in regulating the function of ß cells. However, the role of such cells during ß-cell regeneration is currently unknown. Here, using a zebrafish nitroreductase (NTR)-mediated ß-cell ablation model, we reveal that ghrelin-positive ε-cells in the pancreas act as contributors to neogenic ß-cells after extreme ß-cell loss. Further studies show that the overexpression of ghrelin or the expansion of ε-cells potentiates ß-cell regeneration. Lineage tracing confirms that a proportion of embryonic ε-cells can transdifferentiate to ß-cells, and that the deletion of Pax4 enhances this transdifferentiation of ε-cells to ß-cells. Mechanistically, Pax4 binds to the ghrelin regulatory region and represses its transcription. Thus, deletion of Pax4 derepresses ghrelin expression and causes producing more ghrelin-positive cells, enhancing the transdifferentiation of ε-cells to ß-cells and consequently potentiating ß-cell regeneration. Our findings reveal a previously unreported role for ε-cells during zebrafish ß-cell regeneration, indicating that Pax4 regulates ghrelin transcription and mediates the conversion of embryonic ε-cells to ß-cells after extreme ß-cell loss.

VERNALIZATION1 represses FLOWERING PROMOTING FACTOR1-LIKE1 in leaves for timely flowering in Brachypodium distachyon.

FLOWERING PROMOTING FACTOR1 (FPF1), a small protein without any known domains, promotes flowering in several plants; however, its functional mechanism remains unknown. Here, we characterized 2 FPF1-like proteins, FPL1 and FPL7, which, in contrast, function as flowering repressors in Brachypodium distachyon. FPL1 and FPL7 interact with the components of the florigen activation complex (FAC) and inhibit FAC activity to restrict expression of its critical target, VERNALIZATION1 (VRN1), in leaves, thereby preventing overaccumulation of FLOWERING LOCUS T1 (FT1) at the juvenile stage. Further, VRN1 can directly bind to the FPL1 promoter and repress FPL1 expression; hence, as VRN1 gradually accumulates during the late vegetative stage, FAC is released. This accurate feedback regulation of FPL1 by VRN1 allows proper FT1 expression in leaves and ensures sufficient FAC formation in shoot apical meristems to trigger timely flowering. Overall, we define a sophisticated modulatory loop for flowering initiation in a temperate grass, providing insights toward resolving the molecular basis underlying fine-tuning flowering time in plants.

A programmable protease-based protein secretion platform for therapeutic applications.

Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.

A small-molecule drug inhibits autophagy gene expression through the central regulator TFEB.

Autophagy supports the fast growth of established tumors and promotes tumor resistance to multiple treatments. Inhibition of autophagy is a promising strategy for tumor therapy. However, effective autophagy inhibitors suitable for clinical use are currently lacking. There is a high demand for identifying novel autophagy drug targets and potent inhibitors with drug-like properties. The transcription factor EB (TFEB) is the central transcriptional regulator of autophagy, which promotes lysosomal biogenesis and functions and systematically up-regulates autophagy. Despite extensive evidence that TFEB is a promising target for autophagy inhibition, no small molecular TFEB inhibitors were reported. Here, we show that an United States Food and Drug Administration (FDA)-approved drug Eltrombopag (EO) binds to the basic helix-loop-helix-leucine zipper domain of TFEB, specifically the bottom surface of helix-loop-helix to clash with DNA recognition, and disrupts TFEB-DNA interaction in vitro and in cellular context. EO selectively inhibits TFEB's transcriptional activity at the genomic scale according to RNA sequencing analyses, blocks autophagy in a dose-dependent manner, and increases the sensitivity of glioblastoma to temozolomide in vivo. Together, this work reveals that TFEB is targetable and presents the first direct TFEB inhibitor EO, a drug compound with great potential to benefit a wide range of cancer therapies by inhibiting autophagy.

Assembly of a pangenome for global cattle reveals missing sequences and novel structural variations, providing new insights into their diversity and evolutionary history.

A cattle pangenome representation was created based on the genome sequences of 898 cattle representing 57 breeds. The pangenome identified 83 Mb of sequence not found in the cattle reference genome, representing 3.1% novel sequence compared with the 2.71-Gb reference. A catalog of structural variants developed from this cattle population identified 3.3 million deletions, 0.12 million inversions, and 0.18 million duplications. Estimates of breed ancestry and hybridization between cattle breeds using insertion/deletions as markers were similar to those produced by single nucleotide polymorphism-based analysis. Hundreds of deletions were observed to have stratification based on subspecies and breed. For example, an insertion of a Bov-tA1 repeat element was identified in the first intron of the APPL2 gene and correlated with cattle breed geographic distribution. This insertion falls within a segment overlapping predicted enhancer and promoter regions of the gene, and could affect important traits such as immune response, olfactory functions, cell proliferation, and glucose metabolism in muscle. The results indicate that pangenomes are a valuable resource for studying diversity and evolutionary history, and help to delineate how domestication, trait-based breeding, and adaptive introgression have shaped the cattle genome.

Rice EIL1 interacts with OsIAAs to regulate auxin biosynthesis mediated by the tryptophan aminotransferase MHZ10/OsTAR2 during root ethylene responses.

Ethylene plays essential roles in adaptive growth of rice (Oryza sativa). Understanding of the crosstalk between ethylene and auxin (Aux) is limited in rice. Here, from an analysis of the root-specific ethylene-insensitive rice mutant mao hu zi 10 (mhz10), we identified the tryptophan aminotransferase (TAR) MHZ10/OsTAR2, which catalyzes the key step in indole-3-pyruvic acid-dependent Aux biosynthesis. Genetically, OsTAR2 acts downstream of ethylene signaling in root ethylene responses. ETHYLENE INSENSITIVE3 like1 (OsEIL1) directly activated OsTAR2 expression. Surprisingly, ethylene induction of OsTAR2 expression still required the Aux pathway. We also show that Os indole-3-acetic acid (IAA)1/9 and OsIAA21/31 physically interact with OsEIL1 and show promotive and repressive effects on OsEIL1-activated OsTAR2 promoter activity, respectively. These effects likely depend on their EAR motif-mediated histone acetylation/deacetylation modification. The special promoting activity of OsIAA1/9 on OsEIL1 may require both the EAR motifs and the flanking sequences for recruitment of histone acetyltransferase. The repressors OsIAA21/31 exhibit earlier degradation upon ethylene treatment than the activators OsIAA1/9 in a TIR1/AFB-dependent manner, allowing OsEIL1 activation by activators OsIAA1/9 for OsTAR2 expression and signal amplification. This study reveals a positive feedback regulation of ethylene signaling by Aux biosynthesis and highlights the crosstalk between ethylene and Aux pathways at a previously underappreciated level for root growth regulation in rice.

CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs.

BACKGROUND: Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair. METHODS: The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or green fluorescent protein (GFP), in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals. RESULTS: CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction. CONCLUSIONS: Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

Arterial Smooth Muscle Cell AKAP150 Mediates Exercise-Induced Repression of CaV1.2 Channel Function in Cerebral Arteries of Hypertensive Rats.

BACKGROUND: Hypertension is a major, prevalent risk factor for the development and progression of cerebrovascular disease. Regular exercise has been recommended as an excellent choice for the large population of individuals with mild-to-moderate elevations in blood pressure, but the mechanisms that underlie its vascular-protective and antihypertensive effects remain unknown. Here, we describe a mechanism by which myocyte AKAP150 (A-kinase anchoring protein 150) inhibition induced by exercise training alleviates voltage-dependent L-type Ca2+ channel (CaV1.2) activity and restores cerebral arterial function in hypertension. METHODS: Spontaneously hypertensive rats and newly generated smooth muscle-specific AKAP150 knockin mice were used to assess the role of myocyte AKAP150/CaV1.2 channel in regulating cerebral artery function after exercise intervention. RESULTS: Activation of the AKAP150/PKCα (protein kinase Cα) signaling increased CaV1.2 activity and Ca2+ influx of cerebral arterial myocyte, thus enhancing vascular tone in spontaneously hypertensive rats. Smooth muscle-specific AKAP150 knockin mice were hypertensive with higher CaV1.2 channel activity and increased vascular tone. Furthermore, treatment of Ang II (angiotensin II) resulted in a more pronounced increase in blood pressure in smooth muscle-specific AKAP150 knockin mice. Exercise training significantly reduced arterial myocyte AKAP150 expression and alleviated CaV1.2 channel activity, thus restoring cerebral arterial function in spontaneously hypertensive rats and smooth muscle-specific AKAP150 knockin mice. AT1R (AT1 receptor) and AKAP150 were interacted closely in arterial myocytes. Exercise decreased the circulating Ang II and Ang II-involved AT1R-AKAP150 association in myocytes of hypertension. CONCLUSIONS: The current study demonstrates that aerobic exercise ameliorates CaV1.2 channel function via inhibiting myocyte AKAP150, which contributes to reduced cerebral arterial tone in hypertension.

Thermochromic Energy Efficient Windows: Fundamentals, Recent Advances, and Perspectives.

Thermochromic energy efficient windows represent an important protocol technology for advanced architectural windows with energy-saving capabilities through the intelligent regulation of indoor solar irradiation and the modulation of window optical properties in response to real-time temperature stimuli. In this review, recent progress in some promising thermochromic systems is summarized from the aspects of structures, the micro-/mesoscale regulation of thermochromic properties, and integration with other emerging energy techniques. Furthermore, the challenges and opportunities in thermochromic energy-efficient windows are outlined to promote future scientific investigations and practical applications in building energy conservation.

Atypical behaviour and connectivity in SHANK3-mutant macaques.

Mutation or disruption of the SH3 and ankyrin repeat domains 3 (SHANK3) gene represents a highly penetrant, monogenic risk factor for autism spectrum disorder, and is a cause of Phelan-McDermid syndrome. Recent advances in gene editing have enabled the creation of genetically engineered non-human-primate models, which might better approximate the behavioural and neural phenotypes of autism spectrum disorder than do rodent models, and may lead to more effective treatments. Here we report CRISPR-Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques (Macaca fascicularis) and their F1 offspring. Genotyping of somatic cells as well as brain biopsies confirmed mutations in the SHANK3 gene and reduced levels of SHANK3 protein in these macaques. Analysis of data from functional magnetic resonance imaging revealed altered local and global connectivity patterns that were indicative of circuit abnormalities. The founder mutants exhibited sleep disturbances, motor deficits and increased repetitive behaviours, as well as social and learning impairments. Together, these results parallel some aspects of the dysfunctions in the SHANK3 gene and circuits, as well as the behavioural phenotypes, that characterize autism spectrum disorder and Phelan-McDermid syndrome.

Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

LPS-induced monocarboxylate transporter-1 inhibition facilitates lactate accumulation triggering epithelial-mesenchymal transformation and pulmonary fibrosis.

The epithelial-mesenchymal transformation (EMT) process of alveolar epithelial cells is recognized as involved in the development of pulmonary fibrosis. Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung tissue and elevated lactate concentration are associated with the pathogenesis of sepsis-associated pulmonary fibrosis. However, it is uncertain whether LPS promotes the development of sepsis-associated pulmonary fibrosis by promoting lactate accumulation in lung tissue, thereby initiating EMT process. We hypothesized that monocarboxylate transporter-1 (MCT1), as the main protein for lactate transport, may be crucial in the pathogenic process of sepsis-associated pulmonary fibrosis. We found that high concentrations of lactate induced EMT while moderate concentrations did not. Besides, we demonstrated that MCT1 inhibition enhanced EMT process in MLE-12 cells, while MCT1 upregulation could reverse lactate-induced EMT. LPS could promote EMT in MLE-12 cells through MCT1 inhibition and lactate accumulation, while this could be alleviated by upregulating the expression of MCT1. In addition, the overexpression of MCT1 prevented LPS-induced EMT and pulmonary fibrosis in vivo. Altogether, this study revealed that LPS could inhibit the expression of MCT1 in mouse alveolar epithelial cells and cause lactate transport disorder, which leads to lactate accumulation, and ultimately promotes the process of EMT and lung fibrosis.