G-quadruplex-based CRISPR photoswitch for spatiotemporal control of genomic modulation.

CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of CRISPR editing is essential to overcome its uncontrollable reaction process and excessive activity that leads to off-target editing. To overcome this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) for precisely controlled gene editing and expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated that rational design of the G-quadruplex onto crRNA conferred higher stability and sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization of AZD++ quickly transformed the on state of GqRNA, which facilitated rapid activation of ribonucleoprotein activity for genome editing of on-target sites in cells with excellent editing efficiency. In turn, AZD++-GqRNA promptly refolded to an off state to inhibit genomic cleavage, and limited the generation of off-target effects and by-products. Therefore, the proposed strategy of a photo-reversible modality presents a new opportunity for CRISPR-Cas9 modulation to improve its safety and applicability.

Mumia quercus sp. nov., isolated from the root of Quercus variabilis.

A novel endophytic actinomycete, designated strain NEAU-365T, was isolated from the root of Quercus variabilis collected from Nanjing, Jiangsu, PR China. Comparative 16S rRNA gene sequencing showed that strain NEAU-365T belonged to the genus Mumia but clearly differs from the currently recognized species Mumia zhuanghuii Z350T (99.31 %), Mumia xiangluensis NEAU-KD1T (98.82 %) and Mumia flava MUSC 201T (97.78 %). Phylogenetic tree analysis revealed that strain NEAU-365T clustered with the type strains of the genus Mumia. The genome size was 4.1 Mbp with a DNA G+C content of 71.2 mol%. Digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain NEAU-365T and those of M. zhuanghuii Z350T(27.5 and 84.0 %), M. xiangluensis NEAU-KD1T(23.4 and 80.9 %) and M. flava MUSC 201T(20.9 and 77.7 %) were below the recommended thresholds for species delineation. Cells were observed to be irregular cocci shaped. The cell wall contained ll-diaminopimelic acid and the whole-cell sugars were galactose and rhamnose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid and three unidentified phospholipids. The predominant menaquinone was detected as MK-9(H4). The major fatty acids were C16 : 0, C18 : 0, C18 : 1 ω9c and 10-methyl C18 : 0. On the basis of genotypic and phenotypic differences from members of the genus Mumia, a novel species, Mumia quercus sp. nov. is proposed. The type strain is NEAU-365T (=CCTCC AA 2021033T=JCM 35005T).

Sphaerisporangium perillae sp. nov., isolated from the root of Perilla frutescens (Linn.) Britt.

A novel protease-producing actinomycete, designated strain NEAU-ZS1T, was isolated from the root of Perilla frutescens (Linn.) Britt collected from Jiamusi, Heilongjiang, PR China. Comparative 16S rRNA gene sequencing showed that strain NEAU-ZS1T belonged to the genus Sphaerisporangium and was most closely related to 'Sphaerisporangium corydalis' NEAU-YHS15T (99.2%) and Sphaerisporangium cinnabarinum JCM 3291T (99.0%). Phylogenetic tree analysis revealed that strain NEAU-ZS1T formed a monophyletic clade with 'S. corydalis' NEAU-YHS15T. The genome size was 9.3 Mbp with a DNA G+C content of 70.3 mol%. Digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values between the genome sequence of strain NEAU-ZS1T and those of 'S. corydalis' NEAU-YHS15T (28.6, 83.9 and 79.1 %) and S. cinnabarinum JCM 3291T (18.5, 70.6 and 50.2 %) were below the recommended thresholds for species delineation. The strain formed spherical spore vesicles produced on the aerial hyphae. The cell wall contained meso-diaminopimelic acid and the whole-cell sugars were glucose and madurose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, an unidentified phospholipid and an unidentified glycolipid. The menaquinones were MK-9(H4), MK-9(H6) and MK-9(H2). The major fatty acids were iso-C16 : 0, C16 : 1 ω5c, 10-methy C17 : 0 and C17 : 1 ω7c. On the basis of the results of a polyphasic taxonomic study, it is concluded that strain NEAU-ZS1T represents a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium perillae sp. nov. is proposed. The type strain is NEAU-ZS1T (=CCTCC AA 2021019T= JCM 35655T).

NIR-II-absorbing diimmonium polymer agent achieves excellent photothermal therapy with induction of tumor immunogenic cell death.

Photothermal therapy has shown great promise for cancer treatment and second near-infrared (NIR-II) -absorbing particles could further improve its precision and applicability due to its superior penetration depth and new imaging ability. Herein, high NIR-II-absorbing polymer particles were prepared by using soluble isobutyl-substituted diammonium borates (P-IDI). The P-IDI showed stronger absorption at 1000-1100 nm, which exhibited excellent photostability, strong photoacoustic imaging ability and high photothermal conversion efficiency (34.7%). The investigations in vitro and in vivo demonstrated that the excellent photothermal effect facilitated complete tumor ablation and also triggered immunogenic cell death in activation of the immune response. The high solubility and excellent photothermal conversion ability demonstrated that polymer IDI particles were promising theranostic agents for treatment of tumors with minor side effects.

Surgical outcomes and risk factors for surgical complications after en bloc resection following reconstruction with 3D-printed artificial vertebral body for thoracolumbar tumors.

BACKGROUND: The outcomes of patients with tumors of the thoracolumbar spine treated with en bloc resection (EBR) using three-dimensional (3D)-printed endoprostheses are underreported. METHODS: We retrospectively evaluated patients with thoracolumbar tumors who underwent surgery at our institution. Logistic regression analysis was performed to identify the potential risk factors for surgical complications. Nomograms to predict complications were constructed and validated. RESULTS: A total of 53 patients with spinal tumors underwent EBR at our hospital; of these, 2 were lost to follow-up, 45 underwent total en bloc spondylectomy, and 6 were treated with sagittal en bloc spondylectomy. The anterior reconstruction materials included a customized 3D-printed artificial vertebral body (AVB) in 10 cases and an off-the-shelf 3D-printed AVB in 41 cases, and prosthesis mismatch occurred in 2 patients reconstructed with the off-the-shelf 3D-printed AVB. The median follow-up period was 21 months (range, 7-57 months). Three patients experienced local recurrence, and 5 patients died at the final follow-up. A total of 50 perioperative complications were encountered in 29 patients, including 25 major and 25 minor complications. Instrumentation failure occurred in 1 patient, and no prosthesis subsidence was observed. Using a combined surgical approach was a dependent predictor of overall complications, while Karnofsky performance status score, lumbar spine lesion, and intraoperative blood loss ≥ 2000 mL were predictors of major complications. Nomograms for the overall and major complications were constructed using these factors, with C-indices of 0.850 and 0.891, respectively. CONCLUSIONS: EBR is essential for the management of thoracolumbar tumors; however, EBR has a steep learning curve and a high complication rate. A 3D-printed AVB is an effective and feasible reconstruction option for patients treated with EBR.

Cu2+ -Anchored Carbon Nano-Photocatalysts for Visible Water Splitting to Boost Hydrogen Cuproptosis.

Both hydrogen (H2 ) and copper ions (Cu+ ) can be used as anti-cancer treatments. However, the continuous generation of H2 molecules and Cu+ in specific sites of tumors is challenging. Here we anchored Cu2+ on carbon photocatalyst (Cu@CDCN) to allow the continuous generation of H2 and hydrogen peroxide (H2 O2 ) in tumors using the two-electron process of visible water splitting. The photocatalytic process also generated redox-active Cu-carbon centers. Meanwhile, the Cu2+ residues reacted with H2 O2 (the obstacle to the photocatalytic process) to accelerate the two-electron process of water splitting and cuprous ion (Cu+ ) generation, in which the Cu2+ residue promoted a pro-oxidant effect with glutathione through metal-reducing actions. Both H2 and Cu+ induced mitochondrial dysfunction and intracellular redox homeostasis destruction, which enabled hydrogen therapy and cuproptosis to inhibit cancer cell growth and suppress tumor growth. Our research is the first attempt to integrate hydrogen therapy and cuproptosis using metal-enhanced visible solar water splitting in nanomedicine, which may provide a safe and effective cancer treatment.

Effect of Human Periodontal Ligament Stem Cell-Derived Extracellular Vesicles on Macrophage Pyroptosis and Periodontal Inflammatory Injury in Periodontitis.

Periodontitis is an inflammatory disease resulting from subgingival microorganisms. Human periodontal ligament stem cells (hPDLSCs) can be applied in periodontal tissue regeneration. This study investigated the effect of hPDLSC-derived extracellular vesicles (EVs) on periodontitis. hPDLSC-derived EVs were isolated and identified. The murine model of periodontitis was established by ligation, and the cell model of periodontitis was established by treatment of macrophages with lipopolysaccharide (LPS). The effects of EVs on macrophage pyroptosis and periodontal inflammatory injury were measured by the means of HE staining, detection of LDH content, CCK-8 assay, Calcein-AM/PI staining, ELISA, Western blot, as well as measurement of caspase-1, SOD, and MDA. miR-590-3p expression was detected using RT-qPCR. miR-590-3p expression was then intervened to validate the effect of miR-590-3p on macrophage pyroptosis. The binding relationship between miR-590-3p and TLR4 was verified using dual-luciferase assay. Functional rescue experiment was performed to validate the role of TLR4 in macrophage pyroptosis. The results showed that inflammatory levels and macrophage pyroptosis were enhanced in the in vivo and in vitro models of periodontitis, evidenced by the increased NLRP3, GSDMD-N, caspase-1, IL-1ß, IL-18, TNF-α, and MDA and decreased IL-10 and SOD. EVs alleviated periodontal inflammatory injury and macrophage pyroptosis. Physiologically, EVs carried miR-590-3p into macrophages to upregulate miR-590-3p expression and thereby suppress TLR4 transcription. miR-590-3p silencing or TLR4 overexpression reduced the inhibitory effect of EVs on macrophage pyroptosis. Collectively, EVs carried miR-590-3p into macrophages to subsequently inhibit TLR4 transcription, thereby reducing macrophage pyroptosis and alleviating periodontal inflammatory injury.

Intracellular marriage of bicarbonate and Mn ions as "immune ion reactors" to regulate redox homeostasis and enhanced antitumor immune responses.

BACKGROUND: Different from Fe ions in Fenton reaction, Mn ions can function both as catalyst for chemodynamic therapy and immune adjuvant for antitumor immune responses. In Mn-mediated Fenton-like reaction, bicarbonate ([Formula: see text]), as the most important component to amplify therapeutic effects, must be present, however, intracellular [Formula: see text] is strictly limited because of the tight control by live cells. RESULTS: Herein, Stimuli-responsive manganese carbonate-indocyanine green complexes (MnCO3-ICG) were designed for intracellular marriage of bicarbonate and Mn ions as "immune ion reactors" to regulate intracellular redox homeostasis and antitumor immune responses. Under the tumor acidic environment, the biodegradable complex can release "ion reactors" of Mn2+ and [Formula: see text], and ICG in the cytoplasm. The suddenly increased [Formula: see text] in situ inside the cells regulate intracellular pH, and accelerate the generation of hydroxyl radicals for the oxidative stress damage of tumors cells because [Formula: see text] play a critical role to catalyze Mn-mediated Fenton-like reaction. Investigations in vitro and in vivo prove that the both CDT and phototherapy combined with Mn2+-enhanced immunotherapy effectively suppress tumor growth and realize complete tumor elimination. CONCLUSIONS: The combination therapy strategy with the help of novel immune adjuvants would produce an enhanced immune response, and be used for the treatment of deep tumors in situ.

Composition tunability of semiconductor radiosensitizers for low-dose X-ray induced photodynamic therapy.

Radiation therapy is one of the most commonly used methods in clinical cancer treatment, and radiosensitizers could achieve enhanced therapeutic efficacy by incorporating heavy elements into structures. However, the secondary excitation of these high-Z elements-doped nanosensitizers still imply intrinsic defects of low efficiency. Herein, we designed Bi-doped titanium dioxide nanosensitizers in which high-Z Bi ions with adjustable valence state (Bi3+ or Bi4+) replaced some positions of Ti4+ of anatase TiO2, increasing both X-rays absorption and oxygen vacancies. The as-prepared TiO2:Bi nanosensitizers indicated high ionizing radiation energy-transfer efficiency and photocatalytic activity, resulting in efficient electron-hole pair separation and reactive oxygen species production. After further modification with cancer cell targeting peptide, the obtained nanoplatform demonstrated good performance in U87MG cell uptakes and intracellular radicals-generation, severely damaging the vital subcellular organs of U87MG cells, such as mitochondrion, membrane lipid, and nuclei etc. These combined therapeutic actions mediated by the composition-tunable nanosensitizers significantly inhibited the U87MG tumor growth, providing a refreshing strategy for X-ray induced dynamic therapy of malignant tumors.

Minimally invasive separation surgery for the treatment of spinal metastases with small incision and freehand pedicle screw fixation: the surgical learning curve.

BACKGROUND: Minimally invasive separation surgery (MISS) is a safe and effective surgical technique, the current optimal treatment for spinal metastases. However, the learning curve for this technique has not been analyzed. This study aimed to define and analyze the surgical learning curve of MISS for the treatment of spinal metastases with small incision and freehand pedicle screw fixation. METHODS: A continuous series of 62 patients with spinal metastases who underwent MISS were included. Each patient's operative data were accurately counted. The improvement of the patients' neurological function was followed up after surgery to evaluate the surgical treatment effect. Logarithmic curve-fit regression was used to analyze the surgical learning curve of MISS. The number of cases needed to achieve proficiency was analyzed. Based on this cut-off point, this series of cases was divided into the early phase and later phase groups. The influence of the time sequence of MISS on surgical data and surgical efficacy was analyzed. RESULTS: The operative time decreased gradually with the number of surgical cases increasing and stabilized after the 20th patient. There was no statistical difference in demographic characteristics and preoperative characteristics between the two groups. The mean operative time in the later phase group was about 39 min shorter than that in the early phase group (mean 227.95 vs. 189.02 min, P = 0.027). However, it did not affect other operative data or the surgical treatment effect. CONCLUSION: The learning curve of MISS for spinal metastases is not steep. With the increase of surgeons' experience, the operative time drops rapidly and stabilizes within a certain range. MISS can be safely and effectively performed at the beginning of a surgeon's caree.

Surveilling and Tracking COVID-19 Patients Using a Portable Quantum Dot Smartphone Device.

The ability to rapidly diagnose, track, and disseminate information for SARS-CoV-2 is critical to minimize its spread. Here, we engineered a portable smartphone-based quantum barcode serological assay device for real-time surveillance of patients infected with SARS-CoV-2. Our device achieved a clinical sensitivity of 90% and specificity of 100% for SARS-CoV-2, as compared to 34% and 100%, respectively, for lateral flow assays in a head-to-head comparison. The lateral flow assay misdiagnosed ∼2 out of 3 SARS-CoV-2 positive patients. Our quantum dot barcode device has ∼3 times greater clinical sensitivity because it is ∼140 times more analytically sensitive than lateral flow assays. Our device can diagnose SARS-CoV-2 at different sampling dates and infectious severity. We developed a databasing app to provide instantaneous results to inform patients, physicians, and public health agencies. This assay and device enable real-time surveillance of SARS-CoV-2 seroprevalence and potential immunity.

NIR-II emissive AIEgen photosensitizers enable ultrasensitive imaging-guided surgery and phototherapy to fully inhibit orthotopic hepatic tumors.

Accurate diagnosis and effective treatment of primary liver tumors are of great significance, and optical imaging has been widely employed in clinical imaging-guided surgery for liver tumors. The second near-infrared window (NIR-II) emissive AIEgen photosensitizers have attracted a lot of attention with higher-resolution bioimaging and deeper penetration. NIR-II aggregation-induced emission-based luminogen (AIEgen) photosensitizers have better phototherapeutic effects and accuracy of the image-guided surgery/phototherapy. Herein, an NIR-II AIEgen phototheranostic dot was proposed for NIR-II imaging-guided resection surgery and phototherapy for orthotopic hepatic tumors. Compared with indocyanine green (ICG), the AIEgen dots showed bright and sharp NIR-II emission at 1250 nm, which extended to 1600 nm with high photostability. Moreover, the AIEgen dots efficiently generated reactive oxygen species (ROS) for photodynamic therapy. Investigations of orthotopic liver tumors in vitro and in vivo demonstrated that AIEgen dots could be employed both for imaging-guided tumor surgery of early-stage tumors and for 'downstaging' intention to reduce the size. Moreover, the therapeutic strategy induced complete inhibition of orthotopic tumors without recurrence and with few side effects.

Reprograming the tumor immunologic microenvironment using neoadjuvant chemotherapy in osteosarcoma.

Tumor-infiltrating immune cells play a crucial role in tumor progression and response to treatment. However, the limited studies on infiltrating immune cells have shown inconsistent and even controversial results for osteosarcoma (OS). In addition, the dynamic changes of infiltrating immune cells after neoadjuvant chemotherapy are largely unknown. We downloaded the RNA expression matrix and clinical information of 80 OS patients from the TARGET database. CIBERSORT was used to evaluate the proportion of 22 immune cell types in patients based on gene expression data. M2 macrophages were found to be the most abundant immune cell type and were associated with improved survival in OS. Another cohort of pretreated OS samples was evaluated by immunohistochemistry to validate the results from CIBERSORT analysis. Matched biopsy and surgical samples from 27 patients were collected to investigate the dynamic change of immune cells and factors before and after neoadjuvant chemotherapy. Neoadjuvant chemotherapy was associated with increased densities of CD3+ T cells, CD8+ T cells, Ki67 + CD8+ T cells and PD-L1+ immune cells. Moreover, HLA-DR-CD33+ myeloid-derived suppressive cells (MDSC) were decreased after treatment. We determined that the application of chemotherapy may activate the local immune status and convert OS into an immune "hot" tumor. These findings provide rationale for investigating the schedule of immunotherapy treatment in OS patients in future clinical trials.

Acridine Orange Encapsulated Mesoporous Manganese Dioxide Nanoparticles to Enhance Radiotherapy.

Manganese dioxide (MnO2) nanoparticles are a promising type of radiosensitizer for they can catalyze H2O2 decomposition to produce O2. Combining MnO2 nanoparticles with conventional, small molecule radiosensitizers would further enhance radiotherapy (RT) efficacy due to complementary mechanisms of action. However, solid MnO2 nanoparticles are suboptimal at drug loading, limiting the related progress. Herein we report a facile method to synthesize mesoporous MnO2 (mMnO2) nanoparticles, which can efficiently encapsulate small molecule therapeutics. In particular, we found that acridine orange (AO), a small molecule radiosensitizer, can be loaded onto mMnO2 nanoparticles at very high efficiency and released to the surroundings in a controlled fashion. We show that mMnO2 nanoparticles can efficiently produce O2 inside cells. This, together with AO-induced DNA damage, significantly enhances RT outcomes, which was validated both in vitro and in vivo. Meanwhile, mMnO2 nanoparticles slowly degrade in acidic environments to release Mn2+, providing a facile way to keep track of the nanoparticles through magnetic resonance imaging (MRI). Overall, our studies suggest mMnO2 as a promising nanoplatform that can be exploited to produce composite radiosensitizers for RT.

Aggregation-Induced Emission Gold Clustoluminogens for Enhanced Low-Dose X-ray-Induced Photodynamic Therapy.

The use of gold nanoparticles as radiosensitizers is an effective way to boost the killing efficacy of radiotherapy while drastically limiting the received dose and reducing the possible damage to normal tissues. Herein, we designed aggregation-induced emission gold clustoluminogens (AIE-Au) to achieve efficient low-dose X-ray-induced photodynamic therapy (X-PDT) with negligible side effects. The aggregates of glutathione-protected gold clusters (GCs) assembled through a cationic polymer enhanced the X-ray-excited luminescence by 5.2-fold. Under low-dose X-ray irradiation, AIE-Au strongly absorbed X-rays and efficiently generated hydroxyl radicals, which enhanced the radiotherapy effect. Additionally, X-ray-induced luminescence excited the conjugated photosensitizers, resulting in a PDT effect. The in vitro and in vivo experiments demonstrated that AIE-Au effectively triggered the generation of reactive oxygen species with an order-of-magnitude reduction in the X-ray dose, enabling highly effective cancer treatment.

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model.

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara-C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara-C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara-C were elucidated by whole-exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara-C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.

Clinical Effect of Radiotherapy Combined with Chemotherapy for Non-Surgical Treatment of the Esophageal Squamous Cell Carcinoma.

BACKGROUND Chemoradiotherapy (CRT) is widely accepted and is considered a standard treatment, particularly for unresectable and inoperable esophageal squamous cell carcinoma (ESCC). However, the optimal use of the combined modalities of chemotherapy (CT) and radiotherapy (RT) remains controversial. In addition, no consensus has been reached regarding the exact efficacy of consolidation chemotherapy (CCT) and the most appropriate radiotherapy dose. MATERIAL AND METHODS Clinical data from 262 ESCC patients treated with CRT (n=165) or RT alone (n=97) were collected and reviewed. The long-term outcomes were analyzed, and treatment related acute toxicity reactions were compared. RESULTS The 1-year, 3-year, and 5-year overall survival (OS) rates were 75.3%, 35.6%, and 25.3%, respectively, for the CRT group and 61.5%, 26.7%, and 17.6% for the RT-alone group (P=0.015). The concurrent chemoradiotherapy (CCRT) and sequential chemoradiotherapy (SCRT) groups exhibited similar survival outcomes (for OS, P=0.568; for progression-free survival (PFS,) P=0.145). CCT after CCRT did not influence OS (P=0.236) but was associated with a more favorable PFS (P=0.020). In addition, high-dose of 60-65 Gy tended to prolong OS compared with low-dose (<60 Gy) or excessive-dose (>65 Gy). The incidence of adverse reactions, such as esophagitis and leukopenia, in the CRT group were significantly higher than in the RT-alone group (P=0.019, P=0.001, respectively), and no significant difference was observed between patients treated with CCRT and CCT after CCRT. CONCLUSIONS Treating non-surgical ESCC patients with CCRT conferred a significant survival benefit compared with RT alone. CCT after CCRT prolongs PFS but does not increase acute toxicity. High-dose (60-65 Gy) CCRT could generate more favorable survival outcomes.

[Relative security of health care big data protection].

With China's implementing big data strategy, health care becomes one of the key areas in which the national big data strategy is highly promoted. However, as the health care big data industry grows rapidly, the security risk is increasingly prominent and the internet plus medical care makes the protection of health care big data more complicated. By analyzing the current situation of health care big data security protection, the article proposes the viewpoint of relative security and suggests the strategies and key issues of health care big data protection. From the aspects of legal supervision, talents cultivation, publicity and education, and key data protection, it is necessary to build the prevention and control system for health care big data security, so that the security risk can be reduced and the data utility can be maximized.

Src-mediated ligand release-independent EGFR transactivation involves TGF-ß-induced Smad3 activation in mesangial cells.

A great deal of evidence highlighted the pathophysiologic importance of TGF-ß1/Smad3 pathway in masangial extracellular matrix (ECM) accumulation, but some alternative signaling pathways are also involved. TGF-ß was shown recently to induce rapid and transient epidermal-like growth factor receptor (EGFR) transactivation and subsequent fibronectin expression via heparin-binding epidermal-like growth factors (HB-EGF) release and binding in mesangial cells, which is independent of Smad2 activation. However, whether TGF-ß could induce persistent EGFR transactivation remains to be identified. The present study demonstrates that in addition to transient EGFR transactivation, TGF-ß1 can also induce continuous EGFR transactivation by a non-ligand-dependent pathway in rat mesangial cells. This sustained EGFR transactivation is mainly due to Src kinase-mediated persistent EGFR tyrosine phosphorylation at Y845 rather than Y1173. TGF-ß1-induced early Smad3 phosphorylation is independent of transient EGFR transactivation and ERK1/2 activation initiated by HB-EGF release, whereas Src-mediated chronic EGFR transactivation and ERK1/2 activation participate in Smad3 activation in a relatively modest and delayed manner. Therefore, the present study further clarifies the mechanisms of EGFR transactivation in the TGF-ß-initiated ECM upregulation and raises the possibility that targeting EGFR may provide a viable alternative strategy for inhibiting TGF-ß in chronic kidney disease.