Biology subjects

Dong, X.

Publications and source records attributed to Dong, X..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Microengineered transplantation of human solid tumors for in vitro studies of CAR T immunotherapy

Treatment of solid malignancies using chimeric antigen receptor (CAR) T cells remains a significant challenge, but current efforts to advance this therapy are challenged by our limited capacity to probe and understand cancer-immune interactions in human solid tumors. Here, we present a microengineered platform for in vitro modeling of malignant solid tumors during CAR T therapy. This system makes it possible to vascularize human tumor explants and perfuse them with blood-borne immune cells in a controlled manner. We first present a microphysiological model of human lung adenocarcinomas infused with CAR-T cells and show how this system can be used to simulate, visualize, and interrogate tumor-directed trafficking and effector function of CAR T cells. We then demonstrate the proof-of-principle of testing a chemokine-directed CAR T cell engineering strategy in a model of malignant pleural mesothelioma and validating our in vitro assessment using a matching in vivo mouse model. Finally, we describe a potential therapeutic target discovered by single-cell RNA sequencing that can be pharmacologically modulated to increase the efficacy of CAR T cells for lung adenocarcinoma, for which we also present specific biomarkers identified by global metabolomics analysis. We believe that the bioengineering principle demonstrated here will make important contributions to developing new capabilities for preclinical studies of adoptive cell therapies for cancer and other complex diseases.

bioengineering↗

bioRxiv2025

Longitudinal single-cell RNA sequencing of a neuroendocrine transdifferentiation model reveals transcriptional reprogramming in treatment-induced neuroendocrine prostate cancer

Neuroendocrine transdifferentiation (NEtD) of prostate adenocarcinoma (PRAD) leads to aggressive neuroendocrine prostate cancer (NEPC). The LTL331 patient-derived xenograft (PDX) model consistently progresses to NEPC following castration, mimicking clinical responses to androgen-deprivation therapy. Here we tracked NEtD using longitudinal single-cell RNA sequencing (scRNA-seq) across eight time points in LTL331 from pre- to post-castration. Castration led to the loss of AR-high PRAD cells, expansion of AR-low populations, and emergence of an AR- and NE-negative (AR-/NE-) intermediate state that transitioned into NEPC. We delineate a model in which pre-EMT cells enriched in ciliogenesis and cell-adhesion pathways differentiate into EMT-like populations before branching into distinct ASCL1+ and ASCL1- NEPC states. The EMT-enriched intermediate, marked by progenitor and neural crest stem-cell genes, acts as a transition bridge, suggesting EMT-associated stemness underlies lineage plasticity. A terminal ASCL1-NEPC state also suggests that ASCL1 is not essential for NEPC maintenance. Gene regulatory analysis highlighted key regulators driving NEtD, including MSX1 and ASCL1 in early EMT-like and NEPC states, respectively. These transcriptional states were validated in both patient-derived bulk and scRNA-seq data. Our findings offer insights into intervention strategies to delay or prevent NEtD with the potential of identifying novel prognostic and therapeutic targets.

cancer biology↗

bioRxiv2025

Fucoidans are senotherapeutics that enhance SIRT6-dependent DNA repair

Aging is marked by the accumulation of senescent cells (SnCs), which contribute to tissue dysfunction and age-related diseases. Senotherapeutics, including senolytics which specifically induce lysis of SnCs and senomorphics, which suppress the senescence phenotype, represent promising therapeutic interventions for mitigating age-related pathologies and extending healthspan. Using a phenotypic-based senescent cell screening assay, we identified fucoidans, a class of sulfated polysaccharides derived from brown algae and seaweed, as novel senotherapeutics. In particular, fucoidan from Fucus vesiculosus (Fucoidan-FV) displayed potent senomorphic activity in different types of SnCs, reduced senescence in multiple tissues in aged mice, and extended healthspan in a mouse model of accelerated aging. Fucoidan-FV also enhanced the deacetylation and mono-ADP-ribosylation (mADPr) activity of SIRT6 and improved DNA repair and reduced senescence, in part, through SIRT6-dependent pathways. In addition, Fucoidan-FV downregulated genes associated with inflammation, Wnt signaling, and ECM remodeling pathways in SnCs and increased expression of genes involved with DNA repair. These findings support the translational potential of fucoidans as novel senotherapeutics that also are able to improve SIRT6-mediated DNA repair.

cell biology↗

bioRxiv2025

Shared genomic architecture of the brain white matter structural connectome and intelligence

White matter (WM) connections, which facilitate communication between brain regions, substantially impact intellectual performance. However, the shared genetic underpinnings of WM connections and intelligence remain unclear. In the present study, we first conducted genome-wide association studies on the global and regional topological properties of WM connectomes constructed from diffusion-weighted and T1-weighted magnetic resonance images of 26,655 participants from the UK Biobank. Forty-one independent significant single-nucleotide polymorphisms (SNPs) associated with global WM connectome efficiency and 45 SNPs linked to nodal efficiency for 246 brain regions were identified. Then, regional heterogeneity was demonstrated for the genetic correlations between WM connectome efficiency and intelligence. The nodal efficiency of 128 regions with nominally significant genetic correlations with intelligence was determined. Among these, 44 regions and 3 regions shared SNPs with intelligence within the chromosome 6q21 and 3p21.1 locus respectively, and significant causal effects on intelligence were found for 63 regions, mainly in the orbital gyrus and superior frontal gyrus. Finally, we found that the integration of polygenetic score and WM connectome efficiency data was more accurate for predicting individual intelligence than the use of a single data type. These findings improve our understanding of how genes shape the individual WM connectome and intelligence and the relationship between WM connectomes and intelligence.

genomics↗

bioRxiv2025

DTX4 regulates neural progenitor transitions during cortical development

Neural progenitors drive cellular diversification in the neocortex. Prolongation of their proliferative capacity and increased diversity underpins the evolutionary expansion and morphological complexity of the human neocortex. Here, we investigate the mechanisms that regulate maintenance of the highly proliferative early neural progenitor subtypes and transition to subsequent progenitors of limited proliferative capacity during human and murine neocortical development. We identify DTX4, a Deltex family member, as an evolutionarily conserved molecular determinant of neural progenitor identity in the mammalian neocortex. DTX4 sustains the identity of radial glia, a highly proliferative progenitor subtype. Loss of DTX4, on the other hand, is a prerequisite for the generation of intermediate progenitors which possess low proliferative capacity. Perturbing DTX4 expression in human cerebral organoids and in the murine neocortex, alters progenitor composition, thereby inducing changes in neuronal diversity and cortical morphology. Mechanistically, we reveal that DTX4 controls progenitor identity by regulating the length of the cell cycle. Our findings underscore the critical role of cell cycle dynamics and of DTX4 in determining progenitor identity and thereby defining neuronal outcome during mammalian development. Highlights[bullet] The Deltex family member, DTX4, is expressed in human and murine neuroepithelia and radial glial progenitors but is absent in intermediate progenitor cells. [bullet]DTX4 maintains radial glia identity and prevents generation of intermediate progenitors during murine and human development. [bullet]Disruption of DTX4 expression compromises neuronal composition and neocortical architecture [bullet]DTX4 regulates progenitor identity by regulating cell cycle length

neuroscience↗

bioRxiv2025

Identification of Novel Mutations Causing Clofazimine Resistance in Mycobacterium intracellulare

Clofazimine (CFZ) is a promising repurposed drug for treating Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD), but its resistance mechanisms in M. intracellulare remain poorly understood. In this study, we generated 36 CFZ-resistant M. intracellulare mutants in vitro and identified various mutations in the marR gene (WP_009952290.1) in 61% of resistant mutants through whole-genome sequencing. Mutations were identified in additional genes encoding flavin-dependent oxidoreductase (ssuD, C67A), membrane lipoprotein (lppI, C207 deletion), glucose-methanol-choline oxidoreductase (G157 deletion), MASE1 domain-containing protein (C62G), and PPE family protein (222C deletion). Gene complementation experiments demonstrated that introducing the wild-type marR in CFZ-resistant strains (L72 and L74) with marR mutations reduced CFZ minimum inhibitory concentrations (MICs) from 1 g/mL to the susceptible baseline (0.25 g/mL), confirming its critical role in CFZ resistance. Notably, the M. intracellulare MarR lacks homology to M. tuberculosis MarR family protein Rv0678 (MmpR) whose mutations are involved in CFZ and bedaquiline (BDQ) resistance and is flanked by non-efflux pump genes (dhmA and doxX), and unlike M. tuberculosis, its mutation does not cause bedaquiline cross-resistance, indicating a different MarR and distinct regulatory mechanism for CFZ resistance in M. intracellulare. This work highlights marR as a key determinant of CFZ resistance in M. intracellulare and underscores the need for further mechanistic studies with implications for clinical diagnostics and therapeutic strategies.

microbiology↗

bioRxiv2025

Emergence of a New ST617 Hypervirulent Klebsiella pneumoniaewith ESBL+ and Strong Biofilm Forming Ability in China

The increasing infections caused by hypervirulent Klebsiella pneumoniae (hvKP) resistant to carbapenem, broad-spectrum {beta}-lactam and colistin has posed great challenges to global public health. However, the typing and detailed molecular features of the multi-drug resistant hvKP remain to be improved. Here, we characterized a broad-spectrum {beta}-lactam-resistant K. pneumoniae ST617 isolate that has not been previously reported to cause severe disease in China. We obtained the whole-genome sequence of one K. pneumoniae ST617 isolate--HZ7--from a patient with bile duct infection. Phylogenetic reconstruction was performed to investigate the possible host evolutionary origin of HZ7, and the virulence property was studied in comparison with the nearest ST617 neighbours--YZ strains isolated from patient blood infections. Our studies indicated that the HZ7 is an ESBL+ Kp that is likely evolved from environmental or animal-derived sources. Importantly, the virulence assay showed that HZ7 was highly virulent and compared with the well known hvKP strain--NTUH-K2044 and the highly virulent clinical strain ST11-K64--HZ54--isolated from a patient with severe pneumonia. In addition, HZ7 showed strong biofilm formation ability. Whole-genome sequencing analysis identified seven potential virulence genes in HZ7: kp7_000748, kp7_002156, kp7_002157, kp7_002158, kp7_002159, kp7_003103, kp7_003104, which are absent in K. pneumoniae patient isolates from Yangzhou and the environmental samples. Finally, transcriptomic analysis of HZ7 identified six genes--wcaJ, fimA, mrkH, pgaA, ugd, and gndA that are associated with biofilm formation. Given its high virulence likely associated with the seven unique genes acquired via horizontal transfer and strong biofilm forming ability, this strain should be closely monitored to prevent wider spread in the clinic.

microbiology↗

bioRxiv2025

Foliar gas exchange, morphology, and cannabinoid contents of three hemp varieties in southwest Texas

In the US, a high level ([≥] 0.3%) of intoxicating {Delta}-9-tetrahydrocannabinol (THC) threatens farm-scale production of industrial of Hemp (Cannabis sativa L. ssp. sativa), but the linkage between THC and major physiol-morphological traits of hemp is not well-known. This study aims to characterize the variations in physiological and/or morphological parameters and cannabinoid contents of three hemp varieties, i.e., Berry Blossom, Painted Lady, and Skipper. Diurnal foliar gas exchange, chlorophyll fluorescence, water potential, and canopy temperature were measured on five clear days in the 2022 growing season, and cannabinoids were measured at peak flowering using high-performance liquid chromatography. Allometric equations were developed to use easily measured biomass or morphological variables to predict variables that are more difficult to measure. The diurnal foliar gas exchange of the three hemp varieties was largely unaffected by the high temperatures of southwest Texas, with Berry Blossom and Skipper showing the highest and lowest photosynthesis, respectively, and Painted Lady having the most efficient stomatal control of gas exchange. Although the rooting depth of Berry Blossom was shallower than that of the two other varieties, there was no evidence showing the effect of rooting habit on the physiology of the studied hemp varieties, which was presumably due to the lack of water stress in our experiment. Nor were there significant differences in the cannabinoid contents in relation to environmental and varietal responses, as the measured THC contents of all three varieties were under 0.3%. Overall, the three hemp varieties showed different behavior strategies in southwest Texas.

plant biology↗

bioRxiv2025

Phagosome-mediated anti-bacterial immunity is governed by the proton-activated chloride channel in peritoneal macrophages

The success of phagosome degradation relies on the ability of phagocytes to regulate the maturation of phagosomes. However, its underlying molecular mechanisms remain poorly understood. Here, we identify the proton-activated chloride (PAC) channel as a key negative regulator of phagosome maturation. PAC deletion enhanced phagosomal acidification and protease activities, leading to augmented bacterial killing in large peritoneal macrophages (LPMs) upon Escherichia coli infection in mice. Surprisingly, phagosome degradation also stimulated STING-IRF3-interferon responses and inflammasome activation in LPMs, both of which are enhanced upon PAC deletion. The increased inflammasome activation induced the release of cleaved gasdermin D, which localized to the surface of bacteria in the peritoneum and further contributed to their killing. Finally, enhanced bacterial clearance by PAC-deficient LPMs reduced proinflammatory immune cell infiltration and peritoneal inflammation, resulting in improved survival in mice. Our study thus provides new insights into the molecular mechanism of phagosome maturation and the dynamics of host defense response following phagosome-mediated bacterial degradation in peritoneal macrophages. SummaryThe PAC channel mediates phagosome maturation during bacterial infection in macrophages. PAC deletion promotes phagosome-mediated STING-interferon signaling and inflammasome-mediated gasdermin D secretion during bacterial infection in peritoneal macrophages.

immunology↗

bioRxiv2025

Does sample size of leaf osmotic potential affect its relationship with cotton yield?

Leaf osmotic potential at full turgor ({pi}0) has been used frequently to indicate turgor loss point of plant leaves. However, even a rapid measurement of{pi} 0 using osmometry is time-consuming, if numerous leaf samples need to be measured. Because of this, researchers tend to use a small sample size to determine{pi} 0 and relate it to indices of crop performance. Yet the statistical and agronomic significance of using a small sample size of{pi} 0 to indicate crop performance is not known. We address this question using field measurements and statistical resampling. Six mature leaf samples were collected at the peak bloom stage from each of the 54 cotton plots in Texas, USA in 2024. The{pi} 0 of the collected leaves were measured using an osmometer. Seed cotton yields from the field plots were measured near the end of cotton season. To test the effect of sample size on strength of the linear relation between{pi} 0 and cotton yield, 1-6 resamples of{pi} 0 were randomly drawn with replacement from the original 6 measurements per plot for the 54 plots. The resampled data of{pi} 0 were then used as independent variable to predict cotton yield. We found that, considering the labor and cost, sampling 3 or 6 leaves per plot may not make a significant difference for the linear regression between{pi} 0 and cotton yield.

plant biology↗

bioRxiv2025

Carbon monoxide exposure stimulates growth and activity of primary producers in diverse soil ecosystems

Carbon monoxide (CO) is both a potent poison for many aerobic organisms and a desirable energy source for diverse microorganisms. Atmospheric emissions of this gas have increased since industrialization and their levels are highly elevated in many urban and natural environments; however, it is unresolved whether elevated levels of CO at environmentally relevant concentrations are primarily stimulatory or inhibitory to soil microbial communities. Here, we showed that CO exposure minimally affects microbial abundance, richness, and composition in diverse ecosystem soils, suggesting most microbes are tolerant of this gas. Genome-resolved metagenomic profiling showed that these soils harbored diverse bacteria capable of using CO an electron donor for aerobic respiration and carbon fixation. CO stimulated the growth of several of these putative CO-oxidizing bacteria in a dose-dependent manner, especially widespread proteobacterial and actinobacterial lineages. Additionally, we found a strong relationship between CO oxidation and carbon fixation and observed an enrichment of several carboxydotrophic MAGs capable of carbon fixation in most soil types. These findings highlight that environmentally relevant CO levels do not inhibit soil microbes, but instead foster the growth of distinct carboxydotrophic bacteria, suggesting a robust soil CO sink that could help mitigate anthropogenic CO emissions and contribute to carbon cycling in terrestrial ecosystems.

biochemistry↗

bioRxiv2025

Single cell resolved spatial immune repertoire unveils spatial heterogeneity of lymphoid aggregates in human immune disorders

Adaptive immunity, mediated by T and B cell responses, is essential for defending against infections and cancers while also being implicated in autoimmune diseases. Tracking T and B cell repertoires in situ at single-cell resolution is essential for understanding adaptive immune responses. To address the lack of tools for in situ single-cell T/BCR (XCR) sequencing, we developed Stereo-XCR-seq, an efficient strategy for retrieving and sequencing TCR and BCR from Stereo-seq cDNA libraries at subcellular resolution. Stereo-XCR-seq provides unbiased full-length XCR reads alongside spatial transcriptomics, enabling the identification of heterogeneous lymphoid aggregates with distinct clonal activities in cancers and inflammatory bowel disease (IBD). We identified plasma cell aggregates that differ from tertiary lymphoid structures (TLSs) in both transcriptomic profiles and clonal activities, with spatial positioning potentially mediating unique immune responses. Collectively, Stereo-XCR-seq enables in situ single-cell profiling of T and B cell clonal activities within tissue microenvironments, providing insights into lymphocyte adaption to environmental stimuli. This technology provides potential for advancing our understanding of tissue immunity and the development of therapeutic strategies for immune disorders.

immunology↗

bioRxiv2025

Distinct Classes of Gut Bacterial Molybdenum-Dependent Enzymes Produce Urolithins

Urolithin A is an anti-aging and anti-inflammatory gut bacterial metabolite derived from ellagic acid (EA), a polyphenol abundant in berries and nuts. The conversion of EA to urolithin A involves multiple chemically challenging phenol dehydroxylation steps that produce urolithins with varying bioactivities. Despite their biological and chemical significance, the bacterial enzymes responsible for urolithin production remain unidentified. Here, we use differential gene expression analysis, anaerobic protein expression, and enzyme assays to identify members of two distinct molybdenum enzyme families (the DMSO reductase family and the xanthine oxidase family) capable of regioselective dehydroxylation and urolithin generation. These two enzyme families have distinct substrate requirements, suggesting they employ different catalytic mechanisms for phenol dehydroxylation. Multi-omics analysis of a human cohort uncovers decreased levels of urolithin A and genes encoding urolithin A-producing enzymes in patients with inflammatory bowel disease (IBD), implying reduced health effects of EA consumption in this setting. Together, this study elucidates the molecular basis of urolithin production, expands the known enzymatic repertoire of the human gut microbiome, and suggests a potential link between gut bacterial urolithin production and host inflammation. Significance statementThe human gut microbiome modulates the health effects of dietary compounds by modifying their chemical structures. For example, gut microbes extensively metabolize polyphenols, a group of diverse plant-derived compounds associated with positive health outcomes. Urolithin A, a gut bacterial metabolite derived from a polyphenol abundant in berries and nuts, exhibits potent anti-inflammatory activity. However, the gut bacterial enzymes involved in its production remain largely unknown. Here, we report urolithin-producing gut bacterial enzymes, including four phenol dehydroxylases from two distinct molybdenum-dependent enzyme families. Analyzing human gut microbiomes suggests a potential link between genes encoding these enzymes and host inflammation. Together, our findings fully map urolithin A production to enzymes, increasing our understanding of how the gut microbiome can alter the impacts of diet on human health.

biochemistry↗

bioRxiv2025

Clinical and Taxonomic Characterization of a Novel Urinary Tract Pathogen Providencia lanzhouensis

BackgroundThe genus Providencia includes species of ecological and clinical significance, with some acting as opportunistic pathogens in hospital-acquired infections such as urinary tract infection (UTI). However, overlapping phenotypic traits and genetic similarities pose challenges for accurate species characterization. This study exemplifies this challenge by identifying a novel Providencia isolate through genomic analysis. ResultsIn this study, strain PAZ2 was isolated from the urine sample of a hospitalized patient in Lanzhou, Gansu Province, China. Initial mass spectrometry analysis identified the strain as Providencia alcalifaciens. It exhibits a resistance profile against antibiotics including ampicillin, tetracycline, tigecycline, polymyxin B, colistin, trimethoprim-sulfamethoxazole, and ciprofloxacin. However, whole-genome sequence analysis indicated that this strain is a novel species with an average nucleotide identity (ANI) values ranging from 79.92% to 94.73% and digital DNA-DNA hybridization (dDDH) values between 21.2% and 57.6% when compared with all known Providencia species. Furthermore, strain PAZ2 exhibited unique biochemical properties that differentiated it from all previously described species within the genus. ConclusionsGenomic and biochemical evidence supports PAZ2 as a novel Providencia species, which we named Providencia lanzhouensis sp. nov. This finding enhances the taxonomy of Providencia and underscores its clinical relevance, notably in UTIs and other nosocomial infections. Accurate identification is crucial for guiding effective treatment and preventing misdiagnosis. Further studies should investigate its pathogenicity, antimicrobial resistance, and epidemiology to inform infection control measures.

microbiology↗

bioRxiv2025

An improved rapid and sensitive long amplicon method for nanopore-based RSV whole genome sequencing

BackgroundWhole-genome sequencing (WGS) provides critical insights into the Respiratory syncytial virus (RSV) transmission and any emerging mutations that could impair the efficacy of monoclonal antibodies or vaccines that have been recently licenced for clinical use worldwide. However, the ability to sequence RSV genomes at large scale is limited by expensive and time-consuming sequencing methods. Oxford Nanopore Technology (ONT) offers significant improvements in next generation sequencing (NGS) both in turnaround time and cost, compared to other platforms for viral WGS. MethodsWe have developed and modified an RSV long amplicon-based WGS protocol for the ONT platform using a one-step multiplex RT-PCR assay and the rapid library barcoding kit. 135 RSV positive Australian clinical specimens (91 RSV-A and 44 RSV-B) sampled in 2023 with Cycle threshold (Ct) values between 14 to 35 were tested in this study. This ONT workflow was compared with other recent RSV WGS amplification assays based on short amplicons. ResultsA PCR amplicon clean-up step prior to library preparation significantly improved WGS result for samples with poor amplicon generation, but it is not necessary or beneficial for ones that generated high concentrations of amplicons. Overall, a success rate of 85.9% was achieved for WGS. This method performed as well as the more complex short amplicon methods in terms of genome coverage and sequencing depth. ConclusionsThe workflow described here was highly successful in generating RSV WGS on ONT platform and had improved turnaround times and excellent results with RSV clinical samples with Ct values up to 30.

microbiology↗

bioRxiv2024

Rab2B promotes chaperonin-mediated actin folding and prevents developmental transcription reprogramming and quiescence in trypanosomes

The Rab family of small GTPases are regulators of intracellular membrane trafficking. Rab2B, a Golgi-resident Rab GTPase, has been shown to function in ER-Golgi transport and autophagosome-lysosome fusion in mammalian cells and Drosophila. In Trypanosoma brucei, the autophagy-related function of Rab2B was conserved and depletion of Rab2B blocked autophagosome-lysosome fusion. Intriguingly, depletion of Rab2B induced differentiation of T. brucei from a proliferative slender form to a quiescent stumpy form, a step crucial for parasite transmission. Chaperonin CCT/TRiC was identified as an effector of Rab2B. Depletion of CCT/TRiC subunits or actin (a known CCT/TRiC substrate), or blocking actin polymerisation with Latrunculin A all triggered differentiation, suggesting a role of Rab2B on CCT/TRiC-mediated actin folding. Using an actin chromobody, we identified a pool of nuclear actin that is crucial for transcription reprogramming and life cycle differentiation in T. brucei. The study revealed a function of Rab2B and actin in T. brucei transcription reprogramming leading to cell differentiation, and an unexpected role of Rab2B and CCT/TRiC mediated actin folding.

cell biology↗

bioRxiv2024

Microbial virulome and resistome in deep-sea cold seeps pose minimal public health risks

Deep-sea cold seeps host high microbial biomass and biodiversity that thrive on hydrocarbon and inorganic compound seepage, exhibiting diverse ecological functions and unique genetic resources. However, potential health risks from pathogenic or antibiotic-resistant microorganisms in these environments remain largely overlooked, especially during resource exploitation and laboratory research. Here, we analyzed 165 metagenomes and 33 metatranscriptomes from 16 global cold seep sites to investigate the diversity and distribution of virulence factors (VFs), antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs). A total of 2,353 VFs are retrieved in 689 MAGs, primarily associated with non-pathogenic functions like adherence. Additionally, cold seeps harbor nearly 100,000 ARGs, as important reservoirs, with high-risk ARGs presenting at low abundance. Compared to other environments, microorganisms in cold seeps exhibit substantial differences in VF and ARG counts, with potential horizontal gene transfer facilitating their spread. These virulome and resistome profiles provide valuable insights into the evolutionary and ecological implications of pathogenicity and antibiotic resistance in extreme deep-sea ecosystems. Collectively, these results indicate that cold seep sediments pose minimal public health risks, shedding lights on environmental safety in deep-sea resource exploitation and research. ImportanceUnderstanding pathogenicity and antibiotic resistance in environmental reservoirs like deep-sea cold seeps is critical for assessing public health risks, particularly with increasing human activities such as deep-sea mining. This study offers the first comprehensive analysis of virulome, resistome, and mobilome profiles in cold seep microbial communities. While cold seeps act as reservoirs for diverse ARGs, high-risk ARGs are rare, and most VFs contribute to non-pathogenic ecological functions. These results highlight the minimal threat to public health posed by cold seeps, providing a reference for monitoring the spread of pathogenicity and resistance in extreme ecosystems and informing environmental safety assessments during deep-sea resource exploitation.

microbiology↗

bioRxiv2024

Engineered mitochondrial A-to-G editors with enhanced efficiency and targeting scope

Mitochondrial base editing is a powerful technology, but the current A-to-G mitochondrial DNA (mtDNA) base editors are reluctant to achieve high efficiency, which is critical for mtDNA engineering. Through laboratory evolution, TadA-8e variants were discovered with substantially increased activity and expanded targeting compatibility, especially for previously unfavorite sequence contexts, when applied to both nuclear and mitochondrial ABEs. Further engineering of the mtDNA editors (eTd-mtABEs) dramatically reduced both DNA and RNA off-targeting effects and enhanced strand-selective A-to-G editing with substitution of DddA to DNA nickases. Moreover, the eTd-mtABEs induced up to 145-fold editing frequencies compared to previous mtDNA adenine base editors in rat cells, and installed targeted mutations in all injected rat embryos with up to 74% efficiency in founders, resulting generation of SNHL and Leigh syndrome models with severe defects. It suggests that eTd-mtABEs are promising mtDNA engineering technology for basic research and for translational studies. HighlightsO_LIEngineered TadA variants enhance nuclear and mitochondrial ABE activity in all sequence contexts. C_LIO_LIAdditional mutations in eTd-mtABEs further minimize both DNA and RNA off-targeting effects. C_LIO_LIEngineered TadA variants are compatible to DNA nickase assisted strand-selective mtDNA editing. C_LIO_LIeTd-mtABEs enable highly efficient generation of rat mtDNA disease models with severe phenotypes. C_LI

bioengineering↗