High-resolution vasomotion analysis reveals novel arteriole physiological features and progressive modulation of cerebral vascular networks by stroke.

Spontaneous cerebral vasomotion, characterized by ∼0.1 Hz rhythmic contractility, is crucial for brain homeostasis. However, our understanding of vasomotion is limited due to a lack of high-precision analytical methods to determine single vasomotion events at basal levels. Here, we developed a novel strategy that integrates a baseline smoothing algorithm, allowing precise measurements of vasodynamics and concomitant Ca2+ dynamics in mouse cerebral vasculature imaged by two-photon microscopy. We identified several previously unrecognized vasomotion properties under different physiological and pathological conditions, especially in ischemic stroke, which is a highly harmful brain disease that results from vessel occlusion. First, the dynamic characteristics between SMCs Ca2+ and corresponding arteriolar vasomotion are correlated. Second, compared to previous diameter-based estimations, our radius-based measurements reveal anisotropic vascular movements, enabling a more precise determination of the latency between smooth muscle cell (SMC) Ca2+ activity and vasoconstriction. Third, we characterized single vasomotion event kinetics at scales of less than 4 seconds. Finally, following pathological vasoconstrictions induced by ischemic stroke, vasoactive arterioles entered an inert state and persisted despite recanalization. In summary, we developed a highly accurate technique for analyzing spontaneous vasomotion, and our data suggested a potential strategy to reduce stroke damage by promoting vasomotion recovery.

Ca2+ oscillation in vascular smooth muscle cells control myogenic spontaneous vasomotion and counteract post-ischemic no-reflow.

Ischemic stroke produces the highest adult disability. Despite successful recanalization, no-reflow, or the futile restoration of the cerebral perfusion after ischemia, is a major cause of brain lesion expansion. However, the vascular mechanism underlying this hypoperfusion is largely unknown, and no approach is available to actively promote optimal reperfusion to treat no-reflow. Here, by combining two-photon laser scanning microscopy (2PLSM) and a mouse middle cerebral arteriolar occlusion (MCAO) model, we find myogenic vasomotion deficits correlated with post-ischemic cerebral circulation interruptions and no-reflow. Transient occlusion-induced transient loss of mitochondrial membrane potential (ΔΨm) permanently impairs mitochondria-endoplasmic reticulum (ER) contacts and abolish Ca2+ oscillation in smooth muscle cells (SMCs), the driving force of myogenic spontaneous vasomotion. Furthermore, tethering mitochondria and ER by specific overexpression of ME-Linker in SMCs restores cytosolic Ca2+ homeostasis, remotivates myogenic spontaneous vasomotion, achieves optimal reperfusion, and ameliorates neurological injury. Collectively, the maintaining of arteriolar myogenic vasomotion and mitochondria-ER contacts in SMCs, are of critical importance in preventing post-ischemic no-reflow.

Fate mapping of Spp1 expression reveals age-dependent plasticity of disease-associated microglia-like cells after brain injury.

Microglial reactivity to injury and disease is emerging as a heterogeneous, dynamic, and crucial determinant in neurological disorders. However, the plasticity and fate of disease-associated microglia (DAM) remain largely unknown. We established a lineage tracing system, leveraging the expression dynamics of secreted phosphoprotein 1（Spp1） to label and track DAM-like microglia during brain injury and recovery. Fate mapping of Spp1+ microglia during stroke in juvenile mice revealed an irreversible state of DAM-like microglia that were ultimately eliminated from the injured brain. By contrast, DAM-like microglia in the neonatal stroke models exhibited high plasticity, regaining a homeostatic signature and integrating into the microglial network after recovery. Furthermore, neonatal injury had a lasting impact on microglia, rendering them intrinsically sensitized to subsequent immune challenges. Therefore, our findings highlight the plasticity and innate immune memory of neonatal microglia, shedding light on the fate of DAM-like microglia in various neuropathological conditions.

Roseburia intestinalis sensitizes colorectal cancer to radiotherapy through the butyrate/OR51E1/RALB axis.

The radioresistant signature of colorectal cancer (CRC) hampers the clinical utility of radiotherapy. Here, we find that fecal microbiota transplantation (FMT) potentiates the tumoricidal effects of radiation and degrades the intertwined adverse events in azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC mice. FMT cumulates Roseburia intestinalis (R. intestinalis) in the gastrointestinal tract. Oral gavage of R. intestinalis assembles at the CRC site and synthetizes butyrate, sensitizing CRC to radiation and alleviating intestinal toxicity in primary and CRC hepatic metastasis mouse models. R. intestinalis-derived butyrate activates OR51E1, a G-protein-coupled receptor overexpressing in patients with rectal cancer, facilitating radiogenic autophagy in CRC cells. OR51E1 shows a positive correlation with RALB in clinical rectal cancer tissues and CRC mouse model. Blockage of OR51E1/RALB signaling restrains butyrate-elicited autophagy in irradiated CRC cells. Our findings highlight that the gut commensal bacteria R. intestinalis motivates radiation-induced autophagy to accelerate CRC cell death through the butyrate/OR51E1/RALB axis and provide a promising radiosensitizer for CRC in a pre-clinical setting.

Transcriptional and epigenetic decoding of the microglial aging process.

As important immune cells, microglia undergo a series of alterations during aging that increase the susceptibility to brain dysfunctions. However, the longitudinal characteristics of microglia remain poorly understood. In this study, we mapped the transcriptional and epigenetic profiles of microglia from 3- to 24-month-old mice. We first discovered unexpected sex differences and identified age-dependent microglia (ADEM) genes during the aging process. We then compared the features of aging and reactivity in female microglia at single-cell resolution and epigenetic level. To dissect functions of aged microglia excluding the influence from other aged brain cells, we established an accelerated microglial turnover model without directly affecting other brain cells. By this model, we achieved aged-like microglia in non-aged brains and confirmed that aged-like microglia per se contribute to cognitive decline. Collectively, our work provides a comprehensive resource for decoding the aging process of microglia, shedding light on how microglia maintain brain functions.

Modeling Distal Middle Cerebral Artery Occlusion in Neonatal Rodents with Magnetic Nanoparticles or Magnetized Red Blood Cells.

It is challenging to establish animal models to mimic perinatal arterial ischemic stroke. Here, we provided two approaches that precisely occlude rodent pups' distal middle cerebral artery of rodent pups at any postnatal age. One uses magnetic nanoparticles to generate platelet-rich thrombus, and the other utilizes magnetized red blood cells (mRBCs) to generate an erythrocyte-rich embolus. Both approaches result in focal cerebral ischemia followed by controllable reperfusion while requiring no arterial surgery.

Proteins secreted by brain arteriolar smooth muscle cells are instructive for neural development.

Intercellular communication between vascular and nerve cells mediated by diffusible proteins has recently emerged as a critical intrinsic program for neural development. However, whether the vascular smooth muscle cell (VSMC) secretome regulates the connectivity of neural circuits remains unknown. Here, we show that conditioned medium from brain VSMC cultures enhances multiple neuronal functions, such as neuritogenesis, neuronal maturation, and survival, thereby improving circuit connectivity. However, protein denaturation by heating compromised these effects. Combined omics analyses of donor VSMC secretomes and recipient neuron transcriptomes revealed that overlapping pathways of extracellular matrix receptor signaling and adhesion molecule integrin binding mediate VSMC-dependent neuronal development. Furthermore, we found that human arterial VSMCs promote neuronal development in multiple ways, including expanding the time window for nascent neurite initiation, increasing neuronal density, and promoting synchronized firing, whereas human umbilical vein VSMCs lack this capability. These in vitro data indicate that brain arteriolar VSMCs may carry direct instructive information for neural development through intercellular communication in vivo.

DDX10 promotes the proliferation and metastasis of colorectal cancer cells via splicing RPL35.

BACKGROUND: Colorectal cancer (CRC) has become the second deadliest cancer in the world and severely threatens human health. An increasing number of studies have focused on the role of the RNA helicase DEAD-box (DDX) family in CRC. However, the mechanism of DDX10 in CRC has not been elucidated. METHODS: In our study, we analysed the expression data of CRC samples from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Subsequently, we performed cytological experiments and animal experiments to explore the role of DDX10 in CRC cells. Furthermore, we performed Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network analyses. Finally, we predicted the interacting protein of DDX10 by LC-MS/MS and verified it by coimmunoprecipitation (Co-IP) and qPCR. RESULTS: In the present study, we identified that DDX10 mRNA was extremely highly expressed in CRC tissues compared with normal colon tissues in the TCGA and GEO databases. The protein expression of DDX10 was measured by immunochemistry (IHC) in 17 CRC patients. The biological roles of DDX10 were explored via cell and molecular biology experiments in vitro and in vivo and cell cycle assays. We found that DDX10 knockdown markedly reduced CRC cell proliferation, migration and invasion. Then, we constructed a PPI network with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). GO and KEGG enrichment analysis and gene set enrichment analysis (GSEA) showed that DDX10 was closely related to RNA splicing and E2F targets. Using LC-MS/MS and Co-IP assays, we discovered that RPL35 is the interacting protein of DDX10. In addition, we hypothesize that RPL35 is related to the E2F pathway and the immune response in CRC. CONCLUSIONS: In conclusion, provides a better understanding of the molecular mechanisms of DDX10 in CRC and provides a potential biomarker for the diagnosis and treatment of CRC.

Precise control of embolic stroke with magnetized red blood cells in mice.

Precise embolism control in immature brains can facilitate mechanistic studies of brain damage and repair after perinatal arterial ischemic stroke (PAIS), but it remains a technical challenge. Microhemorrhagic transformation is observed in one-third of infant patients who have suffered PAIS, but the underlying mechanism remains elusive. Building on an established approach that uses magnetic nanoparticles to induce PAIS, we develop a more advanced approach that utilizes magnetized erythrocytes to precisely manipulate de novo and in situ embolus formation and reperfusion in perinatal rodent brains. This approach grants spatiotemporal control of embolic stroke without any transarterial delivery of pre-formed emboli. Transmission electron microscopy revealed that erythrocytes rather than nanoparticles are the main material obstructing the vessels. Both approaches can induce microbleeds as an age-dependent complication; this complication can be prevented by microglia and macrophage depletion. Thus, this study provides an animal model mimicking perinatal embolic stroke and implies a potential therapeutic strategy for the treatment of perinatal stroke.

Ambient temperature structures the gut microbiota of zebrafish to impact the response to radioactive pollution.

Potential nuclear accidents propel serious environmental pollution, and the resultant radionuclide release devastates severely the environment severely and threatens aquatic organism survival. Likewise, ongoing climate change coupled with the gradual increase in global surface temperatures can also adversely impact the aquatic ecosystems. In the present study, we preconditioned zebrafish (Danio rerio) at three different temperatures (18 °C, 26 °C and 34 °C) to investigate the effects of a temperature profile on their radiosensitivity (exposure to 20 Gy of gamma rays) to identify the potential biochemical mechanism responsible for influencing radiosensitivity. We found that preconditioning of zebrafish at different temperatures moulded specific gut microbiota configurations and impacted hepatic glycometabolism and sensitivity to subsequent radiation. Following antibiotic treatment to reduce gut bacteria, these observed differences in the expression of hepatic glycometabolism-related genes and radiation-induced intestinal toxicity were minimal, supporting the hypothesis that the gut bacteria reshaped by different ambient temperatures might be the key modulators of hepatic functions and radiosensitivity in zebrafish. Together, our findings provide novel insights into the connection of radiation injuries with temperature alterations in fish, and suggest that maintaining the stability of gram-positive bacteria may be efficacious to protect aquatic organisms against short or long-term radioactive contamination in the context of global climate change.

Low-Intensity Exercise Modulates Gut Microbiota to Fight Against Radiation-Induced Gut Toxicity in Mouse Models.

Radiation-induced gastrointestinal (GI) tract toxicity halts radiotherapy and degrades the prognosis of cancer patients. Physical activity defined as "any bodily movement produced by skeletal muscle that requires energy expenditure" is a beneficial lifestyle modification for health. Here, we investigate whether walking, a low-intensity form of exercise, could alleviate intestinal radiation injury. Short-term (15 days) walking protected against radiation-induced GI tract toxicity in both male and female mice, as judged by longer colons, denser intestinal villi, more goblet cells, and lower expression of inflammation-related genes in the small intestines. High-throughput sequencing and untargeted metabolomics analysis showed that walking restructured the gut microbiota configuration, such as elevated Akkermansia muciniphila, and reprogramed the gut metabolome of irradiated mice. Deletion of gut flora erased the radioprotection of walking, and the abdomen local irradiated recipients who received fecal microbiome from donors with walking treatment exhibited milder intestinal toxicity. Oral gavage of A. muciniphila mitigated the radiation-induced GI tract injury. Importantly, walking did not change the tumor growth after radiotherapy. Together, our findings provide novel insights into walking and underpin that walking is a safe and effective form to protect against GI syndrome of patients with radiotherapy without financial burden in a preclinical setting.

Caloric restriction alleviates radiation injuries in a sex-dependent fashion.

Safe and effective regimens are still needed given the risk of radiation toxicity from iatrogenic irradiation. The gut microbiota plays an important role in radiation damage. Diet has emerged as a key determinant of the intestinal microbiome signature and function. In this report, we investigated whether a 30% caloric restriction (CR) diet may ameliorate radiation enteritis and hematopoietic toxicity. Experimental mice were either fed ad libitum (AL) or subjected to CR preconditioning for 10 days and then exposed to total body irradiation (TBI) or total abdominal irradiation (TAI). Gross examinations showed that short-term CR pretreatment restored hematogenic organs and improved the intestinal architecture in both male and female mice. Intriguingly, CR preconditioning mitigated radiation-induced systemic and enteric inflammation in female mice, while gut barrier function improved in irradiated males. 16S rRNA high-throughput sequencing showed that the frequency of pro-inflammatory microbes, including Helicobacter and Desulfovibrionaceae, was reduced in female mice after 10 days of CR preconditioning, while an enrichment of short-chain fatty acid (SCFA)-producing bacteria, such as Faecalibaculum, Clostridiales, and Lactobacillus, was observed in males. Using fecal microbiota transplantation (FMT) or antibiotic administration to alter the gut microbiota counteracted the short-term CR-elicited radiation tolerance of both male and female mice, further indicating that the radioprotection of a 30% CR diet depends on altering the gut microbiota. Together, our findings provide new insights into CR in clinical applications and indicate that a short-term CR diet prior to radiation modulates sex-specific gut microbiota configurations, protecting male and female mice against the side effects caused by radiation challenge.

SREBP1/FASN/cholesterol axis facilitates radioresistance in colorectal cancer.

Acquired and intrinsic radioresistance remains a major challenge during the treatment of patients with colorectal cancer (CRC). Aberrant cholesterol metabolism precipitates the development of multiple cancers. Here, we report that exogenous or endogenous cholesterol enhances the radioresistance of CRC cells. The addition of cholesterol protects CRC cells against irradiation both in vitro and in vivo. Sterol response element-binding protein 1/fatty acid synthase (SREBP1/FASN) signaling is rapidly increased in response to radiation stimuli, resulting in cholesterol accumulation, cell proliferation and inhibition of apoptosis. Blocking the SREBP1/FASN pathway impedes cholesterol synthesis and accelerates radiation-induced CRC cell death. Our findings provide novel insights into the role of the SREBP1/FASN/cholesterol axis in radiotherapy and suggest that it may be a potential target for CRC treatment. Clinically, our results suggest that CRC patients undergoing radiotherapy may benefit from a lowered cholesterol intake.

LncRNA HOTAIR enhances breast cancer radioresistance through facilitating HSPA1A expression via sequestering miR-449b-5p.

BACKGROUND: Breast cancer (BRCA) is the leading cause of cancer-related death in women worldwide. Pre- and postoperative radiotherapy play a pivotal role in BRCA treatment but its efficacy remains limited and plagued by the emergence of radiation resistance, which aggravates patient prognosis. The long noncoding RNA (lncRNA)-implicated mechanisms underlying radiation resistance are rarely reported. The aim of this study was to determine whether lncRNA HOX transcript antisense RNA (HOTAIR) modulated the radiosensitivity of breast cancer through HSPA1A. METHODS: A Gammacell 40 Exactor was used for irradiation treatment. Bioinformatic tools and luciferase reporter assay were adopted to explore gene expression profile and demonstrate the interactions between lncRNA, miRNA and target mRNA 3'-untranslated region (3'-UTR). The expression levels of certain genes were determined by real-time PCR and western-blot analyses. in vitro and in vivo functional assays were conducted by cell viability and tumorigenicity assays. RESULTS: The levels of oncogenic lncRNA HOTAIR were positively correlated with the malignancy of BRCA but reversely correlated with the radiosensitivity of breast cancer cells. Moreover, the expression levels of HOTAIR were positively associated with those of heat shock protein family A (Hsp70) member 1A (HSPA1A) in clinical BRCA tissues and HOTAIR upregulated HSPA1A at the mRNA and protein levels in irradiated BRCA cells. Mechanistically, miR-449b-5p restrained HSPA1A expression through targeting the 3'-UTR of HSPA1A mRNA, whereas HOTAIR acted as a competing sponge to sequester miR-449b-5p and thereby relieved the miR-449b-5p-mediated HSPA1A repression. Functionally, HOTAIR conferred decreased radiosensitivity on BRCA cells, while miR-449b-5p overexpression or HSPA1A knockdown abrogated the HOTAIR-enhanced BRCA growth under the irradiation exposure both in vitro and in vivo. CONCLUSIONS: LncRNA HOTAIR facilitates the expression of HSPA1A by sequestering miR-449b-5p post-transcriptionally and thereby endows BRCA with radiation resistance. KEY POINTS: Therapeutically, HOTAIR and HSPA1A may be employed as potential targets for BRCA radiotherapy. Our findings shed new light into the mechanism by which lncRNAs modulate the radiosensitivity of tumors.

Metformin fights against radiation-induced early developmental toxicity.

Nuclear pollution intertwined accidental irradiation not only triggers acute and chronic radiation syndromes, but also endangers embryonic development in sight of uncontrollable gene mutation. Metformin (MET), a classic hypoglycemic drug, has been identified to possess multiple properties. In this study, we explored the radioprotective effects of MET on the developmental abnormalities and deformities induced by irradiation among three "star drugs". Specifically, zebrafish (Danio rerio) embryos exposed to 5.2 Gy gamma irradiation at 4 h post fertilization (hpf) showed overt developmental toxicity, including hatching delay, hatching rate decrease, developmental indexes reduction, morphological abnormalities occurrence and motor ability decline. However, MET treatment erased the radiation-induced phenotypes. In addition, MET degraded inflammatory reaction, hinders apoptosis response, and reprograms the development-related genes expression, such as sox2, sox3, sox19a and p53, in zebrafish embryos following radiation challenge. Together, our findings provide novel insights into metformin, and underpin that metformin might be employed as a promising radioprotector for radiation-induced early developmental toxicity in pre-clinical settings.

Tiling light sheet selective plane illumination microscopy using discontinuous light sheets.

Tiling light sheet selective plane illumination microscopy (TLS-SPIM) improves the 3D imaging ability of SPIM by using real-time optimized tiling light sheets. However, the imaging speed decreases and the raw image size increases due to the tiling process and additional camera exposures. The decreased imaging speed and the increased raw data could cause significant problems when TLS-SPIM is used to image large specimens at high spatial resolutions. Here, we present a novel method to solve the problem. Discontinuous light sheets created by scanning coaxial beam arrays synchronized with the detection camera rolling shutter are used in TLS-SPIM for 3D imaging. It improves the imaging efficiency of TLS-SPIM by reducing the number of tiles required per image plane without influencing the spatial resolution. We investigate the method via numerical simulations and experiments. We demonstrate the imaging ability of the TLS-SPIM using discontinuous light sheets and show the improved imaging efficiency by imaging optically cleared mouse brain.

The deubiquitinating enzyme Usp5 regulates Notch and RTK signaling during Drosophila eye development.

Usp5 belongs to the USP family of deubiquitinating enzymes (DUBs), which comprises the largest class of DUBs. We previously reported that loss of Usp5 impairs development of photoreceptors in Drosophila eyes, although the detailed mechanism remained unclear. In the present study, we demonstrate that Usp5 regulates both Notch and receptor tyrosine kinase (RTK) signaling. Loss of Usp5 results in upregulation of Notch signaling and downregulation of RTK signaling, leading to impaired photoreceptor development. Moreover, genetic rescue experiments with the DNA binding protein Suppressor of Hairless or Notch RNAi indicate that they mediate the regulation of RTK signaling by Usp5. The present study provides mechanistic insight into how Usp5 regulates photoreceptor differentiation by Notch and RTK signaling in the Drosophila eye.