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Abstract Objective Lymphaticovenular anastomosis (LVA) is increasingly utilized in the treatment of lymphedema. This study aims to assess the efficacy and safety of the “Overlapping” LVA technique, which addresses the size mismatch between lymphatic and venous vessels in lymphedema treatment. Methods Between August 2022 and April 2023, seventeen patients diagnosed with lymphedema were enrolled in this study. The severity of lymphedema in these patients was classified according to the International Society of Lymphology (ISL) staging system.All patient underwent LVA procedures, anastomosis techniques including the Overlapping, end-to-end and octopus anastomosis. The techniques of anastomosis, anastomosis time, patency rate, and volume of limb lymphedema were evaluated. Results Our study enrolled 17 lymphedema patients who underwent the LVA procedure. All patients showed significant postoperative improvement in limb edema. The mean drainage volume was 472.29 ml. The Overlapping technique demonstrated a 100% success rate as assessed by clinical observation and intraoperative Indocyanine Green (ICG) lymphography. The average anastomosis time was 5.3 min, reducing operative time compared to traditional methods. Conclusions These findings suggest that the Overlapping technique could serve as a valuable addition to the current LVA technique. This Overlapping anastomosis technique provides a wide range of applications for lymphatic anastomosis treatment and prevention of lymphedema.

Abstract Background Hyperuricaemia is common among obese children and adolescents, and is closely related to insulin resistance. The aim of this study was to explore the relationships between youth insulin resistance and hyperuricaemia, as well as their relationships with lifestyle factors in youths, to provide early guidance on the risk factors for hyperuricaemia in adolescents. Methods This study included 233 adolescents aged 10 to 20 years. Insulin resistance was evaluated via the homeostasis model assessment-insulin resistance (HOMA-IR) method. Binary logistic regression analysis was used to assess the associations of HOMA-IR with hyperuricaemia status and serum uric acid (UA) levels. The participants were subsequently divided into two groups, the noninsulin resistant group (HOMA-IR ≤ 3.2) and the insulin resistant group (HOMA-IR > 3.2), to further explore the factors that may affect the serum UA level. Finally, the predictive ability of different indicators of hyperuricaemia was evaluated via the ROC curve. Results Binary logistic regression analysis revealed a significant increase in the risk of developing hyperuricaemia for individuals with elevated HOMA-IR (p

In a recent study, a research group from Taiyuan University of Technology published their findings in the journal Opto-Electronic Science with a title "Simultaneously realizing thermal and electromagnetic cloaking by multi-physical null medium." This work introduces a structure that can control simultaneously both electromagnetic waves and heat flow. For the first time, a single structure capable of cloaking both electromagnetic waves and heat flow was experimentally demonstrated. This research offers new solutions for the simultaneous control of electromagnetic waves and heat flow, and advances the hybrid design of electromagnetic compatibility and thermal management, which may have important potentials in e.g. medical applications.

Summary: This study investigated host responses to long COVID by following up with 89 of the original 144 cohorts for 1-year (N = 73) and 2-year visits (N = 57). Pulmonary long COVID, characterized by fibrous stripes, was observed in 8.7% and 17.8% of patients at the 1-year and 2-year revisits, respectively, while renal long COVID was present in 15.2% and 23.9% of patients, respectively. Pulmonary and renal long COVID at 1-year revisit was predicted using a machine learning model based on clinical and multi-omics data collected during the first month of the disease with an accuracy of 87.5%. Proteomics revealed that lung fibrous stripes were associated with consistent down-regulation of surfactant-associated protein B in the sera, while renal long COVID could be linked to the inhibition of urinary protein expression. This study provides a longitudinal view of the clinical and molecular landscape of COVID-19 and presents a predictive model for pulmonary and renal long COVID.

Priestia megaterium (P. megaterium PH3) is an endophytic bacterium isolated from peanuts. It has natural resveratrol production ability and shows potential application value. This study analyzed its genetic function and metabolic mechanism through whole-genome sequencing and found that the genome size is 5,960,365 bp, the GC content is 37.62%, and 6132 genes are annotated. Functional analysis showed that this strain contained 149 carbohydrate active enzyme genes, 7 secondary metabolite synthesis gene clusters, 509 virulence genes, and 273 drug-resistance genes. At the same time, this strain has the ability to regulate salt stress, low temperature, and hypoxia. Genomic analysis reveals a stilbene-synthase-containing type III polyketide synthase gene cluster that contributes to resveratrol synthesis. A safety assessment showed that the strain is non-hemolytic, does not produce amino acid decarboxylase, and is not resistant to multiple antibiotics. In the mouse model, P. megaterium PH3 did not have significant effects on body weight, behavior, or physiological indicators. These results provide important basic data and theoretical support for its industrial application and the research and development of plant protection agents.

Abstract Background Harnessing hepatocytes for basic research and regenerative medicine demands a complete understanding of the genetic determinants underlying hepatocyte differentiation and maturation. Single-cell CRISPR screens in organoids could link genetic perturbations with parallel transcriptomic readout in single cells, providing a powerful method to delineate roles of cell fate regulators. However, a big challenge for identifying key regulators during data analysis is the low expression levels of transcription factors (TFs), which are difficult to accurately estimate due to noise and dropouts in single-cell sequencing. Also, it is often the changes in TF activities in the transcriptional cascade rather than the expression levels of TFs that are relevant to the cell fate transition. Results Here, we develop Organoid-based Single-cell CRISPR screening Analyzed with Regulons (OSCAR), a framework using regulon activities as readouts to dissect gene knockout effects in organoids. In adult-stem-cell-derived liver organoids, we map transcriptomes in 80,576 cells upon 246 perturbations associated with transcriptional regulation of hepatocyte formation. Using OSCAR, we identify known and novel positive and negative regulators, among which Fos and Ubr5 are the top-ranked ones. Further single-gene loss-of-function assays demonstrate that Fos depletion in mouse and human liver organoids promote hepatocyte differentiation by specific upregulation of liver metabolic genes and pathways, and conditional knockout of Ubr5 in mouse liver delays hepatocyte maturation. Conclusions Altogether, we provide a framework to explore lineage specifiers in a rapid and systematic manner, and identify hepatocyte determinators with potential clinical applications.

Abstract Osteosarcoma is the most common malignant bone tumor, tending to be aggressive and recurrent. The therapeutic development for treating osteosarcoma has been largely hampered by the lack of effective and specific targets. Using kinome-wide CRISPR-Cas9 knockout screens, we systematically revealed a cohort of kinases essential for the survival and growth of human osteosarcoma cells, in which Polo-like kinase 1 (PLK1) appeared as a specific prominent hit. PLK1 knockout substantially inhibited proliferation of osteosarcoma cells in vitro and the tumor growth of osteosarcoma xenograft in vivo. Volasertib, a potent experimental PLK1 inhibitor, can effectively inhibit the growth of the osteosarcoma cell lines in vitro. It can also disrupt the development of tumors in the patient-derived xenograft (PDX) models in vivo. Furthermore, we confirmed that the mode of action (MoA) of volasertib is primarily mediated by the cell-cycle arrest and apoptosis triggered by DNA damage. As PLK1 inhibitors are entering phase III clinical trials, our findings provide important insights into the efficacy and MoA of the relevant therapeutic approach for combating osteosarcoma.

Prefabricated modular substations are expected to become the mainstream construction type for substations in China. However, there is a lack of scientific basis for structural selection and seismic performance evaluation. Taking a 110 kV substation as an example, this study compares the construction cost of cast-in situ reinforced concrete (RC) substations and prefabricated steel (PS) ones. Two types of PS structures are considered: one with H-section steel columns and the other with box-section steel columns. A seismic vulnerability analysis is performed to compare the probability distribution of various damage states of substation building structures under different seismic damage levels. Results indicate that the construction cost of PS structures is approximately 27.9% higher than that of cast-in situ concrete. When using H-section steel columns, there is a significant difference in the flexural stiffness in two horizontal directions, resulting in reduced seismic performance in the weak-axis direction. The construction cost of using box-section steel columns is slightly higher than that of the H-section steel case, but its seismic performance is significantly improved. Although the probability of slight and moderate damage states for the box-section steel column scheme is generally higher than that of the cast-in situ RC scheme, the probability of collapse is reduced. Thus, box-section steel columns are recommended for prefabricated modular substation building structures.

Abstract Background Patellar dislocation is common in young people. Although isolated anatomic double-bundle reconstruction of the MPFL is a common and effective surgical treatment for patellofemoral instability, concerns about the risk of injury to the epiphysis remain. Methods A total of 21 children and adolescents (9 males, 12 females; mean age: 10.7 years; range: 8 to 13 years) with recurrent patella dislocation or symptomatic instability following a primary dislocation were enrolled in the study. In all patients, double-bundle medial patellofemoral ligament (MPFL) reconstruction and femoral sling procedure were performed under arthroscopy, using an anterior half peroneus longus tendon (AHPLT) autograft. Functional outcomes were evaluated preoperatively and during follow-ups based on Kujala and Lysholm scores. Radiological examinations including radiographs, 3D-CT, and MRI were performed pre- and post-operatively. Results Among two-year postoperative follow-up (range: 24–42 months) showed significant improvement in functional scores (p

Abstract Background Mesenchymal stromal cells (MSCs) have attracted great attention in the application of cell-based therapy because of their pluripotent differentiation and immunomodulatory ability. Due to the limited number of MSCs isolated from donor tissues, a large number of MSCs need to be expanded in a traditional two-dimensional cell culture device to obtain a sufficient therapeutic amount. However, long-term cultivation of MSCs in vitro has been proven to reduce their differentiation potential and change their immunomodulatory characteristics. We aimed to explore the cellular heterogeneity and differentiation potential of different MSCs expanded in vitro and reconstruct the complex cloning track of cells in the process of differentiation. Methods Single cell transcriptome sequencing was combined with ‘CellTagging’, which is a composite barcode indexing method that can capture the cloning history and cell identity in parallel to track the differentiation process of the same cell over time. Results Through the single-cell transcriptome and CellTagging, we found that the heterogeneity of human adipose tissue derived stem cells (hADSCs) in the early stage of culture was very limited. With the passage, the cells spontaneously differentiated during the process of division and proliferation, and the heterogeneity of the cells increased. By tracing the differentiation track of cells, we found most cells have the potential for multidirectional differentiation, while a few cells have the potential for unidirectional differentiation. One subpopulation of hADSCs with the specific osteoblast differentiation potential was traced from the early stage to the late stage, which indicates that the differentiation trajectories of the cells are determined in the early stages of lineage transformation. Further, considering that all genes related to osteogenic differentiation have not yet been determined, we identified that there are some genes that are highly expressed specifically in the hADSC subsets that can successfully differentiate into osteoblasts, such as Serpin Family E Member 2 (SERPINE2), Secreted Frizzled Related Protein 1 (SFRP1), Keratin 7 (KRT7), Peptidase Inhibitor 16 (PI16), and Carboxypeptidase E (CPE), which may be key regulatory genes for osteogenic induction, and finally proved that the SERPINE2 gene can promote the osteogenic process. Conclusion The results of this study contribute toward the exploration of the heterogeneity of hADSCs and improving our understanding of the influence of heterogeneity on the differentiation potential of cells. Through this study, we found that the SERPINE2 gene plays a decisive role in the osteogenic differentiation of hADSCs, which lays a foundation for establishing a more novel and complete induction system.

Osteosarcoma (OS), a common bone malignancy, is highly metastatic and featured by poor prognosis. SUMOylation is deeply involved in tumorigenesis and cancer progression. SUMO-activating enzyme (SAE) inhibitor has been found to play an anti-proliferative role in multiple cancers. However, their functions in osteosarcoma remain unknown. Based on this, it was hypothesized that ML792, an SAE inhibitor, might be an anti-tumor candidate against osteosarcoma in this paper. The role of ML792 in the treatment of osteosarcoma was investigated in vitro and in vivo. The results showed ML792 significantly arrested human osteosarcoma cells (HOCs) at the G2/M phase, inhibited the proliferation of HOCs, and elicited apoptosis. To better assess the inhibitory role of ML792 in human osteosarcoma, we induced HOCs with TGF-β1. Our study showed that ML792 significantly suppressed the migration, invasion, and EMT of HOCs induced by TGF-β1. Moreover, ML792 inhibited TGF-β1-induced phosphatidylinositol- 3-kinase (PI3K) and AKT activation, as well as Smad2/3 phosphorylation in HOCs. In vivo, ML792 inhibits the growth of osteosarcoma. Thus, ML792 is a promising new antitumor agent to suppress the progress of OS cells undergoing proliferation and EMT by targeting the PI3K/Akt and TGF-β1/Smad pathway and may prevent or reduce the growth and metastasis of osteosarcoma.

Stroke is characterized by high morbidity, high mortality and high disability rate, which is a major health problem worldwide. However, most community-based studies report a lack of public knowledge related to stroke. The aim of this study is to investigate stroke-related knowledge and prevention practices among stroke patients in Taizhou, China. A face-to-face survey was conducted and questionnaires were completed by 156 S patients from June 27 to August 30, 2022. A generalized linear model was applied to explore the factors influencing prevention practices. Among the total participants, 36.5% and 40.4% of them had good knowledge of the stroke-related warning signs and risk factors, respectively. Participants who had good stroke prevention practices accounted for 57.7%. The higher score of stroke-related knowledge among inpatients, the better their prevention practices (B = 0.16, 95 %CI: 0.05 ∼ 0.28). In addition, those with age ≥ 60 (B = 1.20, 95 %CI: 0.42 ∼ 1.97), females (B = 0.93, 95 %CI: 0.24 ∼ 1.61), having physical activities (B = 1.01, 95 %CI: 0.33 ∼ 1.68), or without underlying diseases (B = −1.67, 95 %CI:−2.42 ∼ −0.92) were also related with prevention practices. In general, this survey indicated that the stroke-related knowledge and prevention practices of participants were not good enough. Stroke related knowledge, age, sex, physical activity, and underlying disease were significant factors related to stroke prevention practices. These findings suggest the need to focus on stroke health education for stroke patients.

Abstract Prolonged activation of nuclear factor (NF)-кB signaling significantly contributes to the development of colorectal cancer (CRC). New therapeutic opportunities are emerging from targeting this distorted cell signaling transduction. Here, we discovered the critical role of RING finger 138 (RNF138) in CRC tumorigenesis through regulating the NF-кB signaling, which is independent of its Ubiquitin-E3 ligase activity involved in DNA damage response. RNF138−/− mice were hyper-susceptible to the switch from colitis to aggressive malignancy, which coincided with sustained aberrant NF-кB signaling in the colonic cells. Furthermore, RNF138 suppresses the activation of NF-кB signaling pathway through preventing the translocation of NIK and IKK-Beta Binding Protein (NIBP) to the cytoplasm, which requires the ubiquitin interaction motif (UIM) domain. More importantly, we uncovered a significant correlation between poor prognosis and the downregulation of RNF138 associated with reinforced NF-кB signaling in clinical settings, raising the possibility of RNF138 dysregulation as an indicator for the therapeutic intervention targeting NF-кB signaling. Using the xenograft models built upon either RNF138-dificient CRC cells or the cells derived from the RNF138-dysregulated CRC patients, we demonstrated that the inhibition of NF-кB signaling effectively hampered tumor growth. Overall, our work defined the pathogenic role of aberrant NF-кB signaling due to RNF138 downregulation in the cascade events from the colitis switch to colonic neoplastic transformation and progression, and also highlights the possibility of targeting the NF-кB signaling in treating specific subtypes of CRC indicated by RNF138-ablation.

Abstract Aflatoxin exposure is a crucial factor in promoting the development of primary hepatocellular carcinoma (HCC) in individuals infected with the hepatitis virus. However, the molecular pathways leading to its bioactivation and subsequent toxicity in hepatocytes have not been well-defined. Here, we carried out a genome-wide CRISPR-Cas9 genetic screen to identify aflatoxin B1 (AFB1) targets. Among the most significant hits was the aryl hydrocarbon receptor (AHR), a ligand-binding transcription factor regulating cell metabolism, differentiation, and immunity. AHR-deficient cells tolerated high concentrations of AFB1, in which AFB1 adduct formation was significantly decreased. AFB1 triggered AHR nuclear translocation by directly binding to its N-terminus. Furthermore, AHR mediated the expression of P450 induced by AFB1. AHR expression was also elevated in primary tumor sections obtained from AFB1-HCC patients, which paralleled the upregulation of PD-L1, a clinically relevant immune regulator. Finally, anti-PD-L1 therapy exhibited greater efficacy in HCC xenografts derived from cells with ectopic expression of AHR. These results demonstrated that AHR was required for the AFB1 toxicity associated with HCC, and implicate the immunosuppressive regimen of anti-PD-L1 as a therapeutic option for the treatment of AFB1-associated HCCs.

Abstract Background Millions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens. Results To improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons of BRCA1 and BRCA2 genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants for BRCA1/2, including 151 variants in the noncoding part of the genes such as the 5′ untranslated regions. Many of them are identified in cancer patients and are reported as “benign/likely benign” or “variants of uncertain significance” by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer. Conclusions Our results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.

Severity prediction of COVID-19 remains one of the major clinical challenges for the ongoing pandemic. Here, we have recruited a 144 COVID-19 patient cohort, resulting in a data matrix containing 3,065 readings for 124 types of measurements over 52 days. A machine learning model was established to predict the disease progression based on the cohort consisting of training, validation, and internal test sets. A panel of eleven routine clinical factors constructed a classifier for COVID-19 severity prediction, achieving accuracy of over 98% in the discovery set. Validation of the model in an independent cohort containing 25 patients achieved accuracy of 80%. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 0.70, 0.99, 0.93, and 0.93, respectively. Our model captured predictive dynamics of lactate dehydrogenase (LDH) and creatine kinase (CK) while their levels were in the normal range. This model is accessible at https://www.guomics.com/covidAI/ for research purpose.

Abstract Background Most cancer risk-associated single nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) are noncoding and it is challenging to assess their functional impacts. To systematically identify the SNPs that affect gene expression by modulating activities of distal regulatory elements, we adapt the self-transcribing active regulatory region sequencing (STARR-seq) strategy, a high-throughput technique to functionally quantify enhancer activities. Results From 10,673 SNPs linked with 996 cancer risk-associated SNPs identified in previous GWAS studies, we identify 575 SNPs in the fragments that positively regulate gene expression, and 758 SNPs in the fragments with negative regulatory activities. Among them, 70 variants are regulatory variants for which the two alleles confer different regulatory activities. We analyze in depth two regulatory variants—breast cancer risk SNP rs11055880 and leukemia risk-associated SNP rs12142375—and demonstrate their endogenous regulatory activities on expression of ATF7IP and PDE4B genes, respectively, using a CRISPR-Cas9 approach. Conclusions By identifying regulatory variants associated with cancer susceptibility and studying their molecular functions, we hope to help the interpretation of GWAS results and provide improved information for cancer risk assessment.

Tibial plateau fractures are multiple fracture patterns associated with soft-tissue injuries. Among which, the combined existence of posterolateral tibial plateau depression fracture with anterior cruciate ligament (ACL) rupture has been reported rarely. Meanwhile, surgical method for the treatment of depression fracture is fairly complex. The aim of this article is to show a case series of this unusual injury pattern and the therapy of posterolateral tibial plateau depression fracture accompanying ACL rupture. In our treatment, arthroscopy assisted reduction of depression fracture and ACL reconstruction reduces surgical trauma and leads to good functional recovery. We also review the current literature.

Purpose: Sternoclavicular joint is an amphiarthrodial joint formed by the clavicle and sternal manubrium. This joint becomes chronically unstable in case of a medial clavicle dislocation or fracture, and improper treatment could cause malformation and pain. We aimed to determine the efficacy of a novel sternoclavicular hook plate for treatment of unstable sternoclavicular joint dislocation or fracture. Methods: Between June 2011 and December 2013, the sternoclavicular hook plate was used to surgically treat 32 adult patients with unstable sternoclavicular joint dislocation or fracture. Of these, 12 and 5 patients suffered from anterior and posterior dislocation of the sternoclavicular joint, respectively, 10 had medial clavicle fracture, and 5 had fracture dislocation. For anterior fracture dislocation, the standard sternoclavicular hook plate was used, while for the posterior dislocation, screws were added at the distal end of the hook plate, anterior to the sternal manubrium, to prevent postoperative redislocation. Results: No intraoperative complications were observed during the procedure. Postoperative X-ray and computed tomography revealed normal anatomical positions of sternoclavicular joints and excellent positions of internal fixation. About 3–6 months after surgery, all patients achieved primary healing without redislocation of the sternoclavicular joint along with satisfactory restoration of anatomical structures of the medial clavicle; nine patients had swelling but no pain around the sternoclavicular joints. Internal fixation was removed in 29 patients 6–12 months postoperatively and no sequelae were observed. Conclusion: This novel sternoclavicular hook plate demonstrated excellent efficacy and could provide a reliable therapeutic approach for this kind of trauma.

Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway.

Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP) cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19). We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

In this study, we introduce a dual-modal fluorescence hyperspectral micro-CT system developed for, e.g., bioimaging applications. The system integrates an X-ray computed tomography (CT) module with a custom-designed hyperspectral fluorescence imaging module, achieving high-resolution structural imaging with detailed molecular-level insights. With a spectral resolution of 10 nm across the wavelength range of 450-750 nm, the hyperspectral fluorescence imaging module enables a fine compositional analysis. Using surface-modified nanoparticles, we demonstrate the system's capability to capture fluorescence under both X-ray and UV excitation. Imaging experiments on animal models further highlight the system's ability to generate comprehensive four-dimensional (4D) datasets that integrate spatial, spectral, and structural information. To the best of our knowledge, no such a dual-modal system or the like has even been reported before. This dual-modal approach enhances the visualization and analysis of biological tissues, offering promising applications in, e.g., disease diagnosis, surgical guidance, and preclinical research. [ABSTRACT FROM AUTHOR]

Smad2 and Smad3 (Smad2/3) are structurally similar proteins that primarily mediate the transforming growth factor-β (TGF-β) signaling responsible for driving cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provide the key mechanism for regulating the TGF-β signaling pathway, but the details surrounding this phosphorylation remain unclear. Here, using in vitro kinase assay coupled with mass spectrometry, we identified for the first time that nemo-like kinase (NLK) regulates TGF-β signaling via modulation of Smad2/3 phosphorylation in the linker region. TGF-β-mediated transcriptional and cellular responses are suppressed by NLK overexpression, whereas NLK depletion exerts opposite effects. Specifically, we discovered that NLK associates with Smad3 and phosphorylates the designated serine residues located in the linker region of Smad2 and Smad3, which inhibits phosphorylation at the C terminus, thereby decreasing the duration of TGF-β signaling. Overall, this work demonstrates that phosphorylation on the linker region of Smad2/3 by NLK counteracts the canonical phosphorylation in response to TGF-β signals, thus providing new insight into the mechanisms governing TGF-β signaling transduction. [ABSTRACT FROM AUTHOR]