RESUMEN
The ultrafast decay dynamics of N-methyl-2-pyridone upon excitation in the near-ultraviolet range of 261.5-227.9 nm is investigated using the femtosecond time-resolved photoelectron spectroscopy method. Irradiation at 261.5 nm prepares N-methyl-2-pyridone molecules with high vibrational levels in the 11ππ* state. The radiation-less decay to the ground state via internal conversion is suggested to be the dominant channel for the 11ππ* state with large vibrational excess energy, which is revealed by a lifetime of 1.6 ± 0.2 ps. As the pump wavelength decreases, we found that irradiation at 238.5 and 227.9 nm results in the population of the 21ππ* state. This is in agreement with the assignment of the vapor-phase UV absorption bands of N-methyl-2-pyridone. On the basis of the detailed analysis of our measured time-resolved photoelectron spectra at all pump wavelengths, we conclude that the 21ππ* state has an ultrashort lifetime of 50 ± 10 fs. In addition, the S1(11ππ*) state is subsequently populated via internal conversion and decays over a lifetime of 680-620 fs. The most probable whole deactivation pathway of the 21ππ* state is discussed. This experimental study provides new insights into the excitation energy-dependent decay dynamics of electronically excited N-methyl-2-pyridone.
RESUMEN
Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.
Asunto(s)
Acetil-CoA Carboxilasa , Ligasas de Carbono-Nitrógeno , Chloroflexus , Acetil-CoA Carboxilasa/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/química , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/química , Chloroflexus/genética , Chloroflexus/metabolismo , Chloroflexus/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Biotina/metabolismo , Biotina/biosíntesis , Malonil Coenzima A/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Acido Graso Sintasa Tipo IIRESUMEN
Methanol-gasoline blends have emerged as a promising and environmentally friendly bio-fuel option, garnering widespread attention and promotion globally. The methanol content within these blends significantly influences their quality and combustion performance. This study explores the qualitative and qualitative analysis of methanol-gasoline blends using Raman spectroscopy coupled with machine learning methods. Experimentally, methanol-gasoline blends with varying methanol concentrations were artificially configured, commencing with initial market samples. For qualitative analysis, the partial least squares discriminant analysis (PLS-DA) model was employed to classify the categories of blends, demonstrating high prediction performance with an accuracy of nearly 100% classification. For the quantitative analysis, a consensus model was proposed to accurately predict the methanol content. It integrates member models developed on clustered variables, using the unsupervised clustering method of the self-organizing mapping neural network (SOM) to accomplish the regression prediction. The performance of this consensus model was systemically compared to that of the PLS model and uninformative variable elimination (UVE)-PLS model. Results revealed that the unsupervised consensus model outperformed other models in predicting the methanol content across various types of methanol gasoline blends. The correlation coefficients for prediction sets consistently exceeded 0.98. Consequently, Raman spectroscopy emerges as a suitable choice for both qualitative and quantitative analysis of methanol-gasoline blend quality. This study anticipates an increasing role for Raman spectroscopy in analysis of fuel composition.
RESUMEN
Alternative complex III (ACIII) couples quinol oxidation and electron acceptor reduction with potential transmembrane proton translocation. It is compositionally and structurally different from the cytochrome bc1/b6f complexes, but functionally replaces these enzymes in the photosynthetic and/or respiratory electron transport chains (ETCs) of many bacteria. However, the true compositions and architectures of ACIIIs remain unclear, as do their structural and functional relevance in mediating the ETCs. We here determined cryogenic electron microscopy structures of photosynthetic ACIII isolated from Chloroflexus aurantiacus (CaACIIIp), in apo-form and in complexed form bound to a menadiol analog 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Besides six canonical subunits (ActABCDEF), the structures revealed conformations of two previously unresolved subunits, ActG and I, which contributed to the complex stability. We also elucidated the structural basis of menaquinol oxidation and subsequent electron transfer along the [3Fe-4S]-6 hemes wire to its periplasmic electron acceptors, using electron paramagnetic resonance (EPR), spectroelectrochemistry, enzymatic analyses and molecular dynamics (MD) simulations. A unique insertion loop in ActE was shown to function in determining the binding specificity of CaACIIIp for downstream electron acceptors. This study broadens our understanding of the structural diversity and molecular evolution of ACIIIs, enabling further investigation of the (mena)quinol oxidoreductases evolved coupling mechanism in bacterial energy conservation.
RESUMEN
The male reproductive system has a standard immune response regulatory mechanism, However, a variety of external stimuli, including viruses, bacteria, heat, and medications can damage the testicles and cause orchitis and epididymitis. It has been shown that various RNA viruses are more likely to infect the testis than DNA viruses, inducing orchitis and impairing testicular function. It was found that local injection of the viral RNA analog poly(I:C) into the testes markedly disrupted the structure of the seminiferous tubules, accompanied by apoptosis and inflammation. Poly(I:C) mainly inhibited the expression of testosterone synthesis-associated proteins, STAR and MGARP, and affected the synthesis and metabolism of amino acids and lipids in the testis. This led to the disruption of the metabolite levels in the testis of mice, thus affecting the normal spermatogenesis process. The present study analyzed the acute inflammatory response of the testis to viral infection using a multi-omics approach. It provides insights into how RNA virus infection impairs testicular function and offers a theoretical basis for future studies on immune homeostasis and responses under stress conditions in male reproduction.
RESUMEN
The ultrafast decay dynamics of pyridine-N-oxide upon excitation in the near-ultraviolet range of 340.2-217.6 nm is investigated using the femtosecond time-resolved photoelectron imaging technique. The time-resolved photoelectron spectra and photoelectron angular distributions at all pump wavelengths are carefully analyzed and the following view is derived: at the longest pump wavelengths (340.2 and 325.6 nm), pyridine-N-oxide is excited to the S1(1ππ*) state with different vibrational levels. The depopulation rate of the S1 state shows a marked dependence on vibrational energy and mode, and the lifetime is in the range of 1.4-160 ps. At 289.8 and 280.5 nm, both the second 1ππ* state and the S1 state are initially prepared. The former has an extremely short lifetime of â¼60 fs, which indicates that the ultrafast deactivation pathway such as a rapid internal conversion to one close-lying state is its dominant decay channel, while the latter is at high levels of vibrational excitation and decays within the range of 380-520 fs. At the shortest pump wavelengths (227.3 and 217.6 nm), another excited state of Rydberg character is mostly excited. We assign this state to the 3s Rydberg state which has a lifetime of 0.55-2.2 ps. This study provides a comprehensive picture of the ultrafast excited-state decay dynamics of the photoexcited pyridine-N-oxide molecule.
RESUMEN
BACKGROUND: Postoperative thirst is common in patients admitted to the intensive care unit. Existing methods like wet cotton swabs or oral care prove ineffectual or operationally intricate. Currently, an efficacious postoperative thirst alleviation method remains elusive. Exploring a prompt, safe, and efficacious solution is of paramount importance. OBJECTIVE: To assess the effect of ice-cold water spray applied following a symptom management model on postoperative thirst and to establish a framework for mitigating thirst in intensive care unit patients. RESEARCH DESIGN: Single-center randomized controlled study. SETTING: Surgical intensive care unit in a university-affiliated hospital. MAIN OUTCOME MEASURES: 56 intensive care unit patients were selected and equally randomized. The experimental group received ice-cold water spray in conjunction with eight symptom management strategies, while the control group underwent standard care involving wet cotton swabs. Thirst intervention was initiated 0.5 hours after postoperative extubation, followed by subsequent interventions at 2-hour, 4-hour, and 6-hour intervals post-extubation. Thirst intensity, oral comfort, and the duration of relief from thirst were assessed and compared between groups before and 0.5 hours after each thirst intervention. RESULTS: Across different interventions, the experimental group exhibited superior scores in thirst intensity and oral comfort compared to the control group. Additionally, the nursing time required to alleviate thirst in the experimental group was significantly shorter than that in the control group (P < 0.01). CONCLUSION: Ice-cold water spray following the model for symptom management can effectively mitigate the postoperative thirst intensity in intensive care unit patients, improve oral comfort, and reduce the nursing time for relieving thirst. IMPLICATIONS FOR CLINICAL PRACTICE: Clinical nurses can employ ice-cold water spray following the model for symptom management to ameliorate postoperative thirst intensity in ICU patients while enhancing oral comfort. Furthermore, the utilization of ice-cold water spray can reduce the nursing time required for relieving postoperative thirst in intensive care unit patients.
Asunto(s)
Sed , Agua , Humanos , Cuidados Críticos/métodos , Unidades de Cuidados IntensivosRESUMEN
Roseiflexus castenholzii is a gram-negative filamentous phototrophic bacterium that carries out anoxygenic photosynthesis through a cyclic electron transport chain (ETC). The ETC is composed of a reaction center (RC)-light-harvesting (LH) complex (rcRC-LH); an alternative complex III (rcACIII), which functionally replaces the cytochrome bc1/b6f complex; and the periplasmic electron acceptor auracyanin (rcAc). Although compositionally and structurally different from the bc1/b6f complex, rcACIII plays similar essential roles in oxidizing menaquinol and transferring electrons to the rcAc. However, rcACIII-mediated electron transfer (which includes both an intraprotein route and a downstream route) has not been clearly elucidated, nor have the details of cyclic ETC. Here, we identify a previously unknown monoheme cytochrome c (cyt c551) as a novel periplasmic electron acceptor of rcACIII. It reduces the light-excited rcRC-LH to complete a cyclic ETC. We also reveal the molecular mechanisms involved in the ETC using electron paramagnetic resonance (EPR), spectroelectrochemistry, and enzymatic and structural analyses. We find that electrons released from rcACIII-oxidized menaquinol are transferred to two alternative periplasmic electron acceptors (rcAc and cyt c551), which eventually reduce the rcRC to form the complete cyclic ETC. This work serves as a foundation for further studies of ACIII-mediated electron transfer in anoxygenic photosynthesis and broadens our understanding of the diversity and molecular evolution of prokaryotic ETCs.
Asunto(s)
Proteínas Bacterianas , Chloroflexi , Grupo Citocromo c , Citocromos c , Transporte de Electrón , Chloroflexi/química , BacteriasRESUMEN
With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
Asunto(s)
Cabras , Células Madre , Animales , Masculino , Espermatogonias/metabolismo , Proliferación Celular , Citometría de Flujo , Testículo/metabolismoRESUMEN
Spermatogonial stem cells (SSCs) play a crucial role in mammalian spermatogenesis and maintain the stable inheritance of the germline in livestock. However, stress and bacterial or viral infections can disrupt immune homeostasis of the testes, thereby leading to spermatogenesis destruction and infertility, which severely affects the health and productivity of mammals. This study aimed to explore the effect of ubiquitin C-terminal hydrolase L1 (UCHL1) knockdown (KD) in goat SSCs and mouse testes and investigate the potential anti-inflammatory function of UCHL1 in a poly(I:C)-induced inflammation model to maintain microenvironmental homeostasis. In vitro, the downregulation of UCHL1 (UCHL1 KD) in goat SSCs increased the expression levels of apoptosis and inflammatory factors and inhibited the self-renewal and proliferation of SSCs. In vivo, the structure of seminiferous tubules and spermatogenic cells was disrupted after UCHL1 KD, and the expression levels of apoptosis- and inflammation-related proteins were significantly upregulated. Furthermore, UCHL1 inhibited the TLR3/TBK1/IRF3 pathway to resist poly(I:C)-induced inflammation in SSCs by antagonizing HSPA8 and thus maintaining SSC autoimmune homeostasis. Most importantly, the results of this study showed that UCHL1 maintained immune homeostasis of SSCs and spermatogenesis. UCHL1 KD not only inhibited the self-renewal and proliferation of goat SSCs and spermatogenesis but was also involved in the inflammatory response of goat SSCs. Additionally, UCHL1 has an antiviral function in SSCs by antagonizing HSPA8, which provides an important basis for exploring the specific mechanisms of UCHL1 in goat spermatogenesis.
Asunto(s)
Cabras , Espermatogonias , Animales , Masculino , Ratones , Homeostasis , Inflamación/metabolismo , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Células Madre , Testículo/metabolismoRESUMEN
Blocking the PI3K/Akt pathway has been widely recognized as an attractive cancer therapeutic strategy because of its crucial role in cell growth and survival. This study presents the synthesis of 24 new 5-Methoxy-6-substituted-1H-benzimidazole derivatives (4a-4x) and the evaluation of their anti-proliferative activities against A549, Siha, MCF-7, HepG2, PC3, and HCT-116 tumor cell lines through MTT assay. Compound 4w exhibited superior anti-tumor activity against the A549 cells with IC50 values of 1.55 ± 0.18 µM, and better than the BKM120 (IC50 = 9.75 ± 1.25 µM). Further studies indicated that 4w could induce G0/G1 phase arrest, cell apoptosis, and down-regulate expression of p-PI3K and p-Akt. These results indicate that 4w could be served as a lead compound of PI3K inhibitor for the treatment of human lung cancers.
RESUMEN
Astragalus species have a certain capacity to enrich selenium (Se) and are the strongest Se hyperaccumulator legumes known globally at present. The biochar application to medicinal plants has been reported to affect plant metabolites. In this study, we aimed to employ hyperaccumulating Astragalus species in the plant growth of selenium-lacked soil, while also investigating the impact of varying selenium doses and biochar application on legumes growth, selenium content, and secondary metabolite production. Applying biochar to soil, along with a Se concentration of 6 mg/kg, significantly enhanced the growth, Se content, total polysaccharide content, and calycosin-7-glucoside content of Astragalus species (p < 0.05). Importantly, the Se and biochar application also led to a significant improvement in Se content in ABH roots (p < 0.05). Meanwhile, the content of total flavonoids in ABH roots could be promoted by a Se concentration of 3 mg/kg and biochar application in soil. Additionally, the Se enrichment coefficients of Astragalus species under Se treatments were significantly higher than those under control treatment, with a marked difference observed across all treatments, whether roots or above-ground (p < 0.05). Remarkably, the Se transport coefficients of Astragalus species were observed to be lower than one, except for the transport coefficient of AB in the Se concentration of the control treatment (0 mg/kg). This result showed that a medium concentration treatment of Se and biochar application in soil not only promotes the growth of Astragalus species and the uptake of exogenous Se but also increases the active component content, meanwhile enhancing the Se enrichment and transport capacity. Taken as a whole, the present findings offer a more comprehensive understanding of the interplay between distinct Se levels, as well as the addition of biochar in soil, providing valuable insight for the cultivation of Se-rich Astragalus in Se-deficient soil-plant systems.
RESUMEN
Eif2s3y (eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked, Eif2s3y) is an essential gene for spermatogenesis. Early studies have shown that Eif2s3y can promote the proliferation of spermatogonial stem cells (SSCs) and can replace the Y chromosome together with sex-determining region Y (Sry) to transform SSCs into round spermatozoa. We injected lentiviral particles into the seminiferous tubules of mouse testes by sterile surgery surgically to establish overexpressing Eif2s3y testes. And then the mice were intraperitoneally injected with LPS to established the model of testis inflammation. Through RNA sequencing, qRT-PCR analysis, Western blot, co-culture etc., we found that Eif2s3y alleviated LPS-induced damage in mouse testes and maintained spermatogenesis. In testes with Eif2s3y overexpression, the seminiferous tubules were more regularly organized after exposure to LPS compared with the control. Eif2s3y performs its function by negatively regulating Adamts5 (a disintegrin and metalloproteinase containing a thrombospondin-1 motif), an extracellular matrix-degrading enzyme. ADAMTS5 shows a disruptive effect when the testis is exposed to LPS. Overexpression of Eif2s3y inhibited the TLR4/NFκB signaling pathway in the testis in response to LPS. Generally, our research shows that Eif2s3y protects the testis from LPS and maintains spermatogenesis by negatively regulating Adamts5.
Asunto(s)
Lipopolisacáridos , Testículo , Masculino , Ratones , Animales , Testículo/metabolismo , Lipopolisacáridos/toxicidad , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Túbulos Seminíferos , Espermatogonias , Proteína ADAMTS5 , Factores de Transcripción/metabolismoRESUMEN
Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.
Asunto(s)
Alcohol Deshidrogenasa , Chloroflexi , NADP/metabolismo , Microscopía por Crioelectrón , Oxidorreductasas/metabolismo , Chloroflexi/metabolismo , Aldehído Deshidrogenasa , Malonil Coenzima A/metabolismoRESUMEN
Bacterial or viral infections, such as Brucella, mumps virus, herpes simplex virus, and Zika virus, destroy immune homeostasis of the testes, leading to spermatogenesis disorder and infertility. Of note, recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells, leading to male reproductive dysfunction. Due to the many side effects associated with antibiotic therapy, finding alternative treatments for inflammatory injury remains critical. Here, we found that Dmrt1 plays an important role in regulating testicular immune homeostasis. Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) revealed that Dmrt1 positively regulated the expression of Spry1, an inhibitory protein of the receptor tyrosine kinase (RTK) signaling pathway. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) analysis indicated that SPRY1 binds to nuclear factor kappa B1 (NF-κB1) to prevent nuclear translocation of p65, inhibit activation of NF-κB signaling, prevent excessive inflammatory reaction in the testis, and protect the integrity of the blood-testis barrier. In view of this newly identified Dmrt1- Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis, our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.
Asunto(s)
Fertilidad , Homeostasis , FN-kappa B , Testículo , FN-kappa B/metabolismo , Fertilidad/genética , Fertilidad/inmunología , Humanos , Masculino , Testículo/inmunología , Testículo/metabolismo , Homeostasis/inmunología , Animales , Ratones , Células HEK293 , Espermatogénesis , Inflamación , Regiones Promotoras Genéticas/genética , Activación Transcripcional , Técnicas de Silenciamiento del GenRESUMEN
BACKGROUND: Combretastatin A-4 (CA-4) is a natural product isolated from the bark of the South African bush willow tree Combretum caffrum, which exerts tubulin inhibition, but its clinical application is limited due to poor stability and water solubility. 2-aryl benzimidazoles are excellent pharmacological skeletons with many activities, especially in tumor inhibition, and better pharmacokinetic properties. Several scaffold CA-4 analogs have been synthesized to date possessing antitumor activities. OBJECTIVE: The benzimidazole was applied as the core moiety to replace the B ring and unstable linkage of CA-4, and the 5-aryl acetenyl group was introduced to improve the antitumor activity. MCF-7, A549, Caco-2, Siha, and Eca-109 tumor cell lines were used to study inhibition by these agents in vitro. METHODS: The benzimidazole structure was constructed from the oxidation of o-nitroaniline and aldehyde and the following schemes, and the structural characterization was carried out. The antitumor effects were evaluated in vitro through MTT assay, cell cycle arrest, and apoptosis assay. Molecular docking with tubulin (Protein ID: 1SA0) was analyzed for the structure-activity relationship. RESULTS: Among these derivatives, 4a-4h series (with 6-methoxy group) compounds inhibited the tumor cell lines much stronger than the CA-4 and cisplatin, especially compound 4f showed prominently inhibitory activity in Siha cell with IC50 value as 0.61 µmol/L. The further assay showed that the cell cycle was arrested at the G0/G1 phase as well verified in apoptosis assay. Molecular docking indicated that 4f had stronger affinity energy and hydrogen bond than CA-4. CONCLUSION: The compound 4f has the potency to be used as an anti-tubulin agent and the 2-trimethoxyphenyl benzimidazole skeleton deserves further study as an antitumor structure.
Asunto(s)
Antineoplásicos , Bencimidazoles , Humanos , Simulación del Acoplamiento Molecular , Células CACO-2 , Ensayos de Selección de Medicamentos Antitumorales , Relación Estructura-Actividad , Línea Celular Tumoral , Bencimidazoles/química , Tubulina (Proteína)/metabolismo , Antineoplásicos/química , Proliferación Celular , Estructura Molecular , Diseño de FármacosRESUMEN
Metalloproteinases include matrix metalloproteinases and disintegrin metalloproteinases. They are important members of the ECM degradation and reconstruction process and are associated with tissue development and disease. The ECM is a three-dimensional network of large molecules consisting of a variety of proteins. It is a physical scaffold for organs, and all types of cells can be found within the ECM. The testicle, where sperm are produced, is an organ that is constantly in dynamic flux. Metalloproteinases can regulate testicular tissue development and the maturation of sperm by affecting the ECM. Metalloproteinase disorders can lead to cryptorchidism, azoospermia, poor semen quality and other diseases. As a member of the metalloproteinase family, ADAMTS plays an important role in testicular slippage to the scrotum. ADAM is involved in the fertilization process, and excessive MMP can damage the BTB. In the testis, metalloproteinase stability represents the stability of the extracellular microenvironment in which germ cells are located and is associated with reproductive function. Metalloproteinases have a definite relationship with male reproduction, but the underlying mechanism is still unclear. This paper summarizes the literature on various metalloproteinases in testicular tissue physiology and pathology to elucidate their role in reproductive function and male reproductive mechanisms.
Asunto(s)
Análisis de Semen , Semen , Masculino , Animales , Análisis de Semen/veterinaria , Semen/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Mamíferos , Metaloproteinasas de la Matriz/metabolismoRESUMEN
Quasi-2D perovskites have demonstrated great application potential in light-emitting diodes (LEDs). Defect passivation with chemicals plays a critical role to achieve high efficiency. However, there are still challenges in comprehensively passivating the defects distributed at surface, bulk, and buried interface of quasi-2D perovskite emitting films, hindering the further improvement of device performance. Herein, 9,9-substituted fluorene derivatives with different terminal functional groups are developed tactfully to realize comprehensive passivation, which greatly contributes to reducing nonradiative recombination at surface, suppressing ion migration in bulk, and filling interfacial charge traps at buried interface, respectively. Eventually, quasi-2D perovskite LEDs have an increased external quantum efficiency from 18.2% to 23.2%, improved operation lifetime by more than six times and lower turn-on voltage simultaneously. Here the importance of comprehensive passivation is highlighted and guidelines for the design and application of passivators for perovskite optoelectronics are provided.
RESUMEN
A high performance liquid chromatography(HPLC) method was established to analyze the components in Shengjiang Powder(SJP) such as emodin and curcumin and explore its therapeutic effect on experimental autoimmune encephalomyelitis(EAE) mice. To be specific, HPLC was performed to determine the content of compounds in SJP such as emodin and curcumin. A total of 72 female SPF C57 BL/6 mice were randomized into control group(equivalent volume of ultrapure water, ig), model group(equivalent volume of ultrapure water, ig), low-, medium-, and high-dose SJP groups(SJP, ig), and positive control group(prednisone acetate, ig), 12 each group. EAE was induced in mice except the control group. Administration began from the first day after immunization. The general conditions, symptom score, and body weight of the mice were recorded. On the 21 st day, mouse brain tissues were separrated. Then hematoxylin-eosin(HE) staining and Luxol Fast Blue(LFB) staining were used to detect the pathological changes of brain tissues. Immunohistochemistry(IHC) was employed to determine the myelin basic protein(MBP) level, and Western blot the expression of occludin and claudin-5, as well as the levels of interleukin-6(IL-6) and proteins in the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway and their phosphorylation levels. The mRNA expression of IL-6, JAK2, and STAT3 was detected by real-time quantitative polymerase chain reaction(qPCR). Finally, molecular docking of six main active components in SJP, including emodin and curcumin, with IL-6, JAK2 and STAT3 was performed, and the binding affinity was evaluated. The results showed that the established HPLC method demonstrated high precision, reproducibility, stability, and high recovery of samples. Compared with the model group, SJP reduced the clinical symptom score and alleviate the inflammatory infiltration of brain white matter and demyelination of EAE mice. At the same time, SJP increased the expression of occludin and claudin-5, down-regulated the mRNA expression of IL-6, JAK2, and STAT3, as well as the levels of IL-6/JAK/STAT3 proteins and the phosphorylation levels, with significant difference. Molecular docking suggested that the six active components in SJP had high binding energy with IL-6, JAK2, and STAT3 proteins. The established HPLC method is simple, accurate, and highly sensitive, which can simultaneously determine the content of emodin and curcumin in SJP. SJP may alleviate the clinical symptoms of EAE by inhibiting IL-6/JAK2/STAT3 signaling pathway, protecting the blood-brain barrier, and relieving the inflammatory response and demyelinization of brain tissue.
Asunto(s)
Curcumina , Emodina , Encefalomielitis Autoinmune Experimental , Animales , Cromatografía Líquida de Alta Presión , Claudina-5/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Femenino , Interleucina-6/genética , Interleucina-6/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Ocludina/metabolismo , Polvos , ARN Mensajero , Reproducibilidad de los Resultados , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Agua/metabolismoRESUMEN
Coenzyme A (CoA) transferases catalyze reversible transfer of CoA groups from CoA-thioesters to free acids, playing important roles in the metabolism of carboxylic acids in all organisms. An intramolecular CoA transferase, Mesaconyl-CoA C1-C4 CoA transferase (MCT) was identified in the autotrophic CO2 fixation pathway, 3-hydroxypropionic acid cycle of filamentous anoxygenic phototrophs (FAPs). Different from the well-known CoA transferases that catalyze CoA transfer between two distinct substrates, MCT specifically catalyzes the reversible transformation of mesaconyl-C1-CoA to mesaconyl-C4-CoA, a key reaction intermediate for carbon fixation. However, the molecular mechanism of MCT in employing one substrate is enigmatic. Here we determined the crystal structure of MCT from a chlorosome-less FAP Roseiflexus castenholzii at 2.5 Å resolution, and characterized the catalytic mechanisms through structural analyses and molecular dynamic simulations. The structure of R. castenholzii MCT consists of a Rossmann fold larger domain and a small domain that are connected by two linkers. Two MCT subunits are cross interlocked at the linker regions to form a functional dimer in solution, in which the substrate binding pockets are located at the interface of the Rossmann fold larger domain from one subunit and the small domain from the other subunit. In the simulated binding structures, both the substrate mesaconyl-C1-CoA and product mesaconyl-C4-CoA form extensive electrostatic and hydrogen bonding interactions with MCT. But some differences exist in the binding mode of these two CoA analogs, Arg314' from the second subunit of the dimer presenting dramatic conformational changes in binding with mesaconyl-C4-CoA. Together with Arg47 and one water molecule, a strictly conserved residue Asp165 are essential for catalyzing the reversible intramolecular CoA transfer reaction, through the electrostatic and hydrogen bonding interactions with the mesaconic tail of both the substrate and product. This study revealed a previously unrecognized mechanism for the uncommon intramolecular CoA transfer reaction, which will not only broaden the knowledge on the catalytic mechanisms of CoA transferases, but also contribute to enzyme engineering or biosynthetic applications of the 3-HP cycle for synthesis of fine chemicals and important metabolites.