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Glycoside hydrolase family 91 (GH91) inulin fructotransferase (IFTases) enables biotransformation of fructans into sugar substitutes for dietary intervention in metabolic syndrome. However, the catalytic mechanism underlying the sequential biodegradation of inulin remains unelusive during the biotranformation of fructans. Herein we present the crystal structures of IFTase from Arthrobacter aurescens SK 8.001 in apo form and in complexes with kestose, nystose, or fructosyl nystose, respectively. Two kinds of conserved noncatalytic binding regions are first identified for IFTase-inulin interactions. The conserved interactions of substrates were revealed in the catalytic center that only contained a catalytic residue E205. A switching scaffold was comprised of D194 and Q217 in the catalytic channel, which served as the catalytic transition stabilizer through side chain displacement in the cycling of substrate sliding in/out the catalytic pocket. Such features in GH91 contribute to the catalytic model for consecutive cutting of substrate chain as well as product release in IFTase, and thus might be extended to other exo-active enzymes with an enclosed bottom of catalytic pocket. The study expands the current general catalytic principle in enzyme-substrate complexes and shed light on the rational design of IFTase for fructan biotransformation.
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The effect of the starch chain structure on 4,3-α-glucanotransferase's (4,3-α-GTase) catalytic properties was investigated to modulate the digestibility of starch. Three starches with diverse amylose contents were used, and the enzymatic kinetic reaction of 4,3-α-GTase was fitted using the Michaelis-Menten equation. The results revealed that the linear substrate was more suitable for modification by 4,3-α-GTase. Linear starch chains were then selected with various degrees of polymerization (DP) as substrates of 4,3-α-GTase modification. Additionally, the structures and in vitro digestion of 4,3-α-GTase derived α-glucans were studied. The results showed that enzyme catalysis increased the amount of α-1,3 glycosidic linkages in products (highest 33.5%), the digestibility of 4,3-α-GTase derived α-glucans conformed to a first-order two-phase equation, and the equilibrium digestibility was controlled between 43.2-72.1%. It was observed that the structure of α-glucans could be managed to attain low digestibilities (43.2%) by selecting maltodextrin with DE 2 as the substrate. These findings offer valuable insights into the fabrication of α-glucans and their potential applications in various fields.
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Digestión , Glucanos , Sistema de la Enzima Desramificadora del Glucógeno , Glucanos/química , Glucanos/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Sistema de la Enzima Desramificadora del Glucógeno/química , Cinética , Amilosa/química , Amilosa/metabolismo , Almidón/química , Almidón/metabolismo , CatálisisRESUMEN
BACKGROUND: Perceived cognitive impairment is a significant symptom experienced by breast cancer patients and may be affected by sleep quality. Coping styles have potential relevancies with both sleep quality and perceived cognitive impairment. However, the empirical evidence supporting their association among breast cancer patients is limited. OBJECTIVE: This study explored the associations between sleep quality, coping styles, and perceived cognitive impairment and tested the mediating role of coping styles in breast cancer patients. METHODS: A total of 294 breast cancer patients were included in this cross-sectional study. Patients were assessed using the Pittsburgh Sleep Index Scale, the Simplified Coping Styles Questionnaire, and the Functional Assessment of Cancer Therapy-Cognitive Functioning (Version 3) Scale. The data were analyzed using SPSS and Process macros. RESULTS: The direct effect of sleep quality on reported cognitive impairment was significant (ß = -0.245, P < .001). Furthermore, sleep quality was found to have a significant indirect effect on perceived cognitive impairment through positive coping style (ß = -0.026, P < .05) and negative coping style (ß = -0.131, P < .05). CONCLUSIONS: Our research suggests that sleep quality has both a direct effect on perceived cognitive impairment and an indirect effect through positive and negative coping styles in breast cancer patients. Moreover, negative coping style had a more pronounced mediating effect than positive coping style. IMPLICATIONS FOR PRACTICE: Clinical medical staff could reduce the perceived cognitive impairment of breast cancer patients by improving their sleep quality and encouraging them to adopt a more positive coping style.
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AIMS: Lansoprazole is one of the many proton pump inhibitors (PPIs) that acts more strongly with ABCB1 and ABCG2. The present study is to investigate the potential of lansoprazole on reversal of ABCB1/G2-mediated MDR in cancer, in vitro and in vivo. METHODS: Reversal studies and combination evaluation were conducted to determine the synergistic anti-MDR effects on lansoprazole. Lysosomal staining was used to determination of lansoprazole on ABCB1-mediated lysosomal sequestration. Substrate accumulation and efflux assays, ATPase activity, and molecular docking were conducted to evaluate lansoprazole on ABCB1/G2 functions. Western blot and immunofluorescence were used to detect lansoprazole on ABCB1/G2 expression and subcellular localization. MDR nude mice models were established to evaluate the effects of lansoprazole on MDR in vivo. RESULTS: Lansoprazole attenuated ABCB1/G2-mediated MDR and exhibited synergistic effects with substrate drugs in MDR cells. In vivo experiments demonstrated that lansoprazole attenuated ABCB1/G2-mediated MDR and exhibited synergistic effects that augmented the sensitivity of substrate anticancer drugs in ABCB1/G2-mediated settings without obvious toxicity. Lansoprazole impeded lysosomal sequestration mediated by ABCB1, leading to a substantial increase in intracellular accumulation of substrate drugs. The effects of lansoprazole were not attributable to downregulation or alterations in subcellular localization of ABCB1/G2. Lansoprazole promoted the ATPase activity of ABCB1/G2 and competitively bound to the substrate-binding region of ABCB1/G2. CONCLUSIONS: These findings present novel therapeutic avenues whereby the combination of lansoprazole and chemotherapeutic agents mitigates MDR mediated by ABCB1/G2 overexpression.
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Subfamilia B de Transportador de Casetes de Unión a ATP , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Lansoprazol , Lisosomas , Inhibidores de la Bomba de Protones , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Lansoprazol/farmacología , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Ratones Desnudos , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de la Bomba de Protones/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Large language models (LLMs) offer transformative opportunities in bioinformatics education for medical students by creating interactive experiences. The integration of LLMs enhances educational outcomes through providing accessible code templates, clarifying the function of coding elements, and assisting in error troubleshooting. Here, we demonstrate the practical applications of LLMs with a case study on transcriptome sequencing data processing, a vital component of medical research. However, the reliability of the content that LLMs generate requires rigorous validation. Ensuring the accuracy and appropriateness of the LLM-generated information requires integrating innovative LLMs with traditional educational methods to prepare medical students effectively for future challenges in bioinformatics.
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Biología Computacional , Estudiantes de Medicina , Biología Computacional/educación , Humanos , Educación MédicaRESUMEN
Currently, sonodynamic therapy (SDT) has limited therapeutic outcomes and immune responses, highlighting the urgent need for enhanced strategies that can stimulate robust and long-lasting antitumor effects. Microcystis, a notorious microalga, reveals the possibility of mediating SDT owing to the presence of gas vesicles (GVs) and phycocyanin (PC). Herein, a nontoxic strain of Microcystis elabens (labeled Me) is developed as a novel agent for SDT because it generates O2 under red light (RL) illumination, while GVs and PC act as cavitation nuclei and sonosensitizers, respectively. Moreover, algal debris is released after ultrasound (US) irradiation, which primes the Toll-like receptor pathway to initiate a cascade of immune responses. This sono-immune strategy inhibits CT26 colon tumor growth largely by promoting dendritic cell (DC) maturation and cytotoxic T-cell activation. After combination with the immune checkpoint blockade (ICB), the therapeutic outcome is further amplified, accompanied by satisfactory abscopal and immune memory effects; the similar potency is proven in the "cold" 4T1 triple-negative breast tumor. In addition, Me exhibits good biosafety without significant acute or chronic toxicity. Briefly, this study turns waste into wealth by introducing sono-immunotherapy based on Microcystis that achieved encouraging therapeutic effects on cancer, which is expected to be translated into the clinic.
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Microcystis , Animales , Ratones , Línea Celular Tumoral , Terapia por Ultrasonido/métodos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Ficocianina/química , Ficocianina/farmacología , Inmunoterapia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Neoplasias del Colon/terapia , Neoplasias del Colon/inmunologíaRESUMEN
Owing to its remarkable ease of use, ultrasound has recently been explored for stimulating or amplifying immune responses during cancer therapy, termed 'sono-immunotherapy'. Ultrasound can cause immunogenic cell death in cancer cells via thermal and nonthermal effects to regulate the tumor microenvironment, thereby priming anticancer immunity; by integrating well-designed biomaterials, novel sono-immunotherapy approaches with augmented efficacy can also be developed. Here, we review the advances in sono-immunotherapy for cancer treatment and summarize existing limitations along with potential trends. We offer emerging insights into this realm, which might prompt breakthroughs and expand its potential applications to other diseases.
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Inmunoterapia , Neoplasias , Microambiente Tumoral , Humanos , Neoplasias/terapia , Neoplasias/inmunología , Inmunoterapia/métodos , Animales , Microambiente Tumoral/inmunología , Terapia por Ultrasonido/métodosRESUMEN
BACKGROUND: Metachronous second primary lung cancer (MSPLC) is not that rare but is seldom studied. OBJECTIVE: We aim to compare real-world survival outcomes between different surgery strategies and radiotherapy for MSPLC. METHODS: This retrospective study analyzed data collected from patients with MSPLC between 1988 and 2012 in the Surveillance, Epidemiology, and End Results (SEER) database. Propensity score matching (PSM) analyses and machine learning were performed to compare variables between patients with MSPLC. Survival curves were plotted using the Kaplan-Meier method and were compared using log-rank tests. RESULTS: A total of 2451 MSPLC patients were categorized into the following treatment groups: 864 (35.3%) received radiotherapy, 759 (31%) underwent surgery, 89 (3.6%) had surgery plus radiotherapy, and 739 (30.2%) had neither treatment. After PSM, 470 pairs each for radiotherapy and surgery were generated. The surgery group had significantly better survival than the radiotherapy group (P<.001) and the untreated group (563 pairs; P<.001). Further analysis revealed that both wedge resection (85 pairs; P=.004) and lobectomy (71 pairs; P=.002) outperformed radiotherapy in overall survival for MSPLC patients. Machine learning models (extreme gradient boosting, random forest classifier, adaptive boosting) demonstrated high predictive performance based on area under the curve (AUC) values. Least absolute shrinkage and selection operator (LASSO) regression analysis identified 9 significant variables impacting cancer-specific survival, emphasizing surgery's consistent influence across 1 year to 10 years. These variables encompassed age at diagnosis, sex, year of diagnosis, radiotherapy of initial primary lung cancer (IPLC), primary site, histology, surgery, chemotherapy, and radiotherapy of MPSLC. Competing risk analysis highlighted lower mortality for female MPSLC patients (hazard ratio [HR]=0.79, 95% CI 0.71-0.87) and recent IPLC diagnoses (HR=0.79, 95% CI 0.73-0.85), while radiotherapy for IPLC increased mortality (HR=1.31, 95% CI 1.16-1.50). Surgery alone had the lowest cancer-specific mortality (HR=0.83, 95% CI 0.81-0.85), with sublevel resection having the lowest mortality rate among the surgical approaches (HR=0.26, 95% CI 0.21-0.31). The findings provide valuable insights into the factors that influence cumulative cancer-specific mortality. CONCLUSIONS: Surgical resections such as wedge resection and lobectomy confer better survival than radiation therapy for MSPLC, but radiation can be a valid alternative for the treatment of MSPLC.
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OBJECTIVE: Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis. METHODS: We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT-PCR, western blotting, and immunofluorescence assays. RESULTS: DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway. CONCLUSIONS: The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.
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Apoptosis , Dexametasona , Glucocorticoides , Macrófagos , Mitocondrias , Receptores de Glucocorticoides , Receptores de Glucocorticoides/metabolismo , Animales , Ratones , Apoptosis/efectos de los fármacos , Células RAW 264.7 , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ubiquinona/farmacología , Ubiquinona/análogos & derivados , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Compuestos OrganofosforadosRESUMEN
Endoplasmic reticulum stress (ERS) and oxidative stress (OS) are adaptive responses of the body to stressor stimulation. Although it has been verified that Trichinella spiralis (T. spiralis) can induce ERS and OS in the host, their association is still unclear. Therefore, this study explored whether T. spiralis-secreted serpin-type serine protease inhibitor (TsAdSPI) is involved in regulating the relationship between ERS and OS in the host intestine. In this study, mice jejunum and porcine small intestinal epithelial cells (IECs) were detected using qPCR, western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and detection kits. The results showed that ERS- and OS-related indexes changed significantly after TsAdSPI stimulation, and Bip was located in IECs, indicating that TsAdSPI could induce ERS and OS in IECs. After the use of an ERS inhibitor, OS-related indexes were inhibited, suggesting that TsAdSPI-induced OS depends on ERS. When the three ERS signalling pathways, ATF6, IRE1, and PERK, were sequentially suppressed, OS was only regulated by the PERK pathway, and the PERK-eif2α-CHOP-ERO1α axis played a key role. Similarly, the expression of ERS-related indexes and the level of intracellular Ca2+ were inhibited after adding the OS inhibitor, and the expression of ERS-related indexes decreased significantly after inhibiting calcium transfer. This finding indicated that TsAdSPI-induced OS could affect ERS by promoting Ca2+ efflux from the endoplasmic reticulum. The detection of the ERS and OS sequences revealed that OS occurred before ERS. Finally, changes in apoptosis-related indexes were detected, and the results indicated that TsAdSPI-induced ERS and OS could regulate IEC apoptosis. In conclusion, TsAdSPI induced OS after entering IECs, OS promoted ERS by enhancing Ca2+ efflux, and ERS subsequently strengthened OS by activating the PERK-eif2α-CHOP-ERO1α axis. ERS and OS induced by TsAdSPI synergistically promoted IEC apoptosis. This study provides a foundation for exploring the invasion mechanism of T. spiralis and the pathogenesis of host intestinal dysfunction after invasion.
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Estrés del Retículo Endoplásmico , Células Epiteliales , Estrés Oxidativo , Serpinas , Trichinella spiralis , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Trichinella spiralis/fisiología , Ratones , Estrés Oxidativo/efectos de los fármacos , Porcinos , Serpinas/metabolismo , Serpinas/genética , Inhibidores de Serina Proteinasa/farmacología , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Yeyuno/efectos de los fármacosRESUMEN
Introduction: Acetyl-CoA synthetase 2 (ACSS2), one of the enzymes that catalyze the conversion of acetate to acetyl-CoA, has been proved to be an oncogene in various cancers. However, the function of ACSS2 is still largely a black box in melanoma. Methods: The ACSS2 expression was detected in melanoma cells and melanocytes at both protein and mRNA levels. Cell viability, apoptosis, migration and invasion were investigated after ACSS2 knockdown. RNA sequencing (RNA-Seq) technology was employed to identify differentially expressed genes caused by ACSS2 knockdown, which were then verified by immunoblotting analysis. Animal experiments were further performed to investigate the influence of ACSS2 on tumor growth and metastasis in vivo. Results: Firstly, we found that ACSS2 was upregulated in most melanoma cell lines compared with melanocytes. In addition, ACSS2 knockdown dramatically suppressed melanoma cell migration and invasion, whereas promoted cell apoptosis in response to endoplasmic reticulum (ER) stress. Furthermore, tumor growth and metastasis were dramatically suppressed by ACSS2 knockdown in vivo. RNA-Seq suggested that the Hippo pathway was activated by ACSS2 knockdown, which was forwardly confirmed by Western blotting and rescue experiments. Taken together, we demonstrated that ACSS2 enables melanoma cell survival and tumor metastasis via the regulation of the Hippo pathway. Discussion: In summary, this study demonstrated that ACSS2 may promote the growth and metastasis of melanoma by negatively regulating the Hippo pathway. Targeting ACSS2 may be a promising target for melanoma treatment.
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Although decreasing body mass index (BMI) is associated with higher mortality risk in patients undergoing hemodialysis (HD), BMI neither differentiates muscle and fat mass nor provides information about the variations of fat distribution. It remains unclear whether changes over time in fat and muscle mass are associated with mortality. We examined the prognostic significance of trajectory in the triceps skinfold (TSF) thickness and mid-upper arm circumference (MUAC). In this multicenter prospective cohort study, 972 outpatients (mean age, 54.5 years; 55.3% men) undergoing maintenance HD at 22 treatment centers were included. We calculated the relative change in TSF and MUAC over a 1-year period. The outcome was all-cause mortality. Kaplan-Meier, Cox proportional hazard analyses, restricted cubic splines, and Fine and Gray sub-distribution hazards models were performed to examine whether TSF and MUAC trajectories were associated with all-cause mortality. During follow-up (median, 48.0 months), 206 (21.2%) HD patients died. Compared with the lowest trajectory group, the highest trajectories of TSF and MUAC were independently associated with lower risk for all-cause mortality (HR = 0.405, 95% CI 0.257-0.640; HR = 0.537; 95% CI 0.345-0.837; respectively), even adjusting for BMI trajectory. Increasing TSF and MUAC over time, measured as continuous variables and expressed per 1-standard deviation decrease, were associated with a 55.7% (HR = 0.443, 95% CI 0.302-0.649), and 97.8% (HR = 0.022, 95% CI 0.005-0.102) decreased risk of all-cause mortality. Reduction of TSF and MUAC are independently associated with lower all-cause mortality, independent of change in BMI. Our study revealed that the trajectory of TSF thickness and MUAC provides additional prognostic information to the BMI trajectory in HD patients.
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Índice de Masa Corporal , Diálisis Renal , Grasa Subcutánea , Humanos , Diálisis Renal/mortalidad , Masculino , Femenino , Persona de Mediana Edad , Estudios Prospectivos , Grosor de los Pliegues Cutáneos , Brazo/anatomía & histología , Anciano , Pronóstico , Adulto , Músculo Esquelético/patología , Modelos de Riesgos Proporcionales , Estimación de Kaplan-MeierRESUMEN
A total of twenty-two diterpenoid alkaloids, including ten unprecedented ones, namely refractines C-L, were isolated from the roots of Aconitum refractum (Finet et Gagnep.) Hand.-Mazz. Refractine C was the first example of a natural diterpenoid alkaloid wherein C-19 is linked to N position by an oxaziridine ring. Refractine L was a rare glycosidic diterpenoid alkaloid with fructofuranoside. Most of the isolated compounds obtained from a previous study were screened for their anti-inflammatory and myocardial protective activities. The autophagy-inducing effects of some of these compounds on RAW 264.7 cells were evaluated by assessing the expression of microtubule-associated protein 1 light chain 3 (LC3-II/LC3-I). Results revealed that some compounds exerted varying levels of inhibitory effects on the proliferative activity of RAW 264.7 cells.
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Aconitum , Alcaloides , Autofagia , Diterpenos , Aconitum/química , Ratones , Animales , Autofagia/efectos de los fármacos , Células RAW 264.7 , Alcaloides/farmacología , Alcaloides/aislamiento & purificación , Alcaloides/química , Diterpenos/farmacología , Diterpenos/química , Diterpenos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Raíces de Plantas/químicaRESUMEN
The clinical applications of immunocytokines are severely restricted by dose-limiting toxicities. To address this challenge, here we propose a next-generation immunocytokine concept involving the design of LH05, a tumor-conditional anti-PD-L1/interleukin-15 (IL-15) prodrug. LH05 innovatively masks IL-15 with steric hindrance, mitigating the "cytokine sink" effect of IL-15 and reducing systemic toxicities associated with wild-type anti-PD-L1/IL-15. Moreover, upon specific proteolytic cleavage within the tumor microenvironment, LH05 releases an active IL-15 superagonist, exerting potent antitumor effects. Mechanistically, the antitumor efficacy of LH05 depends on the increased infiltration of CD8+ T and natural killer cells by stimulating the chemokines CXCL9 and CXCL10, thereby converting cold tumors into hot tumors. Additionally, the tumor-conditional anti-PD-L1/IL-15 can synergize with an oncolytic virus or checkpoint blockade in advanced and metastatic tumor models. Our findings provide a compelling proof of concept for the development of next-generation immunocytokines, contributing significantly to current knowledge and strategies of immunotherapy.
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Antígeno B7-H1 , Interleucina-15 , Microambiente Tumoral , Interleucina-15/inmunología , Antígeno B7-H1/metabolismo , Antígeno B7-H1/inmunología , Antígeno B7-H1/genética , Animales , Humanos , Ratones , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de los fármacos , Línea Celular Tumoral , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Inmunoterapia/métodos , Ratones Endogámicos C57BL , Femenino , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacologíaRESUMEN
Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH's colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH's EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy.Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl's balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin.
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Actinas , Autofagosomas , Proteínas Relacionadas con la Autofagia , Autofagia , Autofagia/fisiología , Humanos , Autofagosomas/metabolismo , Actinas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Animales , Proteínas Asociadas a Microtúbulos/metabolismo , Beclina-1/metabolismo , Citoesqueleto de Actina/metabolismo , Células HeLa , Proteínas de Transporte Vesicular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismoRESUMEN
Melanoma, arising from the malignant transformation of melanocytes, stands as the most lethal type of skin cancer. While significant strides have been made in targeted therapy and immunotherapy, substantially enhancing therapeutic efficacy, the prognosis for melanoma patients remains unoptimistic. SIRT7, a nuclear-localized deacetylase, plays a pivotal role in maintaining cellular homeostasis and adapting to external stressors in melanoma, with its activity closely tied to intracellular nicotinamide adenine dinucleotide (NAD+). However, its involvement in adaptive resistance to targeted therapy remains unclear. Herein, we unveil that up-regulated SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma. Initially, we observed a significant increase of SIRT7 expression in publicly available datasets following targeted therapy within a short duration. In consistent, we found elevated SIRT7 expression in melanoma cells subjected to BRAF or MEK inhibitors in vitro. The up-regulation of SIRT7 expression was also confirmed in xenograft tumors in mice after targeted therapy in vivo. Furthermore, we proved that SIRT7 deficiency led to decreased cell viability upon prolonged exposure to BRAF or MEK inhibitors, accompanied by an increase in cell apoptosis. Mechanistically, SIRT7 deficiency restrained the upregulation of genes associated with mitochondrial biogenesis and intracellular ATP levels in response to targeted therapy treatment in melanoma cells. Ultimately, we proved that SIRT7 deficieny could sensitize BRAF-mutant melanoma cells to MAPK inhibition targeted therapy in vivo. In conclusion, our findings underscore the role of SIRT7 in fostering adaptive resistance to targeted therapy through the facilitation of mitochondrial biogenesis. Targeting SIRT7 emerges as a promising strategy to overcome MAPK inhibitor adaptive resistance in melanoma.
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Resistencia a Antineoplásicos , Melanoma , Biogénesis de Organelos , Inhibidores de Proteínas Quinasas , Sirtuinas , Melanoma/metabolismo , Melanoma/patología , Melanoma/genética , Melanoma/tratamiento farmacológico , Humanos , Sirtuinas/metabolismo , Sirtuinas/genética , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Inhibidores de Proteínas Quinasas/farmacología , Ratones , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/tratamiento farmacológico , Ratones Desnudos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidoresRESUMEN
This study investigated the impact of ultrasound treatment on dioscorin, the primary storage protein found in yam tubers. Three key factors, namely ultrasound power, duration, and frequency, were focused on. The research revealed that ultrasound-induced cavitation effects disrupted non-covalent bonds, resulting in a reduction in α-helix and ß-sheet contents, decreased thermal stability, and a decrease in the apparent hydrodynamic diameter (Dh) of dioscorin. Additionally, previously hidden amino acid groups within the molecule became exposed on its surface, resulting in increased surface hydrophobicity (Ho) and zeta-potential. Under specific ultrasound conditions (200 W, 25 kHz, 30 min), Dh decreased while Ho increased, facilitating the adsorption of dioscorin molecules onto the oil-water interface. Molecular dynamics (MD) simulations showed that at lower frequencies and pressures, the structural flexibility of dioscorin's main chain atoms increased, leading to more significant fluctuations between amino acid residues. This transformation improved dioscorin's emulsifying properties and its oil-water interface affinity.