Biology subjects

Zhou, L.

Publications and source records attributed to Zhou, L..

At least 145 records · Page 8Linked to original sources

bioRxiv2024

Armed Macrophages as Hunters for Photodynamic Therapy of Systemic Bacterial Infections by Bathing in the Sunshine

There has been a vision to conduct therapy using sunlight since ancient Greece, Egypt, and India. In modern medicine, photodynamic therapy is one popular form of therapy that uses light to excite photosensitizers to eliminate malignant and other diseased cells. It offers highly effective and broad-spectrum therapeutic efficacy. Yet, there are several hindrances to a real treatment of disease through photodynamic therapy, such as the limitation on the irradiation depth and areas, the regulation of side effects, etc. Usually, the patients should be kept in a dark environment during and after the therapeutic process for days to avoid side effects induced by light in daily life, not mention to conduct the phototherapy through sunbathing. Based on the above consideration, we propose an innovative idea to bring photodynamic therapy back to the origin of phototherapy-bathing in the sunshine. Namely, we designed a "live drug", as a smart hunter, named A-RAWs, by loading an "off-on" type of photosensitizer in macrophages to solve two technical problems. Firstly, to find and capture bacteria accurately, and then transport these bacteria to the epidermis through the blood capillary, where sunlight is reachable. Secondly, to minimize the potential side effects and maximize the therapeutic time windows and efficacy by using bacteria as the trigger of the photodynamic effect. Specifically, we first designed and synthesized a lysosome-targeted and inflammation-activated NIR photosensitizer (Lyso710A), and loaded it in the lysosomes of macrophages. These "armed" macrophages were transferred into the infected host to capture deep-tissue bacteria through innate immunity and transport the captured bacteria through the bloodstream to superficial skin. Finally, the pathogenic bacteria are killed by the photodynamic effect when transported to the epidermis and receive sunbathing. In vivo experiment demonstrates a 100% therapeutic efficiency on systemic bacterial infection model. We also demonstrated the working mechanism of this "live drug" using a lung infection model. This hunter demonstrates high intelligence to break the limitations of current photodynamic therapy and executes photodynamic therapy of deep-tissue bacterial infection simply by bathing in the sunshine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/574019v1_ufig1.gif" ALT="Figure 1"> View larger version (92K): org.highwire.dtl.DTLVardef@11331aeorg.highwire.dtl.DTLVardef@122baf6org.highwire.dtl.DTLVardef@1208bd5org.highwire.dtl.DTLVardef@1bc672c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

A spectrum of altered non-rapid eye movement sleep in schizophrenia

BackgroundMultiple facets of sleep neurophysiology, including electroencephalography (EEG) metrics such as non-rapid eye movement (NREM) spindles and slow oscillations (SO), are altered in individuals with schizophrenia (SCZ). However, beyond group-level analyses which treat all patients as a unitary set, the extent to which NREM deficits vary among patients is unclear, as are their relationships to other sources of heterogeneity including clinical factors, illness duration and ageing, cognitive profiles and medication regimens. Using newly collected high density sleep EEG data on 103 individuals with SCZ and 68 controls, we first sought to replicate our previously reported (Kozhemiako et. al, 2022) group-level mean differences between patients and controls (original N=130). Then in the combined sample (N=301 including 175 patients), we characterized patient-to-patient variability in NREM neurophysiology. ResultsWe replicated all group-level mean differences and confirmed the high accuracy of our predictive model (Area Under the ROC Curve, AUC = 0.93 for diagnosis). Compared to controls, patients showed significantly increased between-individual variability across many (26%) sleep metrics, with patterns only partially recapitulating those for group-level mean differences. Although multiple clinical and cognitive factors were associated with NREM metrics including spindle density, collectively they did not account for much of the general increase in patient-to-patient variability. Medication regimen was a greater (albeit still partial) contributor to variability, although original group mean differences persisted after controlling for medications. Some sleep metrics including fast spindle density showed exaggerated age-related effects in SCZ, and patients exhibited older predicted biological ages based on an independent model of ageing and the sleep EEG. ConclusionWe demonstrated robust and replicable alterations in sleep neurophysiology in individuals with SCZ and highlighted distinct patterns of effects contrasting between-group means versus within-group variances. We further documented and controlled for a major effect of medication use, and pointed to greater age-related change in NREM sleep in patients. That increased NREM heterogeneity was not explained by standard clinical or cognitive patient assessments suggests the sleep EEG provides novel, nonredundant information to support the goals of personalized medicine. Collectively, our results point to a spectrum of NREM sleep deficits among SCZ patients that can be measured objectively and at scale, and that may offer a unique window on the etiological and genetic diversity that underlies SCZ risk, treatment response and prognosis.

neuroscience↗

bioRxiv2023

CDC20 determines the sensitivity to spindle assembly checkpoint (SAC) inhibitors

Spindle assembly checkpoint (SAC) inhibitors are a recently developed class of drugs that perturb the regulation of chromosome segregation during division, induce chromosomal instability (CIN), and eventually lead to cell death. While they are currently in clinical trials for solid cancers, biomarkers to predict the response to SAC inhibitors are still lacking. We recently reported that aneuploid cancer cells are preferentially sensitive to SAC inhibition. Here, we investigated the molecular determinants of the response to SAC inhibition that underlies the differential sensitivity of aneuploid cells to these drugs. We found that this response was largely driven by the expression of CDC20, a main activator of the anaphase-promoting complex (APC/C), rather than by APC/C itself. Mechanistically, we discovered that CDC20 depletion prolonged metaphase duration, diminished mitotic errors, and reduced sensitivity to SAC inhibition. Aneuploid cells expressed high levels of CDC20 and experienced shorter metaphases and multiple mitotic errors, resulting in increased long-term sensitivity to SAC inhibition. Our findings propose high CDC20 expression as a favorable biomarker for SAC inhibition therapy and as an aneuploidy-induced therapeutic vulnerability.

cancer biology↗

bioRxiv2023

Netrin4 is a new target specific factor, ensuring adult sympathetic neuron survival via promoting protein synthesis

How mature neurons survive under homeostasis is a question of utmost importance and is known to be different from developing neurons. However, the understanding of this regard remains largely unknown. Here, based on the relationship between the sympathetic cervical ganglia (SCG) and the arterial networks of projecting and targeting organs, we report that the secretome of cerebral, but not peripheral, arterial smooth muscles (SMC) was required for the survival of sympathetic neurons. Among the secretome, we further identified that netrin-4, encoded by the ntn-4 gene, only entered neurons and not glia and played a crucial role both in vitro and in vivo. This was demonstrated with three independent lines of tamoxifen-inducible SMC-specific conditional knockout mice (cKO). Notably, in cKO mice, the local supply of exogenous netrin-4 confined to SCG selectively rescued neuronal necroptosis, which otherwise consistently occurred in a specific subgroup of SCG neurons that innervate cerebral SMCs. Mechanistically, we demonstrated that cerebral netrin-4 was endocytosed at the neurovascular interface and retrogradely long transported to peripheral soma in SCG, where it differentially regulated mRNA translations. This regulation suppressed vacuolization and neuronal necrosis, both of which took place spontaneously in cKO mice. The former is immediately followed by the latter when we injured axons using two-photon laser ablation. The findings revealed a new principle of neurovascular interactions vital for mature neuron survival, implying that under circumstances of cerebral SMC insufficient secretion, such as natural aging, may initiate mature neuronal loss due to uncontrolled vacuolization.

neuroscience↗

bioRxiv2023

Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia

Thrombocytopenia caused by long-term radiotherapy and chemotherapy exists in cancer treatment. Previous research demonstrates that 5-Hydroxtrayptamine (5-HT) and its receptors induces the formation of megakaryocytes (MKs) and platelets. However, the relationships between 5-HT1A receptor (5-HTR1A) and MKs is unclear so far. We screened and investigated the mechanism of vilazodone as a 5-HTR1A partial agonist in promoting MK differentiation and evaluated its therapeutic effect in thrombocytopenia. We employed a drug screening model based on machine learning (ML) to screen the megakaryocytopoiesis activity of VLZ. The effects of Vilazodone (VLZ) on megakaryocytopoiesis were verified in HEL and Meg-01 cells. Tg (itga2b: eGFP) zebrafish was performed to analyze the alterations in thrombopoiesis. Moreover, we established a thrombocytopenia mice model to investigate VLZ administration accelerates platelet recovery and function. We carried out network pharmacology, Western blot and immunofluorescence to demonstrate the potential targets and pathway of VLZ. VLZ has been predicted to have a potential biological action. Meanwhile, VLZ administration promotes MK differentiation and thrombopoiesis in cells and zebrafish models. Progressive experiments showed that VLZ has a potential therapeutic effect on radiation-induced thrombocytopenia in vivo. The network pharmacology and associated mechanism study indicated that SRC and MAPK signaling are both involved in the processes of megakaryopoiesis facilitated by VLZ. Furthermore, the expression of 5-HTR1A during megakaryocyte differentiation is closely related to the activation of SRC and MAPK. Our findings demonstrated that the expression of 5-HTR1A on MK, VLZ could bind to the 5-HTR1A receptor and further regulate the SRC/MAPK signaling pathway to facilitate megakaryocyte differentiation and platelet production, which provides new insights into the alternative therapeutic options for thrombocytopenia.

pharmacology and toxicology↗

bioRxiv2023

An inducible genetic tool for tracking and manipulating specific microglial states in development and disease

Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative region-associated microglia (PAM) in developing white matter and disease-associated microglia (DAM) prevalent in various neurodegenerative conditions. PAM and DAM share a similar core gene signature and other functional properties. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we report the generation of an inducible Cre driver line, Clec7a-CreERT2, designed to target PAM and DAM in the brain parenchyma. Utilizing this tool, we profile labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM/DAM gene expression. Through long-term tracking, we demonstrate surprising levels of plasticity in these microglial states. Lastly, we specifically depleted DAM in cuprizone-induced demyelination, revealing their roles in disease progression and recovery.

neuroscience↗

bioRxiv2023

Aberration correction for deformable mirror based remote focusing enables high-accuracy whole-cell super-resolution imaging

Single-molecule localization microscopy (SMLM) enables three-dimensional (3D) investigation of nanoscale structures in biological samples, offering unique insights into their organization. However, traditional 3D super-resolution microscopy using high numerical aperture (NA) objectives is limited by imaging depth of field (DOF), restricting their practical application to relatively thin biological samples. Here, we developed a unified solution for thick sample super-resolution imaging using a deformable mirror (DM) which was served for fast remote focusing, optimized point spread function (PSF) engineering and accurate aberration correction. By effectively correcting the system aberrations introduced during remote focusing and sample aberrations at different imaging depths, we achieved high-accuracy, large DOF imaging of the whole-cell organelles [i.e. nuclear pore complex (NPC), microtubules, and mitochondria] with a nearly uniform resolution of approximately 30 nm across the entire cellular volume.

bioengineering↗

bioRxiv2023

Isolation and electrochemical analysis of a facultative anaerobic electrogenic strain Klebsiella sp. SQ-1

Electricigens decompose organic matter and convert stored chemical energy into electrical energy through extracellular electron transfer. They serve as significant biocatalysts for microbial fuel cells which have practical applications in green energy generation, effluent treatment, and bioremediation. A facultative anaerobic electrogenic strain SQ-1 is isolated from sludge in a biotechnology factory. The strain SQ-1 is a close relative of Klebsiella variicola. Multilayered biofilms form on the surface of a carbon electrode after the isolated bacteria are inoculated into a microbial fuel cell device. This strain produces high current densities of 625 A cm-2 by using acetate as the carbon source in a three-electrode configuration. The electricity generation performance is also analyzed in a dual-chamber microbial fuel cell. It reaches a maximum power density of 560 mW m-2 when the corresponding output voltage is 0.59 V. The facultative strain SQ-1 utilizes hydrous ferric oxide as an electron acceptor to perform extracellular electricigenic respiration in anaerobic conditions. Since facultative strains possess better properties than anaerobic strains, Klebsiella sp. SQ-1 may be a promising exoelectrogenic strain for applications in microbial electrochemistry.

microbiology↗

bioRxiv2023

TRAIL pathway suppression of cancer cell growth and immune cell-mediated tumor cell-killing in a senescent fibroblast-constructed tumor microenvironment

Cellular senescence and the associated secretory phenotype (SASP) promote cancer in the aging population. During aging or upon chemotherapy exposure, cellular and molecular changes occur in non-cancerous cells and alter responses to cancer therapy, primarily via modifications in the tumor microenvironment (TME) and immune response. Targeting senescent cells through removal, modulation of the SASP, or cellular reprogramming represent promising therapeutic avenues for treating cancer. We elucidate an interplay between cancer cells, immune cells, and senescent fibroblasts and describe the impact of fibroblast senescence on tumor growth and response to cancer therapy. Cytokine profiling reveals dynamic changes in SASP production during etoposide-induced senescence in IMR90 fibroblasts. We show that SASP is partially regulated by p21 (WAF1; CDKN1A), leading to the downregulation of anti-tumorigenic cytokines and upregulation of pro-tumorigenic cytokines. Senescent fibroblasts promote bystander cancer cell growth via a p21-driven SASP. These results provide strategies to target the p21-driven SASP in the TME during cancer therapy. Treatment with TRAIL or TRAIL-inducing Dordaviprone (TIC10/ONC201) reduces cell viability of tumor cells co-cultured with senescent or proliferating fibroblasts and promotes immune-mediated tumor cell-killing in co-culture with senescent IMR90 fibroblasts. ONC201 combined with senolytic drugs (e.g., Navitoclax, Lamivudine) synergizes towards tumor suppression. These results indicate that senolytic therapies may be combined with cancer therapies to target senescence-associated changes in the TME including for modulation of the senescent cytokine landscape.

cancer biology↗

bioRxiv2023

Androgen receptor signaling blockade enhances NK cell-mediated killing of prostate cancer cells and sensitivity to NK cell checkpoint blockade

BackgroundThe blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but many patients progress to metastatic castration-resistant prostate cancer (mCRPC). Treatments for mCRPC include AR inhibitors (ARi), chemotherapy, PARP inhibitors, and radioligands. Checkpoint inhibitor activity is limited to a small subset of MSI-H mCRPC. AR signaling modulates CD8+ T cell function, but its impact on natural killer (NK) cell cytotoxicity is unknown. We investigated the effect of ARi on NK cell activation, cytokine secretion, NKG2A expression, and NK cell-mediated killing of PCa cells in vitro. MethodsPCa cell lines (LNCaP, 22Rv1, DU145, PC3) were co-cultured with NK-92 and treated with ARi (enzalutamide [enza], darolutamide [daro]) alone or in combination with anti-NKG2A antibody monalizumab. Immune cell-mediated tumor cell killing and cytokine secretion were quantified. NK cell expression of NKG2A and PCa cell expression of HLA-E were investigated by flow cytometry. The AR-negative cell lines PC3 and DU145 were stably transduced with an AR expression vector to evaluate the AR modulation of HLA-E. To assess the in vivo combination of NKG2A blockade and ARi therapy in vivo, Cas9 was used to genetically ablate the murine HLA-E ortholog, H2-T23, from RM-1 murine PCa cells. H2-T23 knockout and control cells were grown subcutaneously in castrated C57BL/6 mice and treated with daro or control. The activation status of peripheral blood NK cell isolated from patients with PCa before and after initiation of androgen deprivation therapy (ADT) was evaluated by flow cytometry. ResultsARi activated NK cells and significantly increased immune-mediated NK-92 cell killing of PCa cells. IFN-{gamma} and TRAIL mediated ARi-induced NK cell activation. ARi increased expression of the inhibitory receptor NKG2A on NK cells, and immune killing of PCa cells was enhanced with the combination of ARi and monalizumab. ARi also increased the expression of HLA-E, the ligand of NKG2A, on PCa cell lines. By transducing AR into AR-negative PC3 and DU145, we demonstrated that androgen signaling regulates HLA-E expression. In a mouse model of PCa, HLA-E knockout synergized with darolutamide to increase NK cell activation. NK cells derived from patients with metastatic PCa exhibited increased expression of Granzyme B and Perforin following ARi treatment. ConclusionsARi activates NK cells via IFN-{gamma} and TRAIL and promotes the killing of PCa cells. ARi also upregulates expression of HLA-E on PCa which may suppress the innate immune response against PCa. ARi-mediated NK cell killing of PCa cells was enhanced by NKG2A blockade. These results support novel immunotherapeutic strategies for PCa targeting NK activation through the combination of ARi and monalizumab. Graphical AbstractAndrogen receptor inhibitors (ARi) enhance NK cell-mediated killing of prostate cancer cells and sensitivity to NK cell checkpoint NKG2A blockade. ARi upregulate the NK cell inhibitor ligand (HLA-E) mediating suppression NK cell killing of PCa. This regulation is dependent on a functional AR signal on tumor cell lines. Adding an anti-NKG2a-HLA-E mAb with ARi further enhances the NK cell-mediated killing of PCa. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/567201v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@ba35e6org.highwire.dtl.DTLVardef@128a2e7org.highwire.dtl.DTLVardef@def16aorg.highwire.dtl.DTLVardef@bb72e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

MicroRNA-541-3p alters lipoproteins to reduce atherosclerosis by degrading Znf101 and Casz1 transcription factors

High apoB-containing low-density lipoproteins (LDL) and low apoA1-containing high-density lipoproteins (HDL) are associated with atherosclerosis. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both decreases apoB and increases apoA1 expression by inducing mRNA degradation of two different transcription factors, Znf101 and Casz1. Znf101 enhances apoB expression while Casz1 represses apoA1 expression. The hepatic knockdown of orthologous Zfp961 and Casz1 genes in mice altered plasma lipoproteins and reduced atherosclerosis without causing hepatic lipid accumulation, most likely by lowering hepatic triglyceride production, increasing HDL cholesterol efflux capacity, and reducing lipogenesis. Notably, human genetic variants in the MIR541, ZNF101, and CASZ1 loci are significantly associated with plasma lipids and lipoprotein levels. This study identifies miR-541-3p and Znf101/Casz1 as potential therapeutic agent and targets, respectively, to reduce plasma lipoproteins and atherosclerosis without causing liver steatosis. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/565110v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@741dd1org.highwire.dtl.DTLVardef@151ad9aorg.highwire.dtl.DTLVardef@15c96a9org.highwire.dtl.DTLVardef@1a73b2d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract. A schematic diagram depicting the role of miR-541-3p in the control of plasma lipoproteins and atherosclerosis.Our data show that miR-541-3p downregulates ZNF101 and CASZ1 by enhancing post-transcriptional degradation of mRNAs after interacting with their 3'-UTRs. Furthermore, our data indicate that ZNF101 is an enhancer of APOB, and CASZ1 is a repressor of APOA1. Hepatic knockdown (KD) of Zfp961, an orthologue of ZNF101, reduces plasma apoB-containing lipoproteins, whereas KD of Casz1 increases high density lipoproteins in mice. Furthermore, we show that hepatic KDs of these transcription factors reduces atherosclerosis in mice induced by the expression of mutant PCSK9. C_FIG

physiology↗

bioRxiv2023

SARS-CoV-2 spike protein induces the cytokine release syndrome by stimulating T cells to produce more IL-2

Cytokine release syndrome (CRS) is one of the leading causes of mortality in COVID-19 patients caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more IL-2, which subsequently cooperates with spike protein to facilitate peripheral blood mononuclear cells to release IL-1{beta}, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release TNF- and IFN-{gamma}, as well as T cells to release IFN-{gamma}. Mechanistically, IFN-{gamma} and TNF- enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of TLR4 which serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of NF-{kappa}B. Furthermore, TNF- also activates NF-{kappa}B signaling in monocytes, which further cooperates with IFN-{gamma} and spike protein to modulate NF-{kappa}B-dependent transcription of CRS-related inflammatory cytokines. Targeting TNF-/IFN-{gamma} in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19.

immunology↗

bioRxiv2023

Simulation-Driven Design of Stabilized SARS-CoV-2 Spike S2 Immunogens

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimers opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

biophysics↗

bioRxiv2023

Fast and universal single molecule localization using multi-dimensional point spread functions

The recent development of single molecule imaging techniques has enabled not only high accuracy spatial resolution imaging but also information rich functional imaging. Abundant information of the single molecules can be encoded in its diffraction pattern and be extracted precisely (e.g. 3D position, wavelength, dipole orientation). However, sophisticated high dimensional point spread function (PSF) modeling and analyzing methods have greatly impeded the broad accessibility of these techniques. Here, we present a graphics processing unit (GPU)-based B-spline PSF modeling method which could flexibly model high dimensional PSFs with arbitrary shape without greatly increasing the model parameters. Our B-spline fitter achieves 100 times speed improvement and minimal uncertainty for each dimension, enabling efficient high dimensional single molecule analysis. We demonstrated, both in simulations and experiments, the universality and flexibility of our B-spline fitter to accurately extract the abundant information from different types of high dimensional single molecule data including multicolor PSF (3D + color), multi-channel four-dimensional 4Pi-PSF (3D + interference phase) and five-dimensional vortex PSF (3D + dipole orientation).

biophysics↗

bioRxiv2023

FBXO24 modulates mRNA alternative splicing and MIWI degradation and is required for normal sperm formation and piRNA production

Spermiogenesis is a critical, post-meiotic phase of male gametogenesis, in which the proper gene expression is essential for sperm maturation. However, the underlying molecular mechanism that controls mRNA expression in the round spermatids remains elusive. Here, we identify that FBXO24, an orphan F-box protein, is highly expressed in the testis of humans and mice and interacts with the splicing factors (SRSF2, SRSF3, and SRSF9) to modulate the gene alternative splicing in the round spermatids. Genetic mutation of FBXO24 in mice causes many abnormal splicing events in round spermatids, thus affecting a large number of critical genes related to sperm formation that were dysregulated. Further molecular and phenotypical analyses revealed that FBXO24 deficiency results in aberrant histone retention, incomplete axonemes, oversized chromatoid body (CB), and abnormal mitochondrial coiling along sperm flagella, ultimately leading to male sterility. In addition, we discovered that FBXO24 interacts with MIWI and SCF subunits and mediates the degradation of MIWI via K48-linked polyubiquitination. Furthermore, we show that FBXO24 depletion could lead to aberrant piRNA production in testes, which suggests FBXO24 is required for normal piRNA counts. Collectively, these data demonstrate that FBXO24 is essential for sperm formation and piRNA production by regulating mRNA alternative splicing and MIWI degradation during spermiogenesis.

developmental biology↗

bioRxiv2023

CircSMAD3 represses VSMC phenotype switching and neointima formation via promoting hnRNPA1 ubiquitination degradation

AimsCircular RNAs (circRNAs) are novel regulatory RNAs with high evolutionary conservation and stability, making them attractive therapeutic agents for various vascular diseases. SMAD family is a downstream mediator of the canonical TGF{beta} signalling pathway and has been considered a critical regulator in vascular injury. However, the role of circRNAs derived from the SMAD family members in vascular physiology remains unclear. Therefore, this study aimed to identify a functional circRNA derived from the SMAD family and elucidate its potential as an effective therapeutic agent for vascular proliferative diseases. Methods and ResultsWe initially identified potential functional circRNAs originating from the SMAD family using integrated transcriptome screening. circSMAD3, derived from the SMAD3 gene, was identified to be significantly downregulated in vascular injury and atherosclerosis. Transcriptome analysis was conducted to comprehensively illustrate the pathways modulated by circRNAs. Functionally, circSMAD3 repressed VSMC proliferation and phenotype switching in vitro evidenced by morphological assays and ameliorated arterial injury-induced neointima formation in vivo. Mechanistically, circSMAD3 interacted with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) within the nucleus, enhanced its interaction with E3 ligase WD repeat domain 76 (WDR76) to promote hnRNPA1 ubiquitination degradation, facilitated p53 pre-RNA splicing, activated p53{gamma} signalling pathway, and finally suppressed VSMC proliferation and phenotype switching. ConclusionOur study identifies circSMAD3 as a novel epigenetic regulator that suppresses VSMC proliferation and phenotype switching, thereby attenuating vascular remodelling and providing a new circRNA-based therapeutic strategy for cardiovascular diseases.

molecular biology↗

bioRxiv2023

The apoptotic splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin

Understanding the mechanisms of pre-mRNA splicing and spliceosome assembly is limited by technical challenges to examining spliceosomes in vivo. Here we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. Sequencing these pre-mRNA fragments allowed the transcriptome-wide mapping of branch sites with high sensitivity. In addition to known U2 snRNP proteins, these complexes contained the proteins RBM5 and RBM10. RBM5 and RBM10 are alternative splicing regulators that control exons affecting apoptosis and cell proliferation in cancer, but were not previously shown to associate with the U2 snRNP or to play roles in branch site selection. We delineate a common segment of RBM5 and RBM10, separate from their known functional domains, that is required for their interaction with the U2 snRNP. We identify a large set of splicing events regulated by RBM5 and RBM10 and find that they predominantly act as splicing silencers. Disruption of their U2 interaction renders the proteins inactive for repression of many alternative exons. We further find that these proteins assemble on branch sites of nearly all exons across the transcriptome, including those whose splicing is not altered by them. We propose a model where RBM5 and RBM10 act as components of the U2 snRNP complex. From within this complex, they sense structural features of branchpoint recognition to either allow progression to functional spliceosome or rejection of the complex to inhibit splicing.

molecular biology↗

bioRxiv2023

Preclinical Synergistic Combination Therapy of Lurbinectedin with Irinotecan and 5-Fluorouracil in Pancreatic Cancer

Pancreatic cancer is a devastating disease with a poor prognosis. Novel chemotherapeutics in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin (PM01183; LY-01017) received FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin stalls and degrades RNA Polymerase II and introduces breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedins highly efficient killing of human-derived pancreatic tumor cell lines PANC-1, BxPC-3, and HPAF-II as a single agent. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells. Western blot analysis of combination therapy indicates an upregulation of {gamma}H2AX, a DNA damage marker, and the Chk1/ATR pathway, involved in replicative stress and DNA damage response. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-fluorouracil (5-FU) results in highly efficient killing of tumor cells. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer.

cancer biology↗