comparative-strategies-for-ubiquitination-detection-mammalian-cell

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Ubiquitination, a post-translational modification, is a critical regulator of intracellular protein function. Ubiquitination modulates protein functions ...

Huazhong University of Science and Technology

Choroidal neovascularization (CNV) accounts for 90% of cases of severe vision loss in patients with advanced age-related macular degeneration. Identifying new therapeutic targets for CNV may lead to novel combination therapies to improve outcomes and reduce treatment burden. Our goal was to test whether phosphatidylserine (PS) becomes exposed in the outer membrane of choroidal neovascular endothelium, and whether this could provide a new therapeutic target for CNV.

Phosphatidylserine (PS) Is Exposed in Choroidal Neovascular Endothelium: PS-Targeting Antibodies Inhibit Choroidal Angiogenesis In Vivo and Ex Vivo.

Magnetic resonance imaging (MRI) has gained wide interest in early accurate diagnoses due to the high resolution and low toxicity of magnetic nanoparticles. In order to develop potential alternatives of toxic Gd- or Mn-based chelating agents, we report the synthesis of water soluble ultra-small FeO nanoparticles by a modified co-precipitation method as T-weighted positive contrast agents. The magnetic iron oxide nanoparticles (MIONs) were functionalized by polymer ligand dodecanthiol-polymethacrylic acid (DDT-PMAA) to enhance their colloidal stability. These MIONs have high longitudinal relaxivity ( = 8.18 mM·S) and exhibited good results in the in vitro and in vivo MR imaging. No toxicity was observed in cytotoxicity assay and histology toxicity analysis. The MIONs@DDT-PMAA(magnetic iron oxide nanoparticles @ dodecanthiol-polymethacrylic acid) present great potential as positive contrast agents for tumor diagnosis.

FeO Nanoparticles Functionalized with Polymer Ligand for T-Weighted MRI In Vitro and In Vivo.

Herpes simplex virus type 1 H129 strain has been widely used as a useful anterograde neuronal circuit tracing tool. However, whether H129 is a rigorous anterograde tracer and undergoes anterograde-only spreading are questions of significant interest. In the present study, we evaluated the retrograde labeling efficiency of H129 using a TK and ICP34.5 dual deleted H129 recombinant (named as H306) which was replication-deficient in non-dividing postmitotic neurons. The novel tracer was tested in vitro and in vivo for evaluating its invasion properties and tracing capacities. The results demonstrated that H306 could efficiently label the neurons following intracerebral injection. Notably, H306 could also efficiently infect upstream innervating neurons through axon terminal uptake and displayed obvious retrograde labeling phenotype, regardless of 3 days or 10 days of tracing. The data implied that replication-competent, trans-multisynaptic H129 tracing results might be a mixed neural networks from two types of starter cells, because the retrogradely infected neurons would also replicate H129 and spread virus anterogradely through their axon collaterals (ectopic starter sites), as the local infected neurons in the injection site (true starter site). Therefore, the interpretation of the anterogradely tracing neural networks by current H129 tools at longer post-inoculation intervals need to be cautious, and effective modification strategies are needed to avoid or block the axon terminal invasion process of H129, which is important for rigorous anterograde H129 tracer.

Evaluation of retrograde labeling profiles of HSV1 H129 anterograde tracer.

Previous work have shown several key brain nuclei involved in acute psychological stress and glucose homeostasis. Acute stress influences glucose metabolism via released stress hormones by activating the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. Little is known about the brain nuclei which response to peripheral glucose alteration are either abundant with glucosesensing neurons or the activations are secondary to stress. Here we profile and compare the brain nuclei that response to stress and glucose homeostasis in mouse models of acute restraint stress, glucose and 2-DG injections respectively. Our present work provide a comprehensive depiction on key brain nuclei involved in CNS control of stress and glucose homeostasis, which gives clue for functional identification of brain nuclei that regulate glucose homeostasis under stress.

Profiling of key brain nuclei involved in CNS control of stress and glucose homeostasis.

Understanding the connecting structure of brain network is the basis to reveal the principle of the brain function and elucidate the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although the retrograde trans-synaptic tracing for analyzing the input neural networks with recombinant rabies and pseudorabies virus has been broadly applied in neuroscience, viral tools for analyzing the output neural networks are still lacking. The recombinant vesicular stomatitis virus (VSV) has been used for the mapping of synaptic outputs. However, several drawbacks, including high neurotoxicity and rapid lethality in experimental animals, hinder its application in long-term studies of the structure and function of neural networks. To overcome these limitations, we generated a recombinant VSV with replication-related N gene mutation, VSV-N, and examined its cytotoxicity and efficiency of trans-synaptic spreading. We found that by comparison with the wild-type tracer of VSV, the N mutation endowed the virus lower rate of propagation and cytotoxicity in vitro, as well as significantly reduced neural inflammatory responses in vivo and much longer animal survival when it was injected into the nucleus of the mice brain. Besides, the spreading of the attenuated VSV was delayed when injected into the VTA. Importantly, with the reduced toxicity and extended animal survival, the number of brain regions that was trans-synaptically labeled by the mutant VSV was more than that of the wild-type VSV. These results indicated that the VSV-N, could be a promising anterograde tracer that enables researchers to explore more downstream connections of a given brain region, and observe the anatomical structure and the function of the downstream circuits over a longer time window. Our work could provide an improved tool for structural and functional studies of neurocircuit.

A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency.

Recombinant adeno-associated viruses (rAAVs), particularly those that permit efficient gene transfer to neurons from axonal terminals or across the blood-brain barrier, are useful vehicles for structural and functional studies of the neural circuit and for the treatment of many gene-deficient brain diseases that need to compensate for the correct genes in every cell in the whole brain. However, AAVs with these two advantages have not been reported. Here, we describe a new capsid engineering method, which exploits the combination of different capsids and aims to yield a capsid that can provide more alternative routes of administration that are more suitable for the wide-scale transduction of the central nervous system (CNS). A new AAV variant, AAV9-Retro, was developed by inserting the 10-mer peptide fragment from AAV2-Retro into the capsid of AAV9, and the biodistribution properties were evaluated in mice. By intracranial and intravenous injection in the mice, we found that AAV9-Retro can retrogradely infect projection neurons with an efficiency comparable to that of AAV2-Retro and retains the characteristic of AAV9, which can be transported across the nervous system. Our strategy provides a new tool for the manipulation of neural circuits and future preclinical and clinical treatment of some neurological and neurodegenerative disorders.

AAV9-Retro mediates efficient transduction with axon terminal absorption and blood-brain barrier transportation.

OTU deubiquitinase 6B (OTUD6B) study in tumors is gradually increasing; however, studies on the role of OTUD6B in colorectal cancer (CRC) are rare. OTUD6B was overexpressed in some human CRC and liver metastasis samples. Although OTUD6B facilitated migration and invasion in CRC cells, it exhibited opposite effects on liver metastasis in immunodeficient and immunocompetent mice. We demonstrated that Otud6b enhanced metastasis in nude mice, but it recruited more CD8 T cell infiltration in colorectal liver metastasis (CRLM) mouse model of C57BL/6J to inhibit CRLM through upregulating Cxcl11. Furthermore, we demonstrated that OTUD6B deubiquitinated and stabilized DDX5. Ectopically expressed DDX5 facilitated transcription factor STAT3 activation by resolving the RNA G-quadruplex structure of STAT3, resulting in a higher level of CXCL11 transcription and an increase in tumor-infiltrating CD8 T cells. All-trans retinoic acid inhibited CRLM by upregulating OTUD6B.

ATRA upregulates OTUD6B to recruit CD8 T cells to suppress colorectal liver metastasis by stabilizing DDX5/STAT3/CXCL11 axis.

: Chronic Kidney Disease (CKD) is a global public health burden with a rising prevalence driven by population aging. Existing prediction models, such as the Kidney Failure Risk Equation (KFRE), often lack generalizability across ethnicities and comprehensive systemic indicators. This study aimed to develop and validate a machine learning model for predicting CKD progression by integrating traditional risk factors with novel composite indicators reflecting systemic health. : Data from the China Health and Retirement Longitudinal Study (CHARLS,  = 2500) was used for model training. External validation was performed using independent cohorts from the English Longitudinal Study of Ageing (ELSA,  = 1200) and the Health and Retirement Study (HRS,  = 1500). Multiple machine learning algorithms, including XGBoost, were employed. Feature engineering incorporated composite indicators such as the frailty index (FI), triglyceride-glucose (TyG) index, and aggregate index of systemic inflammation (AISI). : The XGBoost model achieved an area under the curve (AUC) of 0.892 in the training set and maintained robust performance in external validation (AUC 0.867 in ELSA, 0.871 in HRS), outperforming the KFRE (AUC 0.745). SHAP analysis identified the FI as the most influential predictor. Decision curve analysis confirmed the model's clinical utility. : This machine learning model demonstrates high accuracy and cross-ethnicity validity, offering a practical tool for early intervention and personalized CKD management. Future work should address limitations such as the retrospective design and expand validation to underrepresented regions.

A Cross-Ethnicity Validated Machine Learning Model for the Progression of Chronic Kidney Disease in Individuals over 50 Years Old.

Surgical stress, such as hepatic ischemia-reperfusion (I/R) injury, induces excessive inflammation and adversely affects liver surgery outcomes. Regulatory T cells (Tregs) are crucial for immune homeostasis, yet their protective mechanisms against liver I/R injury remain unclear. In this study, we demonstrated that decreased hepatic Treg abundance correlates with increased liver injury in patients undergoing hepatic hemangioma resections. In murine models, Treg depletion worsened liver I/R injury. Bulk RNA-seq of hepatic Tregs showed enrichment of Toll-like receptor (TLR) signaling pathways, with flow cytometry identifying TLR4 as the most increased TLR after I/R. Treg-specific Tlr4 knockout mice (Treg-Tlr4-/- mice) exhibited exacerbated liver injury following I/R. Adoptive transfer of WT Tregs, but not Tlr4-deficient Tregs, alleviated liver injury in both Treg-depleted and Treg-Tlr4-/- mice. Transcriptomic analysis revealed that IL-10 production was impaired in Tlr4-deficient Tregs. Mechanistically, Tlr4-deficient Tregs showed reduced activation of the MyD88/ERK/CREB pathway, resulting in diminished IL-10 production. Myd88-/- and IL-10-/- Tregs failed to confer protection against liver I/R injury, whereas exogenous IL-10 administration rescued the hepatic dysfunction in Treg-Tlr4-/- mice. Our findings implicate the vital role of TLR4 in Tregs to mitigate liver I/R injury and offer a potential therapeutic option to reduce postoperative complications following liver surgery.

Article	Year
Phosphatidylserine (PS) Is Exposed in Choroidal Neovascular Endothelium: PS-Targeting Antibodies Inhibit Choroidal Angiogenesis In Vivo and Ex Vivo. Investigative ophthalmology & visual science\| PubMed ID: 26529048	2015
FeO Nanoparticles Functionalized with Polymer Ligand for T-Weighted MRI In Vitro and In Vivo. Polymers\| PubMed ID: 31091782	2019
Evaluation of retrograde labeling profiles of HSV1 H129 anterograde tracer. Journal of chemical neuroanatomy\| PubMed ID: 31348990	2019
Profiling of key brain nuclei involved in CNS control of stress and glucose homeostasis. Biochemical and biophysical research communications\| PubMed ID: 31672274	2020
A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency. Molecular brain\| PubMed ID: 32197632	2020
AAV9-Retro mediates efficient transduction with axon terminal absorption and blood-brain barrier transportation. Molecular brain\| PubMed ID: 33054827	2020
ATRA upregulates OTUD6B to recruit CD8 T cells to suppress colorectal liver metastasis by stabilizing DDX5/STAT3/CXCL11 axis. Cell death & disease\| PubMed ID: 40651961	2025
A Cross-Ethnicity Validated Machine Learning Model for the Progression of Chronic Kidney Disease in Individuals over 50 Years Old. Journal of clinical medicine\| PubMed ID: 41598767	2026
TLR4 maintains Treg-mediated protection against adverse outcomes in a model of hepatic surgical stress. The Journal of clinical investigation\| PubMed ID: 41766664	2026

Xin Zhong

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