Kaempferol 3-O-Rutinoside, a Flavone Derived from Tetrastigma hemsleyanum Diels et Gilg, Reduces Body Temperature through Accelerating the Elimination of IL-6 and TNF-α in a Mouse Fever Model.

Fever is a serious condition that can lead to various consequences ranging from prolonged illness to death. Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) has been used for centuries to treat fever, but the specific chemicals responsible for its antipyretic effects are not well understood. This study aimed to isolate and identify the chemicals with antipyretic bioactivity in T. hemsleyanum extracts and to provide an explanation for the use of T. hemsleyanum as a Chinese herbal medicine for fever treatment. Our results demonstrate that kaempferol 3-rutinoside (K3OR) could be successfully isolated and purified from the roots of T. hemsleyanum. Furthermore, K3OR exhibited a significant reduction in rectal temperature in a mouse model of fever. Notably, a 4 µM concentration of K3OR showed more effective antipyretic effects than ibuprofen and acetaminophen. To explore the underlying mechanism, we conducted an RNA sequencing analysis, which revealed that PXN may act as a key regulator in the fever process induced by lipopolysaccharide (LPS). In the mouse model of fever, K3OR significantly promoted the secretion of IL-6 and TNF-α during the early stage in the LPS-treated group. However, during the middle to late stages, K3OR facilitated the elimination of IL-6 and TNF-α in the LPS-treated group. Overall, our study successfully identified the chemicals responsible for the antipyretic bioactivity in T. hemsleyanum extracts, and it answered the question as to why T. hemsleyanum is used as a traditional Chinese herbal medicine for treating fever. These findings contribute to a better understanding of the therapeutic potential of T. hemsleyanum in managing fever, and they provide a basis for further research and development in this field.

In vivo evaluation of focused ultrasound ablation surgery (FUAS)-induced coagulation using echo amplitudes of the therapeutic focused ultrasound transducer.

OBJECTIVE: Monitoring sensitivity of sonography in focused ultrasound ablation surgery (FUAS) is limited (no hyperechoes in ∼50% of successful coagulation in uterine fibroids). A more accurate and sensitive approach is required. METHOD: The echo amplitudes of the focused ultrasound (FUS) transducer in a testing mode (short pulse duration and low power) were found to correlate with the ex vivo coagulation. To further evaluate its coagulation prediction capabilities, in vivo experiments were carried out. The liver, kidney, and leg muscles of three adult goats were treated using clinical FUAS settings, and the echo amplitude of the FUS transducer and grayscale in sonography before and after FUAS were collected. On day 7, animals were sacrificed humanely, and the treated tissues were dissected to expose the lesion. Echo amplitude changes and lesion areas were analyzed statistically, as were the coagulation prediction metrics. RESULTS: The echo amplitude changes of the FUS transducer correlate well with the lesion areas in the liver (R = 0.682). Its prediction in accuracy (94.4% vs. 50%), sensitivity (92.9% vs. 35.7%), and negative prediction (80% vs. 30.8%) is better than sonography, but similar in specificity (80% vs. 100%) and positive prediction (100% vs. 100%). In addition, the correlation between tissue depth and the lesion area is not good (|R| < 0.2). Prediction performances in kidney and leg muscles are similar. CONCLUSION: The FUS echo amplitudes are sensitive to the tissue properties and their changes after FUAS. They are sensitive and reliable in evaluating and predicting FUAS outcomes.

TRIM29 modulates proteins involved in PTEN/AKT/mTOR and JAK2/STAT3 signaling pathway and suppresses the progression of hepatocellular carcinoma.

Tripartite motif-containing 29 (TRIM29), also known as the ataxia telangiectasia group D-complementing (ATDC) gene, has been reported to play an oncogenic or tumor suppressive role in developing different tumors. So far, its expression and biological functions in hepatocellular carcinoma (HCC) remain unclear. We investigated TRIM29 expression pattern in human HCC samples using quantitative RT-PCR and immunohistochemistry. Relationships between TRIM29 expression level, clinical prognostic indicators, overall survival (OS), and disease-free survival (DFS) were evaluated by Kaplan-Meier analysis and Cox proportional hazards model. A series of in vitro experiments and a xenograft tumor model were conducted to detect the functions of TRIM29 in HCC cells. RNA sequencing, western blotting, and immunochemical staining were performed to assess the molecular regulation of TRIM29 in HCC. We found that the mRNA and protein levels of TRIM29 were significantly reduced in HCC samples, compared with adjacent noncancerous tissues, and were negatively correlated with poor differentiation of HCC tissues. Survival analysis confirmed that lower TRIM29 expression significantly correlated with shorter OS and DFS of HCC patients. TRIM29 overexpression remarkably inhibited cell proliferation, migration, and EMT in HCC cells, whereas knockdown of TRIM29 reversed these effects. Moreover, deactivation of the PTEN/AKT/mTOR and JAK2/STAT3 pathways might be involved in the tumor suppressive role of TRIM29 in HCC. Our findings indicate that TRIM29 in HCC exerts its tumor suppressive effects through inhibition of the PTEN/AKT/mTOR and JAK2/STAT3 signaling pathways and may be used as a potential biomarker for survival in patients with HCC.

Malat1 regulates PMN-MDSC expansion and immunosuppression through p-STAT3 ubiquitination in sepsis.

Myeloid-derived suppressor cells (MDSCs) expand during sepsis and contribute to the development of persistent inflammation-immunosuppression-catabolism syndrome. However, the underlying mechanism remains unclear. Exploring the mechanisms of MDSCs generation may provide therapeutic targets for improving immune status in sepsis. Here, a sepsis mouse model is established by cecal ligation and perforation. Bone marrow cells at different sepsis time points are harvested to detect the proportion of MDSCs and search for differentially expressed genes by RNA-sequence. In lethal models of sepsis, polymorphonuclear-MDSCs (PMN-MDSCs) decrease in early but increase and become activated in late sepsis, which is contrary to the expression of metastasis-associated lung adenocarcinoma transcript 1 (Malat1). In vivo, Malat1 inhibitor significantly increases the mortality in mice with late sepsis. And in vitro, Malat1 down-regulation increases the proportion of PMN-MDSCs and enhanced its immunosuppressive ability. Mechanistically, Malat1 limits the differentiation of PMN-MDSCs by accelerating the degradation of phosphorylated STAT3. Furthermore, Stattic, an inhibitor of STAT3 phosphorylation, improves the survival of septic mice by inhibiting PMN-MDSCs. Overall, the study identifies a novel insight into the mechanism of sepsis-induced MDSCs and provides more evidence for targeting MDSCs in the treatment of sepsis.

A neural network with a human learning paradigm for breast fibroadenoma segmentation in sonography.

BACKGROUND: Breast fibroadenoma poses a significant health concern, particularly for young women. Computer-aided diagnosis has emerged as an effective and efficient method for the early and accurate detection of various solid tumors. Automatic segmentation of the breast fibroadenoma is important and potentially reduces unnecessary biopsies, but challenging due to the low image quality and presence of various artifacts in sonography. METHODS: Human learning involves modularizing complete information and then integrating it through dense contextual connections in an intuitive and efficient way. Here, a human learning paradigm was introduced to guide the neural network by using two consecutive phases: the feature fragmentation stage and the information aggregation stage. To optimize this paradigm, three fragmentation attention mechanisms and information aggregation mechanisms were adapted according to the characteristics of sonography. The evaluation was conducted using a local dataset comprising 600 breast ultrasound images from 30 patients at Suining Central Hospital in China. Additionally, a public dataset consisting of 246 breast ultrasound images from Dataset_BUSI and DatasetB was used to further validate the robustness of the proposed network. Segmentation performance and inference speed were assessed by Dice similarity coefficient (DSC), Hausdorff distance (HD), and training time and then compared with those of the baseline model (TransUNet) and other state-of-the-art methods. RESULTS: Most models guided by the human learning paradigm demonstrated improved segmentation on the local dataset with the best one (incorporating C3ECA and LogSparse Attention modules) outperforming the baseline model by 0.76% in DSC and 3.14 mm in HD and reducing the training time by 31.25%. Its robustness and efficiency on the public dataset are also confirmed, surpassing TransUNet by 0.42% in DSC and 5.13 mm in HD. CONCLUSIONS: Our proposed human learning paradigm has demonstrated the superiority and efficiency of ultrasound breast fibroadenoma segmentation across both public and local datasets. This intuitive and efficient learning paradigm as the core of neural networks holds immense potential in medical image processing.

Hydrophilic Peptide and Glycopeptide as Immobilized Sorbents for Glycosylation Analysis.

Hydrophilic interaction liquid chromatography (HILIC) is widely used for glycopeptide enrichment in shot-gun glycoproteomics to enhance the glycopeptide signal and minimize the ionization competition of peptides. In this work, we have developed a novel hydrophilic material (glycoHILIC) based on glycopeptides and peptides to provide hydrophilic properties. GlycoHILIC was synthesized by oxidizing cotton and then reacting the resulting aldehyde with the N-terminus of the glycopeptide or peptide by reductive amination. Due to the large amount of hydrophilic carbohydrates and hydrophilic amino acids contained in glycopeptides, glycoHILIC showed significantly better enrichment of glycopeptides than cotton itself. Our results demonstrate that glycoHILIC has high selectivity, a low detection limit, and good stability. Over 257 unique N-linked glycosylation sites in 1477 intact N-glycopeptides from 146 glycoproteins were identified from 1 µL of human serum using glycoHILIC. Serum analysis of pancreatic cancer patients found that 38 N-glycopeptides among 21 glycoproteins changed significantly, of which 7 N-glycopeptides increased and 31 N-glycopeptides decreased. These results demonstrate that glycoHILIC can be used for glycopeptide enrichment and analysis.

MS-CFNet: a multi-scale clinical studying-based and feature extraction-guided network for breast fibroadenoma segmentation in ultrasonography.

Segmenting breast tumors in ultrasonography is challenging due to the low image quality and presence of artifacts. Radiologists' studying and diagnosis skills are integrated with artificial intelligence to establish a clinical learning-based deep learning network in order to robustly extract and delineate features of breast fibroadenoma. The spatial local feature contrast (SLFC) module captures overall tumor contours, while the channel recursive gated attention (CRGA) module enhances edge perception through high-dimensional information interaction. Additionally, full-scale feature fusion and enhanced deep supervision are applied to improve model stability and performance. To achieve smoother boundaries, we introduce a new loss function (cosh-smooth) that penalizes and finely tunes tumor edges. Our dataset comprises 1016 clinical ultrasound images of breast fibroadenoma with labeled masks, alongside a publicly available dataset of 246 ones. Segmentation performance is evaluated using the Dice similarity coefficient (DSC) and mean intersection over union (MIOU). Extensive experiments demonstrate that our proposed MS-CFNet outperforms state-of-the-art methods. Compared to TransUNet as a baseline model, MS-CFNet improves by 1.47% in DSC and 2.56% in MIOU. The promising result of MS-CFNet is attributed to the integration of radiologists' clinical diagnosis procedure and the bionic mindset, enhancing the network's ability to recognize and segment breast fibroadenomas effectively.

Two somatic mutations in the androgen receptor N-terminal domain are oncogenic drivers in hepatocellular carcinoma.

The androgen receptor (AR) plays an important role in male-dominant hepatocellular carcinoma, and specific acquired somatic mutations of AR have been observed in HCC patients. Our previous research have established the role of AR wild type as one of the key oncogenes in hepatocarcinogenesis. However, the role of hepatic acquired somatic mutations of AR remains unknown. In this study, we identify two crucial acquired somatic mutations, Q62L and E81Q, situated close to the N-terminal activation function domain-1 of AR. These mutations lead to constitutive activation of AR, both independently and synergistically with androgens, making them potent driver oncogene mutations. Mechanistically, these N-terminal AR somatic mutations enhance de novo lipogenesis by activating sterol regulatory element-binding protein-1 and promote glycogen accumulation through glycogen phosphorylase, brain form, thereby disrupting the AMPK pathway and contributing to tumorigenesis. Moreover, the AR mutations show sensitivity to the AMPK activator A769662. Overall, this study establishes the role of these N- terminal hepatic mutations of AR as highly malignant oncogenic drivers in hepatocarcinogenesis and highlights their potential as therapeutic targets for patients harboring these somatic mutations.

RNA m6A methylation modulates airway inflammation in allergic asthma via PTX3-dependent macrophage homeostasis.

N6-methyladenosine (m6A), the most prevalent mRNA modification, has an important function in diverse biological processes. However, the involvement of m6A in allergic asthma and macrophage homeostasis remains largely unknown. Here we show that m6A methyltransferases METTL3 is expressed at a low level in monocyte-derived macrophages from childhood allergic asthma patients. Conditional knockout of Mettl3 in myeloid cells enhances Th2 cell response and aggravates allergic airway inflammation by facilitating M2 macrophage activation. Loss and gain functional studies confirm that METTL3 suppresses M2 macrophage activation partly through PI3K/AKT and JAK/STAT6 signaling. Mechanistically, m6A-sequencing shows that loss of METTL3 impairs the m6A-YTHDF3-dependent degradation of PTX3 mRNA, while higher PTX3 expression positively correlates with asthma severity through promoting M2 macrophage activation. Furthermore, the METTL3/YTHDF3-m6A/PTX3 interactions contribute to autophagy maturation in macrophages by modulating STX17 expression. Collectively, this study highlights the function of m6A in regulating macrophage homeostasis and identifies potential targets in controlling allergic asthma.

Sulodexide improves vascular permeability via glycocalyx remodelling in endothelial cells during sepsis.

Background: Degradation of the endothelial glycocalyx is critical for sepsis-associated lung injury and pulmonary vascular permeability. We investigated whether sulodexide, a precursor for the synthesis of glycosaminoglycans, plays a biological role in glycocalyx remodeling and improves endothelial barrier dysfunction in sepsis. Methods: The number of children with septic shock that were admitted to the PICU at Children's Hospital of Fudan University who enrolled in the study was 28. On days one and three after enrollment, venous blood samples were collected, and heparan sulfate, and syndecan-1 (SDC1) were assayed in the plasma. We established a cell model of glycocalyx shedding by heparinase III and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Sulodexide was administrated to prevent endothelial glycocalyx damage. Endothelial barrier function and expression of endothelial-related proteins were determined using permeability, western blot and immunofluorescent staining. The survival rate, histopathology evaluation of lungs and wet-to-dry lung weight ratio were also evaluated. Results: We found that circulating SDC1 levels were persistently upregulated in the non-alive group on days 1 and 3 and were positively correlated with IL-6 levels. Receiver operating characteristic curve analysis showed that SDC1 could distinguish patients with mortality. We showed that SDC1-shedding caused endothelial permeability in the presence of heparinase III and sepsis conditions. Mechanistically, sulodexide (30 LSU/mL) administration markedly inhibited SDC1 shedding and prevented endothelial permeability with zonula occludens-1 (ZO-1) upregulation via NF-κB/ZO-1 pathway. In mice with LPS and CLP-induced sepsis, sulodexide (40 mg/kg) administration decreased the plasma levels of SDC1 and increased survival rate. Additionally, sulodexide alleviated lung injury and restored endothelial glycocalyx damage. Conlusions: In conclusion, our data suggest that SDC1 predicts prognosis in children with septic shock and sulodexide may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy.

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/pmrB, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

In Situ Measurement of Acoustic Attenuation for Focused Ultrasound Ablation Surgery Using a Boiling Bubble at the Focus.

OBJECTIVE: Acoustic attenuation in the propagation path of focused ultrasound ablation surgery determines the energy loss toward the focal region and is critical to the consequent treatment outcomes. In situ non-invasive, reliable, and accurate measurement is challenging for multi-layered heterogeneous tissues within the focusing angle. METHODS: A novel measurement approach is proposed and its performance is evaluated using ex vivo porcine tenderloin and bovine heart. A big boiling bubble (i.e., larger than a few millimeters in size) was produced at the focus as a strong reflector inside the tissue, and the echo amplitudes were used to determine the acoustic attenuation. Two models, acoustic ray and energy loss, were developed to derive the equivalent acoustic attenuation coefficient for a focused beam. RESULTS: The measured acoustic attenuation coefficients of ex vivo porcine tenderloin and bovine heart at 0.97 MHz and a thickness of 3 cm are 0.159 ± 0.002 and 0.250 ± 0.005 Np/cm, respectively, which are all within the scope of measured values in the literature. In addition, the echo amplitude is sensitive to the conditions of the propagation path, and the inverse acoustic attenuation coefficient of the silicone gel pad placed in front of the tissue sample was 0.807 ± 0.002 Np/cm, which is comparable to the measurement using the insertion substitution method, 0.766 ± 0.003 Np/cm. CONCLUSION: Our proposed approach could determine the tissue acoustic attenuation for focused ultrasound ablation surgery reliably and accurately in situ. The easy operating protocol may allow clinical translation and adoption for improved safety and efficacy.

The activation of mTOR signalling modulates DNA methylation by enhancing DNMT1 translation in hepatocellular carcinoma.

BACKGROUND: Both dysregulation of mechanistic target of rapamycin (mTOR) signalling and DNA methylation patterns have been shown to be closely associated with tumor progression and serve as promising targets for hepatocellular carcinoma (HCC) therapy. Although their respective roles in HCC have been extensively revealed, the existence of molecular interactions between them remains largely unknown. METHODS: The association of DNA methylation and mTOR signalling in HCC tissues and cell lines was assessed. A Kaplan‒Meier analysis was applied to estimate the overall survival (OS) and recurrence-free survival (RFS) of HCC patients. The modulation of DNMT1 by mTOR in HCC cell lines was determined. The effect of the drug combination in cell lines and mouse models was examined. RESULTS: The results showed that the DNA methylation level was positively associated with the activation of mTOR signalling in HCC tissues and cell lines. Moreover, HCC patients with higher DNA methylation levels and enhanced activation of mTOR signalling exhibited the worst prognosis. Then, we screened methylation-related enzymes and found that the activation of mTOR signalling increased DNMT1 expression and activity. In addition, mTOR enhanced the translational efficiency of DNMT1 in a 4E-BP1-dependent manner, which is based on the pyrimidine rich translational element (PRTE)-containing 5'UTR of DNMT1. Moreover, we demonstrated that the combined inhibition of mTOR and DNMT synergistically inhibited HCC growth in vitro and in vivo. CONCLUSIONS: In addition to some already identified pro-cancer downstream molecules, the activation of mTOR signalling was found to promote DNA methylation by increasing the translation of DNMT1. Furthermore, combined targeting of mTOR and DNMT1 has been demonstrated to have a more effective tumor suppressive function in HCC.

Isolation of biofluids from tissues using a vacuum-assisted filtration biomedical device.

A biopsy is usually used to remove a piece of tissue from a patient for laboratory testing. The interstitial fluid is taken out at the same time as the tissue sample. Since interstitial fluid flows between cells and capillaries in tissues, similar to blood plasma, it is necessary to separate interstitial fluid from tissues in order to study them separately. Vacuum blood sampling has been used to draw blood into vacuum-sealed tubes, while interstitial fluid can be removed directly from the skin using microneedles with standard pumps. However, no methods are available to separate blood or interstitial fluid from the tissue itself for molecular characterization. In this study, we designed a biomedical device that can separate interstitial fluid from tissue using a vacuum-assisted filtration method. The device has a chamber that collects fluid extracted from the tissue that remains on top of the filter. We characterized the weight change and glycan profiles of tissues before and after vacuum-assisted filtration. The results demonstrate that the biomedical device can remove interstitial fluid and facilitate the analysis of tissue-specific molecules while minimizing information from the interstitial fluid.

Multidimensional Analysis of Lung Lymph Nodes in a Mouse Model of Allergic Lung Inflammation following PM2.5 and Indeno[1,2,3-cd]pyrene Exposure.

BACKGROUND: Ambient particulate matter with an aerodynamic diameter of ≤2.5 µm (PM2.5) is suggested to act as an adjuvant for allergen-mediated sensitization and recent evidence suggests the importance of T follicular helper (Tfh) cells in allergic diseases. However, the impact of PM2.5 exposure and its absorbed polycyclic aromatic hydrocarbon (PAHs) on Tfh cells and humoral immunity remains unknown. OBJECTIVES: We aimed to explore the impact of environmental PM2.5 and indeno[1,2,3-cd]pyrene (IP), a prominent PAH, as a model, on Tfh cells and the subsequent pulmonary allergic responses. METHODS: PM2.5- or IP-mediated remodeling of cellular composition in lung lymph nodes (LNs) was determined by mass cytometry in a house dust mite (HDM)-induced mouse allergic lung inflammation model. The differentiation and function of Tfh cells in vitro were analyzed by flow cytometry, quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, chromatin immunoprecipitation, immunoprecipitation, and western blot analyses. RESULTS: Mice exposed to PM2.5 during the HDM sensitization period demonstrated immune cell population shifts in lung LNs as compared with those sensitized with HDM alone, with a greater number of differentiated Tfh2 cells, enhanced allergen-induced immunoglobulin E (IgE) response and pulmonary inflammation. Similarly enhanced phenotypes were also found in mice exposed to IP and sensitized with HDM. Further, IP administration was found to induce interleukin-21 (Il21) and Il4 expression and enhance Tfh2 cell differentiation in vitro, a finding which was abrogated in aryl hydrocarbon receptor (AhR)-deficient CD4+ T cells. Moreover, we showed that IP exposure increased the interaction of AhR and cellular musculoaponeurotic fibrosarcoma (c-Maf) and its occupancy on the Il21 and Il4 promoters in differentiated Tfh2 cells. DISCUSSION: These findings suggest that the PM2.5 (IP)-AhR-c-Maf axis in Tfh2 cells was important in allergen sensitization and lung inflammation, thus adding a new dimension in the understanding of Tfh2 cell differentiation and function and providing a basis for establishing the environment-disease causal relationship. https://doi.org/10.1289/EHP11580.

Degradation reduces greenhouse gas emissions while weakening ecosystem carbon sequestration of Moso bamboo forests.

Moso bamboo (Phyllostachys heterocycla cv. Pubescens) is well known for its high capacity to sequester atmospheric carbon, which has a unique role to play in combating global warming. Many Moso bamboo forests are gradually degrading due to rising labor costs and falling prices for bamboo timber. However, the mechanisms of carbon sequestration of Moso bamboo forest ecosystems in response to degradation are unclear. In this study, a space-for-time substitution approach was used to select Moso bamboo forest plots with the same origin and similar stand types, but different years of degradation, and four degradation sequences, continuous management (CK), 2 years of degradation (D-I), 6 years of degradation (D-II) and 10 years of degradation (D-III). A total of 16 survey sample plots were established based on the local management history files. After a 12-month monitoring, the response characteristics of soil greenhouse gases (GHG) emissions, vegetation, and soil organic carbon sequestration in different degradation sequences were evaluated to reveal the differences in the ecosystem carbon sequestration. The results indicated that under D-I, D-II, and D-III, the global warming potential (GWP) of soil GHG emissions decreased by 10.84 %, 17.75 %, and 31.02 %, while soil organic carbon (SOC) sequestration increased by 2.82 %, 18.11 %, and 4.68 %, and vegetation carbon sequestration decreased by 17.30 %, 33.49 %, and 44.76 %, respectively. In conclusion, compared to CK, the ecosystem carbon sequestration was reduced by 13.79 %, 22.42 %, and 30.31 %, respectively. This suggests that degradation reduces soil GHG emissions but weakens the ecosystem carbon sequestration capability. Therefore, in the background of global warming and the strategic goal of carbon neutrality, restorative management of degraded Moso bamboo forests is critically needed to improve the carbon sequestration potential of the ecosystem.

SEPT2 crotonylation promotes metastasis and recurrence in hepatocellular carcinoma and is associated with poor survival.

BACKGROUND: Hepatocellular carcinoma (HCC) metastasis and recurrence lead to therapy failure, which are closely associated with the proteome. However, the role of post-translational modification (PTM) in HCC, especially for the recently discovered lysine crotonylation (Kcr), is elusive. RESULTS: We investigated the correlation between crotonylation and HCC in 100 tumor tissues and performed stable isotope labeling by amino acids and liquid chromatography tandem mass spectrometry in HCC cells, and we found that crotonylation was positively correlated with HCC metastasis, and higher crotonylation in HCC cells facilitated cell invasiveness. Through bioinformatic analysis, we found that the crotonylated protein SEPT2 was significantly hypercrotonylated in highly invasive cells, while the decrotonylated mutation of SEPT2-K74 impaired SEPT2 GTPase activity and inhibited HCC metastasis in vitro and in vivo. Mechanistically, SIRT2 decrotonylated SEPT2, and P85α was found to be the downstream effector of SEPT2. Moreover, we identified that SEPT2-K74cr was correlated with poor prognosis and recurrence in HCC patients, thus indicating its clinical potential as an independent prognostic factor. CONCLUSIONS: We revealed the role of nonhistone protein crotonylation in regulating HCC metastasis and invasion. Crotonylation facilitated cell invasion through the crotonylated SEPT2-K74-P85α-AKT pathway. High SEPT2-K74 crotonylation predicted poor prognosis and a high recurrence rate in HCC patients. Our study revealed a novel role of crotonylation in promoting HCC metastasis.

Construction of a red emission fluorescent probe for selectively detection of cysteine in living cells.

Cysteine (Cys) is a vital amino acid in the body, and its abnormal expression level is associated with many diseases. In this study, a novel fluorescent probe ACHB was synthesized, showing high selectivity, anti-interference ability and achieving accurate detection of cysteine. Different from most previous off-on probes, ACHB showed an on-off fluorescence response to Cys. Acrylic ester was used as a recognizer while green fluorescence protein (GFP) chromophore derivative 4-hydroxybenzylidene-imidazolinone (HBI) was used as the fluorophore. The addition of Cys leads to the hydrolysis of the red-emitting probe (613 nm), releasing a precursor with a lower fluorescent signal and showing an on-off spectral signal, which was ideal for obtaining sensitive detection with high specificity. Furthermore, the probe was successfully applied for simultaneous determination of cysteine (Cys) in living cells and biological sample (mouse serum). In conclusion, probe ACHB is a promising tool to display the intracellular cysteine concentration level, providing a good visualization method for clinical diagnosis and scientific basic research.

Impacts of resistance exercise on performance in swimmers / Impactos do exercício de resistência sobre o desempenho em nadadores / Impacto del ejercicio de resistencia en el rendimiento de nadadores

ABSTRACT Introduction: The need for strength of the lower limbs to provide absolute speed in competitive-level swimmers requires methodological and evidence-based training. Resistance training is an effective way to increase muscle strength and it is believed that it can be adapted to benefit swimmers. Objective: Study the impacts of lower-limb resistance exercise on the sport performance of swimming athletes. Methods: Twelve volunteer athletes were selected for the experiment, randomly divided between experimental and control groups. There was no significant difference between the two groups. The experimental group received intervention with lower limb resistance training, while the control continued with routine physical training. Results: The thigh circumference of the experimental group increased from 56.01±5.40 cm to 57.14±5.06 cm; the Dive angle decreased from 44.85±6.74 to 43.23±7.71; the Entry distance increased from 3.51±0.36 m to 3.69±0.39 m; flight time was reduced from 0.33±0.05 s to 0.31±0.07 s; freestyle performance was reduced from 28.90±2.40 s to 27.18±2.72 s. Conclusion: The training with resistance exercise in the lower limbs showed evident improvements in the swimmers' physical performance, besides the evident gain of muscle mass. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: A necessidade de força dos membros inferiores para proporcionar velocidade absoluta na natação dos esportistas de nível competitivo exige treinamentos metodológicos e embasados em evidências. O treinamento de resistência é uma maneira eficaz de aumentar a força muscular e acredita-se que ele possa ser adaptado para beneficiar os nadadores. Objetivo: Estudar os impactos do exercício de resistência de membros inferiores sobre o desempenho esportivo dos atletas de natação. Métodos: Foram selecionados 12 atletas voluntários para o experimento, divididos aleatoriamente entre grupo experimental e controle. Não houve diferença significativa entre os dois grupos. O grupo experimental recebeu intervenção com treinamento de resistência dos membros inferiores, enquanto o controle prosseguiu com o treinamento físico rotineiro. Resultados: A circunferência da coxa do grupo experimental aumentou de 56,01±5,40 cm para 57,14±5,06 cm; o ângulo de mergulho diminuiu de 44,85±6,74 para 43,23±7,71; a distância de entrada foi aumentada de 3,51±0,36 m para 3,69±0,39 m; o tempo de voo foi reduzido de 0,33±0,05 s para 0,31±0,07 s; o desempenho no estilo livre foi reduzido de 28,90±2,40 s para 27,18±2,72 s. Conclusão: O treinamento com exercício de resistência nos membros inferiores mostrou evidentes melhoras sobre o desempenho físico dos nadadores, além do evidente ganho de massa muscular. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: La necesidad de fuerza de los miembros inferiores para proporcionar velocidad absoluta en la natación de los atletas de nivel competitivo requiere un entrenamiento metodológico y basado en la evidencia. El entrenamiento de resistencia es una forma eficaz de aumentar la fuerza muscular y se cree que puede adaptarse para beneficiar a los nadadores. Objetivo: Estudiar el impacto del ejercicio de resistencia de las extremidades inferiores en el rendimiento deportivo de los atletas de natación. Métodos: Se seleccionaron 12 atletas voluntarios para el experimento, divididos aleatoriamente entre los grupos experimental y de control. No hubo diferencias significativas entre los dos grupos. El grupo experimental recibió intervención con entrenamiento de resistencia de miembros inferiores, mientras que el control continuó con entrenamiento físico rutinario. Resultados: La circunferencia del muslo del grupo experimental aumentó de 56,01±5,40 cm a 57,14±5,06 cm; el ángulo de inmersión disminuyó de 44,85±6,74 a 43,23±7,71; la distancia de entrada aumentó de 3,51±0,36 m a 3,69±0,39 m; el tiempo de vuelo se redujo de 0,33±0,05 s a 0,31±0,07 s; el rendimiento en estilo libre se redujo de 28,90±2,40 s a 27,18±2,72 s. Conclusión: El entrenamiento con ejercicios de resistencia en los miembros inferiores mostró mejoras indiscutibles en el rendimiento físico de los nadadores, además de la evidente ganancia de masa muscular. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Ezetimibe Induces Paraptosis through Niemann-Pick C1-like 1 Inhibition of Mammalian-Target-of-Rapamycin Signaling in Hepatocellular Carcinoma Cells.

Currently, hepatocellular carcinoma (HCC) is characterized by its unfavorable prognosis and resistance to conventional chemotherapy and radiotherapy. Drug repositioning, an approach aimed at identifying novel therapeutic applications for existing drugs, presents a cost-effective strategy for developing new anticancer agents. We explored the anticancer properties of Ezetimibe, a widely used oral lipid-lowering drug, in the context of HCC. Our findings demonstrate that Ezetimibe effectively suppresses HCC cell proliferation through paraptosis, an apoptotic-independent cell death pathway. The examination of HCC cells lines treated with Ezetimibe using light microscopy and transmission electron microscopy (TEM) showed cytoplasmic vacuolation in the perinuclear region. Notably, the nuclear membrane remained intact in both Ezetimibe-treated and untreated HCC cell lines. Probe staining assays confirmed that the cytoplasmic vacuoles originated from dilated endoplasmic reticulum (ER) compartments rather than mitochondria. Furthermore, a dose-dependent accumulation of reactive oxygen species (ROS) was observed in Ezetimibe-treated HCC cell lines. Co-treatment with the general antioxidant NAC attenuated vacuolation and improved cell viability in Ezetimibe-treated HCC cells. Moreover, Ezetimibe induced paraptosis through proteasome activity inhibition and initiation of the unfolded protein response (UPR) in HCC cell lines. In our in vivo experiment, Ezetimibe significantly impeded the growth of HCC tumors. Furthermore, when combined with Sorafenib, Ezetimibe exhibited a synergistic antitumor effect on HCC cell lines. Mechanistically, Ezetimibe induced paraptosis by targeting NPC1L1 to inhibit the PI3K/AKT/mTOR signaling pathway. In conclusion, our study highlights the potential of Ezetimibe as an anticancer agent by triggering paraptosis in HCC cells.