Biology subjects

Dong, F.

Publications and source records attributed to Dong, F..

At least 19 recordsLinked to original sources

bioRxiv2026

A genetically encoded redox bottleneck constrains human developmental rate

The intrinsically slow pace of human development poses challenges for regenerative medicine and disease modeling. This trait is attributed to low metabolic rates, yet the endogenous mechanisms determining species-specific metabolic flux remain unknown. Here, we identify coupling between glycolytic NADH production and mitochondrial oxidation through the glycerol-3-phosphate (G3P) shuttle as a genetic bottleneck constraining human developmental tempo. Using stem cell-derived models of the segmentation clock, an oscillator whose period reflects developmental rate, we show that low expression of the G3P shuttle enzyme GPD1L limits NADH oxidation in human progenitors compared to mouse. Overexpressing GPD1L boosts metabolic flux, accelerating the segmentation clock, cell cycle, and differentiation across germ layers. G3P-mediated redox coupling is thus a genetically encoded, rate-limiting mechanism that sets the tempo of human development.

developmental biology↗

bioRxiv2026

Protein Phosphatase 2A Activation Attenuates Acute Myocardial Injury in Takotsubo Syndrome by Modulating Ferroptosis and Mitochondrial Injury in Cardiomyocytes

BackgroundTakotsubo syndrome (TTS) is an acute stress-induced cardiomyopathy characterized by transient left ventricular dysfunction. Despite its reversible nature, TTS is associated with substantial morbidity and mortality in the acute phase, and no specific treatments are available. The precise molecular mechanisms that connect catecholamine stress to reversible myocardial injury are not fully understood. PP2A, a holoenzyme with serine/threonine phosphatase activity, plays a vital role in normal cardiac development, and its dysregulation has been associated with heart disease. Its role in TTS and its potential as a therapeutic target remain completely unknown. MethodsAnalysis of public multi-omics datasets from stress cardiomyopathy (SCM) and experimental models of TTS, along with treatment of cardiomyocytes with human TTS plasma, was used to investigate a potential role for protein phosphatase 2A (PP2A) in stress-induced myocardial injury. To clarify the functional impact of manipulating PP2A activity in TTS, we used a series of disease relevant cell based and in vivo models, leveraging both genetic and pharmacological approaches to modulate PP2A activity in cardiomyocytes and in mice. To gain mechanistic insights into how PP2A influences TTS pathology and downstream signaling pathways, RNA sequencing, stress-responsive iron handling, mitochondrial function, and cardiac phenotypes were thoroughly evaluated in both in vivo and in vitro studies. ResultsPP2A activity was markedly reduced in cardiac tissues from mice with isoprenaline-induced TTS, as well as in isoprenaline-treated cardiomyocytes. Genetic or pharmacological inhibition of PP2A worsened catecholamine-induced cardiac dysfunction and myocardial injury. Most notably, pharmacological activation of PP2A using an orally bioavailable small-molecule activator strongly mitigated myocardial damage and enhanced cardiac function in TTS models. Mechanistically, PP2A inactivation promoted JNK-MAPK signaling and dysregulated stress-responsive iron-handling pathways, leading to ferritinophagy-mediated ferroptosis and mitochondrial dysfunction. Pharmacological JNK inhibition effectively rescued myocardial injury caused by PP2A deficiency in two TTS animal models and in cardiomyocytes. ConclusionsPP2A inactivation is a key molecular event linking catecholamine stress to myocardial injury in TTS. Restoring PP2A activity or inhibiting downstream JNK attenuates ferritinophagy-dependent stress responses and mitochondrial dysfunction, providing a unifying mechanistic framework and highlighting the PP2A-JNK axis as a potential target for short-term intervention during the acute phase of TTS. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Protein phosphatase 2A (PP2A) activity is acutely suppressed during the early phase of Takotsubo syndrome (TTS), with preferential involvement of the apical myocardium, closely mirroring the diseases characteristic clinical phenotype. * Downstream of PP2A inactivation, catecholamine stress triggers JNK-MAPK signaling activation and dysregulated stress-responsive iron handling, including ferritinophagy-mediated ferroptosis and mitochondrial dysfunction in cardiomyocytes. * Pharmacological restoration of PP2A activity or downstream inhibition of JNK mitigates acute myocardial injury and preserves cardiac function, thereby establishing the PP2A-JNK axis as a central stress-responsive pathway in TTS. What Are the Clinical Implications?* Identification of PP2A inactivation as a key molecular event highlights a novel opportunity for targeted intervention during the acute phase. * Studies in preclinical animal TTS models provide the first evidence that illuminates the therapeutic potential of a small-molecule PP2A activator as a strategy for acute TTS and may serve as a broad cardioprotective strategy in settings where cardiac dysfunction is implicated. * Targeting downstream JNK signaling, which is activated by PP2A inactivation, emerges as an additional mechanism-based strategy for attenuating acute myocardial injury in TTS.

molecular biology↗

bioRxiv2026

A cinnamyl alcohol dehydrogenase scaffold organizes monoterpenoid indole alkaloid biosynthesis

Biosynthesis of ~3,000 monoterpenoid indole alkaloids (MIAs), including the anticancer drug vinblastine, involves the highly unstable intermediate strictosidine aglycone. Its formation by strictosidine {beta}-glucosidase (SGD) and subsequent conversion by geissoschizine synthase (GS) occur in spatially separated compartments, representing a major biosynthesis bottleneck. Here we discover VinBLAST, a cinnamyl alcohol dehydrogenase-like protein repurposed as a scaffold for efficient processing of this labile intermediate. VinBLAST physically mediates SGD and GS interaction in the nucleus and allosterically enhances GS catalytic efficiency. VinBLAST homologues from diverse plant families enhance biosynthesis of several representative MIAs, with the production of catharanthine increased to ~160 mg L-1 in yeast, nearly 1,000-fold higher than previous studies. Our discovery provides a missing link in organizing MIA biosynthesis and enables scalable bioproduction of geissoschizine-derived therapeutics.

synthetic biology↗

bioRxiv2026

An Energy Landscape Approach to Miniaturizing Enzymes using Protein Language Model Embeddings

AO_SCPLOWBSTRACTC_SCPLOWWe present a general approach to find amino acid sequences corresponding to the most compact enzyme likely to retain the structure of a given catalytic site. Our approach is based on using Monte Carlo (MC) simulations to sample an energy landscape where minima correspond, by construction, to sequences with the aforementioned properties. Building on previous work (Wu et al., 2025) and with the BAGEL package (Lala et al., 2025), we implement a route to achieve this goal using only the information extracted from a protein language model (PLM), without structural information. After generating a set of candidate sequences with this PLM-guided BAGEL optimization, we further filter potential candidates for downstream experimental validation using a two-stage protocol. First, deep-learning-based structure prediction models (ESMFold, Chai-1, Boltz-2) are used to identify a structural consensus among designs with highly conserved active-site geometries, yielding many candidates with active-site RMSD below a few angstroms relative to the wild-type and pLDDT scores above 80. Second, molecular dynamics simulations are performed on a filtered subset of sequences (based on active-site RMSD and SolubleMPNN log-likelihoods) to evaluate active-site stability when including thermal fluctuations. For the most promising enzymes, these yield RMSF values in the active site below 1.0 [A] and an active-site RMSD drift between 0.5 and 1.5 [A], making these mini-variants comparable to the wild type, though outcomes vary across enzymes. Given the protocols generality, we believe these results represent a step forward in AI-guided enzyme design. To facilitate rapid experimental validation by the broader community, we open-source all sequences generated by our computational pipeline. These include designs for four representative enzymes of this study: PETase, subtilisin Carlsberg (serine protease), Taq DNA polymerase, and VioA.

bioinformatics↗

bioRxiv2026

Interkingdom glycine conjugates of indole-3-carboxylates are Ah receptor ligands

Substituted indoles are conserved metabolites across all kingdoms of life and may function as a mediators of inter- and intra-species communication. Indole-3-carboxylates (indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA)) represent abundant tryptophan-derived AHR agonists in human serum, potentially influencing AHR-dependent physiology. LC-MS analysis of mouse serum, urine and cecal/fecal contents reveals that both IAA and IPA undergo host and microbial mediated glycine conjugation to facilitate urinary elimination. Notably, at physiologically detectable human serum concentrations (M), IAA-Glycine retains human AHR activation potential. Comparative in silico docking simulations corroborate IAA-Glycine as a direct ligand for the human AHR. Data suggest, in contrast to xenobiotic ligands, AHR activation by endogenous tryptophan metabolites is greater in humans than in mice. These results underscore the role of microbial and host-derived amino acid conjugation in generating bioactive metabolites. Thus, positioning interkingdom auxin chemistry within human physiology and revealing an unexpected link between plants, microbes, and humans.

biochemistry↗

bioRxiv2026

4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity

Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for >90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease. In vitro and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism. Significance StatementInborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease - MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) - and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.

biochemistry↗

bioRxiv2025

Mitochondrial depolarization stabilizes the vitamin B12 chaperone MMADHC in the cytosol to increase MTR activity

Of the [~]1100 mitochondrial proteins, only a handful like PINK1 and ATFS-1 are known to stabilize and relocalize upon collapse of the proton motive force (PMF) to execute signaling roles. To systematically identify genes that increase exclusively at the protein level upon PMF collapse, we performed a joint proteomic and RNA-seq screen. The screen revealed 10 candidates (six mitochondrial), including the vitamin B12 chaperone MMADHC and cytosolic B12-dependent methionine synthase (MTR). MMADHC is short-lived across cell types and we show that its levels increase with PMF collapse. MMADHC stabilization precedes PINK1 activation in a time course of increasing mtDNA depletion, suggesting greater sensitivity to PMF collapse. MMADHC accumulates in mitochondria with LONP1 inhibition but in the cytosol upon PMF collapse, likely due to mitochondrial import failure. Cytosol-stabilized MMADHC increases MTR levels and activity. Altogether, the mitochondrial PMF regulates the cytosolic B12-dependent MTR, integral to one-carbon metabolism, by controlling the stability and compartmentalization of the B12 chaperone MMADHC. Significance StatementHumans have only two vitamin B12-dependent enzymes - mitochondrial MMUT and cytosolic MTR - and both require a common B12 chaperone MMADHC. We discover that MMADHC is a low abundant, short-lived protein that is continuously imported and degraded by energized mitochondria. Upon collapse of the mitochondrial proton motive force, MMADHC accumulates in the cytosol and increases the levels and activity of MTR, critical for one-carbon metabolism. This PMF-dependent regulation of MMADHC stability and localization is important for understanding cofactor rationing and spatiotemporal compartmentalization of B12 metabolism.

biochemistry↗

bioRxiv2025

PLiCat: Decoding protein-lipid interactions by large language model

MotivationProtein-lipid interactions are essential for many cellular processes, as proteins associate with diverse lipid molecules to exert distinct functions. However, existing approaches are limited in discriminating among lipid categories in these interactions. Recent advances in protein language models enable discovery of novel sequence insights. ResultsWe introduce PLiCat (Protein-Lipid interaction Categorization tool), a predictive framework designed to classify lipid categories interacting with proteins. By integrating ESMC and BERT models into a hybrid architecture, PLiCat achieves accurate and interpretable predictions to distinguish lipid-binding signatures across eight major lipid categories. Furthermore, we investigated the potential of PLiCat to identify lipid-binding sites and assess the impact of pathogenic mutations on lipid-binding events. Collectively, PLiCat provides a powerful framework for elucidating the lipid-binding codes, offering new opportunities for exploring lipid-binding specificity and guiding rational protein design. Availability and implementationThe PLiCat source code and processed datasets are available at https://github.com/Noora68/PLiCat.

bioinformatics↗

bioRxiv2025

Pleistocene demographic histories dominate contemporary genomic diversity in a continental radiation of Himalayan-Hengduan songbirds

While population genetic theory expects genetic diversity to scale predictably with population size, mounting empirical evidence has challenged this fundamental prediction. This striking inconsistency has prompted a paradigm shift in our understanding of the determinants of genetic diversity, driving increased efforts to disentangle the relative contributions of Pleistocene demography and linked selection. Here, based on systematic sampling and a unified analytical pipeline, we de novo assembled genomes for 120 songbird species breeding in the Himalayas-Hengduan Mountains (HHMs) and conducted population genomic analysis to examine the drivers of their genomic diversity. We observed a 6.5-fold variation in genome-wide heterogeneity and a 16.4-fold variation nucleotide diversity and across species. Notably, these measures of genomic diversity showed no correlation with recent population dynamics, current population size, or other contemporary factors--such as natural selection, elevational distribution, or life-history traits. Instead, historical demography strongly predicted genetic diversity, with ancestral population size during the late Pleistocene emerging as the sole correlate: larger ancestral sizes consistently coincided with higher diversity. These findings underscore the critical influence of historical demography on contemporary genetic diversity in natural populations--an insight essential for designing effective conservation strategies.

genetics↗

bioRxiv2025

Longitudinal Trajectories of Cognition and Neural Metrics as Predictors of Persistent Distressing Psychotic-Like Experiences Across Middle Childhood and Early Adolescence

ObjectivesPsychotic-like experiences (PLEs) may arise from genetic and environmental risk leading to worsening cognitive and neural metrics over time, which in turn lead to worsening PLEs. Persistence and distress are factors that distinguish more clinically significant PLEs. Analyses used three waves of unique longitudinal Adolescent Brain Cognitive Development Study data (ages 9-13) to test whether changes in cognition and structural neural metrics attenuate associations between genetic and environmental risk with persistent distressing PLEs. MethodsMultigroup univariate latent growth models examined three waves of cognitive metrics and global structural neural metrics separately for three PLE groups: persistent distressing PLEs (n=356), transient distressing PLEs (n=408), and low-level PLEs (n=7901). Models then examined whether changes in cognitive and structural neural metrics over time attenuated associations between genetic liability (i.e., schizophrenia polygenic risk scores/family history) or environmental risk scores (e.g., poverty) and PLE groups. ResultsPersistent distressing PLEs showed greater decreases (i.e., more negative slopes) of cognition and neural metrics over time compared to those in low-level PLE groups. Associations between environmental risk and persistent distressing PLEs were attenuated when accounting for lowered scores over time on cognitive (e.g., picture vocabulary) and to a lesser extent neural (e.g., cortical thickness, volume) metrics. ConclusionsAnalyses provide novel evidence for extant theories that worsening cognition and global structural metrics may partially account for associations between environmental risk with persistent distressing PLEs.

neuroscience↗

bioRxiv2024

Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1+ TAMs and CD8+ T cells

Administration of selective serotonin reuptake inhibitors (SSRIs) is associated with a reduced cancer risk and shows significant anti-tumor effects across multiple tumor types, suggesting the potential for repurposing SSRIs in cancer therapy. Nonetheless, the specific molecular target and mechanism of action of SSRIs remain to be fully elucidated. Here, we reveal that citalopram exerts an immune-dependent anti-tumor effects in hepatocellular carcinoma (HCC). Interestingly, the anti-HCC effects of citalopram are not reliant on its conventional target, the serotonin transporter. Through various drug repurposing approaches, including global reverse gene expression profiling, drug affinity responsive target stability assay, and molecular docking, the complement component 5a receptor 1 (C5aR1) is identified as a new target of citalopram. C5aR1 is predominantly expressed by tumor-associated macrophages (TAMs), and citalopram treatment enhances local macrophage phagocytosis and elicits CD8+ T anti-tumor immunity. C5aR1 deficiency or depletion of CD8+ T cells hinders the anti-HCC effects of citalopram. Collectively, our study reveals the immunomodulatory roles of citalopram in inducing anti-tumor immunity, and provides a basis for considering the repurposing of SSRIs as promising anticancer agents for HCC treatment.

cancer biology↗

bioRxiv2024

Efficient generation of CAR NK cells from human umbilical cord blood CD34+ stem and progenitors for democratizing affordable immunotherapy

Chimeric antigen receptor (CAR) natural killer cells (CAR NK) cells, leveraging safety and not requiring HLA match in adoptive infusion, have emerged as promising alternative cells to CAR-T cells for immunotherapies. High and multiple doses of CAR NK cell infusions are essential to maintain therapeutic efficacy in clinical trials. This requires efficient methods for generating large-scale CAR NK cells and significantly reducing CAR engineering costs. In this study, we develop a three-step strategy to generate highly high yields of induced NK (iNK) and CAR iNK cells from human umbilical cord blood CD34+ hematopoietic stem and progenitor cells (CD34+ HSPCs). Starting from a single umbilical cord blood CD34+ HSPC, our reliable method efficiently produces 14-83 million mature iNK cells or 7-32 million CAR iNK cells with high expression levels of CD16 and zero T cell contaminations. Introducing CAR expression elements at the HSPC level reduces the quantities of CAR pseudoviruses to 1 / 140.000 - 1 / 600,000 compared to engineering CARs in mature NK cells. The iNK and CAR iNK cells, including fresh cells and thawed cells from cryopreserved conditions, demonstrate remarkable tumoricidal activities against various human cancer cells and significantly prolong the survival of human tumor-bearing animals. The high yields of CAR NK cells and negligible costs of CAR engineering of our method support the broad applications of CAR NK cells for treating cancer patients.

cell biology↗

bioRxiv2024

SSRI antidepressant citalopram reverses the Warburg effect to inhibit hepatocellular carcinoma by directly targeting GLUT1

Although there is growing appreciation for effective repurposing of selective serotonin reuptake inhibitors (SSRIs) for cancer therapy, particularly hepatocellular carcinoma (HCC), efforts are hampered by limited knowledge of their molecular targets and mechanism of action. Global inverse gene-expression profiling method, drug affinity responsive target stability assay, and in silico molecular docking analysis was performed to identify the targets of SSRIs. Murine subcutaneous, orthotopic models, and patient-derived xenograft were employed to explore the therapeutic effects and underlying mechanisms of SSRIs in HCC. The clinical relevance of SSRI use was verified with real world data. SSRIs exhibit significant anti-HCC effects independent of their known target serotonin reuptake transporter. The glucose transporter 1 (GLUT1) is identified as a new target of SSRIs. Citalopram binds to and antagonizes GLUT1, resulting in reduced glycolytic flux and ATP generation. Mutant GLUT1 in the binding site E380 of citalopram compromises the inhibitory effects of citalopram on the Warburg effect and tumor growth. In preclinical models, citalopram dampens the growth kinetics of GLUT1high liver tumors and displays a synergistic effect with anti-PD-1 therapy. Retrospective analysis of health records found that SSRIs use is associated with a lower risk of metastasis among HCC patients. Our study reveals an unprecedented role of SSRIs in cancer metabolism, and establishes a rationale for repurposing SSRIs as potential anticancer drugs for HCC.

cancer biology↗

bioRxiv2024

The maternal vGluT2 and embryonic mGluR3 signaling relay system controls offspring wing dimorphism in pea aphid

Transgenerational phenotypic plasticity (TPP) refers to the phenomenon that environmental conditions experienced by one generation can influence the phenotype of subsequent generations to adapt to the environment without modification of their DNA sequences. Aphid wing dimorphism is a textbook example of TPP by which a maternal aphid perceives the environmental cues to decide the wing morph of her offspring. However, the signaling mechanism from mother to daughter remains unclear. In this study, we showed that the population density and physical contact caused high proportion of winged offspring in the pea aphid Acyrthosiphon pisum. Its vesicular glutamate transporter 2 (ApvGluT2) and metabotropic glutamate receptor 3 (ApmGluR3) were identified by tissue-specific RNA-seq as differentially expressed genes in the head and embryo respectively between solitary and more densely housed maternal aphids. Elevated expression of brain ApvGluT2 and embryonic ApmGluR3 led to increases in the winged proportion. Knockdown of either gene inhibited phosphorylation of ApFoxO in embryos. Furthermore, EMSA showed that dephosphorylated ApFoxO directly bound to the promotor of hedgehog (ApHh), a morphogen gene for wing development, to repress its transcription in stage 20 embryos, causing a lower winged proportion. Our results demonstrated that brain vGluT2 and embryonic mGluR3 coordinately relayed the maternal physical contact signals and control wing development in offspring, showcasing a novel regulatory mechanism underlying physical contact-dependent, transgenerational wing dimorphism in aphids.

zoology↗

bioRxiv2024

Genetic purging of strongly deleterious mutations underlies black-necked crane's unusual escape from an extinction vortex

Many species are undergoing rapid demographic declines, necessitating an examination of the resulting genetic impacts. The prevailing small population paradigm posits an elevated genetic load and extinction risk. However, instances of fast recovery from severe population bottlenecks suggest alternative outcomes. To investigate this issue, we performed a population genomic analysis on the black-necked crane, analyzing 42 modern and 11 historical genomes. This study revealed substantial evidence of large-effect allele purging underlying the unexpectedly rapid population recovery following an abrupt bottleneck during the 1980s. Nevertheless, forward simulations supposing a prolonged bottleneck (e.g., five generations) predicted a reversion with negative prospects, implying that rapid population recovery served as both the cause and consequence of the species escaping from an extinction vortex. These findings shed light on a potential positive microevolutionary response to current widespread population collapses and underscore the urgency of implementing active and effective conservation strategies to reverse this trend before it becomes irreversible.

evolutionary biology↗

bioRxiv2023

Urine proteomic analysis of patients with schizophrenia

We tried to explore the difference of urinary proteome between unmedicated schizophrenia patients and normal people through a small number of cases. The results showed that a total of 35 differential proteins were screened in the schizophrenia group compared with the healthy control group. Through random grouping evaluation, it has 91.4 % credibility. Fifteen of the differentially expressed proteins were reported to be related to schizophrenia mechanism, drug target or nervous system regulation. Among them, the aromatic amino acid decarboxylase related to the pathogenesis of schizophrenia can distinguish all 10 patients and 9 normal people with 100 % accuracy in this study, and the AUC value of 17 proteins is greater than or equal to 0.9. The biological pathways enriched by differentially expressed proteins include ephrin receptor signaling pathway, positive regulation of long-term potentiation (LTP), etc. This study shows that urine proteomics can reflect the difference between schizophrenia and healthy controls, and has the potential as a diagnostic marker.

biochemistry↗

bioRxiv2023

Study on the preparation of bovine milk exosomes and the stability of lyophilized powder

Bovune milk exosomes (MK-exo) have bioactive effects on the health of consumers and can also serve as excellent drug delivery vehicles. However, long-term preservation and transportation remain major challenges at present. In this report, high-quality MK-exo are prepared on a large scale. We then developed a lyophilization method belonging to MK-exo, and performed acceleration and long-term stability experiments on the lyophilized MK-exo to evaluate the effect of lyophilization. The results showed that the lyophilized MK-exo can be stored at room temperature for at least 3 months and 2-8{degrees}C for 15 months. These findings provide data support for further understanding of the properties and applications of MK-exo.

bioengineering↗

bioRxiv2023

Genome-wide Perturbation Analysis Screening System for Exosomes-Related Genes Based on the CRISPR/Cas9 Platform

As reported previously, exosomes have significant impacts on the physiological and pathological state in vivo. Exosomes have been extensively studied as a drug delivery carrier and some exosomes drugs have been undergoing clinical research. The mechanisms of exosome production, transport, and secretion remain to be studied in depth. Thus, here, we developed a novel CRISPR-UMI-based gene screening system, with which we can reveal the exosomes biogenesis, transport, and uptake mechanisms on a genome-wide scale. This system consists of two parts: one is a gene knockout component; the other part is a unique molecular identifiers (UMI) labeling component that can label the exosomes produced by the knockout cells, in which each sgRNA corresponds to a specific UMI. In this way, by detecting the UMI loaded in the exosomes, we can trace the knockout gene. In this study, we first verified the function of each component using plasmids and lentiviruses respectively. Next, we simulated the infection of cells with lentiviral libraries using a single lentivirus to validate the functionality of the screening system. Finally, we constructed a CRISPR-UMI-based library targeting 15 genes (genes with clear effects on exosomes biogenesis) to further validate the reliability of the screening system. The development of this screening system is of indispensable importance for the in-depth study of the mechanisms of exosome production and secretion, as well as for the improvement of exosomes production and the advancement of exosomes industrialization.

bioengineering↗